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+{"text": "Between November 2 and 10, 2002 several patients with psoriasis and personnel staying in the health centre in Gran Canaria, Spain fell ill with diarrhoea, vomiting or both. Patient original came from Norway, Sweden and Finland. The patient group was scheduled to stay until 8 November. A new group of patients were due to arrive from 7 November.A retrospective cohort study was conducted to assess the extent of the outbreak, to identify the source and mode of transmission and to prevent similar problems in the following group.Altogether 41% (48/116) of persons staying at the centre fell ill. Norovirus infection was suspected based on clinical presentations and the fact that no bacteria were identified. Kaplan criteria were met. Five persons in this outbreak were hospitalised and the mean duration of diarrhoea was 3 days. The consequences of the illness were more severe compared to many other norovirus outbreaks, possibly because many of the cases suffered from chronic diseases and were treated with drugs reported to affect the immunity (methotrexate or steroids).During the two first days of the outbreak, the attack rate was higher in residents who had consumed dried fruit  and strawberry jam  than those who did not. In the following days, no association was found. The investigation suggests two modes of transmission: a common source for those who fell ill during the two first days of the outbreak and thereafter mainly person to person transmission. This is supported by a lower risk associated with the two food items at the end of the outbreak.We believe that the food items were contaminated by foodhandlers who reported sick before the outbreak started. Control measures were successfully implemented; food buffets were banned, strict hygiene measures were implemented and sick personnel stayed at home >48 hours after last symptoms. On 5 November 2002, the Department of Infectious Disease Epidemiology at the Norwegian Institute of Public Health (NIPH) was contacted by a dermatologist at Rikshospitalet, a tertiary care university hospital in Oslo, Norway. He reported several cases of gastro-enteritis in a medical care centre in Arg\u00faineg\u00fain, Gran Canaria, Spain where Nordic patients with psoriasis undergo climate therapy. The symptoms (diarrhoea and vomiting) were reported to be of short duration (1\u20132 days). The patient group was scheduled to stay until 8 November. A new group of patients were due to arrive from 7 November. An outbreak management team was created in order to investigate and control the current outbreak, and to prevent similar problems in the following group. The outbreak management team consisted of investigators working in public health institutes in the countries involved . The investigation was organised and co-ordinated from the NIPH in Oslo, Norway.The objectives of this investigation were to assess the extent of the outbreak, identify the mode of transmission, the vehicle and the causative pathogen and recommend appropriate control measures.The health centre is a private institution owned by the Norwegian Asthma and Allergy Association. Every three weeks the health centre receives a group of 100\u2013110 Nordic patients suffering from skin diseases (mainly psoriasis). Rikshospitalet, Oslo, Norway administers the project. Patients are residents of Norway, Sweden and Finland. On site, Nordic medical staff  provides health services for the patients. The centre also hosts private individuals and employees of the Asthma and Allergy Association.We conducted a retrospective cohort study among all Nordic patients attending dermatological care and employees staying at the health centre between 2 and 10 November. The persons from the Asthma and Allergy Association, and employees eating and staying outside the centre in the same time period were not included since there were no reported cases among this group and they did not eat or use the same facilities as the dermatological patients.A case was defined as a person who 1) attended dermatological care or worked at the Health Centre in Gran Canaria, 2) took meals at the Centre and 3) fell ill between the 2 and 10 November 2002, with symptoms of diarrhoea (3 or more loose stools in 24 hours), vomiting or both.Health personnel at the health centre provided a guest list with name, address, age, sex and bungalow number. For cases they also recorded date of onset of symptoms and stool sampling and results of microbiological analysis.A standard questionnaire was developed at the Norwegian Institute of Public Health, translated in each national centre and mailed to the entire cohort, with instructions to fill it in and send it back to the public health institute in their country of origin.The local health authorities in Gran Canaria enquired about gastro-intestinal illness in the community. They contacted health centres, churches and schools in the area.An exposure was defined as a food item eaten or an environmental factor present within the 2 days before onset of illness . Possible exposures included consumption of all food items served in the health centre's restaurant, consumption of food served outside the centre, brushing teeth in tap water, drinking tap water, consumption of ice cubes, swimming in one of the two pools, swimming in the ocean, having had contact with an ill person, hand washing habits before meals, and the sharing of bungalow with symptomatic persons. We recorded the number of times food items were eaten to enable the study of potential dose-response relationships.Day by day, we compared food-specific attack rates (AR) for each item for the exposed and the non-exposed. Suspecting the etiological agent to be norovirus and knowing it has a short incubation period (24\u201348 hours)  only fooWe used EpiData software  for data collection and analysed them with SPSS version 10.0 . Attack rates, relative risk and 95% confidence intervals were calculated for each of the food items and other exposures. Only variables with RR greater than 2.0 are presented in this report. A multivariable analysis was run to assess potential confounding, including age, gender and the pooled variables with RR greater than 2.0 in the univariable analysis.All cases in the cohort presenting gastro-enteric symptoms were encouraged to deliver a stool sample. The samples were sent to a local private laboratory in Gran Canaria. Both virological and bacterial tests were requested.Food sampling of some of the served items was performed on 5, 21 and 22 November by the local health authorities in Gran Canaria. The health authorities also took samples on 12 November from water taps and from the pools. Both stool and environmental samples were to be sent to Madrid for virology testing. Due to misunderstandings the samples were never forwarded to Madrid. Virological analyses were therefore not performed.We mailed 110 questionnaires to the patients who stayed at the Centre between 17 October and 8 November 2002, and employees (n = 6) eating at the centre. Ninety-one questionnaires (response rate = 78%) were returned .Among the 91 respondents there were 47 men and 44 women. The median age was 48 years (range 18\u201380).Forty-eight persons fulfilled the case definition (attack rate (AR) = 53%). The AR was not significantly different by genders or nationalities. The AR was higher among those above 70 years of age (80%).The outbreak peaked on November 4, with 16 cases , AR for those sharing room with one person 38% (3/8) and AR among those 9 living in a single room was 44%. Two cases were employees living private or in a separate section. Six cases fell ill subsequent (more than 10 hours later and within two days) to their roommate.There were no gastro-enteritis cases reported in the community concurrently to the outbreak in the health centre.From the daily analysis of food consumption, strawberry jam, dried fruit (eaten on both 1 and 2 November) and butter eaten on 31 October) were associated with risk of illness (RR> 2.0) for those who became ill on 2 and 3 November  and strawberry jam  were independently associated with disease  were reported negative for bacteria at the private laboratory in Gran Canaria. Virological analyses were not performed.Six food items and water from the two pools were analysed for bacteria: chicken croquettes, frozen chicken breast and leg, sausage, cheese and salmon. Results were negative.We initiated an epidemiological investigation of an outbreak of gastro-enteritis in a health centre in Gran Canaria, Spain. Based on Kaplan criteria, our findings suggest that the aetiological agent was norovirus. Results of the cohort study suggests that the outbreak was initiated by ingestion of dried fruits, strawberry jam or both, followed by person-to-person transmission. There is no supporting microbiological or environmental evidence.Norovirus outbreaks can often be diagnosed presumptively on clinical grounds from their characteristic epidemiological features . Kaplan A high proportion of persons in this outbreak reported that they received IV-treatment , were hospitalised n = 5) and the mean duration of diarrhoea was 3.7 days. The reported consequences, especially the duration of the illness was severe compared to many other norovirus outbreaks  and the  and in hWe believe there were two modes of transmission: a common source for those who fell ill on 2 and 3 November and thereafter mainly person to person transmission. Low RR for all food items at the end of the outbreak and the short incubation period for noroviruses, together with the fact that norovirus outbreaks usually have high rates of person-to-person transmission , supportWe did however not find a higher attack rate among those who shared room or bungalow. Only nine persons had a single room.There were no cases among those persons who stayed at the health centre, but did not eat in the health centre restaurant or in the community during the outbreak. This suggests a foodborne outbreak with its origin at the centre's restaurant.Dried fruits, strawberry jam or both were the probable vehicles of contamination. Strawberry jam and dried fruits were handled and kept at the kitchen in the Health Centre and not supplied by the catering service. They were served in buffet style on plates. Both food items are biologically plausible vehicles. Contaminated hands or silverware could explain contamination of these food items, but these modes of transmission were not verified. One hypothesis was that the food items were contaminated by the foodhandlers who reported being ill before the outbreak started. Both foodhandlers were involved in preparing the food for the buffet. Very few organisms of norovirus are needed to transmit the disease . The jamDose-response analyses give further support for contamination of the strawberry jam. Those who ate jam twice doubled their risk of developing gastro-enteritis.Suspecting norovirus, with a short incubation period, as the causal agent , we treaWhen looking at the whole period as a cohort and thus looking at all persons falling ill between 2 and 10 November, then, none of the exposures seemed to increase the risk of disease. The association between food items and disease was probably masked by a high number of cases infected by person-to-person transmission.Eight cases had not eaten dried fruit during the 2 days before falling ill. The concept of dried fruit is different in Spain from Scandinavia. In Spain, dried fruit means raisins, which were served in the restaurant. In the Nordic countries, dried fruit is in general understood to be a mixture of different types of dried fruits. This difference in concepts may have introduced an information bias.We asked for food history for 5 days. The food was always served buffet-style. The menus included more than 300 different food items. To reduce potential recall bias, we pooled the food items consumed on either of the 2 days prior to onset. The fact that strawberry jam and dried fruit remained associated for those with onset 2 and 3 November , we recommended that this group should travel as planned.Salmonella was not found in any of the 22 stool-samples from our cohort. This, together with the reported symptoms, suggests that there may have been two different outbreaks at the centre during this period.Among persons arriving 7 November (n = 100), 18 became ill with gastro-enteritis. There were no cases after 14 November. Four of seven stool samples were positive for Salmonella. This finding did not change the belief that norovirus caused the gastro-enteritis among persons in the outbreak described in this article. Between 2 and 7 November, 66 persons fell ill with diarrhoea, vomiting or both in a health centre for Nordic patients with skin diseases at Gran Canaria. Our data suggest that norovirus caused the outbreak.Our findings suggest that individuals who consumed dried fruit  or strawberry jam  were more likely to contract the disease. One hypothesis is that the food items were contaminated by foodhandlers that had had gastro-enteritis shortly before the outbreak started.Improved hygiene measures and individually served food were successfully implemented.Norwegian Institute of Public Health; NIPHAttack rate; ARRelative risk RRThe author(s) declare that they have no competing interests.HME: In charge of the investigation, data handeling and writing of the articlePJG: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article.KN: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article.MH: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article.BdJ: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article.AMCR: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article.MK: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article.UD: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article.AGR: Working locally with the outbreak, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article.CM: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article.PA: In the outbreak management team, contributed in writing and distribution of the questionaire, and review and comment on the different versions of the article.The pre-publication history for this paper can be accessed here:"}
+{"text": "Noroviruses  being a leading cause of acute gastroenteritis outbreaks, the impact of NV infection is at present unknown and little information is available about strains circulating in Italy. In April 2002 an outbreak of gastroenteritis occurred in the province of Bari , involving several households.Despite A retrospective cohort study was performed in order to assess risk factors associated with illness. All households where a case occurred were included in the study. Faecal specimens were collected from ill individuals. NV-specific RT-PCR was performed. Eleven samples of mussels were collected from fish-markets involved in the outbreak. A nested PCR was used for mussel samples.One hundred and three cases, detected by means of active surveillance, met the case definition. Raw shellfish eating was the principal risk factor for the disease, as indicated by the analytic issues . NVs were found by means of RT-PCR of all the stool specimens from the 24 patients tested. Eleven samples of shellfish from local markets were tested for the presence or NVs; six were positive by nested PCR and genotypes were related to that found in patients' stools.This is the first community outbreak caused by NVs related to sea-food consumption described in Italy. The study confirms that the present standards for human faecal contamination do not seem to be a reliable indicator of viral contaminants in mussels. Norovirus , one of four genera in the Caliciviridae family, includes a group of morphologically similar but genetically different single-stranded RNA viruses. NVs represent the most important cause of non-bacterial gastroenteritis worldwide. In industrialised countries NVs may be responsible for up to 80% of all outbreaks of gastroenteritis [nteritis . OutbreaContaminated food or water commonly represents the main source of infection. Epidemics spread by the faecal-oral route, even if transmission may also occur through direct person-to-person contact or aerosolised viral particles.The incubation period of NV gastroenteritis is 24\u201348 hours and symptoms include vomiting, diarrhoea, abdominal pain, low-grade fever, headache and myalgia.Italy has no specific surveillance system for viral gastroenteritis and laboratory diagnosis is only carried out in a few cases. Therefore the impact of NV infection is currently unknown and little information is available about circulating strains. Outbreak investigations are usually performed by local health units. Gastroenteritis notifications are often too delayed to identify the etiologic agent and source of infection correctly.Puglia, a region in the South-East of Italy, has about four million inhabitants. Bari  is the capital. Raw mussels are largely consumed in Bari and, in general, in Puglia, especially at Christmas and Easter. In 1994 raw seafood consumption was the source of a small cholera outbreak. Moreover, hepatitis A is endemic in this region.This report describes a large outbreak of NV gastroenteritis that involved several households in Bari province.st and April 7th 2002, in the province of Bari . In Italy, at Easter many people eat in restaurants and the day after Easter most people go picnicking. In the province of Bari seafood  are often consumed on these occasions. The Regional Epidemiological Office, alerted by the Local Health Unit, performed a field study, collecting data from all General Practitioners and all Emergency Units in the province. Active surveillance was conducted until April 15th 2002 (one week after the last case onset). The aim of the investigation was to describe the outbreak and to identify the etiologic agent, the source of infection and the means of transmission.The outbreak occurred during the Easter holidays, between March 31st \u2013 April 7th, who had diarrhoea (three or more loose stools in any 24 hour period) or vomiting (at least one episode). Fever greater than or equal to 38\u00b0C, abdominal pain or nausea were considered additional symptoms. A \"probable\" secondary case was defined as illness in a household with onset of symptoms more than 24 hours after the primary case.A case was defined as illness in a resident in the area during the period March 31A retrospective cohort study was performed in order to assess risk factors associated with illness.All households where a case occurred were included in the study.Information, collected by means of a standardised questionnaire, included: 1) demographic individual characteristics such as age, gender, occupation; 2) type of food consumed during the last 72 hours before onset of symptoms; 3) if ill, type, date and time of onset of symptoms.To assess the association between food consumption and disease, relative risks (RR) and 95% confidence intervals (95% CI) were calculated. Age and gender were compared between ill and unaffected individuals by the chi squared and Mann-Whitney tests. A p-value less than 0.05 was considered significant. Variables associated to the illness in the univariate analysis were included in the stratified analysis; summary RR and 95% CI were calculated from the formulas of Greenland and Robins . CollectFaecal specimens were collected from ill individuals. Part of the specimens was refrigerated and processed within 12 hours to detect ova and parasites by direct microscopy and common bacteria by standard methods. The rest was stored at -20\u00b0C until examination by NV-specific reverse transcription-polymerase chain reaction (RT-PCR).Viral nucleic acid extraction and purification from stool specimens was performed as previously reported . RT-PCR Eleven samples of mussels were collected from two fish-markets from which the cases had bought the shellfish they consumed. Mussel samples were also processed within 12 hours to detect common bacteria by standard methods.Then a nested PCR was used. The procedure for mussel processing as well as viral RNA extraction and purification has been previously described in full .The primers used for first round PCR were JV12 and SM31. Nested PCR was carried out with the use of the primers SR33 for negative strand DNA synthesis and SR48 and SR46 for positive strand synthesis of genogroup I (GI) and genogroup II (GII) sequences, respectively [The 333-bp or 123-bp amplification products from cases and from mussels were subjected to sequencing with PCR primers. When required, cloning was carried out on PCR products. Sequences obtained were aligned with those available in the GenBank.One hundred and three subjects met the case definition; 22 of them were defined as \"probable\" secondary cases. Fifty eight (56.3%) of the 103 cases were female; the mean age was 42.6 years , nausea (58.3%), diarrhoea (53.4%), abdominal pain (47.6%), fever (16.5%). No difference was observed in the clinical pattern by gender. Age was significantly higher in ill individuals with severe diarrhoea (lasting more than 24 hours or undergoing specific treatment) . No difference in age was found for the other symptoms.st at 5,00 pm and lasted eight days. The epidemic curve shows a single peak on April 2nd (35 cases between 1:00 and 12:00 AM) and a right tail probably due to secondary cases  in the days before the onset of symptoms of relatives. All 36 healthy subjects were interviewed. Nineteen (52.8%) of them were female, the mean age was 42.5 years. Gender and age were not significantly different between healthy and ill individuals (table An attack rate of 74.1% (103/139) was observed. In the univariate analysis, the association between raw mussel consumption and illness was significant . The attack rate according to raw mussel consumption was 86.3% (69/80).Even cooked mussel consumption was associated with illness in the univariate analysis . No other food was associated with illness . Such association was not significant in the \"ate raw mussels\" stratum . The relative risk according to cooked mussel consumption was 1.38 (95% CI: 1.00 \u2013 1.91) after correction for raw mussels consumption .Both stool and mussel samples were negative for parasites and bacterial enteropathogens. All stool samples from 24 cases were positive for NV by RT-PCR and 5 of 11 mussel samples by nested PCR. The sequences of strains revealed great heterogeneity. In fact, the simultaneous occurrence of GI and GII viruses within the same outbreak was observed. One sample of mussels showed the presence of a mixed genogroup. Sequence analysis showed that strains from 19 cases and 3 mussel samples had identical sequences and belonged to GII, clustering narrowly to the strain NLV/Tarrag/238/2001/Sp, thus strengthening the epidemiologic link of the mussels to the cases. The nucleotide sequences from three cases and 2 mussel samples formed two other distinct clusters showing the best fit with the strains Saitama U25 and Khs1-1997-JP. Sequences from two further cases and one mussel sample were assigned to genogroup I and showed a high degree of identity with the strain NLV/Steinbach/EG/2001/CA.Three familial groups consisting of 7 cases and three members of a further family group from the outbreak showed identical sequences and were classified as NV GII (the main cluster of 19 cases). A fourth member of the latter family group was positive for one of the two GI strains. The other strains investigated by sequence analysis were unrelated to those from these familial groups.In Italy a national NV outbreaks database does not exist, although NVs are the leading cause of gastroenteritis outbreaks in Europe as in the rest of the western countries . MoreoveThis investigation showed the causative role of mussel consumption in the outbreak, confirmed by laboratory tests on both stools and food samples.The heterogeneity of viral strains associated with the consumption of contaminated mussels is no surprise due to the peculiar features of shellfish. In fact, bivalve shellfish are filter feeders and tend to accumulate whatever pollutants are in the water which can result in viral contamination from a multitude of possible sources affecting many individuals. In any case, further studies should be carried out to clarify this issue . The lowIn the 1990s, NVs were identified as the primary pathogen associated with shellfish-borne gastroenteritis in the United States . Since tRaw shellfish consumption is very frequent among the people of Puglia. Mussels sold in Puglia come from a large number of suppliers: from countries within the European Union (EU) and from other Mediterranean countries. The frequent shellfish consumption had already been blamed for the cases of cholera that occurred in Puglia in 1994, during the Christmas period, when a major epidemic was reported in Albania, on the opposite coast of the Adriatic sea ; moreoveMoreover, our data showed cooked mussels also played a significant role. In fact cooking might not completely inactivate NVs and because of the low infectious dose, even a limited residual contamination can result in illness .The epidemic curve shows a usual pattern consistent with a common point source. However, person to person transmission may have played a role in the last phase of the outbreak. On the other hand, such a means of transmission of NV is well documented.This investigation confirms the importance of field study in gastroenteritis outbreaks, as well as obtaining stool specimens from patients and food samples for laboratory analysis in a short time .The present standards for human faecal contamination do not seem to be a reliable indicator of viral contaminants in mussels . SeafoodThis episode confirms that large NV outbreaks occur in Italy but only an accurate investigation can recognise this pathogen and that current regulations and commercial practices need to be revised to assure the safety of shellfish consumption and to improve control of future outbreaks.None declared.MC and GB carried out microbiological assays and drafted the manuscript. CG participated in the design of the study and performed the statistical analysis. MQ conceived the study as well as participating in its design and coordination. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "This report describes a large international chocolate-associated Salmonella outbreak originating from Germany.Salmonella (S.) Oranienburg isolates were subtyped by the use of pulsed-field gel electrophoresis (PFGE).We conducted epidemiologic investigations including a case-control study, and food safety investigations. S. Oranienburg notifications was registered in Germany. Simultaneously, an increase in S. Oranienburg infections was noted in other European countries in the Enter-net surveillance network. In a multistate matched case-control study in Germany, daily consumption of chocolate , having shopped at a large chain of discount grocery stores , and consumption of chocolate purchased there  were associated with illness. Subsequently, two brands from the same company, one exclusively produced for that chain, tested positive for S. Oranienburg. In two other European countries and in Canada chocolate from company A was ascertained that also contained S. Oranienburg. Isolates from humans and from chocolates had indistinguishable PFGE profiles. No source or point of contamination was identified. Epidemiological identification of chocolate as a vehicle of infections required two months, and was facilitated by proxy measures.From 1 October 2001 through 24 March 2002, an estimated excess of 439 Salmonella-containing products. Particularly in diffuse outbreak-settings, clear associations with surrogates of exposure should suffice to trigger public health action. Networks such as Enter-net have become invaluable for facilitating rapid and appropriate management of international outbreaks.Despite the use of improved production technologies, the chocolate industry continues to carry a small risk of manufacturing  Salmonella spp. is a substantive cause of human gastroenteritis in many parts of the world [Salmonella reports  received at the federal level by the Robert Koch-Institut (RKI) accounted for 31% of all notifications for the 54 notifiable conditions [Salmonella enterica subspecies enterica serotype Enteritidis (S. Enteritidis) is the predominating serotype followed by S. Typhimurium. They represented 65% and 23% of the reported cases of non-typhoidal salmonelloses with known serotype in 2001. Thus, the remaining ~250 serotypes reported to the RKI in that year, including S. Oranienburg, accounted for only 12%.Non-typhoidal he world . In Germnditions . SalmoneSalmonella and Other Enteric Pathogens (NRC) in Hamburg noted an unusual increase in the number of S. Oranienburg isolates received in October. At that time, no increase was noticeable in the national database for statutorily reportable infectious diseases; 50 S. Oranienburg notifications (median: 1 per week) had been registered for 2001. On November 19, the NRC in Wernigerode informed the RKI that it had received a S. Oranienburg isolate in September. The isolate was submitted by a private laboratory for serotyping and had come with the additional source information \"confectionery sample\". Upon inquiry, a large German chocolate manufacturer (company A), which produced a broad variety of chocolates and products made thereof, called the RKI on November 27, and confirmed that it had sent in the confectionery sample. According to company A, the positive sample originated from an in-house control of a chocolate product and the pertaining batch, due to be exported to the United States, was completely destroyed and not distributed. Notwithstanding, the number of statutory S. Oranienburg notifications had sharply increased and continued to rise. This report describes the epidemiologic, food safety, and microbiological investigations of this outbreak.In mid-October 2001, the National Reference Center for S. Oranienburg infections about chocolate consumption in the seven days before disease onset, and also to send any remaining chocolate to a food safety laboratory.A standard exploratory questionnaire was distributed on 20 November 2001 via state health departments to all local health departments to aid the collection of data on food and environmental exposure from cases. In addition, local health departments were asked to immediately interview patients with newly reported A request was distributed to participants of the Enter-net surveillance network  on DecemS. Oranienburg isolates from patients and from the in-house chocolate control were found to be indistinguishable by pulsed-field gel electrophoresis (PFGE). On the same day, a multistate case-control study was initiated and coordinated by RKI to test the hypothesis that at least one product from company A was associated with S. Oranienburg-infections. As we were denied a product list from company A, we resorted to the company's web-site and included in our food history evaluation all the products listed there. Some products from company A, e.g., bars of chocolate (brand A), were exclusively sold at a large chain of discount grocery stores (chain X). We found that the majority of chocolates sold at chain X were produced by company A. Therefore, for the analysis we constructed a variable for chocolate(s) purchased at chain X (\"chain-X-chocolate\") as a proxy for chocolate-products from company A because most patients could remember the flavor of the purchased chocolate, but seldom the brand name.On December 3, while exploration of patients were ongoing and results inconclusive, S. Oranienburg in other countries [S. Oranienburg infection to a public health department before December 6. Case-patients were excluded from the analysis, if they could have been secondary, i.e., if they reported to having had contact with a person with diarrhea in the seven days prior to symptom onset. Cases were selected from the national reportable database by simple random sampling; in Lower Saxony an attempt to interview all case-patients was launched. Case selection was done irrespective of whether patients also had been interviewed with an exploratory questionnaire. At least one age and telephone exchange-matched control subject was selected for each case-patient by sequentially adding 2 to each case-patients' telephone number. Control subjects were eligible if they were in the same age-group as their matched case-patient , had no gastrointestinal symptoms after 1 October 2001, and had not traveled abroad in the seven days prior to the onset of symptoms of the matched case. Telephone interviews were conducted by state health departments, local health departments, and the RKI. Data were analyzed with Epi Info V6.04c .The hypothesis-testing questionnaire collected data on the consumption of chocolates, and some other foods, particularly those previously associated with outbreaks of ountries -5. Food-Salmonella positive chocolate leftovers and five chocolates from grocery stores using the most probable number technique [The local food safety authority inspected company A's production facility and took samples from already packaged (\"in-house\") chocolates, and from ingredients from its suppliers. Beginning December 11, a nationwide chocolate sampling of German chocolates in grocery stores was initiated by the Federal Ministry of Consumer Protection, Food and Agriculture, and was assisted by the Federal Institute for Risk Assessment . On December 18, when a chocolate leftover from brand A tested positive, the investigations were tailored to German chocolates from company A. The BfR examined quantitatively four echnique .S. Oranienburg isolated from stool specimens were sent to the NRC from laboratories in Germany and, on Enter-net request, from other countries. Furthermore, isolates from chocolates were submitted from state or private food laboratories in Germany as well as from Canada and the Czech Republic. PFGE-analysis was carried out according to Prager et al [For comparison by the use of PFGE, er et al .S. Oranienburg up to reporting week 42, but 462 reports in the following 23 weeks , 240 (52%) patients were female. There was no difference in the gender distribution within the single 10-year age-bands (P = .51). All 16 states of Germany reported S. Oranienburg cases during the outbreak period, with the highest incidence in the state of Schleswig-Holstein  bordering on Denmark. In total, 206 of the 440 German counties were affected with a median of one report and a maximum of 16 from the city of Hamburg during the outbreak period.In 2001, the RKI received 50 reports of S. Oranienburg.Sixty exploratory questionnaires were received from eight states by the end of 2001. Forty-three (88%) of 49 patients with information on chocolate consumption had a symptom onset after 1 October 2001. Of the 34 who gave information as to where they bought the chocolate, 21 (62%) explicitly reported chain X. Some reported exclusively having eaten chocolate bars from brand A, among them a two-year-old child. On 18 December 2001, two months after the initial outbreak alert, a leftover consumed reportedly by this child in the seven days before symptom onset tested positive for S. Oranienburg had been reported in Denmark from October 18 through December 10, compared with only two cases in 2001 before October 18. None of the clustered cases were travel-related [S. Oranienburg problem, independently suspected German chocolate bought in chain X as the source of the Danish outbreak. Chocolate was the only food item that all patients reported eating. The majority stated purchasing chocolate in chain X, which, although German, operates internationally [S. Oranienburg infections was reported from other countries such as Austria, Belgium, Finland, Sweden, The Netherlands  after disease onset in case-patients. Twelve case-control pairs were excluded from chocolate-specific analysis, nine because the case-patients could have been secondary, one due to illness in September, and two where the control subjects could not remember whether they had eaten chocolate. Of the 48 cases and 50 controls that were analyzed, 24 (50%) case-patients and 32 (67%) control subjects were female, 22 (46%) case-patients were younger than 10 years. Ten (21%) of the case-patients reported to have suffered from bloody diarrhea and 14 (29%) were hospitalized  of 381 chocolates that were tested and reported to BfR during the outbreak period. S. Oranienburg was isolated from two different brands of company A; all positive chocolates were produced during the same week in August 2001. Estimates of the number of Salmonella in the tested chocolates ranged between 1.1 and 2.8 per gram.The local food safety authority in Germany did not identify hygienic deficiencies at the production facility. Samples obtained in the beginning of December 2001 from in-house chocolates (n = 12), as well as from cocoa (n = 3) and cocoa powder (n = 7) from a supplier of company A tested negative. This applied also to German chocolates sampled in grocery stores until 18 December (on that day a leftover tested positive). Overall, S. Oranienburg isolates from human cases of gastroenteritis originating in Germany (n = 52), Austria (n = 19), Belgium (n = 8), Canada (n = 6), Denmark (n = 4), The Netherlands (n = 4), Sweden (n = 4), and the Czech Republic (n = 1). Furthermore, 15 chocolate isolates were sent to the NRC for PFGE-analysis from Germany (n = 12), Canada (n = 2), and the Czech Republic (n = 1). They came from an in-house sample, from leftovers of chocolates consumed by patients in their incubation period, and from chocolates sampled in grocery stores in Germany. The PFGE profiles of S. Oranienburg isolates from patients with symptom onset after 1 October 2001 (outbreak period) in Germany and in the other countries mentioned above, except Canada, were indistinguishable  in the week prior to symptom onset, but not in the seven days before the interview. Case-patients were more likely than control subjects to report eating chocolate daily, likely indicating an increased probability of having been exposed to contaminated chocolate. Furthermore, patient isolates from the outbreak period shared a PFGE profile with isolates from chocolates but differed from isolates of patients who became sporadically diseased with S. Oranienburg before the outbreak. In addition, the food histories and microbiological results from S. Oranienburg patients in several other countries pointed to the same source [We describe an international outbreak of e source ,10.Salmonella infections after consumption of contaminated chocolate, although rare, have been known since the 1960's [Salmonella have been recovered from chocolates in these outbreaks, suggesting a very low infectious dose. Estimates of the number of S. Oranienburg cells per gram in this outbreak ranged from 1.1\u20132.8. However, we cannot exclude that the bacteria were unevenly distributed in the chocolate(products) and that those parts carrying many viable cells were not tested quantitatively. In chocolate, the low moisture (water activity aw: 0.4\u20130.5) and high sugar content does not favor bacterial growth, but significantly increases thermal resistance [Salmonella against the acidic conditions of the stomach [e 1960's . Common e 1960's -17  can carry Salmonella spp., the low water activity and high fat content in chocolate increases thermal resistance so that temperatures reached during chocolate production  do not  a small number of Salmonella may be sufficient to cause disease, even with low-level contamination, chocolate can affect large number of persons (often children) scattered over a wide area, and thus, has the potential to cause serious public health consequences.It is noteworthy that the case-control study did not identify further products as risk factors. This applied also to the second contaminated brand of company A, which was included in the recall. The small proportion of study cases (25%) mentioning having eaten chain-X-chocolate lends support to the hypothesis of more contaminated brands (even from other manufacturers), which could be one explanation for the continuing case-occurrence. However, inaccuracies due to lack of brand awareness may have played a particular role in this outbreak, and the time-delay between disease onset and interview (median: 37 days) may have contributed to an inaccurate recall of cases and their guardians. Furthermore, cases with a disease onset in 2002 may have occurred as a result of a diminished impact of the public warning due to the Christmas season. For example, chocolate gifts received or given for Christmas may not have been thoroughly enough checked for best-before dates stated in the public warning.Identification of vehicles in foodborne outbreaks can become difficult if the exposure is common. Consumption of a wide variety of German chocolates was reported by all case-patients (and 88% of explored patients), but also from 86% of the control subjects in the week prior to onset of illness. When groups are (nearly) universally exposed or a more specific hypothesis cannot be tested, often the best one can do is to establish a \"dose-response-relationship\" , i.e., uS. Eastbourne outbreak in Canada/USA in 1973/74, 286 environmental samples and 98 chocolate samples from the production-line were examined. No in-line chocolate sample tested positive and overall only 6 (1.6%) samples were positive  were obtained. In a An Enter-net urgent inquiry was sent after the first results of molecular subtyping suggested a link between human cases and chocolate from company A. Until then, investigators in Germany and Denmark had worked independently unaware that the outbreak extended outside of their respective countries. An earlier inquiry, ideally as early as an outbreak was suspected by the investigating countries (or as an increase was noted in the Enter-net database), may have speeded up hypothesis generating, and thus, may have helped in earlier identification of the vehicle, thereby preventing illnesses.Finally, a public warning or recall of company A products did not occur before a brand A leftover tested positive although the confluence of information \u2013 the results of the case-control study, the Danish investigations, and the subtyping comparison between human isolates and the in-house sample \u2013 had already pointed to company A products as the source of the outbreak. Yet, no specific product or lot had been identified at the time. For this reason, a recall or a public warning were considered excessive responses by the German food safety authority. However, relying on microbiological confirmation in leftovers, if available for testing, is disputable  and is dangerous in unopened food packages because critical time can elapse before a positive culture in food is obtained . TherefoSalmonella-containing products. For the future, awareness among German food safety authorities must be heightened for the need to base public health action not exclusively on laboratory confirmation in food, and to conduct timely and comprehensive source investigations to enhance food safety in the long-run. The international scale of this outbreak shows how easy it is to distribute a contaminated product across many countries. This underlines the necessity of mechanisms for international surveillance and information dissemination such as Enter-net to ensure that international outbreaks can be dealt with rapidly and in an appropriate manner. Similar networks should be set up or, if existing, should be connected (possibly overseen by WHO), to allow rapid communications to other parts of the world when it is clear that a contaminated product is distributed internationally.To our knowledge, this is the largest reported chocolate-associated outbreak, the seriousness being emphasized by the hospitalizations (29%) and self-reported bloody diarrhea (21%) of the study cases. Despite the use of improved production technologies, the chocolate industry continues to carry a small risk of manufacturing The author(s) declare that they have no competing interests.Salmonella in chocolate. EW coordinated food safety investigations in this outbreak. AE conducted the Canadian part of this outbreak investigation. AS conducted the Finnish part of this outbreak investigation. YA conducted the Swedish part of this outbreak investigation. MHK was instrumental in the design of the case-control study. AA coordinated the German part of this outbreak investigation, broadened the scope of this outbreak by prompting an urgent Enter-net inquiry, and helped in designing the case-control study and drafting the manuscript.DW was the principal investigator of the German part of this outbreak; he carried out the statistical analysis of the case-control study, and drafted the manuscript. JD and FF were responsible for the outbreak investigation including the case-control study for the state (\"Bundesland\") of Lower Saxony. UvT was responsible for the outbreak investigation including the case-control study for the state (\"Bundesland\") of Northrhine-Westfalia. GF was responsible for the outbreak investigation including the case-control study for the state (\"Bundesland\") of Hamburg. SE conducted the Danish part of this outbreak investigation. AMH was responsible for the outbreak investigation including the case-control study for the state (\"Bundesland\") of Hesse. PR detected the outbreak and conducted microbiological investigations. RP and HT conducted the PFGE-analysis. ISTF coordinated the Enter-net inquiries and investigations. SB conducted interviews in the case-control study, designed the database and helped in the analysis. EB conducted quantitative analysis of All authors participated in revising the manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "An increase in the number of attendees due to acute gastroenteritis and fever was noted at one hospital emergency room in Taiwan over a seven-day period from July to August, 2001. Molecular and epidemiological surveys were performed to trace the possible source of infection.An epidemiological investigation was undertaken to determine the cause of the outbreak. Stool and blood samples were collected according to standard protocols per Center for Disease Control, Taiwan. Typing of the Salmonella isolates from stool, blood, and food samples was performed with serotyping, antibiotypes, and pulsed field gel electrophoresis (PFGE) following XbaI restriction enzyme digestion.Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) was isolated from the stool samples of 28 of 32 individuals and from a recalled bread sample. All S. Enteritidis isolates were of the same antibiogram. PFGE typing revealed that all except two of the clinical isolates and the bread isolates were of the same DNA macrorestriction pattern.Comparison of the number of patients with and without acute gastroenteritis  during the six weeks before the outbreak week revealed a significant increase in the number of patients during the outbreak week  (relative risk (RR): 1.44, 95% confidence interval (CI): 1.22\u20131.70, P value < 0.001). During the week of the outbreak, 34 of 162 patients with gastroenteritis were positive for Salmonella, and 28 of these 34 cases reported eating the same kind of bread. In total, 28 of 34 patients who ate this bread were positive for salmonella compared to only 6 of 128 people who did not eat this bread . These breads were produced by the same bakery and were distributed to six different traditional Chinese markets., S. Enteritidis was confirmed as the vehicle of infection. Alertness in the emergency room, surveillance by the microbiology laboratory, prompt and thorough investigation to trace the source of outbreaks, and institution of appropriate control measures provide effective control of community outbreaks.The egg-covered bread contaminated with  Salmonellosis, resulting from the ingestion of contaminated poultry, beef, pork, eggs, and milk , is an iSalmonella species are relatively uncommon in Taiwan [Salmonella food-borne disease outbreaks but serovar Enteritidis has emerged as a new serovar in Taiwan [Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) infections [Food-borne disease outbreaks due to n Taiwan  comparedn Taiwan  and Japan Taiwan . Thirty-n Taiwan . Serovarn Taiwan , consistfections ,24,25.S. Enteritidis that was performed after notification of the rapid increase of the number of patients with febrile gastroenteritis in an emergency room and an unusually high percentage of Group D Salmonella isolated since it comprised only 7.4% of the various Salmonella groups in the hospital one year before the outbreak.We report the findings of the investigation of an outbreak of Salmonella infection. A cohort study of all patients attending the ER with acute gastroenteritis between July 28 and August 3, 2001 was performed in order to test the hypothesis that illness was caused by a specific food.A rapid increase in the number of attendees due to acute gastroenteritis and fever was noted to have begun on July 28, 2001 at the emergency room (ER) of Kaohsiung Municipal Hsiao-Kang Hospital, and this increase continued for the following six days. Clinical and demographic features and the food reported to have been consumed three days before development of gastrointestinal symptoms for all patients with acute gastroenteritis at the ER from July 28 to August 3, 2001 were reviewed by ER personnel and the infection control team. The infection control team conducted patient interviews and reviewed charts using a standardized case record form. Stool and blood cultures were performed once specimens were available. These specimens were further analyzed by the hospital's clinical microbiology laboratory and the laboratory of the Center for Disease Control, Taiwan  in Kaohsiung, Taiwan. Charts of all patients with and without acute gastroenteritis who visited the ER from six weeks before until two weeks after July 28, 2001 were reviewed by infection control nurses for the baseline data of the ER. An outbreak-associated case was defined as a patient visiting the ER with the diagnosis of acute gastroenteritis and having Methods for sample collection, cultivation and isolation were conducted according to the standard protocol of the CDC, Taiwan for food-borne disease outbreaks as described previously .Salmonella serotypes were determined with the use of antiserum  according to the manufacturer's instructions. Serogrouping and serotyping were performed by the slide agglutination method and tube agglutination method to identify the somatic O antigen and flagellar H antigen, respectively [ectively .Escherichia coli ATCC 25922 was used as the quality control organism.Antimicrobial susceptibility was determined by the disk diffusion method according to the National Committee for Clinical Laboratory Standards . The antXbaI  was used at the manufacturer's suggested temperature. Restriction fragments were separated by PFGE in 1% agarose gel  in 0.5X TBE buffer  for 25 h at 200 V at a temperature of 14\u00b0C, with ramped times of 2 to 40 s using the Bio-Rad CHEF-DRII apparatus . Gels were then stained with ethidium bromide and photographed under ultraviolet light. The resulting genomic-DNA profiles, or \"fingerprints,\" were interpreted according to established guidelines [Total DNA was prepared and PFGE was performed as described previously ,13. The Salmonella infection were suspected as the vector of infection. A sample of the implicated food was collected from a patient's home within six hours of the patient having symptoms of febrile gastroenteritis and it was stored at 4\u00b0C. Further tracing the source of the suspected food and the subsequent environmental investigation were undertaken by the Bureau of Health, Kaohsiung County and CDC, Taiwan.Food items having a significant relationship with gastroenteritis cases with t-test. Significance was considered at a P value < 0.05 (Epi Info. Version 3.2.2 Centers for Disease Control and Prevention (CDC), USA).Differences between groups were compared using Chi-Square analysis. Relative risk and 95% confidence intervals were also calculated when Chi-Square analysis was used. Continuous variables were analyzed with Student's An increase in the number of patients admitted due to acute gastroenteritis and fever was noticed beginning from the early morning of July 28, 2001 with a return to normal levels one week later. Reviewing daily case-visits to the ER due to all causes and due to gastroenteritis six weeks before this outbreak period revealed significantly more cases due to all causes visiting the ER on Saturday and Sunday than on weekdays (From Monday to Friday) (mean \u00b1 standard deviation (S.D.): 140.3 \u00b1 38.3 compared with 109.6 \u00b1 16.7, P = 0.004). The number of patients visiting due to gastroenteritis on Saturday and Sunday were also higher than those visiting on weekdays   of 942 patients visiting the ER during the outbreak period had acute gastroenteritis. Compared with 85 (9.7%) of 872 patients in the week before the outbreak, significantly higher percentages of patients with gastroenteritis were observed in the outbreak week (Relative risk (RR): 1.92, 95% confidence interval (CI): 1.38 \u2013 2.26, P < 0.001). A comparison of the number of patients with and without acute gastroenteritis  during the six weeks before the outbreak week and during it also revealed a significant increase in the number of patients of gastroenteritis during the outbreak week .Salmonella compared to only 6 of 128 people who did not eat this bread . The association of consuming the kind of bread and having Salmonella infection was significant . No other identified food or restaurant exposure was significantly associated with the outbreak.Investigation of food consumed by gastroenteritis patients during the outbreak period revealed 34 (21%) had consumed the same kind of egg covered bread decorated with mayonnaise and fried seasoned pork fiber from six traditional Chinese markets (three located in Kaohsiung City and three in Kaohsiung County). There were 2, 2, 2, 3, 3, and 22 gastroenteritis patients that consumed the bread purchased from 6 markets, respectively. In total, 28 of 34 patients who ate this bread were positive for Salmonella cases that consumed the implicated bread, 12 were male. Their age ranged from three to 71 years old. Eleven cases (39.3%) were under 18 years old and one case was over 65. The incubation periods ranged from 4 to 17  hours after consumption of the bread. Twenty-seven of the 28 patients were hospitalized. The clinical symptoms among the 28 cases included abdominal pain (100%), fever (100%), diarrhea (100%), vomiting (85.7%), chills (35.7%), and headache (7.1%). The laboratory data showed eight (28.6%) cases had white blood cell counts higher than 10000 /mm3. Positive occult blood reaction was found in stool specimens of 24 of 27 (88.9%) patients tested. Colitis was found in all four patients who received colonoscopy examination. The two bacteremia patients' fever subsided one day and two days after admission, respectively, without antimicrobial therapy. No mortality or sequellae occurred among these cases during hospitalization or in the three months' follow-up by infection control nurses.Regarding the 28 Salmonella and surveillance culture of the workplace surfaces were not performed.Further investigation by the city public health administration found the incriminated bread from the six markets was all produced by the same bakery that was prohibited from production of all kinds of bread on August 1, 2001. Because the bakery had stopped their production one day before the official prohibition, no further samples of the components of bread, such as the egg and mayonnaise, were available. Investigation of the bakery staff with stool cultures for Salmonella isolates. Twenty-eight Salmonella isolates were cultured from 32 available stool specimens from 34 patients who consumed the implicated food. These isolates were all of group D and were further identified as S. Enteritidis. This pathogen grew in two of the 34 patients' blood cultures. A Salmonella isolate of the same serovar was cultured from bread provided by a patient. The sample had been stored in the refrigerator because the patient had not finished eating it.In the week of this outbreak, there was an extraordinarily high percentage (94.1%) of group D of Salmonella was isolated from six of the 25 patients' stool specimens. For the six Salmonella isolates from cases not consuming the implicated food during the outbreak period, two were of group B and four were of group D. Further serovar analysis and PFGE analysis of the four group D isolates was not performed. Among the four group D isolates, two were resistant to ampicillin and trimethoprim/sulfamethoxazole, which was different to the antibiogram of the isolates from cases consuming the implicated bread.A total of 30 stool specimens from 25 patients not consuming the implicated bread were collected and All 28 stool isolates, two blood isolates and one food isolate were susceptible to the nine antimicrobial agents tested. Only two stool isolates showed unrelated PFGE patterns with more than six band differences compared to the epidemic PFGE pattern of the other isolates. All the other stool, blood, and food isolates had the same PFGE pattern indicating a clonal relationship  had not consumed the implicated bread, Fig.  suggestiS. Enteritidis [S. Enteritidis [Salmonella isolates.Compared with phage typing  and plaseritidis ,19 thougeritidis . AnalytiSalmonella groups and knowledge that the percentage of group D Salmonella patients was low before. Thus, the unusually high percentage of group D Salmonella seen within the week of the outbreak led to the investigation. The implicated vehicle of infection in the outbreak was an egg-covered bun topped with mayonnaise and fried seasoned pork fiber which had been distributed to different markets after production at the same bakery. Epidemiological study, antibiograms, and serologic and molecular typing patterns revealed that almost all cases of S. Enteritidis infection during the period were the same as that of the bread isolate. Only the implicated baked good from the same bakery caused Salmonella infections while other items from the bakery were not found to be epidemiologically related with the outbreak. This finding suggests contamination of the pathogen did not occur during the common routes of production, transportation, or selling of goods from the bakery. Although the bread sold in the traditional Chinese markets frequently does not conform to sanitary requirements, and although the staff involved in distribution and selling are not subject to the routine hygiene inspections, in food stores, the possibility of contamination of a specific bakery product simultaneously at six markets in the same time period is very low, suggesting that the contamination occurred during the production process. Food contamination in the outbreak was traced to the same bakery but an investigation of infection among the bakery staff and sources of the contents of the bread was not conducted. Although the baking process involves high temperatures sufficient to kill pathogens, the manual addition of toppings or flavors, such as mayonnaise, eggs, and meat products provide possibilities for contamination with food-borne pathogens. In addition, insufficient baking may be a risk factor for human health because it may not destroy microbial contamination.Outbreaks can be missed easily if the increase in case number is not noticed due to the wide distribution of outbreak sources in different areas or if a relatively small outbreak occurs among a large population and cases are exposed to the outbreak source at different times. Recognition of the outbreak reported in this study was aided by the routine surveillance of Salmonella serovars have been related specifically with some foods. S. Enteritidis is particularly related to eggs [S. Enteritidis outbreaks related to baked goods[Salmonella isolated from the bread had the same serovar, antibiogram, and PFGE pattern as isolates from patients and the significant relationship between consumption of this baked good and the isolation of Salmonella implicated this food product as the vehicle of contamination.Different  to eggs -28. In tked goods,5,7,8, aSalmonella outbreaks [This outbreak had a relatively short incubation compared to the 24\u201372 hour range reported for other utbreaks ,30. WhetSalmonella Enteritidis outbreaks.For many food-borne outbreaks, the pathogens and transmission vehicles are often not identified, usually because of delayed collection of epidemiologic and microbiologic information . InitiatThe author(s) declare that they have no competing interest.Po-Liang Lu and L. K. Siu were in charge of the investigation, data handling and writing of the manuscript. Po-Liang Lu and Shang- Jyh Hwang participated in the design of the study. Po-Liang Lu, In-Jane Hwang, Ya-Lina Tung and Shang-Jyh Hwang were in the investigation team for data collecting and data analysis. Po-Liang Lu performed the statistical analysis. Chun-Lu Lin and L. K. Siu carried out microbiological assays. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "The purpose of this study was to identify the agent and the source of the outbreak in order to develop control measures and to avoid similar outbreaks in the future.On the 202 test.A case-control analytical approach was employed with 100 randomly selected cases and 66 controls. Patients completed standardized questionnaires, odds ratios were calculated and statistical significance was determined using \u03c7In addition, to identify the source of the infection, we performed bacteriological examination of food samples  collected from the cuisine of the military unit.Staphylococcus aureus was detected in a sample of raw beef  and in two samples of grated cheese; leftover cheese from lunch  and an unopened package purchased from the market .More than 600 out of the 1,050 individuals who ate lunch that day, became ill. The overall attack rate, as the military doctor of the unit estimated it, was at least 60%. The overall odds ratio of gastroenteritis among those who had lunch was 370 (95% CI: 48\u20137700) as compared to those who didn't eat lunch. Among the symptoms the most prominent were watery diarrhoea (96%) and abdominal pain (73%). The mean incubation period was 9 h and the median duration of the symptoms was 21 h. In the bacteriological examination, S. aureus. The food vehicle was the grated cheese, which was mixed with the beef and served for lunch in the military unit. This outbreak highlights the capacity of enterotoxin-producing bacteria to cause short term, moderately-severe illness in a young and healthy population. It underscores the need for proper food handling practices and reinforces the public health importance of timely notification of such outbreaks.The findings of this study suggest that the aetiological agent of this outbreak was  Foodborne diseases are considered a major public health problem because they affect individuals of all ages and socioeconomic status . DespiteSalmonella species, Clostridium perfringens, Escherichia coli strains, Campylobacter jejuni, Listeria, Yersinia as well as toxins of Staphylococcus aureus and Bacillus cereus [Campylobacter, Cryptosporidium, Shiga-toxin producing E. coli (STEC) O157, Listeria, Salmonella and Yersinia. As for the suspected food vehicles, these are usually dairy products, meat-based dishes, sauces and seafood [Numerous studies published recently, indicate that most outbreaks of acute gastroenteritis occur in places of mass feeding such as institutions, schools, restaurants and military units -8. The Cs cereus -15. Furt seafood -15.In Greece, recording of outbreaks of foodborne gastroenteritis is limited and there is a considerable lack of recorded epidemiological data. Most cases are not reported, resulting in inadequate monitoring and evaluation of outbreak trends, based on aetiological agents, food vehicles and venues. Thus, prevention and control measures for food safety are difficult to establish.The present study focused on a reported outbreak of gastroenteritis in an Air Force base in Western Greece, where during an operative training day, soldiers and staff experienced an outbreak of acute gastroenteritis. Following the assessment of descriptive epidemiology, we performed a case-control analytic approach. Using project-designed standardized questionnaires the main objective of the study was to determine the vehicle of the contamination. Furthermore, forced by a public health perspective and taking into account the possible malpractice of employees, we tried to develop control measures in order to prevent future contamination and underline the importance of adherence to high quality hygiene standards for food preparation.th September 2005. From personal interviews and the questionnaires collected, those who had not developed any relevant symptom became 'controls', while all others became 'cases'.On 20 September 2005, during an operative training day at the Air Force base in Western Greece, soldiers and staff experienced an outbreak of acute gastroenteritis. A case-control analytic approach was utilized, wherein 100 randomly selected subjects out of about 600 taken ill and 66 controls (unaffected soldiers and staff) took part. A questionnaire study was performed 5 days after the outbreak. All subjects completed the same questionnaire during a personal interview by the military doctor. Specifically, there was an effort to interview as many people as possible and to record the same number of cases and controls. Unfortunately, this was not feasible because of the special characteristics of the incident. During that operative training day there were many individuals (soldiers and staff) at the base from other military units as well, who could not be reached afterwards. Thus, it was impossible for the doctor to collect the necessary information from all people who had lunch in the base on 20Data recorded included setting of the outbreak, dates of onset, frequency and types of symptoms, incubation period, duration of illness and consumption of food items served on the base during the implicated training day.2) test.Odds ratios were calculated for consumption of various food items and statistical significance was determined using the Chi-square  and the military doctor. Verbal consent was obtained from all study subjects.Salmonella spp.,S. aureus, E. coli and Heterotrophic Plate Count (HPC) at 22\u00b0C and 37\u00b0C. Meat samples were further tested for the detection of Clostridium spp.Samples of the food served to the soldiers were taken from the cuisine of the military unit and were transferred to the lab for microbiological analysis. Food samples included a) 1 kg of raw meat (beef), b) 200 g of cooked minced meat (derived from the beef), c) 200 g of grated cheese and d) a sample of the same cheese in its unopened original package. Food items were transported to the laboratory under refrigerated conditions (5 \u00b1 1\u00b0C) and analyzed upon arrival. All samples were examined for the detection of Salmonella spp. was performed according to Standard Methods of the American Public Health Association.The detection of S. aureus Baird-Parker's medium (Oxoid) and Manitol salt agar (Oxoid) were used. Surface inoculated plates were incubated at 37\u00b0C for 48 h. The typical S. aureus colonies were counted and tested for coagulase reaction (Oxoid Staphytect Plus kit). For E. coli Tryptone Bile X-Glucuronide medium \u2013 T.B.X. (Oxoid) was used and the plates were incubated at 44\u00b0C for 24 h. HPC was evaluated using Tryptone Yeast Extract Agar (Oxoid) incubated at 22\u00b0C and 37\u00b0C for 72 and 48 h, respectively. Finally, TSC agar (Oxoid) was used for the enumeration of Clostridium spp. colonies after incubation at 44\u00b0C for 24 h under anaerobic conditions.For the other analyses, a homogenate of each sample was prepared by diluting 10 g of sample in 90 ml Peptone Water (Oxoid). For E. coli and Enterococcus spp, as imposed by the Greek legislation.Water samples from the military unit were also examined applying filtration through nitrocellulose membranes . A 100 ml of each sample was filtered for the determination and enumeration of total coliforms, The overall estimated attack rate was at least 60% among the approximately 1050 attendees. The outbreak started abruptly in the late afternoon on 20 September, peaked at midnight and ended about 25 hours later, as demonstrated by the epidemic curve  Figure . The medAmong the symptoms reported, the most prominent were watery diarrhoea (96%) and abdominal pain (73%), whereas vomiting and nausea developed in 8% and 7% of the cases, respectively Table . Of the S. aureus was detected in the sample of raw beef  and in the two samples of grated cheese. In total 7,800 cfu per g were detected in sample \"c\" (opened bag containing the rest of cheese that had been served to the subjects during lunch) and 3,000 cfu per g in sample \"d\" (unopened package purchased from the market).The results of the microbiological analyses showed that only E. coli, Salmonella spp. and Colstridium spp. and HPC ranged in low levels, between 40 cfu and 1500 cfu per g. The analysis of water samples showed negative results for the parameters tested, indicating that the suspected vehicle of the pathogen was among the food items served to the subjects.All food samples cultured were negative for the presence of S. aureus was considered to be the most likely cause. Although mortality and longer-term morbidity are uncommon with food poisoning caused by enterotoxin-producing bacteria [The outbreak of acute gastroenteritis, reported on 20 September 2005 on the Air Force base in Western Greece, was originally presumed to be caused by an enterotoxin-producing bacterium. This speculation was based on the short incubation period with abrupt onset, the symptomatology and the short, self-limiting nature of the illness. bacteria , this ouS. aureus, detected in the cheese was the main aetiological agent of the reported outbreak. As far as the possible cause of the contamination is concerned, the most likely scenario is that the food-handler who packaged grated cheese into the bags in the market was the source, because the pathogen was detected in packaged cheese purchased directly from the market. S. aureus colonies were also detected in the beef sample, but that was probably due to cross-contamination from the cheese during lunch preparation.In the present study our main concern was to determine the food vehicle of this reported foddborne gastroenteritis. For this purpose suspected food items were collected and examined. By that procedure we identified the aetiological agent and proceeded with the isolation and withdraw of the contaminated food item. Nevertheless, a limitation of this investigation was the fact that stool samples were not obtained for microbiological diagnosis. The reasons for this omission include the short duration of the symptoms and the overall lack of coordination in the military base, although they are important in suspected foodborne outbreaks, as reported . The datS. aureus [S. aureus which survives well in cheese during production and handling processes [S. aureus over other bacteria [S. aureus causes a wide array of disease symptoms, ranking staphylococcal enterotoxins as one of the most common causes of reported food-borne illnesses [According to epidemiological studies, a wide range of foods are sources of staphylococci food poisoning, resulting from enterotoxins produced primarily by . aureus -21. Focurocesses ,23,24. Ibacteria . The varllnesses ,19,21. SS. aureus may be more costly to treat than many other food-associated pathogens [S. aureus proliferation and contamination [In developed countries athogens . The chemination . These dmination .S. aureus is an indicator of post process contamination by food handlers, which most likely occurred in our case. Humans are the main reservoir for staphylococci. Hands and the mucous membranes of the nasopharynx are predominant sites of colonization. High rates of carriage and large cells numbers are two major factors that explain the important role of food workers in the epidemiology of staphylococcal food poisoning outbreaks [S. epidermidis and S. aureus [On the other hand, utbreaks . The maj. aureus .Reducing the number of foodborne outbreaks will require continued and coordinated efforts by many different agencies, including those of disease surveillance, consumer education and food handling, processing and marketing . To begiIn Greece supervision authorities regarding foodservices are not fully developed and there is a considerable lack of epidemiological evidence, resulting in an unsatisfactory insight in the incident and pathogens of gastroenteritis caused by contaminated food. Thus, limited collected data of outbreaks do not allow the evaluation of outbreak trends and the proper surveillance by the Greek food-hygiene system.S. aureus. The pathogen was detected and isolated from grated cheese, which served as food vehicle of transmission.Information obtained from the questionnaires with the results of microbiological analysis of food items led to the conclusion that the aetiological agent of this outbreak was This outbreak highlights the capacity of enterotoxin-producing bacteria to cause short term, moderately-severe illness in a young and healthy population. Foodborne outbreaks like this one recorded in the present investigation should be reported to the associated authorities in order to accomplish appropriate monitoring in time. Thus, food-hygiene services could improve the surveillance of outbreaks, particularly those occurring in institutions or affecting the community at large and protect public health.The author(s) declare that they have no competing interests.EJ had the original idea for the study, developed the questionnaire and organized the case-control study, carried out the statistical analysis, formed the layout of the manuscript and wrote the first draft of the manuscript.DV carried out the microbiological analysis, contributed on interpretation of the results and co- wrote the final manuscript.GK carried out the microbiological analysis and contributed to the preparation of the manuscript.TK participated in the data collection and interpretation.TCC contributed in the data interpretation and the final manuscript.CB carried out and supervised the data collection at the air force baseAll authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Pseudomonas aeruginosa and linked to the use of contaminated mouth swabs called Dent-O-Sept. Here we describe the extent of contamination of the swabs, and identify critical points in the production process that made the contamination possible, in order to prevent future outbreaks.In 2002 we investigated an outbreak comprising 231 patients in Norway, caused by Environmental investigation with microbiological examination of production, ingredients and product, molecular typing of bacteria and a system audit of production.P. aeruginosa was detected in 76 swabs from 12 batches produced in 2001 and 2002. In total more than 250 swabs were contaminated with one or more microbial species. P. aeruginosa was detected from different spots along the production line. The audit revealed serious breeches of production regulations. Health care institutions reported non-proper use of the swabs and weaknesses in their purchasing systems.Of the 1565 swabs examined from 149 different production batches the outbreak strain of P. aeruginosa over a period of at least 30 weeks. When not abiding to production regulations fatal consequences for the users may ensue. For the most vulnerable patient groups only documented quality-controlled, high-level disinfected products and items should be used in the oropharynx.Biofilm formation in the wet part of the production is the most plausible explanation for the continuous contamination of the swabs with  Pseudomonas aeruginosa is a gram-negative, obligate aerobe rod-shaped bacterium with minimal nutritional requirements. It is often found in moist environment and can cause infections in immunocompromised or otherwise susceptible hosts [le hosts ,2. Numerle hosts -9, contale hosts -16. Crosle hosts ,17,18.Pseudomonas aeruginosa infection in 24 Norwegian hospitals [P. aeruginosa from the period November 2000 to December 2002, of which 39 were blood culture positive. Seventy-one infected patients, all of whom had severe underlying diseases, died while hospitalized. The outbreak strain was susceptible to all anti-pseudomonas antibiotics . However, some of the isolates cultured late in the outbreak had developed intermediate susceptibility or full resistance to ceftazidime or aztreonam .We have reported a major, nationwide outbreak of ospitals . The outP. aeruginosa was traced to a mouth swab called Dent-O-Sept. This is a clean, non-sterile, moist sponge-on-a-stick produced in Norway, which according to the Norwegian text on the wrap is an antiseptic single-use swab for mouth hygiene  This swab was the dominant product of its kind on the Norwegian market, being widely used in hospitals, long-term care facilities and in home care. Approximately one million swabs were sold in Norway per year. Small quantities were also exported to Denmark and Sweden. As soon as the connection between the swab and the outbreak was identified the company ceased production at its single facility and recalled the product.The outbreak strain of e Figure .  and Bacillus spp. were to be noted and reported.The Norwegian Institute of Public Health (NIPH) coordinated the national outbreak investigation. Immediately after the recall of the product, we asked all hospitals to report to NIPH which batches of the Dent-O-Sept swab they had in store. The rest of the health care services were asked in a newsletter from NIPH to do the same. A batch number printed on the wrap indicated the week and year of production. Up to 10 swabs of each available batch of the product were examined at the microbiological laboratory which the health care institution normally used. We asked the laboratories to identify and deep freeze monocultures of all findings of gram-negative rods, P. aeruginosa were sent for genotyping as described below.The Directorate for Health and Social Affairs organized a system audit of the manufacturer on 12 \u2013 15 April 2002 by studying documents, interviewing selected personnel and inspecting the premises, including microbiological sampling of tap water, swabbing of different places along the production lane and culturing of stored and packed samples of the product. These samples were cultured at the municipal Food Control Authority. Isolates of P. aeruginosa was performed on the moisturizing liquid of 16 wrapped swabs taken from four boxes with swabs produced on the same day and from 15 swabs from three boxes with swabs produced at different times during two consecutive days.On request from the producer Sn\u00f8gg Industri AS, the laboratory at the municipal Food Control Authority performed environmental sampling in addition to what had been performed during the system audit described above. The production site had been left untouched after the production had ceased on 9 April 2002. In May, quantitative analysis of From the bottom of the steel tank a blue pipe connects to a level measuring device Figure . In May P. aeruginosa was performed [Between 28 May and 5 June 2002 water samples were taken from water taps located several places on the production site and from a 1 m chipped, rubber hose leading from a faucet with municipal water to the steel tank The hose had not been replaced in an estimated seven years. Quantitative analysis of erformed .P. aeruginosa were tested in each of the liquids. Then the moisturizing liquid undiluted and in 1:10 dilution were tested for their effect on the outbreak strain and a reference strain of P. aeruginosa (ATCC 9027 -MicroBioLogics) [We performed microbiological analysis ,23 of eaoLogics) .Culturing of samples of Dent-O-Sept swabs was performed at local laboratories according to our instruction: \"Brush the swab against both a lactose and blood agar dish in a rotating manner so all sides of the foam tip touches the agar. It is not necessary to place the swab in a growth broth\". The isolates were identified by standard procedures in use by the laboratories.Culturing of samples from the system audit and the additional investigation of the production site were performed at the laboratory of the municipal Food Control Authority. The qualitative analysis of the samples was performed by direct seeding (except for dry Dent-O-Sept swabs) and seeding after enrichment overnight in a heart infusion broth on Kings Agar B and on blood agar. The quantitative analysis was performed by direct seeding of 0.1 mL undiluted or \u2013 if heavy contamination was expected \u2013 diluted liquid on Kings Agar B and for some samples also on blood agar. The plates were incubated at 37\u00b0C overnight before reading.P. aeruginosa from each contaminated production batch of swabs and all isolates identified as P. aeruginosa from the system audit were sent for genotyping and compared with the outbreak strain at one or more of three reference laboratories. The strain of P. aeruginosa found in the product batch 47.2001 on 8 April 2002 was defined as the outbreak strain. Isolates of P. aeruginosa found in the additional investigation of the production site were not genotyped.One isolate of Two of the reference laboratories used a pulsed field gel electrophoresis (PFGE) method developed at St. Olav's Hospital and the third laboratory used an augmented fraction length polymorphism (AFLP) The methods are described elsewhere . The AFLP. aeruginosa was detected in 76 swabs from 12 different batches of the Dent-O-Sept swab produced from week 38 in 2001 to week 15 in 2002 when production ceased. These 76 swabs were sent for examination from 13 different hospitals, one private person and from the producer. All genotyped strains of P. aeruginosa were identical to the outbreak strain. The outbreak strain was isolated from six patients up to ten months before the production of the first swab found to be contaminated  and week 22 in 1999 (nine swabs). The strains from the two batches were genotypically identical. In total, more than 250 swabs were found to contain one or more species of micro-organisms, mainly gram-positive bacteria which were predominantly discovered in the earlier batches. Gram-negative rods including Acinetobacter baumanii were isolated in swabs produced in 1999 and 2001.In addition to The production process of the swabs was described as follows: The foam rubber heads and sticks were glued together manually in private homes of employees who were following moderate hygiene guidelines.One batch of moisturizing liquid consisted of: Tap water (147 liters), 96% ethanol (3 litres), Glycerol (16 litres) and Vademecum, a commercially available mouth rinse (6 litres). The main ingredients of the mouth rinse are ethanol (44%) and sodium benzoate 5.25%). The final concentration in the Dent-O-Sept moisturizing liquid was calculated to be 2.3% ethanol; 9.3% glycerol and 0.18% sodium benzoate [%. The fiA new batch of the liquid was prepared in the following way every week of production: The tap water was filled into a large steel tank with a lid on Friday, and then heated to 95\u00b0C the following day in an automated but uncontrolled process Figure . On MondPacking, moisturizing and sealing of the swabs were done in an automated packing machine, Fuji Wrapper II, FW 3400. The moisturizing liquid was led from an orifice at the bottom of the steel tank through a pipe, via a pump and a dosing valve ending in the packing machine close to the heat sealer. This piping system was approximately 3.5 meters long and a locally made adaptation to the packing machine. The tank and pipe were made of acid resistant stainless steel.Dry swabs were fed on a conveyer belt and approximately 2.0\u20132.5 grams of liquid were sprayed into the aluminium-plastic laminate wrap right before heat sealing. Staff tested the bags for air tightness by squeezing ten bags at the time when coming out of the machine before packing.At the end of each working day, the remaining liquid was kept in the steel tank. Before start of production the next morning the moisturizing liquid was stirred using a steel rod that first was washed with soap and water and then disinfected. On Fridays when the week's production was finished the remaining moisturizing liquid was emptied from the tank. The tank was then flushed with high pressured hot water (> 60\u00b0C). Then 30 litres of lukewarm water was added together with 1.5 dL of disinfectant . The tank and lid were scrubbed and the solution pumped into the pipes of the packing machine until all moisturizing liquid was assumed to be replaced. After standing for 10\u201315 minutes the tank was emptied and flushed again with high pressured hot water. Then 30 litres of hot water (73\u201380\u00b0C) was filled in the tank and pumped through the pipes of the packing machine until the disinfectant was assumed to be rinsed out. The producer had no quality assurance routines for checking temperatures, composition of the moisturizing liquid during the week or effect of the cleaning and disinfection process.P. aeruginosa was identified after it had been incubated in a growth broth. P. aeruginosa was not detected from other points along the production line, the drain or at other points sampled in the production room. However, the outbreak strain was detected in packed samples of the product batch 15.02 stored on the premises.A sample taken from the end capillary nozzle in the production facilities was negative on initial culturing, but growth was noted and the outbreak strain of The system audit concluded that the production deviated from the existing regulations in several areas:\u2022 The production process, including the recipe for Dent-O-Sept, did not ensure that the product had the qualities and properties stated by the producer nor that the risk of contamination was avoided or reduced to a minimum.\u2022 Neither the boxes nor wraps of the Dent-O-Sept gave the user the necessary information. The CE (Communaut\u00e9 Europ\u00e9enne) marking was unjust because the producer's declaration of conformity with the regulations, including the risk analysis, was poorly based and documented. The technical documentation did not give a third party a basis for assessing whether the device was in accordance with the demands of the regulations.\u2022 The producer did not comply with the obligation to report defects and deficiencies in medical devices to national health officials and had not adequately followed up errors in the production demonstrated in an external review in 1999.P. aeruginosa in swabs produced on the same day and even at the same time of day ranging from 50 to 10000 CFU per mL liquid in the five positive swabs of the 16 examined  declare that they have no competing interests.BGI headed the outbreak investigation and the conception, drafting and revision of the manuscript. HME performed the logistics in collecting and collating the results from the microbiological examination of the swabs. GB headed the additional investigation of the production site. KH was in charge of the system audit. TJ participated in the outbreak investigation, adapted the PFGE method to detecting the outbreak strain and analyzed many of the samples. EE was in charge of the analysis and antimicrobial effect of the ingredients for the moisturizing liquid. JL participated in the microbiological aspects of the outbreak investigation and performed some of the analyses. PA was over all in charge of the outbreak investigation and participated in the conception, drafting and revision of the manuscript. All authors read and approved of the final manuscript."}
+{"text": "From June to November 2005, 18 cases of community-acquired Legionnaires' disease (LD) were reported in Rapid City South Dakota. We conducted epidemiologic and environmental investigations to identify the source of the outbreak.Legionella. Clinical and environmental isolates were compared using monoclonal antibody subtyping and sequence based typing (SBT).We conducted a case-control study that included the first 13 cases and 52 controls randomly selected from emergency department records and matched on underlying illness. We collected information about activities of case-patients and controls during the 14 days before symptom onset. Environmental samples (n = 291) were cultured for Legionella. Six of 11 case-patients (matched odds ratio (mOR) 32.7, 95% CI 4.7-\u221e) reported eating in Restaurant A versus 0 controls. Legionella pneumophila serogroup 1 was isolated from four clinical specimens: 3 were Benidorm type strains and 1 was a Denver type strain. Legionella were identified from several environmental sites including 24 (56%) of 43 cooling towers tested, but only one site, a small decorative fountain in Restaurant A, contained Benidorm, the outbreak strain. Clinical and environmental Benidorm isolates had identical SBT patterns.Case-patients were significantly more likely than controls to have passed through several city areas that contained or were adjacent to areas with cooling towers positive for This is the first time that small fountain without obvious aerosol-generating capability has been implicated as the source of a LD outbreak. Removal of the fountain halted transmission. Legionella, Legionella pneumophila, is responsible for 90% of reported cases of LD in the United States and the majority of these are caused by Legionella pneumophila serogroup 1 (Lp1) [On average, 1675 cases of legionellosis are reported to the Centers for Disease Control and Prevention (CDC) annually by state health departments, the majority of which are Legionnaires' disease (LD) . It is estimated that between 8,000 and 18,000 persons are hospitalized with LD in the United States each year, suggesting that under-diagnosis or under-reporting are common [ 1 (Lp1) ,3.Legionella for these cases is rarely known. In contrast, outbreaks, while rare, provide an opportunity to identify common sources of Legionella. Community LD outbreaks have frequently been associated with cooling towers [Eighty percent of cases of LD are sporadic  and the g towers -13, and g towers -17, and g towers , air-scrg towers , supermag towers , potableg towers ,22, and g towers .In a three week period in June and July 2005, seven cases of LD among residents of Rapid City were reported to the South Dakota Department of Health (SDDH). In the previous 10 years, only two confirmed cases of LD had been reported to the SDDH from Rapid City.  No common exposures among case-patients were initially apparent. In July 2005, SDDH and CDC began an epidemiological and environmental investigation to identify the source of the outbreak and to prevent additional cases.Legionella, by urinary antigen test for Lp1, by a four-fold or greater rise in serum antibody titer to Lp1, or detection of specific Legionella antigen by direct fluorescent antibody staining.We defined a case as a resident of, or visitor to, Rapid City, South Dakota who was diagnosed by a physician, either clinically or radiographically, with community-acquired pneumonia (CAP) with onset after May 1, 2005 and who had laboratory confirmation of LD by culture of Legionella urine antigen testing. Hypothesis generating interviews were held with the first six case-patients or their surrogates in an attempt to identify any common exposures.To enhance surveillance for additional cases of LD associated with Rapid City, alerts regarding the increase in cases were made locally (July 2005), statewide (July 2005), and nationwide (August 2005), to public health practitioners, physicians and health facilities. Clinicians were encouraged to perform urine testing for LD for all hospitalized patients with CAP and to report any cases to SDDH. To determine whether the increase in cases represented an outbreak or simply increased diagnostic testing, we examined trends in admissions for CAP to Rapid City Regional Hospital, and We conducted a matched case-control study that included the first 13 of the 18 case-patients to identify exposures associated with LD. The remaining five case-patients were not enrolled as they were reported after the source of the outbreak had been identified and publicized. Four controls per case (n = 52) were matched on underlying illness category  and smoking status. As all enrolled case-patients were white, older than 50 years, and residents of Rapid City, we restricted our controls to white persons over the age of 50 years from Rapid City. Controls were selected randomly from among persons attending the local hospital's emergency department within seven days before or after the illness onset date of the corresponding case-patient. Potential controls who had a diagnosis of CAP in the last six months, who were residents of a long-term care facility, or who had been away from home for five or more nights during the two weeks of interest were excluded. We interviewed all case-patients or their surrogates in person and interviewed controls either in person, or by phone if they were unwilling to meet an investigator in person.2, and the grid coordinates were recorded.During interviews with case-patients and controls, we used a map of Rapid City and a calendar to prompt recall of activities during each two-week period of interest. Case-patients and controls were asked to refer to their diaries, check-books, and receipts to further assist with recall. Questions were general, such as \"did you visit any restaurants?\" or \"did you visit any supermarkets?\" If the answer was in the affirmative then the person was asked to specify which ones were visited. Reported routes of travel throughout the city were traced on a map which was divided into 180 grids, where each grid represented 1.29 kmMatched analysis of the matched sets using exact methods in SAS version 9.1  was used to compute matched odds ratios (mOR) and 95% confidence intervals (CI).Legionella, we discontinued this sampling as we concluded that acute dissemination of Legionella via the municipal water system was an unlikely explanation for this outbreak.We conducted an environmental assessment of the city to locate aerosol-generating devices. The devices were identified by aerial, driving, and walking surveys of the city and through contacts with city managers, local property managers, water treatment companies, and industrial plumbing contractors. In September 2005, SDDH contracted an engineering consultant (TK) to assist in the environmental investigation and remediation efforts. Sample collection and analysis were performed according to standard procedures . BetweenLegionella Laboratory at CDC  or to the Special Pathogens Laboratory, Pittsburgh Veterans Administration Medical Center where Legionella was identified using previously described methods [A one-liter water sample and, where appropriate, a biofilm swab were taken from each location. Water samples were collected in sterile plastic bottles containing a 0.1% sodium thiosulfate solution. Samples were shipped to the  methods . Clinica methods  and sequ methods .Research was conducted in compliance with guidelines of the U.S. Department of Health and Human Services as they apply to epidemic investigations by the U.S. Public Health Service, CDC.Lp1 from respiratory secretions; 3 were Benidorm strain , a common outbreak strain, and one was Denver strain , a relatively rare strain not previously associated with outbreaks  of 18 patients were hospitalized, and 1 (5.6%) died. Onset dates ranged over a five-month period from May 26, 2005 to October 29, 2005  compared to 61 years (range 50\u201392) for controls. All case-patients and controls were residents of Rapid City and none were current smokers. Seven (53.9%) of the 13 case-patients enrolled in the case-control study had a chronic underlying condition or were immunosuppressed.Legionella  of 123 potential environmental sources and from 95 of 291 (32.6%) samples (Table Legionella pneumophila was the most common species identified and Lp1 was the predominant serogroup with 25 (58.1%) of 43 sources positive for Lp1. Among the Lp1 isolates that were typed, strains reacting with MAb 1 were most common, suggesting the presence of type strain Camperdown (MAb 1) and/or Oxford 4032E . Due to a shortage of MAb 6 we were unable to distinguish between Camperdown and Oxford for all isolates although the few that were tested were Oxford.flaA, pilE, asd, mip, mompS, proA, i.e., the pattern 4,7,11,3,11,12, respectively. The decorative fountain had a water Legionella colony count of 3000 cfu/ml. Of the remaining water sources positive for Legionella that had colony counts recorded, 10 had counts less than 50 cfu/ml, 10 had counts between 50 and 499 cfu/ml, eight had counts between 500 and 1499 cfu/ml, and three had counts above 1500 cfu/ml.The outbreak strain, Benidorm , was isolated from only one source, a decorative fountain in Restaurant A. Swabs were taken from the fountain basin, nozzle, and internal lamp and a water sample was taken. All were positive for Benidorm strain. We compared all Benidorm clinical and environmental isolates and found identical SBT patterns for the genes The decorative fountain located in the lobby of Restaurant A was sampled on October 24, 2005, turned off at that time of sampling, and removed a few days later. The diameter of the basin of the fountain was approximately 1.2 m with a central water column of approximately 30 cm. The basin was covered with mesh and three colored lights were submerged in the water , analysis of potential exposures where more than half the case-patients had visited did not initially show any significant differences between case-patients and controls. This hypothesis was strengthened by the timing of the outbreak at the start of summer with many towers bearing heavier cooling loads and running in excess of 100% capacity due to a hotter than usual summer in South Dakota. Thirdly, as outlined in the background, cooling towers are common sources of community outbreaks of LD.Our initial hypothesis was that a cooling tower was the source of this outbreak for three reasons. Firstly, we could find no common exposures and could not initially place more than two case-patients in any one building, leading us to believe that the source was outdoors. Previous studies have shown infections occurring as far as 6 km from an external source indicating that the wind is capable of transporting a plumes . HoweverLegionella urine antigen testing therefore increased prior to detection of this outbreak. This highlights the importance of appropriate diagnostic testing in patients with CAP. While many patients with LD can be treated successfully and empirically with antibiotics currently recommended for CAP, this outbreak shows that the use of etiology-specific diagnostic tests can lead to a public health intervention that prevents future cases of LD. Furthermore, increasing physician awareness and use of the urinary antigen testing, through our recommendation during the outbreak to test all new cases of CAP for LD, may have improved patient management and contributed to the relatively low case-fatality rate. The availability of clinical isolates of Legionella from 4 case-patients was critical in allowing us to identify the source of the outbreak. In the United States there has been a steady decline in the proportion of LD cases diagnosed by culture since the introduction of urine antigen testing [Legionella whenever possible. This recommendation is included in the recently updated Infectious Disease Society of America/American Thoracic Society (IDSA/ATS) guidelines on the management of CAP in adults [Clinicians at the local hospital in Rapid City had adopted a policy in November 2004 of more intensive diagnostic testing for patients with CAP so that antibiotic therapy could be targeted. n adults .Legionella was cultured from over half (55%) of the cooling towers tested, several of which were positive for Lp1. Although the outbreak was assumed to be caused by Benidorm strain, which was not found in any of the cooling towers, the presence of Legionella in so many sources, some with high colony counts, and with the potential to aerosolize is worrying. Other studies have shown that detectable levels of Legionella are present in many cooling towers and other building water systems [Legionella, we applied the precautionary principle and recommended that all positive towers be remediated regardless of their colony count. SDDH employed a Legionella consultant (TK) to manage remediation of the cooling towers in accordance with published guidelines [ systems ,31. Some systems . Howeveridelines , and to Our investigation had certain limitations. While it is understandable that individuals recovering from serious illnesses would have some difficulties with recall of activities 1 to 2 weeks earlier, poor recall delayed our ability to identify Restaurant A as a potential source of the outbreak. We could have asked our case-patients and controls whether they visited each individual shop, restaurant, etc. in Rapid City. However, this would have made our already lengthy questionnaire unwieldy. Case-patients were re-interviewed about their exposure to Restaurant A but controls were not re-interviewed. Therefore, we have not presented new matched odds ratios as they may be biased. Given the magnitude of the odds ratio (mOR 32.7) based on the initial case-patients' reported attendance at Restaurant A and given that the Benidorm strain was found only in the fountain in Restaurant A, we believe that not re-interviewing controls has not weakened our conclusions that the fountain in Restaurant A was the source of this outbreak. We excluded the last five cases from the case-control study because they were reported after the source of the outbreak was published and we were concerned that their responses may have been biased. We believe that these cases appeared rather suddenly because media attention may have led to increased medical care-seeking, increased diagnostic testing, increased reporting, or all three.We were unable to explain the presence of Denver strain in the clinical isolate of one case-patient. Although this patient ate at Restaurant A, it is possible that this case-patient was infected by a different source and was a sporadic case unrelated to the outbreak. This assumes that the case-patients that were not culture confirmed were infected with Benidorm strain. It is also possible that Denver strain was present in the fountain but was masked by the predominance of Benidorm strain, or that Denver was present in the fountain in early August when this case-patient became ill, but was no longer present in late October when the fountain was sampled. These hypotheses also make it possible that Benidorm strain may have been present in cooling towers but was not present at the time of sampling. If that were the case however, one might have expected more people who had not eaten at Restaurant A to be case-patients. We interviewed approximately half of the controls by phone whereas all case-patients were interviewed in person. This may have led to better recall among cases. Interviews with controls by phone facilitated more rapid recruitment of controls and thus a more timely analysis.Most of the fountains we sampled had little or no routine maintenance although this is recommended by fountain manufacturers and by the American Society of Heating, Refrigeration, and Air-conditioning Engineers (ASHRAE). ASHRAE guidelines are non-specific for fountain maintenance and are not disseminated widely in the restaurant industry . Proper Legionella.Small decorative fountains pose a previously unrecognized source of Legionnaires' disease outbreaks as we believe this is the first time that such a small fountain with apparently limited aerosol-generating capability has been implicated as the source of an LD outbreak. Investigations of future community outbreaks of LD should consider exposures to indoor decorative fountains, including small ones such as those that might be present in restaurants, hotels, or other businesses, as potential sources of Legionella remediation. His consultancy has no conflict of interest with any information contained in this paper.TK was contracted by SDDH to assist with this outbreak investigation as an environmental engineering consultant specializing in The author(s) declare that they have no competing interests.ROL, LK, MCW, CH, TK, BF and MRM participated in the conception and design of the study. ROL, LK, MCW, EB, VS, TK, RB, BF and MRM participated in the analysis and interpretation of data. ROL, MM and LK drafted the article. All authors critically revised the article for important intellectual content and all authors have seen and approved the final version. The corresponding author (ROL) had access to all data and had final responsibility for the decision to submit for publication.The findings and conclusions in this report are those of the authors and do not necessarily represent the view of the Centers for Disease Control and Prevention.The pre-publication history for this paper can be accessed here:"}
+{"text": "In 2004, a major outbreak of hepatitis A among tourists returning from Egypt involved 351 case-patients from 9 European countries who were infected with a single strain (genotype 1b). The case-control study identified orange juice as the most likely infection vehicle. Vaccination against hepatitis A virus is strongly recommended before travel to disease-endemic areas. In Germany, approximately half of the 1,400\u20132,300 cases of laboratory-confirmed HA reported annually since 2001 were acquired abroad. In mid-August 2004, infectious disease surveillance in Germany showed a strong increase of HAV infections in tourists returning from Egypt, where HA is highly endemic (The line listing of HA patients included persons with laboratory evidence of recent HAV infection (anti-HAV immunoglobulin M [IgM]) who stayed at hotel X after June 1, 2004. Also listed were hotel guests with HA disease , without laboratory confirmation, who had traveled with persons with laboratory-confirmed cases.A case-control study was performed among hotel X guests >17 years of age residing in 3 German states. The time span between the earliest arriving case-patient's last day at hotel X and the latest arriving case-patient's first day there was defined as the \"minimum period of transmission\" (MPT). Case-patients came from the line listing. Healthy hotel guests who stayed at the hotel during the MPT who had neither been vaccinated against HAV nor previously infected with HAV were eligible as controls. Telephone interviews were conducted with a standardized questionnaire that elicited information on demographic factors, foods and drink consumption, and participation in recreational activities such as swimming, day trips, etc.2 tests) and multiple logistic regression were performed ; p<0.05 was considered statistically significant. The Egyptian authorities' investigation included testing all hotel employees for HAV antibodies , and scrutiny of food suppliers. Serum samples were obtained from German case-patients for testing by reverse transcription\u2013PCR (RT-PCR) in the VP1\u20132A junction and sequencing of a 160-bp-long PCR fragment of the VP1 region.For statistical analysis, exposures were dichotomized into \"ever\" versus \"never,\" and the \"number of days exposed\" was calculated. Univariate analysis  persons. Case-patients were 2 to 67 years of age (median 34 years) and 54% were male. Overall, 47% of case-patients were hospitalized. Risk of hospitalization rose with increasing age (p for trend = 0.001). The MPT lasted from June 24 to July 23 . No moreSixty-nine HA case-patients (60% response among the 115 case-patients in the 3 states) and 36 controls were included in the statistical analysis. Eighty-seven percent of the case-patients reported absence from work for 3 to 56 days (median 26 days), and 54% of the case-patients were hospitalized for 2 to 25 days (median 9 days).Case-patients and controls did not differ significantly by age, sex, recreational activities, consumption of ice cream or salads, or other foods consumed or behavioral characteristics. Case-patients were significantly more likely to have drunk orange juice served at the breakfast buffet (82%) than were controls (64%) . In multivariate analysis, no other exposures were retained. A dose-response relationship became apparent between number of days of orange juice consumption and HA . Case-paThe on-site investigations in Egypt did not identify hotel staff positive for HAV IgM. Minimal fluctuation among hotel staff renders it unlikely that an HAV-positive employee was missed. Investigations at the orange juice producing plant found significant hygiene problems. In addition, the finished product did not undergo heat treatment. This producer did not cater to other hotel chains in Hurghada.This large outbreak demonstrates risk and clinical impact of HA for nonimmune travelers to HA-endemic countries. In Germany, the outbreak accounted for 12% of all HA case-patients notified in the year 2004.The results of the outbreak investigation strongly point to orange juice as the infection vehicle. In the case-control study, among a broad range of foodstuff, beverages, and recreational activities queried, the consumption of orange juice was the only exposure significantly associated with HA, with higher doses of juice significantly increasing HA risk. These findings are corroborated by the inspection of the hygienic conditions under which the juice was produced in Egypt. The juice was most likely contaminated during the manufacturing process, e.g., by an infected worker with imperfect hand hygiene or by contact of fruit or machinery with sewage-contaminated water.,\u2013>15 days (Citrus fruit and citrus juices have only rarely been implicated as vehicles of HA outbreaks, with contamination typically described during preparation just before consumption (The fact that juice was consumed by 60% of healthy controls may be explained in part by fluctuating virus concentration within the juice, which resulted in varying degrees of infectiousness during the 4-week period. A contaminated lot may have been phased-in and out slowly by gradual mixture with other lots. Also, the study design did not allow the exclusion of controls who did not know they were immune.The Hurghada outbreak-strain clearly differs from strains that have caused nontravel-associated outbreaks in Germany in recent years. Two large autochthonous outbreaks were caused by HAV type 1a strains (Vaccination against HA is recommended for all nonimmune travelers to HA-endemic areas ("}
+{"text": "Patients in Goalando were likely infected by direct contact with fruit bats or their secretions, rather than through contact with an intermediate host. Nipah virus (NiV) is a paramyxovirus that causes severe encephalitis in humans. During January 2004, twelve patients with NiV encephalitis (NiVE) were identified in west-central Bangladesh. A case\u2013control study was conducted to identify factors associated with NiV infection. NiVE patients from the outbreak were enrolled in a matched case-control study. Exact odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by using a matched analysis. Climbing trees  and contact with another NiVE patient  were associated with infection. We did not identify an increased risk for NiV infection among persons who had contact with a potential intermediate host. Although we cannot rule out person-to-person transmission, case-patients were likely infected from contact with fruit bats or their secretions. Paromyxoviridae, genus Henipavirus) are enveloped RNA viruses that cause respiratory illness in pigs and horses and respiratory illness and encephalitis in humans (Pteropodidae) , occurred between September 1998 and April 1999 in Malaysia and Singapore and was associated with an outbreak of severe respiratory illness in pigs  or serum by enzyme immunoassay (EIA). A probable case of NiVE was defined as a patient with a diagnosis of encephalitis in whom fever developed and who was living in the same village as a patient with a confirmed case of NiVE after December 15, 2003. Cases remained in the probable category if the patient died and a specimen for laboratory confirmation could not be obtained.We conducted a population census of the affected area in February 2004; this census was the basis for selecting controls. We identified 3 controls for each case-patient. The controls were selected randomly from the population and then matched to each case-patient on the basis of gender and age group. All households identified during the census, including houses of case-patients and controls, were mapped by Global Positioning System, and data were uploaded into ERDAS Imagine 8.5  and merged with a November 2000 IKONOS Geo 1-m satellite image of the outbreak area .Participation was strictly voluntary, and written informed consent was obtained for all participants; for those <18 years of age, individual and parental consent was obtained. The Bangladesh Ministry of Health and Family Welfare that requested this investigation reviewed and approved all protocols.Probable and confirmed cases identified in 2 contiguous villages of Goalando township  were incSerum samples and CSF were tested as previously described  (<15 years of age (Four (33%) cases were confirmed by EIA; the remaining 8 (67%) case-patients, from whom a diagnostic specimen was not available, were considered probable cases. Among all 13 (36%) controls who consented to blood collection, results of serologic tests for NiV-specific antibodies were negative. Furthermore, none of the controls reported having had a perceived fever or symptoms compatible with NiVE from December 15, 2003, through the week the study was conducted . In addition, an antibody prevalence study conducted among persons (n = 300) living in the outbreak site showed no evidence of asymptomatic or mild infection, which suggested that controls entered into the study were likely uninfected . Proxy interviews were administered to equal proportions of case-patients (83%) and controls . The meds of age . Residens of age .In the matched case-control analysis, a greater percentage of case-patients (60%) than controls (34%) had observed or touched dead animals, although this finding was not statistically significant . We obseA greater proportion of case-patients (83%) than controls (51%) reported having climbed trees between December 15, 2003, and February 3, 2004  visiting a hospital and/or 2) having had contact with a probable or confirmed NiVE patient . In one In contrast to the patients in the Malaysian and Singapore outbreaks, which occurred primarily among adults, a preponderance of the NiV patients in the January/February 2004 Bangladesh outbreak were young boys. These findings, in the absence of high infection rates among adults or evidence of antibodies to NiV in the general population , suggest an association between NiV infection and some childhood activity or specific behavior. The odds of NiV infection were significantly elevated among persons who climbed trees, an activity observed almost exclusively among boys <15 years of age. This behavior is quite common among children because they gather fruit from trees. Therefore, these children may have had contact with partially eaten fruit from fruit bats or the secretions/excretions of these animals. Or, the children may have contacted contaminated fruit bat guano or urine in the trees. The percentages of case-patients playing hide-and-seek, hunting, and fishing\u2014all of which were typical behaviorial traits of local boys\u2014were not significantly different than those for controls. These activities generally occur outdoors; however, they do not place a child in direct contact with bat excretions or secretions, as may be true for tree climbing. Therefore, infection was apparently related to a specific behavior, tree climbing, rather than age or outdoor activities in general. Furthermore, although other exposures that may have placed persons in closer contact with bat secretions  were observed more often among case-patients than controls, these findings were not statistically significant; perhaps because of the small sample size. Nonetheless, our findings can and have been used to help guide NiV outbreak investigations, leading investigators to similar conclusions as ours  during fruit-tree climbing.Numerous investigators have found serologic evidence suggesting that fruit bats of the genus Although indirect contact with bats may have been the primary means of infection for this outbreak, Hsu and others  may have occurred through contact with bat secretions rather than contact with an intermediate host. A limitation of our study is that we were unable to identify a specific mechanism by which person-to-person transmission may have occurred. NiV has been isolated from the respiratory secretions and urine of patients in the Malaysia, Singapore, and current Bangladesh outbreaks  may have resulted in a much larger outbreak. Indeed, a study among health workers in Bangladesh did not find evidence of incidental transmission to persons caring for patients hospitalized with Nipah-related illnesses (In summary, tree climbing, a behavior largely engaged in by young boys, was associated with an increased risk for NiV infection; although the exact mode of transmission is unclear. Our data do not rule out the potential for person-to-person transmission. If person-to-person transmission were extremely efficient, the conditions and population density of Bangladesh (\u22481,000 persons/kmllnesses .Pteropus, including parts of China and Australia, most of the Indian subcontinent, and Southeast Asia (As a prevention measure, we recommend avoiding contact with fruit bats and their secretions/excretions. We also encourage persons to wash and/or peel fruit, in addition to washing their hands, before preparing meals or consuming fruit. Greater understanding of the relationships between pteropid fruit bats, NiV, and its transmission to humans might offer additional strategies for safe coexistence and disease prevention for Bangladesh and other countries where fruit bats reside. Finally, because the geographic range of this highly lethal pathogen may correspond to the distribution of the genus"}
+{"text": "This paper describes the third large outbreak of Norovirus (NoV) gastroenteritis reported in the Southern Italy region of Puglia.A matched case control study was conducted, on 19 July 2005, for investigating risk factors, using a structured questionnaire on food consumption. A multivariate analysis was conducted to estimate the adjusted Odds Ratios. Laboratory and environmental investigation were also performed.On the day of the study 41 cases were identified and 41 controls were enrolled. Controls were matched for age and gender. The mean age of the cases was 26 years old, and 58% were female. The clinical pattern of the disease was characterised by the presence of diarrhoea (95%), vomiting (70%), abdominal pain (51%) and fever (32%). Of the 41 cases included in the study, the majority (65%) were residents of Northern Italian regions. No food samples were available for testing. The matched univariate analysis revealed that cases were more likely to have consumed raw mussels, eggs or ice cubes made of tap water than controls. In the multivariate conditional logistic regression analysis, having eaten raw mussels or ice became more strongly associated with illness.All of the 20 faecal samples collected were tested for NoVs. Eighteen stools  were positive by RT-PCR, and sequence analysis performed onto 3 samples confirmed the presence of a GGII NoV. No test specific for NoV was performed on water or food samples.The most likely hypothesis supported by the findings of the epidemiological investigation was that illness was associated with raw mussels and ice, made with tap water. These hypothesis could not be confirmed by specific microbiologic testing for NoV in food or ice. The lack of clear knowledge of NoV as a major causative agent of epidemic outbreaks of gastroenteritis in Italy is due to the absence of timely reporting of the cases to the local public health offices and the uncommon practice of saving clinical samples for virological analysis after bacteriological testing. Caliciviridae family [Norovirus (NoV) constitutes one of the four genera of the e family . Based oe family . GGIV ine family ,3, and Ge family .NoV is the major cause of non-bacterial gastroenteritis in industrialised countries, and has been shown to account for up to 80% of outbreaks of gastroenteritis in people from all age-groups ,6. It isIn Italy, according to the statutory system for notification of infectious diseases, all outbreaks are to be reported . NeverthIn the present study, we describe the investigation of a large outbreak involving approximately 400 guests in a Resort of Puglia region during a three-week period (2\u201319 July 2005). This represents the third GE NoV outbreak reported in this area of Southern Italy since 2000.The outbreak occurred at a Hotel in Ugento, a small town in the province of Lecce, Puglia, in Southern Italy during July 2005 Figure . The HotOn July 19, 2005, the Regional Centre for Surveillance of Enteric Pathogens in Puglia was alerted by the local health unit that an outbreak of gastroenteritis had occurred at the Hotel. The outbreak continued for about 1 further week. An epidemiological investigation was performed in order to assess risk factors associated with illness.A case of GE was defined as a guest who had stayed at the resort between 2 to 19 July, and presented two or more loose stools and/or vomiting in 24 hours before interview. A convenience sample of 41 cases and 41 controls were interviewed. Case and control finding was done by a door-to-door search within the Hotel.th of July and trained interviewers collected data using a structured questionnaire. The questionnaire included information on food consumption for investigating risk factors during the last 72 hours before onset of symptoms, and nature, time of onset and duration of symptoms. One matched control was selected for each case  matched for age (+/-2 years) and sex. Controls were excluded if they reported to have experienced an episode of gastrointestinal illness  within seven days prior to the onset of illness in the matched case. If cases or controls were below 16 years of age, their parents or guardians were interviewed.A matched case control study was conducted on the 192 test; continuous variables were compared using the Wilcoxon Mann-Whitney test. In the univariate analysis, exposure to potential risk factors was compared between cases and controls calculating matched \"Mantel-Haenszel\" Odds Ratios (mOR) and exact 95% confidence intervals (95% CI).All questionnaires were collected in the Regional Centre for Surveillance of Enteropathogens in Puglia, where data were entered into a File-Maker Pro database . Categorical variables were compared using the chiA multivariate conditional logistic regression model was then performed to assess independent effects of the exposure variables and to estimate adjusted odds ratios (aOR); risk factors associated with the outcome (P < 0.20) in the univariate analysis, after testing for multi-colinearity, were considered eligible to be included in the multivariate model, and retained in the final model, together with matching variables, according to a log likelihood-ratio test for goodness-of-fit. For each variable, the model excluded records with missing values. Analysis was carried out using STATA 8.2 .Yersinia enterocolitica, Staphylococcus aureus, and enteropathogenic E. coli were sought by standard methods [Clostridium perfringens enterotoxin (CPE) was investigated either by assaying the cytopathic effect on Vero cells or by reverse passive latex agglutination (RPLA) test  according to the manufacturer's instructions [Twenty faecal samples were collected from 41 guests whose symptoms matched the case definition. Ova and parasites were investigated by direct microscopy, and Salmonella, Shigella, Campylobacter,  methods . The preructions .All faecal samples were tested for NoV by ELISA test  to detect NoV antigen and by RT-PCR. ELISA assays were performed using a 1:10 (w/v) dilution of stools in kit sample buffer following the Manufacturer's instructions.Viral RNA was extracted from 150 \u03bcl of 10% water suspensions of the stool specimens with the Nucleospin RNA Virus extraction kit , according to manufacturer's instructions. RNA was stored at -80\u00b0C until use in RT-PCR assays.Complementary DNA (cDNA) was prepared from 1.5 \u03bcl of eluted nucleic acid by reverse transcription using iScript Reverse Transcriptase and random hexamers  at 42\u00b0C for 45 min. One \u03bcl of cDNA was then amplified by PCR, using 20 pmol of primers JV12 and JV13 ,24 targeThree positive RT-PCR products obtained were purified by silica membrane using the QIAquick Gel Extraction Kit , and sequenced by the ABI PRISM BigDye Terminator Cycle Sequencing Reaction Kit, version 3.1, . The sequences obtained were compared with those of the FBVE data bank  using DNth, water samples were consecutively collected from various sites, in sterile condition, inside the resort and examined by the Local Agency for Environment. Water samples were subjected to culture tests for enteric bacterial pathogens, according to standard methods; viral contamination was not checked. No food leftover was available for microbiological investigations, at the time of the epidemiological investigation.After July 20th) approximately 150 guests were present in the hotel, and 41 (27.3%) cases were found by the door-to-door search.Four hundred individuals were guest of the hotel, during the entire outbreak period (from 2\u201319 July). On the day of the epidemiological investigation , and 58% of cases were female. The clinical pattern of the disease was characterised by the presence of diarrhoea (95%), vomiting (70%), abdominal pain (51%) and fever (32%). Of the 41 cases included in the study, the majority (65%) were residents of Northern Italian regions.The matched univariate analysis revealed that the cases were more likely to have eaten eggs, raw mussels or ice cubes made of tap water than the controls. They were less likely to have eaten ham, grilled sausages, snacks, or grilled meat Table . In the Two stool samples obtained from 20 patients, resulted positive for GII antigens using the NoV-specific ELISA test. The 20 stool samples were also examined by NoV-specific RT-PCR. Eighteen (90%) samples, including the two positive with the ELISA test, had an amplified DNA of the expected size for both the viral RNA-dependent RNA polymerase and the major capsid protein. All 18 samples resulted positive by genogroups II-specific ORF2 RT-PCR, whereas none was positive for genogroup I.Three of the amplified DNAs were subjected to sequencing of the diagnostic fragment in the ORF1 polymerase gene and/or in the ORF2 capsid gene. Sequences obtained were aligned with those present in the FBVE database and in NCBI. Analysis revealed that all sequences had a best fit with a GII Norovirus recombinant strain sharing ORF2 GII.3 and ORF1 GII.b characteristics. All samples from the outbreak were identical suggesting the involvement of a single virus strain.Water samples collected on July 20 from faucets in the bar, the kitchen, and a guest room had high levels of coliforms (up to 130 CFU/mL) and fecal streptococci (up to 22 CFU/mL). After microbiological analysis for routine bacterial enteropathogens, samples were discarded impairing possible virological investigation.This is the third outbreak described in a tourist resort in Southern Italy ,18 to daInfection appears to have spread rapidly within the resort, hitting persons within just a few days after their arrival, similarly to our previous observations during the 2000 epidemics in Italy .During the period of the outbreak, no other NoV outbreaks were notified outside the resort in the same area. Moreover, a recent study in a close area  of the 20 samples were positive for GII antigens. The RT-PCR test detected NoV RNA in 18 (90%) samples, including the two positive by ELISA, out of the 20 cases. These results fit with previous findings that ELISA assays have a lower sensitivity as RT-PCR methods , althougThe delay in reporting the outbreak is the main cause why it was not possible to identify the source of infection, since most of the patients during the first period of the epidemic were no longer available for interviewing. The data collected by interviews suggest that NoV had spread through consumption of ice made by tap water and raw mussels. Before July 23, no control measures to limit the spread of the infection were implemented, probably because the local health unit did not address the point source and failed to prevent person-to-person transmission. After July 23, the consumption of tap water was banned, because of the faecal contamination, and only bottled mineral water was served in the resort restaurant and used to wash vegetables. Water from the main tank, however, continued to be used for showers. The water inside the resort tank underwent superchlorination. However, NoV does survive high levels of chlorination , and theIn Italy, obtaining knowledge about the relative role of NoV as causative agent of outbreaks of gastroenteritis is hampered by the huge underreporting as well as late reporting of cases, and by disregard to collect and save clinical and environmental samples for virological testing. Moreover, this outbreak clearly shows the lack of laboratory capacity locally to test water and food for NoV, that is presently available only at the National level . The surThe author(s) declare that they have no competing interests.CR designed the epidemiological study, performed the statistical analysis and drafted and edited the manuscript. MS, FMC, RM performed the laboratory test on human samples. MS, IDB carried out the molecular studies, participated in the sequence alignment and drafted the manuscript. DDV, FMR and GR conceived the study, and participated in its design and coordination and helped to draft the manuscript. All the authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Veterinary staff were at high risk; standard veterinary infection-control guidelines should be followed. We determined factors associated with occupational transmission in Wisconsin during the 2003 outbreak of prairie dog\u2013associated monkeypox virus infections. Our investigation included active contact surveillance, exposure-related interviews, and a veterinary facility cohort study. We identified 19 confirmed, 5 probable, and 3 suspected cases. Rash, headache, sweats, and fever were reported by >80% of patients. Occupationally transmitted infections occurred in 12 veterinary staff, 2 pet store employees, and 2 animal distributors. The following were associated with illness: working directly with animal care (p = 0.002), being involved in prairie dog examination, caring for an animal within 6 feet of an ill prairie dog (p = 0.03), feeding an ill prairie dog (p = 0.002), and using an antihistamine (p = 0.04). Having never handled an ill prairie dog (p = 0.004) was protective. Veterinary staff used personal protective equipment sporadically. Our findings underscore the importance of standard veterinary infection-control guidelines. During May\u2013June 2003, an outbreak of monkeypox virus (MPXV) infections, initially detected in Wisconsin, occurred in the midwestern United States  titers in a specimen obtained within 56 days after rash onset or seroconversion in paired acute- and convalescent-phase specimens. The cohort study had no probable or suspected-case definitions and, hence, no probable or suspected cases.Signs and symptoms surveyed were rash, fever, chills, sweats, headache, joint pain, or lymphadenopathy within 21 days of most recent exposure to an ill prairie dog. Cohort members with a history of vaccinia vaccination or unknown vaccination status and birth date before 1972 were defined as vaccinia-vaccinated.A standardized questionnaire was used to determine exposure to prairie dogs, general work practices, demographic information, and medical history. Questions to assess contact with prairie dogs during the reception, initial examination, ongoing medical care, and discharge of the prairie dogs had possible answers of yes, no, unknown, or not applicable. Cohort members who did not work within 48 hours after the prairie dog\u2019s veterinary visit were excluded from the exposures analysis but included in the remainder of analyses. Questions about general work practices such as sanitizing, hand hygiene , and animal bedding changing practices had possible answers of yes, no, unknown, or not applicable; or they used Likert-scale responses of always, usually, sometimes, rarely, never, or not applicable.WDPH or local health department personnel administered the confidential questionnaire in person or by telephone. Data were entered into Microsoft Office Access 2003  and analyzed using Epi Info version 3.3 . Likert-scale responses of always and usually were dichotomized from sometimes, rarely, and never. Responses of unknown or not applicable were excluded.Willing participants provided acute- and convalescent-phase serum specimens, which were tested for nonspecific orthopox virus IgM and IgG levels at the CDC poxvirus laboratory  confirmed, 5 (19%) probable, and 3 (11%) suspected. Illness onsets occurred during May 15\u2013June 13, 2003 . Based o>80% of case-patients were rash, headache, sweats, and fever. Those reported by 60%\u201370% of case-patients were chills, sore throat, cough, or lymphadenopathy. All other signs and symptoms were reported by <23% of case-patients. No statistically significant differences in signs and symptoms were reported between confirmed and probable or suspected case-patients. Five (19%) patients were hospitalized; none died.Signs and symptoms reported by In terms of exposure settings, 12 (44%) cases, including 10 confirmed, occurred in staff of veterinary facilities where ill prairie dogs had received care . Other cSymptom diaries were completed by 258 persons, including 28 pet store employees and 7 veterinary staff. Local health department personnel monitored 243 other persons by telephone, including 77 veterinary staff. Among 501 persons followed up, 10 (2%) experienced illness; all 10 were veterinary staff.Two pet stores had received outbreak-associated prairie dogs. Of 28 employees , 19 (68%) completed the questionnaire: 11 had handled prairie dogs, 9 had fed prairie dogs, 2 had been bitten by a prairie dog, and 2 had been scratched by a prairie dog. One store A employee had a confirmed case, and 1 store B employee had a suspected case; both had handled ill prairie dogs. The store A employee had a tissue diagnosis of MPXV confirmed by viral culture and PCR and was positive for orthopox IgM and IgG antibodies in acute- and convalescent-phase serum specimens. The store B employee had prior vaccinia vaccination; a convalescent-phase serum specimen was negative for orthopox IgM and positive for IgG. Approximately 2 months after the last prairie dog exposure, serum specimens were obtained from 12 noncase pet store employees, 4 of whom had handled ill prairie dogs. All 12 had negative orthopox IgM antibody results.Two Wisconsin exotic animal distributors  distributed outbreak-associated prairie dogs and housed animals in their home. Distributor 2 had a confirmed case of MPXV infection, distributor 3 had a suspected case, and an immunocompromised household member who had no direct animal contact had a confirmed case.Four veterinary facilities had treated 3 outbreak-associated prairie dogs. These facilities employed 81 (range 3\u201359) persons during the outbreak; 74 (91%) participated in the cohort study (cohort members), and 44 (54%) participated in the serosurvey. Fever, sweats, chills, rash, lymphadenopathy, and headache were each associated (p<0.001) with confirmed MPXV infection . Among c>30 minutes handling the prairie dog approached significance (p = 0.09), 7 (41%) cohort case-patients reported never having handled a prairie dog. All of these 7 cases resulting from indirect exposure occurred in employees of facility 4. Five of these 7 case-patients reported having been within 3 feet of prairie dog C, 1 reported having been in the same room as prairie dog C but not within 3 feet, and 1 reported not being in facility 4 while prairie dog C was there but being there within 48 hours of its death. Multivariate analysis was not possible because of the small number of cohort members with each type of exposure.By using the number of cohort members from each facility as the denominator, we calculated veterinary facility attack rates as follows: facility 1, 25%; facility 2, 67%; facility 3, 7%; and facility 4, 25%. The attack rate among cohort members for all 4 facilities combined was 23%. All cohort case-patients had been in the veterinary facility within 48 hours of the prairie dog\u2019s visit. The only factor protective against MPXV infection  was neveUsing antihistamines during the prairie dog visit (p = 0.04) was associated with being a cohort case-patient . AntihisNo general work practice was a protective or risk factor for being a cohort case-patient. General work practices were not outbreak specific and were used to assess overall risk for communicable disease transmission. Several cohort members reported hand-to-mouth activities  in animal care areas . Only 12The 44 serosurvey participants included 9 of 10 patients with tissue-confirmed cases and 35 of 64 persons without tissue-confirmed cases (p = 0.04). Cohort members with direct animal care jobs were not more likely (p = 0.19) than those without such jobs to have participated in the serosurvey. MPXV infection was serologically confirmed for 13 (65%) persons who provided paired serum specimens and 2 (10%) who provided only acute-phase serum specimens. No evidence of asymptomatic seroconversion was found. Among serosurvey participants, only feeding a prairie dog was statistically associated (p = 0.02) with having a confirmed case of illness, and no personal medical history factors were associated with illness. A history of vaccinia vaccination was not protective against MPXV infection (p = 0.35). Nineteen (43%) serosurvey participants had been vaccinated. Of these, 6 (32%) had MPXV infection; 5 had multiple signs or symptoms; and 1 had only a nonpustular, nonvesicular rash. Four (67%) of the vaccinia-vaccinated serosurvey participants with confirmed cases had serologic confirmation only (no tissue confirmation was attempted), and 1 (17%) had both tissue and serologic confirmation of illness. Two serosurvey participants (A and B) with confirmed cases and previous vaccinia vaccination had no acute elevation of IgM. Participant A had symptomatic illness confirmed by IgG seroconversion and multiple high-risk exposures, including participation in 2 prairie dog examinations and having provided care to an animal within 6 feet of the prairie dog. Participant A\u2019s IgM levels were not elevated at 15, 36, and 50 days after exposure. Participant B had symptomatic, pathologically confirmed illness and multiple high-risk exposures, including participation in 4 prairie dog examinations, having fed the prairie dog, and having provided care to an animal within 6 feet of the prairie dog. Participant B\u2019s IgM results were not elevated, and IgG results were positive without a boost in titer at 16 and 157 days after exposure. Participant B had a history of bone marrow ablation and an allogenic bone marrow transplant.An Illinois animal distributor (distributor 1) obtained prairie dogs from a Texas distributor . During April\u2013May, 2003, distributor 1 housed \u2248200 prairie dogs with African rodents that had been purchased on April 21 and subsequently implicated in MPXV introduction  for 10 mThe 2003 outbreak of MPXV infections affected Wisconsin residents who had been exposed in multiple settings; however, 59% of cases occurred among occupationally exposed persons. Our cohort study demonstrates that veterinary staff were particularly at risk (23% attack rate). Pet store employees were at lower risk (7% attack rate). Infected prairie dogs were probably more ill and shedding more virus while in veterinary facilities than in pet stores, which would account for more observed infections among veterinary staff. Both Wisconsin distributors of ill prairie dogs became ill. The preponderance of occupationally acquired cases was unique to Wisconsin during this outbreak. Among other involved states, 1 veterinarian from Indiana had a suspected case, and cases occurred in 2 employees of distributor 1 (Most outbreak cases (59%) and veterinary cohort cases (82%) were associated with exposure to prairie dog C. We found no intrinsic differences in the monkeypox infection of prairie dog C compared with that of prairie dogs A or B and no explanation for this association, although length and type of exposures in facility 4 were likely accountable. Facility 4 had many more employees than the other facilities, and prairie dog C was hospitalized there for a relatively prolonged period (3 days) and received extensive treatments there. In addition, the fact that 7 (54%) facility 4 cohort case-patients reported never having handled prairie dog C indicates that other transmission modes  could have contributed to that facility\u2019s large number of cases. Nebulization treatments, which prairie dog C received \u22484\u00d7 at facility 4, could have exposed employees to MPXV. Although nebulization was performed in an enclosed plastic chamber, the nature of the treatment would foster aerosolization, coughing, and possibly mobilization of respiratory secretions with which employees could have unknowingly come into contact.Although our Wisconsin investigation showed no definitive evidence of human-to-human transmission of MPXV, which concurs with other states\u2019 findings during this multistate outbreak . Furthermore, cohort members reported general work practices that foster hand-to-mouth activities in animal care areas. Few (12%) cohort members reported having used gloves when cleaning ill animals\u2019 cages, a task that can contaminate staff hands with animal dander, urine, and fecal matter. We cannot determine whether infection-control guidelines would have prevented MPXV infections among veterinary staff, but use of personal protective equipment might have limited viral transmission. The National Association of State Public Health Veterinarians recently released the Veterinary Standard Precautions Compendium (In contrast with results from a previously published study (Our case definition for the cohort study differed from that of the overall outbreak investigation. Among veterinary staff, the 2 definitions resulted in different case numbers: the outbreak case definition resulted in 10 confirmed and 2 probable or suspected cases; the cohort study case definition resulted in 17 confirmed cases. Serologic results were not a confirming criterion in the outbreak case definition because their results were not validated at the time and because serologic specimens were not systematically gathered throughout the multistate outbreak.For unknown reasons, 2 cohort case-patients had no elevation of orthopox IgM. Although the IgM response might have been missed, this is unlikely given the timing of specimen collections. Also, previous vaccinia vaccination might have altered the immune response to the MPXV infection. It is also possible that participant B\u2019s past medical history might have affected the immune response to this infection.Our study has several potential limitations. Although 91% of employees at the 4 affected veterinary facilities participated in the cohort study, only 54% participated in the serosurvey. Persons with tissue-confirmed illness were more likely than persons without such illness to have participated in the serosurvey. These factors might have resulted in an underestimation of overall cases and limited the detection of asymptomatic seroconversion. Recall bias, which might have overestimated the association between prairie dog contact and illness, was likely limited by relatively brief intervals between exposures and data collection. Finally, statistical analysis beyond univariate analysis was limited because of the small number of cohort members involved in each of the high-risk prairie dog exposures.Our investigation and cohort study demonstrate that occupational exposure, especially among veterinary staff, was a critical factor during this outbreak. This outbreak highlights the importance of standard infection-control guidelines developed for veterinary settings and the need to encourage their use.Case Definitions for Human Monkeypox Virus Outbreak, Wisconsin, 2003"}
+{"text": "Salmonella enterica serotype Mbandaka occurred in Oregon, Washington, Idaho, and California. Based on epidemiologic and pulsed-field gel electrophoresis evidence from 87 confirmed cases, the outbreak was linked to contaminated alfalfa seeds grown in California\u2019s Imperial Valley. Trace-back and trace-forward investigations identified a single lot of seeds used by five sprout growers during the outbreak period. Cases of salmonellosis were linked with two sprout growers who had not employed chemical disinfection; no cases were linked to three sprout growers who used disinfection. This natural experiment provides empiric evidence that chemical disinfection can reduce the human risk for disease posed by contaminated seed sprouts.Based on in vitro data, the U.S. Food and Drug Administration recommends chemical disinfection of raw sprout seeds to reduce enteric pathogens contaminating the seed coats. However, little is known about the effectiveness of decontamination at preventing human disease. In 1999, an outbreak of  Salmonella enterica serotypes Newport and Stanley in 1995 and 1996  who follow these guidelines, as well as the proportion of sprouts grown from disinfected seeds, is unknown.Methods proposed to reduce the risk to human consumers include testing seeds, irrigation water, and sprouts for pathogens  conducted an outbreak investigation in response to a sharp increase in cases of S. Mbandaka in what soon proved to be a multistate outbreak. This investigation ultimately provided insight into the efficacy of seed disinfection.S. Mbandaka infection and onset of acute illness from January 1 to April 15, 1999. Cases were excluded if they were subsequently shown to have a pulsed field gel electrophoresis (PFGE) pattern that differed from the outbreak pattern. If the illness onset date was unknown, the specimen collection date minus 2 days was used instead.We defined case-patients as persons with culture-confirmed Salmonella during the preceding week.To generate hypotheses about potential exposure vehicles, ODHS conducted open-ended interviews with the first 10 Oregon case-patients. Based on these interviews, we performed a 2:1 (controls:cases) age-matched case-control study using these cases. Age-matched controls were identified by sequentially adding or subtracting one digit from each case-patient\u2019s telephone number prefix. Matched ranges were: 1\u201318, 19\u201350, and 51\u2013100 years of age. Using a standardized food questionnaire, we asked respondents about consumption of food that typically carries After alfalfa sprouts were implicated, we traced the source of sprouts from the case-patients to the brand of sprout\u2019s point of production to identify a common source for the contaminated sprouts. Onsite investigations of an implicated sprouter were conducted in partnership with the Washington Department of Agriculture and the FDA. Seed, water, and sprout samples were collected for culture with sterile swabs and containers.Because alfalfa seeds are often produced in bulk, we conducted trace-back and trace-forward investigations using purchase invoices and shipping records to identify other sprouters who may have purchased and used potentially contaminated seeds from the same production lot. Other sprouters who purchased this lot were asked about their use of the implicated lot, including quantities sprouted and their seed-disinfection practices. To identify additional cases, we queried health departments throughout the United States and Canada, with specific attention to states where seed from the implicated lot had been distributed.Salmonella isolates from patients were serotyped at the Oregon, California, Washington, and Idaho state laboratories. Salmonella cultures were performed according to standard methods , was used to corroborate genetic relatedness of S. Mbandaka isolates according to the method of Tenover et al. , Washington (19 cases), Idaho (7 cases), and California (21 cases) . Two cases clustered within a household, but were not considered to represent secondary spread of the disease, as they occurred concurrently. No patients died.From January 1 to April 15, 1999, a total of 89 cases of 1 cases) ; 74% of Nine of the first 10 case-patients and none of 20 matched control-patients reported eating or handling alfalfa sprouts . No other food vehicles were statistically associated with illness. From the binomial theorem, given an expected frequency of 10%, the probability of 9 out of 10 persons reporting sprout consumption within the past week was <1/1000 . Trace-back investigation showed that the common link between most cases was sprouter X . Eight oS. Mbandaka provided a leftover sample of sprout X brand alfalfa sprouts. Cultures from this sample subsequently grew S. Mbandaka.The index patient of Based on these investigations, representatives of the Washington Department of Health and FDA inspected the sprouter X facility on February 12, 1999, and determined that alfalfa sprouts grown during the outbreak period came from a single seed lot (lot L). Trace-forward information from the seed distributor and interviews with the other area sprout growers confirmed that sprouter X was the only grower in the Pacific Northwest to have purchased lot L seeds. Moreover, the illness onset date for the Oregon index case occurred 2 weeks after this sprouter received a shipment of lot L and 1 week after sprouts grown from lot L first reached market.S. Mbandaka was isolated from 11 of 12 alfalfa sprout samples, and one of two lot L seed samples obtained from the sprouter X facility. No pathogens were identified from this sprouter\u2019s samples of radish, onion, and clover sprouts, seed samples from non\u2013lot L seeds, or samples of irrigation water.Inspectors determined that no seed disinfection had been used at the sprouter X facility. The owner of the facility informed us that disinfection was \u201cunnecessary\u201d because seed samples had tested negative for pathogens before being shipped from the seed broker.After this inspection, sprouter X issued a voluntary recall of all products. Existing stocks of alfalfa sprouts were impounded, and the company was instructed to cease further production of sprouts using lot L seeds. A press release from the Oregon Department of Human Services was issued on February 12, 1999, advising consumers to return brand X sprouts and retailers to return unsold stocks.S. Mbandaka isolates from stool samples from the initial 10 case-patients, the partially consumed box of sprout X retrieved from case-patient 1, and the onsite sprout and seed sample cultures were indistinguishable but clearly differed from the two S. Mbandaka reference samples obtained in 1997 and 1998. Of the final 89 cases, 87 had identical PFGE patterns; 2 case-patients included in the total were not tested for technical reasons.The PFGE patterns for the Sprouter X purchased lot L seeds from a California broker. This broker, in turn, purchased seeds from California seed producer Z. Purchase invoices indicated that 5,454 kg of seed from 18,000 kg of lot L had been distributed among five sprout growers other than sprouter X: sprouters E, F, G, H, and Y . Four spS. Mbandaka outbreak. Isolates from Washington, Oregon, and Idaho were traced to sprouter X in Washington State; all California cases were traced to sprouter Y. Neither sprouter X nor Y had used seed disinfection during the outbreak period. Of the three sprouters  using lot L seed but not linked to cases of S. Mbandaka, all reported disinfecting their seeds with 2,000\u201320,000 ppm Ca(OCl)2, or 200 ppm sodium hypochlorite  were linked to cases of The outbreak was limited to consumers of sprouts grown by two sprouters who used the same seed lot. Neither sprouter used the FDA-recommended seed-disinfection process. Of the three sprouters who disinfected their seeds, none were linked to cases, although they had used 32% of the lot L seed that was sprouted. This finding suggests that seed disinfection does reduce the risk for human disease posed by seed sprouts.Since a formal randomized trial of seed disinfection in the community would be unethical, our understanding of the usefulness of disinfection is based largely on observations made in the course of outbreak investigations. In the current study, this outbreak fortuitously served as a natural experiment of the effectiveness of seed disinfection.Few other published outbreak investigations have addressed the value of disinfection at preventing human disease. Clover sprouts were implicated in a 1999 outbreak in Colorado Salmonella . Accordingly, future investigations should include careful reviews of the disinfection records of implicated sprouters. Receipts for seed-disinfection chemicals, protocols for disinfection, and disinfection logs may prove useful clues to actual disinfection practices. Asking the sprouter to simulate the disinfection process may also be instructive.The inoculum size needed to cause infection is known to vary between different serotypes of Given these limitations, a more reliable method of decontamination is needed if eating sprouts is to be safe. Gamma irradiation of sprout seeds or mature product reduces or eliminates colonizing bacteria. However, the prohibitive expense of irradiating machinery places this technology beyond the means of most sprouters. Moreover, irradiating seeds reduces the germination yield and affects the appearance of mature sprouts that germinate . Regardless of the assay\u2019s sensitivity, this technique appears highly vulnerable to sampling error unless the pathogen is uniformly distributed throughout the seed. Notably, research to date indicates that pathogens are dispersed heterogeneously and at low densities on seeds , a private trade group, has responsibly encouraged adherence among its members by allowing sprouters who use 20,000-ppm Ca(ClO)Given repeated outbreaks, seed testing that is falsely reassuring, ineffectiveness and incompleteness of seed disinfection, and a lack of commitment to disinfected product from retailers, we think that consistently rendering raw sprouts free of enteric pathogens is not practical. Persons at increased risk for invasive salmonellosis and those wishing to reduce their risk for foodborne infections may be well advised to avoid sprouts entirely."}
+{"text": "Salmonella (S.) Enteritidis infections in a large Hamburg nursing home was investigated.During August 2006, a protracted outbreak of Salmonella. Among nursing home residents a cohort study was carried out focusing on foods consumed in the three days before the first part of the outbreak. Instead of relying on residents' memory, data from the home's patient food ordering system was used as exposure data. S. Enteritidis isolates from patients and suspected food vehicles were phage typed and compared.A site visit of the home was conducted and food suppliers' premises tested for Within a population of 822 nursing home residents, 94 case patients among residents  and 17 among staff members were counted 6 through 29 August. The outbreak peaked 7 through 9 August, two days after a spell of very warm summer weather. S. Enteritidis was consistently recovered from patients' stools throughout the outbreak. Among the food items served during 5 through 7 August, the cohort study pointed to afternoon cake on all three days as potential risk factors for disease. Investigation of the bakery supplying the cake yielded S. Enteritidis from cakes sampled 31 August. Comparison of the isolates by phage typing demonstrated both isolates from patients and the cake to be the exceedingly rare phage type 21c.S. Enteritidis were the likely vehicle of the outbreak in the nursing home. While the cakes were probably contaminated with low pathogen dose throughout the outbreak period, high ambient summer temperatures and failure to keep the cake refrigerated led to high pathogen dose in cake on some days and in some of the housing units. This would explain the initial peak of cases, but also the drawn out nature of the outbreak with cases until the end of August. Suggestions are made to nursing homes, aiding in outbreak prevention. Early outbreak detection is crucial, such that counter measures can be swift and drawn-out outbreaks of nosocomial food-borne infections avoided.Cake (various types served on various days) contaminated with  Salmonella enterica ssp.enterica serotype Enteridis (S. Enteritidis). This is the Salmonella serotype most frequently reported in Germany , especially in later summer. As elsewhere, poultry and poultry products like eggs are considered the most important food vehicles for S. Enteritidis infections  \u00d7 [(# case patients exposed)/(# case patients)].Preliminary interviews indicated that residents were unlikely to remember correctly what they ate two weeks previously. Thus instead of information from interviews, data from the central kitchen's computerized patient food ordering system and from the peripheral kitchens' sets of note cards  were used. Data analysis was conducted in SPSSNo data on the background incidence for diarrhoea and vomiting were available for the senior care facility. The epidemic curve shows a first peak of cases (n = 53) between 7 and 9 August followed by approximately two weeks with zero to eight cases daily figure . The outThe entire population of the complex is approximately 1200 inhabitants. A total of 822 persons live in nine nursing units, cared for by 330 staff. The remainder resides in eight houses for assisted living. Only inhabitants of the nursing units (\"residents\") were affected by the outbreak, albeit with different AR  to a small number of businesses in the area. No complaints about diarrhoeal illnesses were received from these customers.Unit nursing staff distributed food into residents' rooms for breakfast, lunch and dinner, including the cake on the lunch tray. Staff members were supposed to deposit the \"vulnerable\" layered cream cake directly into the small patient room refrigerators. Time of consumption of the cake was up to the residents, except for in unit 8. There the pieces of cake were refrigerated in the unit kitchen until staff took the cake to the individual rooms and assisted residents in eating it.Salmonella. Five kitchen employees fell ill with salmonellosis through the course of the outbreak. The employee affected on 6 August worked in the central kitchen but was not involved in cake or light meal processing. Four other kitchen employees fell ill between 11 and 19 August. In addition, three asymptomatically infected staff members tested positive for S. Enteritidis. Two of them, one unit kitchen helper (stool positive on 21 August) and a central kitchen chef (stool positive on 22 August) were involved with food handling, the third (stool positive on 29 August) did not handle food.All reference food samples available  tested negative for Units 1 and 9 housed 181 residents. Of 114 residents with available information on sex 86 (75%) were women. Mean age in men and women was 88 years . Nineteen (10.5%) residents were case-patients with symptom onset before 11 August. Units 2 and 4 housed 197 residents (83% women). Mean age was 87 years with a range from 66\u2013104 years. Twentythree (11.7%) residents were case-patients with onset before 11 August. Thus, in all four units there were 42 (11.1%) case-patients with symptom onset before 11 August. The only meals or food items that were consistently statistically significant or close to significance were the light meals for lunch and the afternoon cake on 5, 6 and 7 August, respectively  and 2 cake isolates submitted to phage typing were all phage type (PT) 21c, a rare PT not seen in the NRC for years (table S. Enteritidis PT 21c contaminated bakery products (cake) delivered daily to the nursing home. A combination of the apparent S. Enteritidis PT 21c contamination of baked goods from the baker's premises and the findings of the cohort study clearly point to the implicated cakes delivered to the nursing home as the ultimate source of the introduction into the institution. Within the home, both the cakes directly, and likely also cross contaminated other food vehicles and person-to-person transmission served as sources of infection. The association between the consumption of the light meal and cake and the result of a stratified analysis were additional indications that the light meal was no independent risk factor. The meal included a sweet chicken curry with fruits, which may have been particularly attractive to persons who also like cake and sweets in general. It is also possible that the correlation arises from the high proportion of diabetics among the residents, who would have received none or special cake and also may have avoided the sweet curry.The outbreak was caused by S. Enteritidis outbreak in a Spanish hospital [S. Enteritidis cases among the assisted-living inhabitants who ate at the campus restaurant, the unaffected unit 8, or the implicated bakery's local customers. As phage typing is not available for the cases in the bakery's home county, it remains unclear if problems extended to local customers, although given the overall low number of S. Enteritidis cases compared to the same month in other years, the problem could not have been significant.Pathogen dose in the cakes upon delivery likely was low. As suspected in a similar hospital , nursingSalmonella within the nursing units, especially if the cake had been left standing at room temperatures for hours before consumption. The fact that staff were prone to forget depositing the cake into the fridge upon delivery to the room may be indicated by a hand scribbled remainder photographed during the site visit on a unit kitchen white board , asymptomatic infections could have lead to transmission. Person-to-person infection with escribed ,6, as weescribed .As many more residents ate the cake than became case-patients, asymptomatic infections are likely to have occurred. Some employees, both from the central kitchen and from unit kitchens, were affected as well, but apparently were excused from work promptly upon developing symptoms. Since the positive cake samples were taken directly from the baker's premises, nursing home kitchen staff members were likely to have become infected from handling the cake rather than being the source of the outbreak. The kitchen employee with onset on the first day of the outbreak developed symptoms in the evening after work and did not return to work the next day. His only contact to the implicated food items would have been that he possibly consumed them. It is feasible, that during the portioning of the cake onto individual plates in the central kitchen cross contamination of other food items occurred, which may explain some of the other food items associated with an increased risk of developing salmonellosis.Salmonella enterica serotype Enteritidis is the predominant Salmonella serovar identified by national reference laboratories in Western Europe. It is important that routine phage typing is carried out such that outbreaks can be recognised and investigated, and trends can be identified. Approximately half of the S. Enteritidis outbreaks recognized by the German NRC in the past years were caused by a variety of comparatively rare phage types other than type PT 4, rendering phage typing by itself (as well as in combination with molecular methods) an excellent tool to pick up associations among cases, and between human infections and S. Enteritidis in foods.The method of utilizing stored data on food orders instead of querying actual consumption introduces some uncertainty into exposure assessment. However, in circumstances such as these, with a population largely not competent to remember their exposure it may be a useful method to filter suspicious food items from a long list of potential exposures. In using the method, measures of association have to be interpreted with even more care than usual. Comparatively low RR in the context of infectious diseases, as well as borderline significant p values need to be evaluated in view of the imprecise exposure assessment. Analysis of food consumption ought to be complemented by food trace back and scrutiny of HACCP  protocol in order to stop current outbreaks and avoid repeated exposures.Salmonella outbreaks, commonly caused by S. Enteritidis [Salmonella outbreaks [Nursing homes, with their population of vulnerable residents, are at a particular risk of eritidis ,9. Bakedutbreaks ,11 and wRecommendations for nursing homes to avoid similar outbreaks are:- to make sure baked goods subject to bacterial contamination are refrigerated until time of consumption, especially during the summer,- to not delegate responsibility for refrigeration to residents, and- to avoid serving certain \"risky\" foods like layered cream cake to those most at risk for serious disease consequences.In addition, the following measures would have greatly enhanced outbreak detection and investigation in this outbreak, and appear useful to establish in similar institutions on a continuous basis:- the storage of reference food samples covering ALL non-packaged food items served to the inhabitants, not only those prepared in the nursing home's kitchen, and- strong infectious disease surveillance including the keeping of daily symptom logs, establishing the background rate of diarrhoeal symptoms (as well as respiratory symptoms) among residents of large facilities in order to more clearly identify the cessation of protracted outbreaks, and to promptly pick up outbreaks of norovirus, influenza etc. such that counter measures can be initiated without delay .The author(s) declare that they have no competing interests.CF and UB initiated the epidemiological investigation, designed the study, participated in the analysis and drafted the manuscript. MM assisted in the outbreak investigation, in the data collection, the epidemiological description and manuscript preparation. AS and KHB collected data on cases, information about exposure and risk factors of cases and non cases, implemented control measures and aided in writing the manuscript. PGD conducted the environmental investigations in the bakery implemented control measures and helped writing the manuscript.WR conducted a large part of the laboratory investigation, participated in the analysis and helped to draft the manuscript. GF conducted major parts of the data analysis, wrote the formal final report of the investigation and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Norovirus is often transmitted from person-to-person. Transmission may also be food-borne, but only few norovirus outbreak investigations have identified food items as likely vehicles of norovirus transmission through an analytical epidemiological study.During 7-9 January, 2009, 36 persons at a military base in Germany fell ill with acute gastroenteritis. Food from the military base's canteen was suspected as vehicle of infection, norovirus as the pathogen causing the illnesses. An investigation was initiated to describe the outbreak's extent, to verify the pathogen, and to identify modes of transmission and source of infection to prevent further cases.For descriptive analysis, ill persons were defined as members of the military base with acute onset of diarrhoea or vomiting between 24 December 2008, and 3 February 2009, without detection of a pathogen other than norovirus in stools. We conducted a retrospective cohort study within the headquarters company. Cases were military base members with onset of diarrhoea or vomiting during 5-9 January. We collected information on demographics, food items eaten at the canteen and contact to ill persons or vomit, using a self-administered questionnaire. We compared attack rates (AR) in exposed and unexposed persons, using bivariable and multivariable logistic regression modelling. Stool specimens of ill persons and canteen employees, canteen food served during 5-7 January and environmental swabs were investigated by laboratory analysis.Overall, 101/815 (AR 12.4%) persons fell ill between 24 December 2008 and 3 February 2009. None were canteen employees. Most persons (n = 49) had disease onset during 7-9 January. Ill persons were a median of 22 years old, 92.9% were male. The response for the cohort study was 178/274 (72.1%). Of 27 cases (AR 15.2%), 25 had eaten at the canteen and 21 had consumed salad. Salad consumption on 6 January  and 7 January  were independently associated with increased risk of disease.Norovirus was detected in 8/28 ill persons' and 4/25 canteen employees' stools, 6/55 environmental swabs and 0/33 food items. Sequences were identical in environmental and stool samples (subtype II.4 2006b), except for those of canteen employees. Control measures comprised cohort isolation of symptomatic persons, exclusion of norovirus-positive canteen employees from work and disinfection of the canteen's kitchen.Our investigation indicated that consumption of norovirus-contaminated salad caused the peak of the outbreak on 7-9 January. Strict personal hygiene and proper disinfection of environmental surfaces remain crucial to prevent norovirus transmission. Noroviruses are highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhoea with acute onset. Symptoms usually resolve within 2 or 3 days. The incubation period ranges from 6-48 hours. Infections typically peak during the colder months ,2. NorovApproximately 50% of all gastroenteritis outbreaks in developed countries are caused by norovirus . GloballIn Germany, norovirus has been the most frequently reported pathogen causing an illness since 2004. In 2008, 80% of all reported outbreaks were caused by norovirus, and 59% of all reported norovirus infections occurred in clusters .On 8 January 2009, the Bundeswehr Medical Service (German Armed Forces) reported an outbreak of acute gastroenteritis on a military base in Leipzig to the Robert Koch Institute. This specific military base had 815 members, and five divisions were stationed there. A total of 36 persons had fallen ill on 7 and 8 January with vomiting and diarrhoea. Food from the military base's canteen was suspected as the vehicle of infection. Norovirus was assumed to be the infectious agent due to the reported typical symptoms and the sudden onset of disease.An outbreak investigation was initiated on 8 January to describe the outbreak's extent, verify norovirus as the causative pathogen, identify mode of transmission and source of infection, and to implement infection control measures to prevent further cases.We collected information on demographics, clinical symptoms, disease onset, and division memberships of persons with acute gastroenteritis. All members from the military base are obliged to visit a doctor of the Medical Service if ill. For this study, all persons with diarrhoea or vomiting between 24 December 2008 and 3 February 2009 were retrospectively and prospectively recorded. Data was entered in EXCEL (Microsoft 2003) and analysed using Stata . We described illnesses by onset of disease, age, sex and clinical symptoms, and calculated division-specific attack rates (AR).In order to test the hypothesis that food items served in the canteen were the vehicles of infection we conducted a retrospective cohort study. Through the head of the division, we distributed a self-administered standardised questionnaire  for data entry and Stata  for data analysis.For each exposure, we calculated the relative risk (RR) of becoming a case using the chi-square test and the attributable fraction . Identified risk factors for illness and protective factors with a P value < 0.2 in the bivariable analysis were included in a multivariable logistic regression model to assess independent association with illness. In a stepwise backward procedure, we excluded exposures with P > 0.1 from the model. The exposure variable \"contact to ill persons or to vomit during the incubation period\" was consistently kept in the model to adjust for possible person-to-person transmission.In order to assess possible dose-response relationships for food items independently associated with disease, we calculated AR and performed the chi-square test for trend.Salmonella, Shigella, Campylobacter, Yersinia, Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, enterohaemorrhagic Escherichia coli (EHEC), adeno- and rotavirus. For norovirus detection, stool samples were ten-fold diluted in phosphate-buffered saline  and centrifuged for 1 minute at 13,500 \u00d7 g. Viral nucleic acid was extracted from 140 \u03bcl of supernatant using QIAamp Viral RNA Mini Kit  according to manufacturer's instruction.We collected stool samples from persons with acute gastroenteritis and from all canteen employees. Stool samples were tested for norovirus and, if negative, for 2 of each environmental surface were wiped as described elsewhere [On 9 January, we sampled various surfaces of the military base's Medical Clinic facilities and cohort isolation rooms, the mail administrating centre, the canteen's kitchen, as well as the living and working areas. Using sterile swabs  moistened in PBS, 100 cmlsewhere . Viral RAt German military base canteens, meal items are routinely sampled and samples are kept for 48 hours. According to the instructions, sampling is performed between preparation and distribution of food. The specific food items served and sampled in the canteen on 6 and 7 January were tested for norovirus if the results from the analytical study suggested an independent association with disease. Samples from food served on 5 January had already been discarded. For testing, we used a method adapted from a previously reported elution-precipitation protocol . ViscousAY485642) for the second round PCR. The amplicons were separated on ethidium bromide-stained 1.5% agarose gels and visualised under UV light.The extracted RNA was tested for norovirus by real-time reverse-transcription polymerase chain reaction (RT-PCR)  in an ABThe amplicons of the ORF1 (213 bp) and the ORF1/2 junction region (215 bp) considered for sequence analysis were purified using the Qiaquick DNA purification kit . Amplicons of the ORF 1/2 junction region were directly sequenced using the amplification primers of the second round PCR. Amplicons of the ORF1 region were sequenced using generic norovirus primers MON431 and MON432  or cloneCampylobacter and EHEC in their stools, see laboratory results.Overall, 104 of 815 military base members (AR 12.8%) fell ill with vomiting or diarrhoea between 24 December 2008 and 3 February 2009. Three norovirus-negative persons shed rotavirus, In the following, we describe the 101 ill persons without detection of any other pathogen than norovirus Figure . Zero toOverall, 178 of 247 (72.1%) persons from the headquarters company completed the questionnaire. Among those, 160 stated the date of questionnaire completion: 31.9% completed it before 12 January (within seven days after the earliest date of food consumption we queried in our questionnaire), 64.4% before 15 January (within seven days after the latest date of food consumption queried) and further 3.8% until 22 January. Information on non-responders was not available.Overall, 27 persons met the case definition (AR 15.2%). Two persons fell ill on 5 January, eight on 7 January, 11 on 8 January and six on 9 January. In cases with available information, the median duration of illness was two days , the median duration of sick leave was four days  and four of eighteen were hospitalized (22%). Cases and non-cases did not differ with regard to age and sex .Overall, 25 of 133 (18.8%) persons who ate any meal at the military base's canteen and two of 45 (4.4%) persons who did not eat there during 5-7 January met the case definition . In bivariable analyses, persons consuming any lunch during 5-7 January at the canteen had an increased risk of disease compared to those who did not. Eating breakfast or eating dinner at the canteen, contact to ill persons or vomit during the incubation period or staying at the military base overnight were not associated with disease  respondents were potentially exposed. Among those, 11 (AR 10.4%) met the case definition. Eating salad from a self-service salad buffet was associated with higher risk of disease in bivariable and multivariable analysis Table .During lunch on 7 January, 108 of 178 (60.1%) respondents were potentially exposed. Of those, 19 (AR 17.6%) met the case definition. In bivariable and multivariable analysis, consumption of salad was again associated with increased risk of disease Table .We assessed a possible dose-response relationship between salad consumption and disease by including only persons in our analysis who ate lunch at the canteen on all days between 5 and 7 January. The risk of disease increased with the number of days on which salad was consumed Table .Within the entire cohort, persons eating salad  had a 4.8-times higher risk  of being a case than persons not consuming salad . Among persons who had lunch in the canteen on 6 and 7 January (n = 119), the AR among salad consumers  was 4.4-times higher  than among non-salad eaters .Campylobacter or EHEC.Overall, 8 of 28 (28.6%) tested stool samples from ill persons were norovirus genogroup II-PCR-positive. Samples were taken between 12 January and 3 February, a median of 4 days after onset of illness (range 1-10 days). Only one sample per person was taken into consideration. Sequencing of five randomly selected specimens revealed an identical subtype (GII.4-2006b) and identical nucleotide sequences in the polymerase and the ORF1/2 gene region, respectively. Stool specimens from one ill person, respectively, tested norovirus-negative but positive for rotavirus, GU017736, GU017737, and GU017738).Stool samples of 4 of 25 canteen employees (16%) taken during 12-29 January tested norovirus-positive. Two of the norovirus-positive persons had worked in the canteen's kitchen between 5 and 7 January. One of them reported to have suffered from nausea with onset on 5 January. She had continued working through 6 January. The other reported not to have had any symptoms. She had cleaned the salad bar on 6 and 7 January. Norovirus sequences found in stool samples of these two persons belonged both to subtype GII.4-2006b but differed between 1.4% and 2.4% from those detected in ill persons Figure . NucleotThe other two canteen employees shedding norovirus did not work at the canteen in January 2009 at all. EHEC was isolated in one other symptomatic canteen employee. All other stool samples from canteen employees tested negative for bacterial or viral pathogens.Norovirus was detected in 6 of 55 (10.9%) environmental samples taken on 9 January. Positive swabs stemmed from the mail administrating centre , the living or working areas , and the cohort isolation rooms (1 of 12: doorknob). Norovirus was neither found in any swabs from the kitchen (0 of 15) nor from the canteen employees' toilets (0 of 4). Surfaces from the salad bar and the dining hall were not sampled. Since PCR products were mostly too weak for confirmation by sequencing, only three environmental samples  were sequenced. They belonged to norovirus subtype II.4-2006b and were of identical sequence to the ill persons' clinical samples.None of the 33 tested food items was norovirus PCR-positive. Investigated food items included salads and salad dressings.On 8 January, the heads of all five military divisions were briefed on the outbreak and on preventive measures. Ill persons were either cohort isolated in assigned rooms or went on sick leave for one week. Hand disinfectants were distributed to ill persons. Healthy persons were sent home for two days (8 and 9 January) to interrupt the outbreak. Specific soldiers were designated and instructed for cleaning and disinfection of areas contaminated with vomit and faeces.The canteens' kitchen was routinely cleaned on 8 and 9 January. On 9 January, the military unit responsible for hygiene inspected the kitchen. Cleaning and disinfection was performed on 10 January using virucidal agents. Canteen employees with positive stool samples were excluded from work until tested negative for the respective infectious agent.We investigated an outbreak of acute gastroenteritis that affected 12% of the members of a military base and that was caused by norovirus of subtype II.4-2006b. The outbreak lasted for 5 weeks. Half of the illnesses occurred within 3 days between 7 and 9 January, strongly indicating a point source of infection. Results of the cohort study suggested food-borne transmission for most of these cases. Eating any meal, having any lunch, and consuming salad from a self-service buffet at the military base's canteen were all highly associated with disease. Frequency of salad consumption correlated positively with risk of disease. Of the 27 cases identified in the cohort study, 25 had eaten lunch and 21 had eaten salad at the canteen supporting the hypothesis that infections were acquired during canteen visits. Person-to-person transmission was tested for but not identified as risk factor for becoming a case. However, for the time period before and after 5-9 January, both person-to-person and environmental-to-person transmission is plausible. Norovirus outbreaks with initial food-borne transmission further propagated by person-to-person or environmental-to-person transmission have been reported .Our findings of a causal role for salad in this outbreak are consistent with other studies in which salad had been involved in norovirus transmission ,17,19,30All analysed food samples including salad were tested norovirus PCR-negative. Reasons for the norovirus PCR-negative food samples could be the presence of substances inhibiting molecular detection such as phenolics and polysaccharides, low virus concentration, or a heterogeneous distribution of viruses in the food. Other studies confirm that norovirus detection in food items remains challenging ,32.Norovirus genome sequence analysis suggests that all ill persons belonged to the same outbreak. However, the specific source of contamination for the salad, the likely vehicle of infection, remains unclear. While norovirus genome sequences from ill persons and environmental swabs were identical, those from canteen employees differed slightly.Therefore it its unlikely that canteen employees contaminated the salad. Since norovirus infections peak every winter, occurrence of norovirus-infected persons in the military base due to virus circulation in the general population is plausible. However, persons shedding several different norovirus strains at the same time have been reported . Hence, Norovirus-contaminated environmental surfaces might have played a role in transmission. Their implication in virus transmission has been documented in other studies -38. In oThe date of sampling might explain why environmental swabs from the canteens' kitchen were norovirus PCR-negative. Samples were drawn on 9 January. If the contamination occurred on 6 or 7 January, norovirus could have been already removed on 9 January due to the daily regular cleaning.Cases might have remembered information on food consumption better than non-cases. Since we provided the list of served food items in the questionnaire and most participants completed the questionnaire within ten days after possible exposure, we assume the impact of recall bias as minor. Possibly, the low proportion of norovirus PCR-positive cases was partly due to the ordinance of sick leave of one week for ill persons in contrast to special leave of two days for healthy persons. This might have tempted some soldiers to pretend being ill resulting in an overestimation of case numbers. However, this potential non-differential misclassification would probably have led to an underestimation of the true association between salad consumption and risk of disease. Another reason for the low proportion of norovirus PCR-positive cases could be that people did not shed virus anymore at the time of sampling.Generally, military bases provide good conditions for epidemiological investigations: the source population is distinct, soldiers are instructed to visit the same doctor for medical advice, food samples are collected routinely from each meal and are kept for 48 hours, and the military is well equipped for timely investigations, which is especially helpful in food-borne disease outbreaks.Early implemented preventive measures like cohort isolation and sick leave for ill persons, special leave for healthy military base members, distribution of hand disinfectants, cleaning and disinfection of the Medical Clinic and the canteens' kitchen as well as identification and exclusion of norovirus PCR-positive canteen employees from work may have successfully averted further cases.In addition to recommendations generally applicable to outbreaks of acute gastroenteritis such as timely and sustained implementation of preventive measures, we derive the following recommendations from the experience in this outbreak. Surfaces that are touched by many people, such as doorknobs or computer equipment, should be cleaned and disinfected daily or more often than usual to avert environmental-to-person transmission.Protective measures should be communicated in oral and written forms. Posters displayed clearly visible in buildings with many passers-by should prompt military base members and visitors to wash their hands with water and soap and use disinfectants after toilet visits and before food consumption. Adequate hand disinfectants should be placed next to sinks for both healthy and ill persons.As we found asymptomatic norovirus-shedding canteen employees, the implications of hand washing and disinfection before handling food or touching surfaces used for food preparation, especially after going to the toilet, should be communicated more frequently to staff members. If canteen employees fall ill, they must stop working immediately. Resumption of work should be earliest two days after remission of symptoms.Sequence analysis of predominant norovirus genotypes like GII.4 should be performed in order to identify chains of infection. Further research on methods for norovirus detection in food is needed.Our investigation indicated that consumption of norovirus-contaminated salad caused the peak of the outbreak on 7-9 January. Strict personal hygiene and proper disinfection of environmental surfaces remain crucial to prevent norovirus transmission.The timely initiated analytical epidemiological investigation proved valuable to identify the likely vehicle of infection and to guide targeted intervention measures.The authors declare that they have no competing interests.MW participated in the design of the study, drafted questionnaires, performed data entry, epidemiological analysis and interpretation of the data, participated in coordinating the study and wrote the manuscript draft. KSc carried out the virological analysis, interpreted the laboratory results and wrote the manuscript draft. SN participated in the design of the study, drafted questionnaires, took part in the analysis of the data and contributed to the manuscript. SD participated in molecular analysis of human stool samples. LE has made contributions to conception and design and organizing the acquisition of data. CF was involved in study planning, data analysis and contributed to manuscript writing.RG carried out microbiological and virological analysis and contributed to the manuscript. MH performed sequencing and phylogenetic analysis of norovirus from human stool samples. RJ was involved in study coordination, analysis and interpretation of the study, and contributed to the manuscript. GK was involved in analysis and interpretation of the study regarding laboratory aspects. JK participated in the design of the study, assisted the outbreak investigation and revised the manuscript critically. JS participated in coordinating the study and revised the manuscript critically. UT participated in coordinating the study in the field and contributed to manuscript writing. KSt was involved in study conception, design, and coordination of the study, data analysis and contributed to manuscript writing. HB was involved in study conception, design, and coordination, data entry, data analysis, data interpretation, and manuscript writing. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/30/prepubQuestionnaire distributed to members of the headquarters company, norovirus-outbreak at a military base, Germany, 2009. The questions focus on potential risk factors for norovirus-outbreaks like food items eaten at the canteen and contact to ill persons or vomit.Click here for file"}
+{"text": "In 2006, a total of 178 cases of acute Chagas disease were reported from the Amazonian state of Par\u00e1, Brazil. Eleven occurred in Barcarena and were confirmed by visualization of parasites on blood smears. Using cohort and case\u2013control studies, we implicated oral transmission by consumption of a\u00e7a\u00ed palm fruit. Trypanosoma cruzi, which is transmitted by bloodsucking triatomine insects. Other modes of transmission are transfusional, congenital, and oral (foodborne)  chronically infects \u224810 million persons in Latin America . Eleven of these cases occurred in Barcarena  , panel BT. cruzi or positive serologic results and clinical evidence of acute Chagas disease. A non-case was any person who participated in the meeting and had negative test results for T. cruzi. We also conducted a 1:3 case\u2013control study (11 case-patients and 34 controls matched by sex and age) that included patients with laboratory-confirmed cases from Barcarena. A case-patient was any person in whom during September 1\u2013October 15 T. cruzi was found by direct parasitologic examination, irrespective of signs or symptoms of disease, or who had positive serologic results and clinical evidence of disease. This interval was based on date of symptom onset of the first and last case-patient and a reported incubation period of 3\u201322 days for orally transmitted disease. Controls were age- and sex-matched residents of case-patient neighborhoods who had negative serologic results for T. cruzi.We conducted a retrospective cohort study of staff members at the health post who participated in the meeting on September 15. A case-patient was any person who participated in the meeting and had a positive direct parasitologic examination result for >40 was considered positive. Controls had nonreactive IgM and IgG titers. We ruled out leishmaniasis in all persons with positive serologic results for T. cruzi by using an immunofluorescent test for IgM to Leishmania spp. . Serologic tests were conducted by using indirect hemagglutination test, ELISA, or indirect immunofluorescent test. An immunoglobulin (Ig) M titer We conducted an entomologic investigation during December 11\u201316, 2006, at the homes of 5 case-patients and in forested areas near the homes of 2 case-patients; at the commercial establishment where a\u00e7a\u00ed consumed by the case-patients linked to the health post was prepared and served; at an a\u00e7a\u00ed juice production and sale establishment reported to be frequented by other case-patients; and at the river dock market where a\u00e7a\u00ed delivered to Barcarena is unloaded. At this market, we searched baskets used to transport a\u00e7a\u00ed in river boats. We applied an insect-displacing compound  to the interior and exterior of buildings at investigation sites and placed traps . We measured relative risk in the cohort study and matched odds ratios in the matched case\u2013control study, with 95% confidence intervals and \u03b1 = 5%. Fisher exact, McNemar, Mantel-Haenszel, and Kruskall-Wallis tests were used as needed. Study power (1 \u2013 \u03b2) was 5%.T. cruzi by direct examination of blood  of whom were case-patients. The remaining persons were seronegative for otective . This shotective . Among eOur study findings implicated a\u00e7a\u00ed in an outbreak of acute Chagas disease. Oral transmission of this disease in the Amazon region has been reported since the 1960s. A\u00e7a\u00ed has long been the principal suspected food vehicle, but characteristics of outbreaks, small groups with universal exposure and high attack rates, have precluded epidemiologic implication of this food. There are no reports of timely collection of a\u00e7a\u00ed for laboratory testing in an outbreak.In this outbreak, vectorborne, transfusional, transplant-associated, and transplacental transmission were excluded. Incubation periods of cohort case-patients were compatible with those of previous reports. A shared meal was the only event linking case-patients, and cohort and case\u2013control studies demonstrated an association between a\u00e7a\u00ed consumption at this meal and infection. These findings indicate an outbreak of orally transmitted disease from contaminated a\u00e7a\u00ed.T. cruzi in a\u00e7a\u00ed, along with the tree-to-bowl continuum of a\u00e7a\u00ed, to identify sources of contamination. Because a\u00e7a\u00ed is a major dietary component in the Amazon region and a component of the local economy, identifying practical prevention measures is essential.Limitations of this study are possible recall bias caused by delay between illness and investigation and failure to collect food samples for testing. Studies are needed to determine viability of"}
+{"text": "Escherichia coli O157:H7. A case\u2013control study of 22 case-patients found that consuming bagged spinach was significantly associated with illness (p<0.01). The outbreak strain was isolated from 3 bags of 1 brand of spinach. Nationally, 205 persons were ill with the outbreak strain.In 2006, Utah and New Mexico health departments investigated a multistate cluster of  Escherichia coli O157:H7 infections and a suspected association with spinach. E. coli O157:H7 expresses 1 of 2 types of Shiga toxin and can cause severe gastrointestinal infections and hemolytic uremic syndrome (HUS).On September 13, 2006, health officials from several states independently notified the Centers for Disease Control and Prevention (CDC) about clusters of A multistate outbreak investigation, involving 26 states, was initiated on September 14. The US Food and Drug Administration (FDA) and CDC advised consumers not to eat bagged spinach . This urgent outbreak investigation did not require institutional review board approval.The case definition for a laboratory-confirmed illness was culture-confirmed The UDOH Enteric Disease Case Report Form and a supplemental CDC questionnaire on spinach consumption were administered by local or state public health officials to all participants by telephone. Information collected included date of disease onset, symptoms, treatment, community-based exposures, and a food-item history. Questions referred to the 8\u201310 days before case-patient symptom onset. Case-patients were first interviewed 3\u201323 days after illness onset (mean = 11.6 days); follow-up interviews for the questionnaire were completed within 23 days of illness onset.<4 years, 5\u201312 years, 13\u201318 years, 19\u201364 years, and >65 years. Controls were selected by using sequential-digit telephone dialing based on the matched case-patient\u2019s telephone number. Controls reported no gastrointestinal illness 3 days before and after symptom onset date of their matched case-patient.Two controls per case-patient were matched by sex and age group to prevent confounding from potential differences in diet (Exact matched odds ratios (mOR) and confidence intervals (CI) were calculated by using conditional logistic regression in SAS 9.1 . An \u03b1 of 0.05 was used. No statistical analyses were performed for the categories \u201cspinach brand\u201d or \u201clocation spinach was eaten\u201d because of insufficient data and inability to generate point estimates; we provide only descriptive evaluations of these variables. Only persons who indicated definite exposure to a single brand of spinach were included in the evaluation of brand.E. coli O157:H7 from both clinical and spinach samples .The Utah Public Health Laboratories (UPHL) and New Mexico Scientific Laboratory Division (NMSLD) provided analytic testing services for all clinical and spinach samples. Public health officials collected spinach samples in their original packaging from confirmed case-patients. One spinach sample in New Mexico was frozen; all other samples were refrigerated. Modified FDA Bacterial and Analytical Manual methods were used to recover E. coli O157 test kit  and DIFCO H7 antiserum . All clinical isolates were tested for Shiga toxin genes (stx1 and stx2) by PCR. Clinical and spinach isolates were tested for Shiga toxin expression with the Premier EHEC enzymatic immunoassay Shiga-toxin test kit . PFGE was performed on E. coli O157:H7 cultures from clinical and spinach samples by using standard CDC PulseNet operating procedures ; NMSLD used Oxoid Dryspot Eighteen cases were confirmed in Utah and 5 in New Mexico. Onset dates ranged from August 22 to September 11 . Shiga tE. coli were not significantly associated with case status.Matched analyses were performed on 22 case-patients and 44 matched controls. Consumption of bagged spinach  was significantly associated with case status . Food itNo patients, versus 6 controls, reported only eating spinach at a restaurant; 16 patients and 10 controls reported only eating spinach in a private home . WashingE. coli O157:H7, matching the national outbreak strain, was detected by PCR and culture in 2 Utah spinach bags and the New Mexico unfrozen bag. All 3 were open bags of brand A baby spinach. Lot codes were available on 2 bags; both were packed on August 15, 2006, at the same plant, on the same shift, but on different machines.Seven bags of spinach  were provided by 7 case-patients. Five bags had been opened and their contents partially consumed. E. coli O157:H7 was not detected were also brand A baby spinach and were packed on August 15, 22, 23, and 28, 2006. Two bags that tested negative were eaten by case-patients who reported eating from multiple bags before illness onset.The 4 bags from which E. coli O157:H7 infection with both epidemiologic and laboratory data. Washing spinach before consumption did not affect odds of being a case-patient. Possible reasons for this include 1) E. coli could be internalized into the plant structure by entering through the roots (Consumption of fresh brand A spinach was associated with E. coli O157:H7 outbreaks (15%\u201320%) (E. coli with a higher incidence of HUS (The percentage of case-patients in whom HUS developed (29%) was high when compared with that in prior Our study was subject to certain limitations. One is potential recall bias since controls had a longer time lag between consumption and interview and less motivation to accurately recall what foods they had eaten. However, overestimation of the association between spinach consumption and illness is unlikely because more controls reported having eaten spinach than were previously identified in surveys of the general population (E. coli O157:H7 infections with matching PFGE patterns among 205 persons in 26 states (E. coli O157 infections (This investigation was conducted in response to a national outbreak of"}
+{"text": "In July 2000, an outbreak of gastroenteritis occurred at a tourist resort in the Gulf of Taranto in southern Italy. Illness in 344 people, 69 of whom were staff members, met the case definition. Norwalk-like virus (NLV) was found in 22 of 28 stool specimens tested. The source of illness was likely contaminated drinking water, as environmental inspection identified a breakdown in the resort water system and tap water samples were contaminated with fecal bacteria. Attack rates were increased (51.4%) in staff members involved in water sports. Relative risks were significant only for exposure to beach showers and consuming drinks with ice. Although Italy has no surveillance system for nonbacterial gastroenteritis, no outbreak caused by NLV has been described previously in the country. Norwalk virus is the prototype of the genus Norwalk-like virus (NLV) in the Caliciviridae family, which includes a large number of genetically related strains that together represent the most important cause of gastroenteritis outbreaks worldwide , outbreaks are characterized by a high secondary attack rate In Italy, which has no surveillance system for nonbacterial gastroenteritis, the impact of NLV infection is unknown, and no previous outbreaks of confirmed NLV infection have been reported. We describe a large outbreak of gastroenteritis caused by NLV at a resort in Italy.The outbreak occurred at a tourist resort in the Gulf of Taranto, southern Italy, during July 7\u201331, 2000 . The resThe resort\u2019s water tank is supplied via a 1-km pipe connected to the main public water supply . On JulyOn July 18, 2000, the local health unit and the Institute of Hygiene of the Faculty of Medicine in Bari were notified about an outbreak of gastroenteritis at the resort. An epidemiologic investigation was initiated the same day to identify the agent and the mode and vehicle of transmission and to implement control measures. By July 20, when the local health unit notified the Istituto Superiore di Sanit\u00e0 in Rome, the outbreak had already been in progress for approximately 2 weeks and >150 persons were ill.A case was defined as illness in any guest or employee who stayed at the tourist resort during the period July 1\u201331 and who had diarrhea (three or more loose stools in any 24-hour period) or vomiting (at least one episode) or both, in the same period. Case finding was done by checking records of the resort medical center; after July 20, a door-to-door search was initiated. Demographic data and information on symptoms were collected in face-to-face interviews by the medical staff of the local health unit and the University of Bari.Because of the high number of cases in resort staff members, a retrospective cohort study was performed to assess risk factors associated with illness in this group. Persons eligible for the study were staff members employed at the resort from July 1 to 31. Standard questionnaires were sent to all 224 staff members in the first week of August. Information requested included name, date of birth, sex, room number, job type, date of onset and type of symptoms, and water and food preferences. A month had elapsed between onset of symptoms and distribution of the questionnaires. We did not inquire about actual food history and activities of staff members during the outbreak but rather about their food preferences and usual activities.The questionnaires from guests and staff members were returned to the Istituto Superiore di Sanit\u00e0, where the data were analyzed by using SPSS Base 10.0  and Epi-Info 6.04 . Information collected on cases was used to construct the epidemic curve and describe the clinical presentation of the disease. Attack rates, denominator data, personal characteristics, and clinical symptoms of cases were compared between guests and staff members by chi square or Fisher exact test when appropriate; the Mann-Whitney U-test was used for comparisons of age. The room location of ill persons was plotted on a map of the resort that included water pipelines in an attempt to identify any clustering of cases along the pipeline. Statistical test for clustering was performed by the cluster k-means method with SPSS Base 10.0 (SPSS Inc.).In the cohort study, the attack rate was calculated for the total staff and also by specific job type. Relative risks and 95% confidence intervals were also calculated for job type, behaviors and activities, and food preferences.From July 18 to 28, samples  were collected from 30 participants whose illness met the case definition. Part of each specimen was stored at -20\u00b0C until examination for viral particles and free fecal cytotoxins, and the rest was refrigerated and processed within 12 hours of collection.Salmonella, Shigella, Campylobacter, Yersinia enterocolitica, Staphylococcus aureus, and enteropathogenic E. coli were sought by standard methods Clostridium perfringens enterotoxin (CPE) was determined either by assaying the cytopathic effect on Vero cells or by reverse passive latex agglutination (RPLA) test  according to the manufacturer\u2019s instructions Ova and parasites were detected by direct microscopy, and Stool and vomit suspensions were examined by NLV-specific reverse transcription/polymerase chain reaction (RT/PCR) with generic primers JV12\u2013JV13 to a consensus sequence on the RNA polymerase segment of the genome shared by most NLV strains After July 13, water samples were repeatedly collected from the main public water supply and from various points outside and inside the resort. Samples from food in the kitchen and the refrigerators were collected and sent to the University of Bari on July 18. Water and food samples were subjected to culture tests for enteric bacterial pathogens, according to standard methods.Of 344 cases identified from July 1 to 31, 69 (20%) were in staff members. Information on personal characteristics and clinical presentation was available for 248 ill persons . DiarrheThe epidemic curve shows three distinct peaks in each of the 3 weeks, beginning on July 12 (70 cases), July 18 (26 cases), and July 27 (55 cases). Over the total outbreak period, 275 cases occurred in guests and 58 in staff . Fifty-sBecause of the rapid turnover at the resort, attack rates for guests were calculated separately for each week: an attack rate of 102 (10.5%) of 970 was observed in week 1; 66 (8.7%) of 760 in week 2; and 105 (10.1%) of 1,034 in week 3. Ill guests occupied 157 of the resort\u2019s 456 rooms. No significant evidence of either clustering by the cluster k-means methods (p=0.392) or increased frequency of cases in rooms near the water pipeline was observed. Attack rates by sex, age group, and week of stay were similar.For the analysis of risk factors in the cohort study, 181 questionnaires from 224 staff members were completed and analyzed. The attack rate in this group was 69 (38.1%) of 181. The lowest attack rates were observed in staff members who worked in the kitchen or the office, and the highest were in waiters, sports trainers, entertainers, and cleaning staff  . Staff mStool samples from 28 patients were negative for ova and parasites and bacterial enteropathogens. Of the 28 stool samples examined by NLV-specific RT-PCR, 22 had an amplified DNA of the size expected for NLV. The 327-bp amplification product was also confirmed for all samples by Southern blot hybridization with NLV-specific probes. Vomit specimens from two other subjects were negative.A readable common sequence of 290 bp was obtained with sequence analysis and found to be the same for eight samples, indicating a single outbreak virus strain. The sequence was analyzed against the European Molecular Biology Laboratory Nucleotide Data Bank, yielding a best fit with the RNA polymerase sequence of the Lordsdale strain of NLV C. perfringens enterotoxin was induced by seven samples. The RPLA test confirmed the presence of C. perfringens enterotoxin in all seven samples. The positive specimens had been collected July 18\u201321, and all were also positive for NLV  that could have been the source of infection by C. perfringens remained unknown. However, the presence of C. perfringens enterotoxin in a small, defined cluster of patients (7 of 28 stool samples) and the concomitant presence of NLV in the 7 positive stools suggests that C. perfringens played only a minor role, if any, in the outbreak.Some specimens showed evidence of simultaneous infection with NLV and enterotoxigenic Control measures to limit the spread of the infection had no effect, probably because they did not address the point source and failed to prevent person-to-person transmission. After July 15, 2000, the consumption of tap water was banned, and only bottled mineral water was served in the resort restaurant and used to wash vegetables. Water from the main tank, however, continued to be used for showers, to make ice for consumption (through July 28), and for irrigation. Furthermore, on July 22, the bypass pipe was removed, the water inside the resort tank underwent superchlorination, and the pipe connecting the resort to the public water supply was shut down. However, NLV do survive high levels of chlorination  was significant. Our analysis found no evidence that contaminated food was the source of infection: no food preference was associated with an increased risk of being ill, and personnel working in the kitchen had the lowest attack rate.In addition to water contamination, person-to-person transmission may have played a role in this outbreak. Typically staff members of tourist resorts share the same living quarters and have frequent contact with guests during meals, sport training, entertainment, and other activities. Person-to-person transmission may also explain the fact that the time between arrival and onset of symptoms in guests was longer than the incubation period expected for NLV. Person-to-person transmission of NLV infection is well documented This investigation had several limitations. Since the cohort study was carried out after the outbreak had ended, we could inquire only about food preferences and usual activities rather than actual food histories and activities before the outbreak. Recall bias may have occurred, which may have led to nondifferential misclassification of exposure and underestimation of the observed relative risks. If NLV had spread through water and person-to-person transmission had occurred, virtually everyone in the resort would have been exposed to the agent and any epidemiologic association would be difficult to find. Finally, no test specific for NLV was performed on water samples, and the hypothesis of water as the actual source of infection cannot be confirmed.In conclusion, this event confirms that large outbreaks due to NLV may be occurring in Italy, but without the use of appropriate diagnostic methods this pathogen may go unrecognized. This occurrence highlights the need for a surveillance system of such outbreaks in cooperation with laboratories capable of diagnosing viral gastrointestinal infections."}
+{"text": "The incidence of melioidosis in Singapore decreased during 1998\u20132007, with the exception of the first quarter of 2004. After heavy rainfalls, an increase in pneumonic cases with a high case-fatality rate was detected. We show that melioidosis has the potential to reemerge following adverse climate events. Burkholderia pseudomallei. It is endemic to southeast Asia and northern Australia, and cases are increasingly being reported in countries elsewhere in Asia, the Pacific, the Americas, the Caribbean, Africa, and the Middle East, and in travelers returning from tropical countries . Biivariate analysis was performed by using \u03c7A total of 693 cases of melioidosis were reported during 1998\u20132007; of these, 83% were diagnosed by culture and 17% by serologic analysis. We observed a decreasing trend in the annual incidence rate, with the exception of an increase in 2004 . PatientB. pseudomallei demonstrated antimicrobial sensitivity to imipenem (100.0%), ceftazidime (99.1%), doxycycline (99.0%), amoxicillin/clavulanate (94.2%), and chloramphenicol (96.1%).A total of 112 deaths were reported during the 10-year period; overall case-fatality rate was 16.2% (range 8.8%\u201327.1%). Of the reported cases, 75.5% had co-existing diseases, with diabetes (47.9%), hypertension (26.4%), renal impairment (13.3%), and ischemic heart disease (12.0%) being the most common. Patients with co-illnesses had a significantly higher case-fatality rate (19.3%) compared to those without (6.5%) (p<0.0005). Approximately half (50.4%) of the melioidosis cases were associated with bacteremia. Patients with bacteremic melioidosis had a significantly higher case-fatality rate (25.8%) than those without bacteremia (5.5%) (p<0.0005). Clinical isolates of Our study showed that male gender, old age, and diabetes mellitus were risk factors for melioidosis. The presence of bacteremia and co-illnesses were risk factors for death in patients with melioidosis, consistent with findings in other endemic countries. The overall case-fatality rate in this study was much lower compared to cases during 1989\u20131996 (39.5%) (B. pseudomallei was isolated from only 1.8% of soil samples and from none of the water samples in Singapore collected during epidemiologic investigations of reported cases (Unlike patients in Australia or Thailand, most of the case-patients in our study could not recall any occupational or recreational exposure to wet soil. The only reported episodes of percutaneous inoculation in Singapore were a few young adults with localized cutaneous infections and abscesses caused by occupational exposure to soil (B. pseudomallei may result in more fulminant disease and a higher case-fatality rate (Rapid molecular typing of the bacteria during the outbreak in 2004 showed that the isolates were genetically heterogeneous, thus excluding the possibility of a common source (Melioidosis is emerging as a serious public health problem in many countries. Although the incidence and case-fatality rate of melioidosis in Singapore has decreased, it has the potential to resurface with adverse climate events such as heavy rainfall and flash floods."}
+{"text": "Epidemiology of severe disease caused by this organism has changed, with increased incidence and different risk groups. Streptococcus pyogenes, the United Kingdom undertook enhanced population-based surveillance during 2003\u20132004. A total of 3,775 confirmed cases of severe S. pyogenes infection were identified over 2 years, 3.33/100,000 population, substantially more than previously estimated. Skin/soft tissue infections were the most common manifestation (42%), followed by respiratory tract infections (17%). Injection drug use was identified as a risk factor for 20% of case-patients. One in 5 infected case-patients died within 7 days of diagnosis; the highest mortality rate was for cases of necrotizing fasciitis (34%). Nonsteroidal antiinflammatory drugs, alcoholism, young age, and infection with emm/M3 types were independently associated with increased risk for streptococcal toxic shock syndrome. Understanding the pattern of these diseases and predictors of poor patient outcome will help with identification and assessment of the potential effect of targeted interventions.As part of a Europe-wide initiative to explore current epidemiologic patterns of severe disease caused by  Streptococcus pyogenes are among the most varied in terms of clinical spectra and severity, ranging from the ubiquitous pharyngitis to rarer life-threatening manifestations such as necrotizing fasciitis. Interest in these diseases was renewed after the United States and several countries in Europe reported increasing numbers of cases of invasive S. pyogenes disease during and since the 1980s   or another severe manifestation . STSS was defined according to US specifications that differentiate between confirmed and probable cases on the basis of recovery of a sterile or nonsterile site isolate, respectively  initiated enhanced surveillance of severe To maximize case ascertainment, cases were identified from 2 sources: isolate referrals to the national reference laboratory (Streptococcus and Diphtheria Reference Unit [SDRU]) and surveillance reports made to the Communicable Disease Surveillance Centre (CDSC)  calculated according to the Poisson distribution. The 2001 census data were used as denominators for calculating rates according to ethnic group. All denominators were obtained from the Office for National Statistics. Stepwise unconditional logistic regression analysis was conducted to examine the independence of explanatory variables and development of STSS; the likelihood ratio test was used to evaluate significance of explanatory variables within each model.Responses to completed questionnaires were entered and stored in a custom-made Access  database. All reports were checked to ensure they met the case definition. Repeat episodes were defined as those occurring in the same patient >30 days after the initial episode; reports received within 30 days were considered part of the same episode. Data were extracted for statistical analysis into STATA statistical software release 8.2 . Descriptive statistics were undertaken on confirmed cases with \u03c7All analyses were made on data from the United Kingdom, Channel Islands, and Isle of Man, except for estimated rates of infection, which were calculated for the areas with dual reporting . The last 2 areas were omitted for age-, sex-, and ethnicity-specific rate calculations because of unavailability of these population denominators.S. pyogenes disease meeting the case definition were reported from laboratories across the United Kingdom, Channel Islands, and Isle of Man. Of these cases, 21% were identified from isolate referrals only, without a corresponding surveillance report. Among the 3,821 reports were 46 repeat episodes, 5 of which were third episodes. Excluding repeat episodes, severe S. pyogenes disease was diagnosed for 3,775 patients in the United Kingdom, Channel Islands, and Isle of Man in 2003 and 2004.From January 1, 2003, through December 31, 2004, a total of 3,821 cases of severe S. pyogenes was isolated from a sterile site from 3,742 (99%) case-patients, primarily from blood culture . Thirty-three cases without sterile site isolates were included on the basis of >1 of the following clinical indicators: probable toxic shock syndrome (13 cases), necrotizing fasciitis (15), pneumonia (4), and puerperal sepsis (3).emm/M type and erythromycin resistance), although a slightly higher proportion of case-patients for whom a questionnaire was returned had disease onset in December\u2013April .Questionnaires were received for 2,647 (70%) of 3,775 case-patients. Information available for case-patients for whom questionnaires were or were not returned indicated their similarity in terms of age , sex , and strain characteristics  than Wales  or Northern Ireland  but not higher than rates in the Channel Islands and Isle of Man . Substantial variations were also apparent between the English regions, with rates higher in Yorkshire and Humber  than in any other English region: East Midlands (3.25), East of England (2.98), London (2.75), North East (3.66), North West (3.70), South East (2.79), South West (3.92), and West Midlands (3.51). Report rates decreased in 2004  compared with 2003 ; decreases in Yorkshire and Humber, and London accounted for 85% of this decrease.In 2003 and 2004 combined, the overall rate of severe S. pyogenes infection reports were highly concentrated in elderly persons  and the young  (S. pyogenes disease observed were significantly higher for whites (3.29) than for those of Indian subcontinent  or black African or Caribbean origin .Severe 100,000) . Rates fS. pyogenes infection were observed in both years. Cases gradually increased from the end of October and first peaked near the end of January  before peaking again (higher) toward the end of March   .S. pyogenes infection  severe cases of nfection . ApproxiS. pyogenes infection . Use of nonsteroidal antiinflammatory drugs was positively associated with development of STSS .Confirmed STSS developed in 196 (8%) patients who had a diagnosis of severe nfection ; 28% of Multivariable analysis of patient, clinical, and microbiologic factors associated with development of STSS identified age to be a strong predictor for STSS; risk for STSS was 5-fold greater for persons 15\u201344 years of age  than for the reference group (children <15 years of age). Persons 45\u201364 years of age had a 5-fold increased risk for STSS . Patients >65 years of age had no increased risk for STSS compared with the pediatric reference group. Regardless of age, patients with necrotizing fasciitis had a 7-fold increased risk for STSS .emm/M3 type, which was the only strain associated with STSS, had a 3-fold increased risk for STSS compared with patients infected with the reference group (emm/R28) .Four risk factors  were independently associated with development of STSS. Patients who used nonsteroidal antiinflammatory drugs had a 3-fold increased risk for STSS . Alcoholism was associated with a 2-fold increased risk for STSS . Conversely, patients with malignancies had a much lower risk for STSS , as did injection drug users . Patients infected with an S. pyogenes infection was the main underlying or contributory cause of death died within 7 days of initial microbiologic diagnosis. Necrotizing fasciitis was the most severe clinical manifestations for patients, according to specified markers; patients with this condition were most likely to be admitted to an intensive care unit (ICU) (77%) or to die within 7 days of diagnosis (34%). However, ICU admission (20%) and surgical intervention (24%) were not uncommon among other patients. Case-fatality rates were also high for patients with pneumonia, 32% of whom died within 7 days. Of the 58 young women in whom puerperal sepsis developed, 2 died. Development of STSS was strongly linked to risk for death; 84 (45%) of 185 patients with STSS died from their infection compared with 329 (16%) of 2,007 without STSS.Overall, 413 (19%) patients for whom S. pyogenes infection. Of these, lesions or wounds to the skin were reported for 31% (719). Skin is the most likely portal of entry recorded overall, especially among persons >60 years of age, 40% of whom had skin lesions. Information on the nature of these lesions was available for 617 case-patients. The 2 most common types were traumatic lesions (188) and chronic wounds (161). Traumatic lesions were most common among young adults ; chronic wounds were most common among elderly persons (14%). Less common types of wounds were recorded that included insect bites  and animal-associated traumatic wounds .Information on risk factors was available for 61% of case-patients with severe S. pyogenes infections, 459 (40%) were injection drug users . Other conditions commonly reported that could have predisposed persons to infection included malignancies (161) and diabetes (158), each noted for 7% of cases overall and 11% and 13%, respectively, among elderly persons. Nine percent (204) of infections were associated with healthcare, mostly postsurgical infections (118). Among pediatric case-patients (<16 years of age), varicella was the next most common predisposing factor noted after skin lesions, reported for 41 (14%) children. Overall, 566 (25%) case-patients did not have any particular predisposition, or risk for severe S. pyogenes infection on the basis of the common factors outlined .Marked seasonal patterns in severe Clinical information provided for these case-patients highlights the severity of these infections; 19% died within 7 days of the initial culture-positive specimen being obtained. This finding is consistent with overall case-fatality estimates during enhanced surveillance in the United Kingdom in 1994\u20131997 (25%) and estimates reported in other countries  (emm/M3 strain was associated with an increased risk for STSS. This finding is consistent with previous (unadjusted) findings from the United States  of cases with no identified focus, several of these cases may have originated from respiratory tract colonization. This colonization could lead to transient bacteremia, which in turn seeded local tissue sites, possibly in the presence of local trauma.Questionnaires concerning possible predisposing factors have highlighted skin lesions as the most commonly identified potential source of infection, which is similar to findings in other countries (S. pyogenes disease in injection drug users (S. pyogenes infection in injection drug users (In contrast to preliminary findings from other Strep-EURO participants (S. pyogenes cases in our study occurred sporadically in the community; only 9% were associated with healthcare interventions. One fourth of all case-patients and nearly 1 in 6 young adults had no particular risk factors identified. These findings highlight the likely economic effect of these infections and the challenges in developing any effective prevention measure.Most severe emm/M type distribution, a key virulence factor inducing immunologic memory, will assist in assessment of the potential effect of vaccines currently under development. Changes in the epidemiology of severe S. pyogenes infections since the last period of enhanced surveillance in the mid-1990s underline the need for periodic monitoring to detect changes in disease manifestations, risk groups, and microbiologic characteristics to develop strategies for control and management of these infections.Analysis of information collected in this study has yielded some unique insights into these infections and has begun to provide an evidence base for mounting public health initiatives in the United Kingdom ("}
+{"text": "During the containment phase, regions of exposure for imported infections changed rapidly. In June 2009, during Singapore\u2019s pandemic influenza plan containment phase, pandemic (H1N1) 2009 was introduced into the country through imported cases. To understand how travel patterns affected the initial outbreak, we examined epidemiologic and travel data for the first 116 case-patients admitted to Tan Tock Seng Hospital, Singapore, with travel-associated infection. Sixty-one percent and 54% of patients, respectively, met US Centers for Disease Control and Prevention and World Health Organization temperature criteria for influenza-like illness. One fourth of the case-patients traveled after illness onset, and 15% became ill while traveling. Regions of exposure for imported infections changed rapidly; case-patients initially arrived from North America, followed by Australasia and Southeast Asia. Case-patients on longer flights were more likely to become ill before arrival; those with shorter flights tended to become ill after arrival. Thermal scanners detected fevers in 12% of the arriving case-patients, resulting in a shorter time to isolation. On April 24, 2009, international authorities reported cases of infection with a novel influenza A virus (H1N1) strain of swine origin, now known as pandemic (H1N1) 2009 virus; 7 cases in the United States and 3 clusters in Mexico were confirmed, and surveillance indicated influenza-like-illness (ILI) had been increasing in Mexico since March 18, 2009 (The role of air travel in the transmission and dissemination of respiratory infections has been examined for severe acute respiratory syndrome (SARS), pneumonic plague, and extensively drug-resistant tuberculosis  2009 was introduced into the country. Public health policy during this phase was to isolate infected case-patients and quarantine exposed persons to prevent local transmission for as long as possible. During this initial period, travel from a pandemic (H1N1) 2009\u2013affected area was the major risk factor for infection; air travel was the main route of introduction. The containment phase was in effect for >2 months until epidemiologic surveillance indicated sustained community spread had begun during epidemiologic week 25 . At that time, a gradual transition to mitigation measures was implemented.During the containment phase, airport thermal scanners were used to detect fevers in arriving passengers at Singapore\u2019s Changi International Airport, and health advisories were used to encourage travelers in whom influenza-like symptoms developed after disembarkation to seek medical care. Ambulances, dedicated for this purpose only, were used to transport suspected case-patients from the airport to hospitals for screening. Adults with appropriate travel histories and ILI were referred to the designated screening center at Tan Tock Seng Hospital (TTSH) for treatment and isolation at the Communicable Disease Centre. During the mitigation phase, the focus of clinical and public health efforts shifted to caring for patients with severe illness and conditions that put them at risk for complications and to reducing transmission in the community through health education and voluntary self-isolation of persons with ILI.We analyzed epidemiologic and travel data as well as data regarding source of referral for case-patients in relation to time of illness onset, time of arrival in Singapore, and time to isolation. Understanding how travel patterns affected propagation of the first pandemic wave of pandemic (H1N1) 2009 virus could yield insights into how the anticipated next wave might be disseminated and provide data on the effectiveness and limitations of different interventions used to slow dissemination.am, 8:00 am to 3.59 pm, or 4:00 pm to 11:59 pm] if the patient was unsure). Symptom onset time was further categorized as before embarkation, during travel, or after disembarkation in Singapore. We also categorized hospitalized case-patients on the basis of whether their symptoms met US Centers for Disease Control and Prevention (CDC) ILI criteria  and WHO ILI criteria  (We obtained demographic data and travel-related information (last port of embarkation and flight times) by direct interview and retrospective review of clinical notes. Duration of travel, which was calculated from flight times and transit time within airports, was categorized into 4 groups: short haul (<3 hours), medium haul (3\u20136 hours), long haul (6\u201315 hours), and extra-long haul (>15 hours). Clinical data were collected for symptoms and date and time of symptom onset (to the nearest hour in most instances and to the nearest 8-hour block [midnight to 7:59 A confirmed case of pandemic (H1N1) 2009 was defined as an ILI in a patient with a temperature >37.5\u00b0C, cough, rhinorrhea, sore throat, or myalgia and with laboratory confirmation of infection by real-time reverse transcription\u2013PCR performed on respiratory samples . An imported, travel-associated case was defined as above but occurred in a person with recent travel outside Singapore who had arrived in Singapore during the containment period and had illness onset within 10 days of arrival.2 test. Means were compared by use of t tests for dichotomous variables and 1-way analysis of variance for multichotomous variables. Stata 10.0 for Windows  was used for all statistical analyses.We compared proportions by using the \u03c7From April 27 through June 27, 2009 (epidemiologic week 25), 152 persons with confirmed pandemic (H1N1) 2009 were admitted to TTSH. Of the 152 patients, 116 met the definitions for having an imported, travel-associated case; the rest either did not have a history of travel or had onset of symptoms >10 days after their travel ended. These 116 cases span 5 weeks (epidemiologic weeks 21\u201325) from when the first confirmed case-patient arrived in Singapore on May 26, 2009.Demographic and travel data for patients with cases of imported pandemic (H1N1) 2009 are shown in As shown in The first cases of pandemic (H1N1) 2009 detected among travelers arriving from the Philippines (week 22), Thailand (week 23), and Indonesia (week 25) indicated potential community transmission in those countries earlier than official announcements. Infections from Southeast Asian countries accounted for 29% of imported cases during the containment phase; the Philippines were linked to 23% cases, mostly in weeks 24\u201325.Travel duration reflects the distance between the regions of exposure and Singapore . Of the Doctors based at the airport referred 15 (12.9%) of the 116 patients to TTSH; thermal scanners used to screen arriving passengers had detected fever in 14 of these 15 patients. Of the remaining 101 patients, 51 (44%) self-reported to the screening center at TTSH and 50 (43%) were referred by doctors in the community.>37.8\u00b0C and 54% had temperatures >38\u00b0C, the temperature criteria used in the US CDC and WHO ILI case definitions, respectively. Considering the entire symptom complex, 51% of patients would have fulfilled ILI criteria as defined by the US CDC, and 44% would have fulfilled the WHO criteria  and there was potential recall bias.Our data demonstrate how swiftly situations can change in a fast-moving pandemic; affected areas shifted from the Americas to Australasia to Asia within a matter of days. These rapid shifts pose a tremendous challenge to health authorities responsible for outbreak management, and they emphasize the narrow window of opportunity during which interventions can slow an epidemic. The monitoring of travelers fulfills a vital sentinel surveillance function, providing an early indicator of community transmission in countries even before transmission has been officially confirmed. Understanding how travel-associated infections propagated the first wave of this pandemic yields rich insights into how health authorities might respond to future outbreaks of emerging respiratory infections."}
+{"text": "Communicating vaccination recommendations may help reduce infections. Approximately 60% of hepatitis A virus infections in Germany occur in persons without a travel history to disease-endemic areas and for whom sources of infection are unknown. Recommendation of pretravel vaccination fails to prevent the remaining imported infections. Using enhanced surveillance in 2007\u20132008, we analyzed epidemiologic patterns of hepatitis A in Germany and appropriateness and adequacy of current immunization recommendations. Young patients with a migration background who had visited friends and family in their ancestral countries accounted for most imported cases. Phylogenetic analysis showed high diversity of sequence data and clustering of strains with similar regions of origin or patient migration backgrounds. Virologic findings are compatible with those of low-incidence countries, where virtually all infections are directly or indirectly imported from other regions. Germans with a migration background are seen as a special risk group so far insufficiently reached by pretravel vaccination advice.After decades of steady decreases  for hepatitis A and often unapparent connections between persons involved in outbreaks (A 1-year study was initiated by the Robert Koch Institute (RKI) to characterize and compare imported and nonimported HAV infections in Germany and determine HAV genotype distribution and sources of infection. We assessed whether the virus is endemic among certain (unrecognized) risk groups, whether (and which) imported infections play a role in secondary autochthonous infections, and which population groups should be targeted for specific prevention approaches .The study was conducted from the 14th calendar week of 2007 through the 13th calendar week of 2008. Acute hepatitis A has been a notifiable disease in Germany for many years. Since 2001, laboratories have reported infections  to local health departments. These departments collect information on case-patients , take preventive measures to avoid further spread , and report standard case information electronically to state health authorities and RKI in a form in which names and addresses are removed. Data can be grouped on all levels to indicate outbreaks.To transcend standard information obtained for each reported infection and add a virologic perspective, we collected additional case information on all HAV infections in Germany over a 1-year period. The 16 state health departments in Germany were requested to participate and coordinate distribution and collection of questionnaires to all local public health departments. These departments recorded additional case information obtained during routine case investigations on paper forms . Data included details of travel, concurrent health conditions, and potential migration background. These investigations consisted of telephone interviews with patients (rarely with their physicians as proxies). Completed forms were sent to RKI without names and addresses but did contain case codes. Accommodations other than hotels were defined as those presumably involving closer contact with the local population or exposure to food prepared under potentially suboptimal hygienic conditions .>18 years of age.Persons with a migration background were defined as those who moved to Germany after 1949 as non-German nationals, children born in Germany to non-German nationals, or children born to at least 1 parent belonging to either of these groups. Adults were defined as persons >10% of persons with HAV infections diagnosed in Germany during the study. To facilitate sequencing and phylogenetic analysis of a representative selection of HAV strains causing infections in Germany during the study, we asked >120 large private laboratories, university clinics, and hospitals in Germany to provide serum samples for patients for whom IgM against HAV was detected at their facilities. Samples were either sent to RKI immediately or stored at \u201320\u00b0C and sent in larger batches.We obtained serum samples from Information recorded on paper forms was entered into a database and matched to electronically transmitted routine data by case codes. All symptomatic cases reported from participating states were analyzed. Clinical specimens were matched to questionnaire and surveillance data according to anonymous patient information  provided by the laboratories.HAV RNA was isolated from serum samples by using a Viral RNA Mini Kit . Reverse transcription and first-round amplification of the capsid protein (VP1)/2A junction region of HAV were performed by using a Onestep RT-PCR Kit (QIAGEN) and primers .Nested PCR was performed by using the HotStarTaq Master Mix Kit (QIAGEN). Purified products of the nested PCR (forward and reverse strands) were sequenced by using a 3130x ABI Prism Genetic Analyzer and a BigDye Terminator version 3.1 Cycle Sequencing Kit .Sequences were processed by using Lasergene SeqMan Pro software , aligned by using the ClustalW algorithm  were in patients with clinical symptoms consistent with acute hepatitis A. Of the 16 states in Germany, 13 participated in the intensified surveillance and contributed 1,037 (85.5%) of all reported infections and 816 (86%) of all reported symptomatic cases . Additional case information sheets were available for 571 (70%) symptomatic cases. Serum samples positive for IgM against HAV were available for 189 (23.2%) cases; 95 (11.6%) were PCR positive.>60 years of age.A total of 74.6% of the cases were reported as single cases; the remainder were in recognized clusters. Among case-patients, 47.1% were male ; age ran>60 years of age (56.5%) when compared with that of younger persons . Among those employed, absences from work ranged from 2 to 32 work days (median 6 days).Of case-patients, 46.4% were hospitalized for a median of 6 days (range 1\u201328 days). Among those with jaundice, no clear trend for age was found in 50.2% who were hospitalized. Among those without jaundice, the proportion of those hospitalized increased among persons Overall, 43.6% of the infections were imported . Nonadults  and male patients  were overrepresented.A migration background was reported by 42.2% of case-patients (78.8% of nonadults and 19.1% of adults), of whom 64.8% had been born in Germany. Among migration background, Turkey was reported most frequently (48.5%), followed by the former Yugoslavia (11.9%), southern and Southeast Asia (9.7%), and the former USSR (8.8%). Nonadults with a migration background lived with a larger number of household members  than those without a migration background . Among adult case-patients, 5.2% were professional food handlers.Among known destinations, Turkey (35.6%) was reported most frequently, followed by the former Yugoslavia, Egypt, and Spain . Most af>40 years of age were almost evenly spread throughout the year.Imported infections were most likely to cause disease from August through October, and a prolonged wave of autochthonous infections then followed from October through March . Among i>60 years of age had autochthonous infections.Case-patients with autochthonous infections were older than case-patients with imported infections and less likely to have migration backgrounds . Almost >40 years of age and 31.1% were >60 years of age, compared with only 24% and 7.5%, respectively, of persons of the same ages with cases of imported infections. Case-patients with imported infections were also slightly more likely to be male and part of case clusters than were autochthonous case-patients.A migration background was more likely among persons with imported infections; 52.8% of case-patients had a background of migration from Turkey. However, 23% of persons with autochthonous infections had a migration background . PatientClose contacts of 58.4% of the patients received prophylactic vaccinations . Contacts were more frequently vaccinated in response to cases in nonadults .>5 infections were detected. The largest (13 symptomatic and 2 asymptomatic infections) was in a school with multiple generations of infection. The ultimate source of infection could not be elucidated. The school index case-patient had not traveled. However, the implicated HAV strain was genetically similar to strains from Turkey. In 4 of the 8 outbreaks with 5\u20137 infected persons, >1 travel-associated index cases were recognized, leading to 1\u20134 secondary infections in Germany.No large outbreaks were reported during the study. In the 13 participating states, 9 clusters with <39 years of age (mean 65%), RNA was detected in only 10% of symptomatic patients >60 years of age. No correlation was seen between duration from symptom onset to day of blood sampling (maximum 29 days) and positive results for HAV RNA.HAV RNA was detected in 95 (50.3%) of 189 samples matching a reported case of symptomatic HAV infection. The likelihood of detecting HAV RNA in serum depended on clinical characteristics of patients . Frequen<8.9%, <7.5%, and <4.9% of positions within genotype IB, IA, and IIIA strains, respectively. Sequence variability was reflected in the diversity of countries or regions from which sequences originated  had genotype IB strains, 36 (28.6%) had genotype IA strains, and 17 (18.6%) had genotype IIIA strains. Sequences differed in iginated .Phylogenetic analysis showed that imported strains clustered according to region or country. Imported genotype IB strains were isolated from patients who traveled to Turkey, the Middle East, and Africa. Strains acquired in South Africa formed a small but distinct subcluster. Patients infected with genotype IA strains reported traveling to eastern Europe, Asia, South America, and northwestern Africa. The small number of imported genotype IIIA strains was obtained from patients who had traveled to eastern and southeastern Europe and Central and southern Asia. Three strains imported from Spain (2 genotype 1A and 1 genotype 3A) were genetically more distant from each other than to strains from other regions .Strains from patients with known migration backgrounds  but no tMost (53.2%) HAV sequences obtained were found only once. Another 18.3% were isolated from smaller outbreaks of hepatitis A, which had already been detected through routine surveillance . RemainiDetailed epidemiologic data for 70% of symptomatic cases reported in participating states during the study  enabled us to characterize incident cases of HAV infection in Germany better than using routine surveillance data alone. Although deaths from hepatitis A are rare, frequent hospitalization and work time missed by patients or adults caring for sick children emphasize the need to focus on hepatitis A.Despite existing vaccination recommendations for travelers to countries with high or intermediate levels of endemicity for hepatitis A, >40% of cases were directly related to travel. Among these cases, persons with any migration background were overrepresented, especially those who had a background of migration from Turkey. The German Statistical Office in 2006 reported that 16% of adults and 26% of children  living in Germany had a migration background, compared with 63.9% of our patients with imported cases. Among those with a migration background who lived in Germany, 22% were from Turkey  of a case-patient, infection chains quickly terminate. This finding is also likely to result in part from satisfactory hygienic conditions in Germany but is also likely a result of effective tracing and vaccination of case-contacts by local health departments. Although the exception, larger outbreaks in daycare centers . If removal of these barriers to vaccination of all travelers is deemed unlikely, the general vaccination of children against HAV should be considered, given the high number of imported infections among children and the evidence of secondary autochthonous transmission.Efforts to communicate recommendations to previously unaware population groups, especially, but not exclusively, persons with migration backgrounds, have the capacity to strongly reduce the number of HAV infections in Germany. However, as long as vaccination recommendations are applied only to travelers and overall immunity in the population remains low or decreases further, risk for secondary transmission of imported infections remains high."}
+{"text": "Gaps in infection control led to biofilm production in central venous catheters and resultant bloodstream infection. Alcaligenes xylosoxidans bloodstream infections by conducting a matched case\u2013control study and a prospective study. Pulsed-field gel electrophoresis (PFGE) was performed on blood culture isolates, and 1 explanted central venous catheter (CVC) was tested for biofilm. We identified 12 cases of A. xylosoxidans bloodstream infection. Case-patients were more likely than controls to have had a CVC . Ten case isolates were indistinguishable by PFGE analysis, and A. xylosoxidans biofilm from the CVC matched the outbreak strain. We observed multiple breaches in infection control, which may have caused contamination of multidose vials used to flush the CVCs. Our study links A. xylosoxidans with CVC biofilm and highlights areas for regulation and oversight in outpatient settings.In 2002, we investigated a cluster of patients with  Alcaligenes xylosoxidans, also known as Achromobacter xylosoxidans, is a gram-negative, water-borne organism that causes healthcare-associated infections  were associated with a single outpatient oncology office, Office B. The other 3 hospitals reported that they had not identified any A. xylosoxidans bloodstream infections in the past 3 months. To identify the source of the outbreak and risk factors for infection and to implement control measures, ACDC then initiated an outbreak investigation.To confirm the presence of an outbreak, ACDC conducted a telephone survey, which asked the microbiology laboratories of Hospital A and 4 surrounding hospitals for a list of all patients who had had positive blood cultures for A. xylosoxidans culture, we requested a review of medical records and laboratory reports.The outbreak investigation focused on Office B. To identify all patients associated with Office B who had had a positive A. xylosoxidans blood culture from November 2001 through January 2002. Controls were defined as patients who had no symptoms or signs of bloodstream infection . For each case-patient, 5\u20137 controls were randomly selected and matched by the closest date of their visits to Office B to the case-patient\u2019s date of visit. Variables included age, sex, underlying diagnosis, intravenous medications received, peripheral white blood cell counts, presence and type of central venous catheter (CVC), clinic visits, hospitalization dates, symptoms, and types of invasive procedures. Data were collected on standardized forms and analyzed by using Epi Info 2000 version 1.1.2 ; odds ratios were used to estimate risk, t tests were performed for continuous variables, and p<0.05 indicated statistical significance.A matched case\u2013control study was performed to determine risk factors for infection. A case-patient was defined as a patient of Office B who had a positive A. xylosoxidans bloodstream infection, ACDC conducted prospective blood culture surveillance. On February 15, 2002, all patients with a CVC who had visited Office B since November 2001 were sent a letter requesting them to have a culture performed on blood drawn through the CVC. CVCs were removed from those whose culture results were positive.To identify possible On January 17, 2002, numerous open containers  were collected and sent to the Los Angeles County Public Health Laboratory for analysis. On February 15, 2002, environmental samples and swabs were collected for culture from work surfaces  and from tap water and hands of healthcare workers who accessed CVCs, collected blood, prepared flushes, or administered chemotherapy.During January and February 2002, we made several site visits to Office B to observe procedures, review medical records, and interview the office staff. Specifically, we observed procedures for preparation and administration of intravenous medications and asked office staff about level of technical training, experience, and license status.A. xylosoxidans from case-patients were obtained from Hospital A\u2019s laboratory. For comparison, all A. xylosoxidans isolated from patients from Los Angeles County in the past 6 months were obtained from a large local reference laboratory. Pulsed-field gel electrophoresis (PFGE) was performed at the Los Angeles County Public Health Laboratory by using standard methods  was surgically removed from an asymptomatic patient identified in the prospective cohort study. Aseptic methods were used. The distal lumen opening was clamped with a sterile hemostat to retain the liquid within the lumen, and the catheter was placed in a sealed, sterile container and shipped overnight to CDC in Atlanta for processing within 24 hours of collection. At CDC, the CVC was placed into a Class II Biological Safety Cabinet, and a 1-cm segment was removed with a sterile scalpel. This segment was cut lengthwise to expose the lumen, and the individual pieces were placed into 5% glutaraldehyde  in 0.67 M cacodylate buffer, pH 6.2, and processed for scanning electron microscopy .A total of 12 patients with ve study . All 12 Of the 9 case-patients identified, 7 who had clear onset date of bloodstream infection symptoms were selected for the case\u2013control study. Case-patients were younger than controls . Case-patients were significantly more likely to have a CVC than controls. Matched case\u2013control analysis showed that all 7 case-patients versus 4 of 47 control patients had a CVC at the time of illness onset (p<0.0001). The 2 other case-patients not included in the case\u2013control study also had CVCs. Patients with CVCs received heparin and saline flushes before and after the CVC was used for blood collection or infusions. No records documented when each of the Office B nurses accessed the CVCs. Patients without CVCs who needed only blood collection for testing did not receive any flushes; however, those without CVCs who needed blood tests before receiving an infusion received a heparin and saline flush after a peripheral intravenous line was placed. Case-patients and controls did not have statistically significant differences in peripheral leukocyte counts, intravenous medications administered, types of chemotherapy received, or underlying diseases.A. xylosoxidans, chart review showed that 2 had been intermittently symptomatic  who had positive culture results for ptomatic .A. xylosoxidans\u2013positive blood cultures, and environmental cultures did not grow A. xylosoxidans. A sample from a sterile saline bottle that was open in the infusion room was positive for Bacillus circulans, and a tap water sample was positive for Moraxella spp.Cultures from available open solutions in the oncology office, collected 6 weeks after the initial cluster of Office B had 10 patient examination rooms and a separate, large, open infusion room where several patients could receive chemotherapy. The infusion room contained a hood and sink for preparation of intravenous medication. Of the 4 staff members at Office B who regularly accessed CVCs; inserted peripheral intravenous catheters; collected blood; and prepared or administered chemotherapy, flushes, or intravenous medications, only 1 was a registered nurse who had a California state license. The 3 nonlicensed staff members were reported to have received nursing training in their native country but did not have documented formal training or education. One nurse wore artificial fingernails but had removed them before hand culture samples were collected; thus, the fingernails were unavailable for culture. The following breaches in infection control were noted: intravenous catheters were inserted by persons not wearing gloves; unlabeled, prefilled syringes were stored in the hood; no documentation of hood cleaning was found; open, multidose heparin vials and saline bottles, some undated, were found throughout the facility; nonhygienic material was stored in the chemotherapy medication preparation hood; and failure to wash hands between patients was noted. No pharmacists were employed at Office B. No documentation of staff training and evaluation for chemotherapy preparation or infection control competency was available.A. xylosoxidans.Scanning electron microscopy of the CVC showed a biofilm that contained rod-shaped bacteria in association with fibrinlike material on the catheter surface . A pure A. xylosoxidans blood culture isolates from case-patients were indistinguishable by PFGE analysis  of 29 patients had A. xylosoxidans\u2013positive blood cultures. Our case\u2013control and prospective studies support the association of A. xylosoxidans bloodstream infection and CVCs, and our molecular biologic studies confirm A. xylosoxidans biofilm of the same outbreak strain on a CVC. A. xylosoxidans outbreaks reported to date have been associated with healthcare and contamination of hospital products (This large outbreak (N = 12) of A. xylosoxidans biofilm on CVCs. Case-patient 1 had been hospitalized from late October through early November at Hospital A. During November 13\u201319, 2001, this case-patient had had blood collected and her CVC line flushed numerous times at Office B; on November 19, culture result indicated A. xylosoxidans infection, which was successfully treated. We observed multiple breaches in infection control at Office B: use of unlabeled, prefilled syringes, poor hand hygiene, and lack of glove use. The CVC of case-patient 1 may have been flushed by using the same syringe and needle inserted into multidose vials, causing contamination of the vials. Another possible route of contamination is through artificial fingernails. A cluster of Serratia marcescens and A. xylosoxidans bacteremia cases linked to multidose heparin vials contaminated by a nurse with artificial fingernails has been reported (A. xylosoxidans and subsequently used on other patients from November 12 through December 18, 2001, when all case-patients had overlapping visits at Office B and received CVC flushes. A culture from an open, supposedly sterile saline bottle grew B. circulans, which suggests possible breaches in infection control. Multidose heparin and saline vials have been reported as the cause of outbreaks of hepatitis C (S. marcescens (Pseudomonas aeruginosa (The cause of this outbreak most likely was the use of contaminated multidose vials of heparin or saline flushes, leading to the formation of an A. xylosoxidans biofilm could have developed on their CVCs and intermittently caused clinical illness when the CVCs were manipulated; i.e., flushing dislodged the biofilm and caused symptomatic bacteremia. Although indwelling catheters are frequently colonized with biofilm shortly after insertion (A. xylosoxidans biofilm supports this. A number of variables may be associated with detachment of microbial cells from a biofilm (Although the heparin and the saline vials could have been contaminated from case-patient 1 in November, case-patients who subsequently received flushes from these vials may not have become immediately ill with symptoms of bloodstream infection. A. xylosoxidans biofilm and the mechanism of bloodstream infection after disruption by catheter flushing suggests that eradication of infection would require catheter removal, as reported by others (A. xylosoxidans bacteremia has been reported in those patients whose indwelling catheters were not removed (A. xylosoxidans biofilm provides an explanation for the organism most commonly causing bacteremia in patients with CVCs (The case-patients in this outbreak had their CVCs removed and were treated with antimicrobial agents. The presence of A. xylosoxidans, further studies are needed to determine whether active surveillance for patients with CVCs would help recognize infections.The California Code of Regulations, Title 17, Section 2500 (A. xylosoxidans bloodstream infections were reported from Office B.Because we noted not only infection control breaches but also that unlicensed office personnel were manipulating the CVCs, line flushes, infusions, and blood collection through CVCs, we reported the situation to the California Medical Board and the California Department of Health Services. Although no California state regulations for infection control in outpatient physician\u2019s offices exist, the California Department of Health Services and Los Angeles County Department of Public Health recommended that the oncology office improve infection control standards; handling, storage, exposure, and access to pharmaceuticals; and improve medical record documentation. New infection control policies were established, and the office subsequently hired new, properly licensed registered nurses and nurse practitioners to handle insertion of intravenous catheters and administration of intravenous medications and chemotherapy. After proper education of the oncology office staff and removal of multidose vials of heparin and saline, no more A. xylosoxidans from the multidose vials. The original vials, used when the outbreak began, had already been discarded and were not available for testing by the time we were notified in January 2002. Our investigation was also limited by the absence of medical records indicating when nursing staff accessed the CVCs. Although the contamination of multidose vials remains suggestive, we suspect that they were the most likely source. The outbreak ended after discontinuing their use and enacting improved infection control practices. The organism could have been introduced into multidose vials by a needle or syringe used on an infected patient or by the artificial fingernails of the nurse, through gaps in infection control.Our investigation has limitations. We did not culture For patients who received infusion therapy at home, receipt of therapy in an outpatient clinic or physician\u2019s office was an independent risk factor for bloodstream infection (A. xylosoxidans bloodstream infection in immunocompromised patients with CVCs. It provides a better understanding of how biofilm formation in a CVC can result in a clinical infectious disease process with this uncommon organism. Substantial illness and death can occur in outpatient settings that lack formal oversight. This outbreak highlights an unaddressed infection control problem in the outpatient setting for regulating agencies to further review.Our investigation helps characterize the mechanisms of"}
+{"text": "To the Editor: The first documented outbreak of salmonellosis linked to consumption of plant products in the Autonomous Province of Vojvodina, Serbia, occurred from March 2007 through September 2008. Fourteen cases of Salmonellaenterica serotype Senftenberg infection were reported.S.enterica serotype Enteritidis  were interviewed by using a standard questionnaire for salmonellosis, which was expanded to include questions regarding tea consumption.After these findings, the Institute of Public Health of Vojvodina conducted an outbreak investigation in collaboration with institutes of public health at the district level. A case was defined as the presence of a laboratory-confirmed A standardized method of enterobacterial repetitive intragenic consensus (ERIC)\u2013PCR, based on the method of Versalovic et al. (S. Enteritidis infection was made). Before feeding it to the infants, the parents had not heated the tea until it boiled, but rather had poured boiled water over the tea. After obtaining that information, we tested 33 samples of the incriminated brand of tea from public grocery stores and supermarkets; 13 samples were positive for S. Senftenberg. The organism\u2019s genetic profile was identical or similar from both tea and human samples . Half had diarrhea and the same proportion had fever >38.5\u02daC. Ten patients were female and 4 were male. All 4 adults had mild infection, except 1 adult who had concomitant Clostridiumdifficile infection. Three infants and an adult with concomitant infection were hospitalized.Demographic characteristics and clinical status of the case-patients were analyzed. Of 14 cases of S. Enteritidis infection. After September 2008, no new cases of S. Senftenberg were reported until July 2009, when 1 case was identified in a 24-year-old man.Most infections were reported in May 2008, including the 3 cases in infants who were recovering from S. Senftenberg is well known and is much higher than that for most other Salmonella serotypes (S. Senftenberg infection resulted from consumption of fresh products. Thus, products that will be used in a fresh state should undergo more rigorous testing for pathogens, or better methods of infection control must be used.The heat resistance of The European Food Safety Authority has noted that all botanicals or botanical preparations could become hazardous as a result of flaws in the production process; therefore, manufacturers should follow the Hazard Analysis and Critical Control Point systematic approach (Salmonella phages could be effective in eliminating or reducing Salmonella contamination of vegetables (In 1999, the US Food and Drug Administration recommended that seeds be disinfected by washing with calcium hypochlorite solution before they sprout. However, this treatment destroys only pathogenic microorganisms on the seed surface ("}
+{"text": "These infections can be prevented by avoiding known risk factors. Escherichia coli (STEC) infection among children in Argentina. We conducted a prospective case\u2013control study in 2 sites and enrolled 150 case-patients and 299 controls. The median age of case-patients was 1.8 years; 58% were girls. Serotype O157:H7 was the most commonly isolated STEC. Exposures associated with infection included eating undercooked beef, living in or visiting a place with farm animals, and contact with a child <5 years of age with diarrhea. Protective factors included the respondent reporting that he or she always washed hands after handling raw beef and the child eating more than the median number of fruits and vegetables. Many STEC infections in children could be prevented by avoiding consumption of undercooked beef, limiting exposure to farm animals and their environment, not being exposed to children with diarrhea, and washing hands after handling raw beef.We evaluated risk factors for sporadic Shiga toxin\u2013producing  Escherichia coli (STEC) infections cause nonbloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS)  serum creatinine concentration >2 standard deviations above the upper limit of normal for age and sex (>2+) or proteinuria (>2+). HUS was considered diarrhea-associated when a diarrheal illness preceded HUS by <3 weeks. We excluded children with a family history of HUS, secondary HUS , or HUS associated with pneumococcal infection. Probable HUS was defined as an illness that met only 2 of the laboratory criteria in a patient with culture-confirmed STEC infection.Definite HUS was defined as acute microangiopathic hemolytic anemia, thrombocytopenia, and renal impairment as determined by 1) hematocrit <30% with microangiopathic changes on peripheral blood smear ; 2) platelet count <150,000/mmFor each patient enrolled, 2 age- and neighborhood-matched control children without gastrointestinal illness in the 2 weeks before the matched case\u2019s illness onset were identified. We neighborhood-matched control children to control for socioeconomic status, which was not a factor of interest, was a possible confounder, and was quite variable in the study areas. In urban areas, a trained interviewer sought controls by walking, starting at the case-patient\u2019s home, going to the third house from the nearest corner, and continuing to every house on the block, then to the block facing the case-patient\u2019s residence, then to other blocks in a clockwise fashion, until 2 eligible and consenting controls were interviewed. In nonurban areas, the interviewer randomly chose a cardinal direction and then sought controls beginning from the third residence from the case patient\u2019s house in that and the opposite direction until controls were found. Informed consent was obtained from the adult primary caregiver, who was interviewed with a standardized questionnaire, administered in person. Most eligible controls were enrolled; information on potential controls who were excluded or chose not to participate was not kept.The questionnaire (available upon request from the corresponding author) had 89 major questions and was divided into 3 major sections: characteristics of and treatment for the illness (19 questions), exposures (55 questions), and demographics (15 questions). Exposure questions were divided into sections that dealt with human contacts , care and feeding of young children, water sources and treatment, beef, other meats, fruits and vegetables, meat handling at home, animal contact, swimming, and travel. Many exposure questions had several parts . Almost all responses were measured discretely . Responses to each question were treated independently, except for those about fruits and vegetables, which were combined and dichotomized at the median; this analysis method was checked against other choices and found robust. All exposure questions for case-patients and controls were about the 7 days before the onset of illness. Interviews of case-patients were conducted a median of 12 days (range 3\u201341) after diarrhea onset; control interviews were conducted a median of 15 days (range 1\u201341) after diarrhea onset in the matched case. Controls were age-matched to case-patients by using the following groups: <12 months; 1\u20135 years \u00b1 1 year; 6\u20139 years \u00b1 2 years; 10\u201315 years \u00b1 3 years.stx1, stx2, and rfbO157 genes by a multiplex PCR  and potassium tellurite (25 mg/mL). The confluent growth zone and colonies were screened for <0.05) and had biologic plausibility were selected for further modeling. In multivariable model-building, we pursued forward, backward, and the best subset selection strategies, as well as manual strategies. Standard methods were used to assess model fit, including residual analyses. Maximum likelihood parameter estimates from these models were used to calculate point estimates and confidence intervals for odds ratios, referred to henceforth as matched odds ratios. Exact analysis was used where small sample size would make asymptotic analysis suspect, and Mantel-Haenszel odds ratios were computed when maximum likelihood estimates did not exist.Data analysis used 3 steps: an initial univariate analysis, a second univariate analysis adjusted for highly significant factors of prior or secondary interest, and a final multivariable model-building analysis. Four factors were ultimately chosen in the second univariate analysis as a fixed set from which to explore further model-building, based on epidemiologic sensibility, strong association, and stability of subsequent adjusted associations. Single- and multiple-variable conditional logistic regression models were used to evaluate associations between the outcome and exposure variables. At each step, risk factors that were statistically significant  and SAS 9.0  software. The study was approved by the hospitals\u2019 ethics committees as well as the institutional review boards of the Ministry of Health of Argentina and CDC.Exploratory and sensitivity analyses were performed on subsets of the data defined by location and subcategory of disease status. Because the subsets by site (Buenos Aires and Mendoza) and by serogroup  were small and most factors examined had already been demonstrated as risky or protective in the larger dataset, we used a p value of 0.10 to assess significance by site and serogroup. For similar reasons, we did not perform multivariable analyses on these subsets. Three patients with mixed STEC infection and 2 Among 157 eligible case-patients, 150 (96%) were enrolled; the parents of 1 child refused, and interviewers could not contact the parents of 6. The hospital \u201cDr. Juan P. Garrahan\u201d in Buenos Aires enrolled 54% of the cases, and the hospital \u201cDr. Humberto Notti\u201d in Mendoza enrolled 46%. Among the 150 enrolled cases, 17 met both entry criteria of culture-confirmed STEC infection and definite HUS, 82 met only the criterion of culture-confirmed STEC infection, and 51 met only the criterion of definite HUS. In addition to the patients with definite HUS, 10 patients with culture-confirmed STEC infection had probable HUS.The median age of case-patients was 1.8 years (range 4 months\u201314 years). The median age of the 299 controls was similar ; 58% of case-patients versus 43% of controls were female (p = 0.01). One hundred and thirty-four (89%) case-patients were urban residents.Among the 150 case-patients, clinical findings included bloody diarrhea (84%) and vomiting (71%). Ninety-four (63%) were hospitalized for a median of 4.5 days (range 1\u201314 days). Among the 78 case-patients with definite HUS (dHUS n = 68) or probable HUS (pHUS n = 10), 37 (47%) had peritoneal dialysis , 2 (3%) had hemodialysis , and 69 (88%) received erythrocyte transfusions .Among the 99 case-patients with culture-confirmed STEC infection, 96 had a single STEC isolated: 58 (60%) were O157:H7, and 38 (40%) non-O157. Among the 99 with STEC isolated, the proportion who had O157 was 82% (14 of 17) among children with definite HUS, 90% (9 of 10) among children with probable HUS, and 53% (38 of 72) among children without HUS. Among the 38 non-O157 isolates, the serotype frequency was 29% O145:NM, 11% O26:H11, 11% O113:H21, 8% O174:H21, 5% O8:H19, 5% O145:H25, 5% ONT:NM, and 3% (1 each) O2:H11, O15:H27, O25:NM, O58:H40, O91:H7, O103:H2, O103:H25, O111:NM, O121:H19, and O171:H2.>2 hours. Many beef-related exposures were significantly associated with STEC infection ; case-patients with this exposure ranged from 7 months to 9 years old. Living in or visiting a place with farm animals, contact with farm animals , and contact with cattle manure were associated with illness. Risky exposures that suggest person-to-person transmission from young children included contact with a child <5 years of age, attending daycare or kindergarten, and contact with a child <5 years of age with diarrhea. Wearing diapers was also linked to illness. No significant differences between case-patients and controls were found in the distribution of most variables that relate to socioeconomic status . However, case households were more likely to have nonparental income.Analysis of single variable associations, when the fixed adjustment factors were controlled for, identified dietary habits and animal exposures linked to illness . Generalnfection , 2. Eatiempanadas (fried or baked pastries with ground beef filling) at home; the child eating meat pie at home; and the respondent always washing hands with soap and water after handling raw beef. The factors controlled for in the adjusted univariate analysis were eating more than the median number of fruits and vegetables, male sex, having a nonparent respondent, and the respondent always washing hands after handling raw beef; all were protective  remained protective . With this model, the fixed adjustment factors had significant estimated protective associations as follows: eating more than the median number of fruits and vegetables , male sex , having a nonparent respondent , and the respondent always washing hands after handling raw beef .On multivariable logistic regression analysis, significant risk factors for STEC infection that remained were eating undercooked beef outside the home , living in or visiting a place with farm animals , contact with a child <5 years of age with diarrhea , and having nonparental household income . Eating ground beef at home , steak, jugo de carne, and salami. Salami and other types of beef are uncommon causes of STEC outbreaks  and diversity (14 serogroups) of the non-O157 STEC subgroup. Others have also reported outbreaks and sporadic cases of non-O157 STEC infections caused by cattle-related items  for STEC infection. However, others have reported female sex as a risk factor for Our finding that eating a wider variety of fruits and vegetables was protective against STEC infection merits further investigation. A varied diet may increase resistance to disease by providing bowel flora that help to protect against colonization with pathogens by providing compounds that block bacterial adhesions, as has been postulated for urinary tract infections . Implementation of prevention measures by industry, government, and consumers could result in a decrease in the incidence of STEC infections in children, and thereby decrease the incidence of childhood kidney disease from HUS in Argentina with its associated human and economic costs.Evidence indicates that measures instituted by industry, in response to government regulations, recalls, and outbreak investigations, are critical in decreasing STEC infections. After a large outbreak due to ground beef in 1993 in which 4 children died ("}
+{"text": "These bacteria have emerged as a hospital problem that appears to be caused by both linezolid exposure and patient-to-patient transmission. Enterococcus faecalis and 2 E. faecium); 7 were vancomycin-resistant. Compared with controls, case-patients had increased in-hospital mortality rates and lengths of stay. Multivariate analysis identified independent predictors of LRE infection: prior cultures positive for methicillin-resistant Staphylococcus aureus (adjusted odds ratio [AOR] 27), hospitalization duration before index culture (AOR 1.1 per day), and duration of preceding linezolid therapy (AOR 1.1 per day). Linezolid exposure and patient-to-patient transmission appear to be responsible for LRE infections, an important emerging hospital problem.Linezolid was approved in 2000 for treatment of gram-positive coccal infections. We performed a case-control study during a hospital outbreak of linezolid-resistant enterococci (LRE) infections, comparing cases of LRE infection (cases) with linezolid-sensitive enterococci infections (controls). Nasal and perirectal swab samples were obtained from all patients in a 1-day point-prevalence survey. We examined antimicrobial drug use and calculated the defined daily dose of linezolid per 1,000 patient-days. Fifteen LRE cases were identified (13  Enterococcus faecalis and E. faecium  and vancomycin-resistant enterococci (VRE) infections (E. faecium (LRVRE) , coronary care unit, and ventilator rehabilitation unit.Cases were identified by manually reviewing hospital A\u2019s microbiology susceptibility testing reports related to all E. faecium, E. faecalis, S. aureus, and S. epidermidis was obtained for the index hospitalization and for the preceding 12 months. Isolates from surveillance cultures (not illness-associated) were not included. Invasive clinical isolates were defined as isolates from any of the following sources: blood; bone; cerebrospinal, joint, pericardial, peritoneal, or pleural fluid; surgical specimen or aspirate; or any other normally sterile site. Noninvasive isolates included isolates from sputum, urine, or wounds. We performed a subset analysis comparing linezolid-sensitive and vancomycin-resistant enterococci (LSVRE) and LRVRE infections (case-control study II). After the initial random selection of controls with LSE infections, we selected additional controls with LSVRE to obtain a 1:4 ratio of LRVRE-infected case-patients to LSVRE controls.Specimen information for clinical isolates of E. faecalis, E. faecium, and S. aureus was performed in hospital A. On March 28, 2005, all patients hospitalized in hospital A were asked if they would give informed consent for the collection and culture of nasal and perirectal swab specimens; specimens were obtained from all patients who gave consent. Identification and antimicrobial drug susceptibilities of nasal and perirectal swab specimen cultures were performed in hospital A\u2019s microbiology laboratory.A 1-day point-prevalence culture survey for >16 \u00b5g/mL for all isolates tested. For the point-prevalence survey, hospital A microbiology staff plated nasal swabs specimens onto mannitol-salt agar plates; linezolid (30 \u00b5g) and oxacillin (1 \u00b5g) disks were placed in the first quadrant to screen for LRSA and MRSA, respectively. Perirectal swabs samples were plated directly onto 2 bile-esculin plates, 1 of which contained 6 \u00b5g vancomycin/mL to screen for VRE. A linezolid disk was placed on the heavy inoculum on the plate without vancomycin to screen for LRE.Hospital A used the Dade Microscan Walkaway 96  SI Pos Combo 21 for all species identification and susceptibility testing of gram-positive clinical isolates  programmed for a molecular weight range of 25\u2013350 kb, an initial switch time of 2.0 s, and a final switch time of 20.0 s. The PulseNet size standard, S. Braenderup strain H9812 (ATCC BAA-664) (Pulsed-field gel electrophoresis (PFGE) subtyping was performed at the Tennessee Department of Health laboratory on available LRE isolates  from hospital A. The PulseNet (CDC) standardized protocol for S. aureus, and VRE isolates cultured from hospital A inpatient blood samples from January 1990 through December 2004. Repeat isolates from the same patient within 30 days were excluded.By using laboratory information system data, we determined the number of MRSA, methicillin-sensitive The defined daily dose (DDD) of linezolid was identified as 1.2 g/day. We reviewed hospital A\u2019s pharmacy purchase data for linezolid for October 2001 through February 2005 and calculated DDD/1,000 patient-days; intravenous linezolid usage was analyzed by patient location (ICU or non-ICU). Oral linezolid use was not tracked by patient location. Hospital A\u2019s antimicrobial drug\u2013prescribing restrictions were reviewed. Hospital A pharmacy staff conducted a drug-usage evaluation of linezolid for January through April 2005. The drug-usage evaluation included prescriber information, duration, and indication for linezolid; patients with active linezolid orders were identified by concurrent computer printouts.Statistical analyses were performed by using Epi Info 3.2.2 (CDC) and SAS v. 9.1 software . Fisher exact test was used to compare categorical variables; the Kruskal-Wallis test was used to compare continuous variables. For univariate analysis, exact methods were used for 95% confidence intervals (CIs); for multivariate analysis, 95% Wald CIs were used. All p values were 2-sided.E. faecium and 13 were E. faecalis infections. Of the 15 case-patients, 12 (80%) were black and 8 (53%) were female; the median age was 54 years (range 38\u201374 years) 4 years) . Two of E. faecalis. LRE isolates were more likely to have been invasive than LSE isolates .Sixty control patients with LSE clinical isolates were identified. Case-patients and LSE patients did not differ significantly by age, race, sex, immunocompromising conditions, or place of origin  . Of the Patients with LRE infection were more likely to die during their hospitalization than those with LSE infection . Case-patients were hospitalized significantly longer than controls . Case-patients were hospitalized for longer periods than control patients before the index culture  and after the index culture .During the preceding 12 months, LRE case-patients had received more cumulative days of antimicrobial drug treatment than LSE patients . Compared with patients with LSE infection, patients with LRE infection had more frequently received linezolid therapy during the prior 14 days . This association remained valid for receipt of linezolid in the prior 2, 3, and 12 months. Patients with LRE infection, compared with patients with LSE infection, also had more cumulative days of linezolid therapy ; among those who had received linezolid, the median was 17.5 days versus 8 days (p = 0.06). Case-patients were less likely than control patients to have received vancomycin during the previous year. Apart from carbapenems, no other antimicrobial drugs were associated with LRE infection; carbapenems were usually prescribed together with linezolid.Case-patients with LRE were also more likely than controls to have had a prior clinical isolation of MRSA : 10 (67%) case-patients and 8 (13%) control patients had an MRSA infection during the 12 months before the index hospitalization. Of the 10 case-patients with a previous MRSA infection, 5 had received linezolid therapy (4 after isolation of MRSA) before their LRE infection.Next, we stratified prior infection with MRSA by linezolid exposure. Among patients who had no linezolid exposure, prior MRSA infection remained strongly associated with subsequent LRE infection . Conversely, when we stratified linezolid exposure by prior MRSA infection, the exposure was associated with LRE infection among those with no previous MRSA infection . The association between prior MRSA infection and hospital stay before the index culture did not reach statistical significance .Case-patients with LRE infection were more likely than control patients to have spent time in 4 locations in hospital A before the index culture occurred. These locations were 3 specific critical care areas  and 1 orthopedic/neurosurgical ward (OR undefined). By multivariate analysis, using forward logistic regression, we identified the following as statistically significant predictors of LRE infection: prior isolation of MRSA , duration of hospitalization before index culture , and duration of preceding linezolid therapy .To compare patients with LRVRE and LSVRE infections, we identified 7 case-patients and 28 controls. Case-patients and controls did not differ significantly by age, race, sex, immunocompromising conditions, instrumentation, or proportion of deaths. The length of hospitalization for case-patients compared with that for controls did not reach statistical significance . During the previous 12 months, the cumulative days of total antimicrobial drug therapy, and of linezolid therapy specifically, did not differ significantly between case-patients with LRVRE and controls with LSVRE .S. aureus was not identified in the cultures. VRE was isolated from 51 (13%) of the 388 perirectal swab specimens, and LRE was isolated from 4 (1%); the 4 LRE isolates also were resistant to vancomycin. Of the 4 patients with LRE, 3 were located on 2 medical-surgical wards and 1 was located in a critical care area.Nasal swab samples were obtained from 393 (93%) of the 424 hospitalized patients, and perirectal swab samples were obtained from 388 (92%). MRSA was isolated from 25 (6%) of the 393 nasal swab specimen cultures; linezolid-resistant E. faecalis) were related but distinguishable . The environmental isolates (E. faecium) were indistinguishable from patient B\u2019s blood culture isolate. A third pattern was shared between patients C and D; the isolates of E. faecium from blood and rectal swab specimens from patient D were identical.LRE isolates from 4 case-patients (patients A\u2013D) and 3 environmental isolates from patient B\u2019s room  after routine cleaning were available for PFGE . The isoHospital A started using linezolid in April 2000; however, pharmacy purchase data were available only from October 2001 onward. DDD increased from 13/1,000 patient-days in 2001 to 35/1,000 patient-days in 2004 . Most liPatients with known VRE infection were placed on contact precautions. Patients with MRSA infection were placed on contact precautions while in critical care areas. Critical care\u2013area staff performed surveillance cultures of urine and sputum on a weekly basis.We describe risk factors and outcomes associated with a large hospital outbreak of LRE, an emerging pathogen. The licensing-to-resistance time interval for linezolid was brief. Linezolid use increased in response to increases in MRSA and VRE infection in this hospital community and probably because of the drug\u2019s convenience compared with vancomycin . Despite being restricted to use by infectious disease and critical care physicians, the linezolid DDD prescribed by hospital A increased nearly 3-fold in 3 years; most use was outside ICUs. In this investigation, we found that exposure to linezolid any time in the preceding 12 months and the increased cumulative days of linezolid use among case-patients suggest that antimicrobial drug pressure contributed to the emergence of multiple clones of LRE at hospital A. De novo resistance has been documented in several instances in which PFGE comparisons have been made between LSE and LRE isolates in the same patient after exposure to linezolid  of 15 patients with LRE infection had been exposed to linezolid; however, 7 (47%) had not been. This finding has been documented previously with nosocomial transmission of LRVRE in a transplantation unit among 7 patients, of whom 6 were linezolid-naive, and isolated from all had the same pattern on A strong risk factor for LRE was prior MRSA infection. In 4 (27%) case-patients, linezolid was used to treat prior MRSA infection. However, the relationship between prior MRSA and LRE goes beyond this expected association of prior MRSA and linezolid exposure. Even among those with no linezolid exposure, MRSA was strongly associated with linezolid resistance . This was confirmed on multivariate analysis (AOR 27). Prior MRSA infection therefore might also be a surrogate marker for patient-to-patient transmission of LRE or increased susceptibility to nosocomial infection.In this outbreak, patients with LRE infection experienced more illness, were hospitalized longer, and were more likely to die than patients with LSE infection. We cannot determine whether the higher number of deaths were attributable to LRE; additional studies are required, including matching of controls to case-patients on severity-of-illness measures. In addition, our sample size and statistical power were limited by the number of cases of LRE and, in particular, LRVRE.>90% for surveillance cultures) and financial . These concerns and obstacles should be addressed; otherwise, the response to 1 emerging resistant infection will breed another emerging infection.Infection-control efforts should focus on preventing infections and interrupting patient-to-patient transmission of multidrug-resistant organisms (MDROs)  and for control-patients with linezolid-sensitive enterococci (LSE) infection*"}
+{"text": "After the 2006\u20132007 epidemic wave of Rift Valley fever (RVF) in East Africa and its circulation in the Comoros, laboratory case-finding of RVF was conducted in Mayotte from September 2007 through May 2008. Ten recent human RVF cases were detected, which confirms the indigenous transmission of RFV virus in Mayotte. Subsequently, to assess the situation of RFV among humans, we retrospectively and prospectively analyzed serum specimens from febrile patients sampled by a routine surveillance program for chikungunya and dengue viruses. We describe the clinical, biological, and epidemiologic characteristics of human RVF cases in Mayotte.Plasmodium spp. by using the rapid diagnostic test OptiMAL , were screened for RVFV. We defined a confirmed recent human case-patient as a person with dengue-like illness and the serum positive for anti-RVFV IgM by antigen-capture ELISA or RVFV RNA by reverse transcriptase\u2013PCR  was found in 10 samples (4.5%). The earliest recorded onset of dengue-like illness was September 27, 2007, and the latest was May 14, 2008. Seven cases (70%) occurred from January through April, during the hot, rainy season  were male , 5 (50%) were French citizens native to Mayotte, and 5 (50%) were Comorian citizens. None of the cases reported recent  travel into countries with RVF endemic transmission.3) and died as a result of gastrointestinal bleeding and hepatic encephalopathy. Case-patient 10 was admitted to the hospital in May 2008 with a 5-day history of high body temperature, body pain, joint pain (particularly in the shoulders), and fatigue, combined with symptoms consistent with right-sided heart failure . The patient was discharged with a diagnosis of pericarditis. He had a follow-up visit 1 month later, and a sample obtained on this occasion tested positive for RVFV IgM and IgG.Although 2 patients were admitted to the intensive care unit for medical management, no severe neurologic manifestations of RVF were observed, and no deaths could be attributed to RVF. Case-patient 6 , with a Predictably, contact with ruminants  was the predominant means of exposure among reported case-patients; this mode was documented in 5 of 9 cases that were fully investigated. Furthermore, only the presence of numerous breeding sites for mosquitoes in the housing environment remained as a possible means of exposure for 3 other case-patients. The mode of transmission remains undetermined for the remaining case-patient, who had no admitted direct contact with ruminants and whose housing environment did not have numerous breeding sites .The laboratory case-finding initiated in response to the 2006\u20132007 RVFV outbreaks in eastern Africa found indigenous transmission in Mayotte. As a result, information was provided to the population, and preventive activities were undertaken, e.g., information for healthcare providers, a campaign to eliminate mosquito-breeding sites, recommendations to avoid contact with mosquitoes and sick animals, and instructions on adopting appropriate protective measures when engaging in activities related to animals, including slaughtering and butchering.Because our investigations were mostly retrospective, we cannot exclude recall bias about exposure and incomplete documentation. Moreover, entomologic investigations in the living areas of the case-patients several months after the first signs of illness occurrence were incomplete.RVF activity in Mayotte appears to be an expansion of the eastern Africa outbreak. As in the case of the chikungunya outbreak in 2005\u20132006 in Mayotte, thought to have started in Lamu, Kenya (This epidemic is a further illustration that it is imperative for healthcare providers, epidemiologists, and policy makers in Mayotte to remain alert each time there are outbreaks in nearby countries because the risk of extension to Mayotte is always plausible. Furthermore, the documentation of RVFV circulation in Mayotte should serve as a further impetus toward the development of surveillance programs for nonspecific febrile illnesses, especially in settings where arboviruses are highly prevalent. To achieve this objective, local laboratory capabilities should be enhanced to enable biological diagnosis of a wide array of arboviruses that actively circulate in the eastern African seaboard region. Furthermore, medical education materials and feedback bulletins should regularly provide information about accessing laboratory services while encouraging patients with febrile illness to seek early medical evaluation. Ultimately, maintaining such programs is essential to promptly detecting any renewed introduction or resurgence of RVF and other pathogens, especially during the hot and rainy season."}
+{"text": "Phylogenetic studies demonstrated that outbreak strains belonged to the East-Central African lineage. Culex poicilipes. Genetic comparison of virion segments indicated little variation among the strains isolated. However, phylogenetic studies clearly demonstrated that these strains belonged to the East-Central African lineage for all segments. To our knowledge, this is the first time viruses of this lineage have been observed in an outbreak in West Africa. Whether these strains were introduced or are endemic in West Africa remains to be determined.In October 2003, 9 human cases of hemorrhagic fever were reported in 3 provinces of Mauritania, West Africa. Test results showed acute Rift Valley fever virus (RVFV) infection, and a field investigation found recent circulation of RVFV with a prevalence rate of 25.5% (25/98) and 4 deaths among the 25 laboratory-confirmed case-patients. Immunoglobulin M against RVFV was found in 46% (25/54) of domestic animals. RVFV was also isolated from the mosquito species  Bunyaviridae, genus Phlebovirus genus, and its genome consists of 3 negative single-stranded RNA segments referred to as L (large), M (medium), and S   is an acute febrile viral disease that affects small domestic ruminants  by using sentinel herds in 5 geographic regions and a notification system of hemorrhagic fever in medical healthcare centers. This NDSS was implemented in collaboration with the Centre National d\u2019Hygi\u00e8ne, the \u201cCentre National d\u2019Elevage et de Recherche V\u00e9t\u00e9rinaire\u201d in Mauritania, and the Institut Pasteur de Dakar in Senegal. The value of the NDSS was further reinforced after an outbreak of Crimean-Congo hemorrhagic fever in Mauritania in February 2003 (>1 of these results: immunoglobulin M (IgM), reverse transcription\u2013PCR (RT-PCR) or virus isolation positive results. A human contact was defined as any person or relative who had been directly in contact with a confirmed human or animal case-patient, or with a person who died who had had overt hemorrhagic fever symptoms from September through December 2003.A suspected human RVFV case-patient was defined as a person with fever associated or not with hemorrhagic, jaundice, or neurologic symptoms or any person who died who had had overt hemorrhagic fever symptoms from September through December 2003. A confirmed human RVFV case-patient was defined as a person for whom laboratory tests confirmed an acute or recent RVFV infection, e.g., by Nine localities belonging to 3 administrative provinces were visited : Keur MaIn affected areas, the investigation was conducted under the supervision of the chief of the sanitary district. For each case, venous or capillary blood samples were collected into dry tubes or onto filter papers, respectively. A thick blood smear was also taken from all suspected case-patients for differential diagnosis of malaria. An interview in which information was gathered about sex, age, date of fever onset, and hemorrhagic signs was conducted for all case-patients and their contacts.All domestic animals living in the close vicinity of suspected or confirmed case-patients were included in the study. Every blood sample was accompanied by an investigation form specifying the species, age, and localization of the animal during the month before the investigation and, for female animals, a history of pregnancies.2, which were placed close to water points or in sheepfolds, respectively; animal-baited traps were placed in the houses of persons with suspected or confirmed cases. Mosquitoes were frozen and subsequently identified on a chilled table by using morphologic keys  and RT-PCR was done by using the Titan One-Step RT-PCR System , according to the recommendations of the manufacturers. The primers NS3a  and NS2g  were used to amplify a 669-nt region of the virus S segment region encoding the NSs protein. The primers MRV1a  and MRV2g  were used to amplify a 809-nt region of the virus M segment region encoding the G2 protein. Primers Wag  and Xg  were used to amplify a 212-nt region of the L segment (www.pasteur.fr/recherche/banques/crora), Institut Pasteur de Dakar on mosquitoes and serum collected from humans by inoculating the virus into suckling mice and a mosquito cell line (AP61). Virus identification was performed by an indirect immunofluorescence assay that used polyclonal and monoclonal antibodies. The identification of virus isolates was confirmed by complement fixation  Collaborating Center for Arboviruses  to leukocytes was estimated in 200 fields based on a mean leukocyte count of 8,000/\u00b5L of blood.To rule out malaria infection, thick blood smears from patients with suspected cases were Giemsa-stained. The ratio of parasite  were included in this study. Of these persons, 25.5% (25/98) had evidence of recent RVFV infection , and 10% had evidence of past infection . Seven viral strains were isolated. For the 25 patients who were recently infected , the median age was 21 years (range 7\u201350 years), 4 died, and 16 had hemorrhagic signs . Among the 23 suspected case-patients from whom blood samples were collected, 10 were infected by RVFV; only 2 patients were positive for malaria parasites.In Assaba Province, 2 confirmed case-patients were recorded before the investigation (index case-patients 1 and 2), and 1 suspected case-patient (S1) was found during the investigation. The index case-patient 1 was dead at the time of the investigation; however, RVFV (strain SHM169867) was isolated from a blood sample taken during his illness. No RVFV infection was noticed in contact persons associated with this patient. However, IgM against RVFV was detected in animals living in and near the residence of index case-patient 1. For index case-patient 2 (infected with the strain SHM169872), a second blood sample was taken, and presence of IgM antibodies against RVFV was confirmed. No evidence of RVFV infection was detected in contact persons or in animals living in or near the residence of this patient. The suspected case-patient 1 was identified in Kiffa Hospital, where he was admitted on November 3, 2003. This 50-year-old patient had onset of fever on October 24, 2003, with asthenia, jaundice, nausea, hematemesis, epistaxis, and gingival hemorrhage. Serologic tests showed IgM against RVFV. No evidence of virus infection was detected in those who had accompanied this patient to the hospital.In Brakna Province, where 3 cases were confirmed before the investigation (index case-patients 3\u20135), 6 suspected case-patients (S2\u2013S7) were found during the investigation. Laboratory testing of samples from the 3 index case-patients showed IgM against RVFV by ELISA and RVF viral RNA by RT-PCR. A virus strain (SHM169898) was isolated from index case-patient 4. Among the suspected case-patients from this province, 3 deaths (S2\u2013S4) were recorded, but no samples were available from those patients. Nevertheless, animals in the vicinity of S1, S2, and S3, were found to be infected . In addition, infection was detected in 4 of 6 persons who had been in contact with suspected case-patient 3. In contrast, no evidence of infection by RVFV was detected in contacts of S2 and S4. The suspected case-patient 5 was a student living in Makhtar Lahjar, who had fever onset on October 20, 2003, was admitted to the National Hospital Center (NHC) of Nouakchott on November 6, and from whose blood RVFV (strain SHM172776) was subsequently isolated. During the investigation, 2 suspected case-patients (S6 and S7) were discovered in the Healthcare Center of Makhtar Lahjar. Suspected case-patient 6 was a 17-year-old female patient who had onset of fever on October 10 and who was admitted to the center on November 1 with headache, abdominal pain, vomiting, hemorrhages, and epistaxis; virus was isolated from this patient (strain SHM172805). The contacts associated with suspected case-patient 6 were also found to be infected with RVFV. Suspected case-patient 7 was a 35-year-old man. He had a fever on October 15 and was admitted to the Health Center of Makhtar Lahjar on November 1 with headache, nausea, vomiting, and epistaxis. The RVF IgM test result for this patient was positive for RVFV.In Trarza Province, 2 confirmed case-patients were observed during the period of surveillance (index case-patients 6 and 7) and 4 suspected case-patients (S8\u2013S11) were identified during the investigation. Viral isolation was positive for the index case-patient 7 (strain SHM169885). Among the suspected case-patients, 2 (S8 and S9) died before blood samples could be obtained. However, blood testing of samples from 2 animals living in the vicinity of S8 and from 2 human contacts of S9 found recent RVFV infection. Suspected case-patient 10 was 28-year-old man, with onset of fever on October 22, who came for consultation to Keur Macene Healthcare Center. He had a prolonged cough without hemorrhagic symptoms, and an IgM ELISA result for RVFV was positive. The suspected case-patient 11 is a 26 year-old woman, with onset of fever on October 22, who was admitted to the healthcare center of Rkiz with headaches, asthenia and anorexia without hemorrhagic signs. The RVF IgM test result of this patient was positive. In Tagant Province, index case-patient 8, whose condition was diagnosed before the investigation, was a man who came for consultation at the provincial hospital on September 24, exhibiting fever and hematemesis. RVFV (strain SHM169868) was isolated from a blood sample taken on September 29.In Gorgol Province, 2 suspected case-patients (S12 and S13) were evacuated to the NHC of Nouakchott. The onset of their symptoms dated to October 23 and October 25, respectively. Samples from each were positive for IgM against RVFV by October 30, and RVFV (strain SHM172768) was isolated from S13, who died on October 30.In Dakar, Senegal, an \u201cimported\u201d case (index case-patient 9) in a person from Rosso, Mauritania, was diagnosed by positive results by ELISA IgM and RT-PCR. This patient was first admitted to the NHC of Nouakchott, Mauritania, before being transferred to the H\u00f4pital Principal de Dakar.Serum samples were obtained and tested from 54 domestic animals (48 goats and 6 sheep) living in the visited localities . The medCulex poicilipes was the most frequent species (43.8%), followed by Cx. antennatus (23%) and Mansonia uniformis (9%). A total of 544 monospecific pools were constituted and submitted for viral isolation. Only Cx. poicilipes was found to be associated with RVFV. Three strains  were isolated from the 146 pools constituted in Guimi Province, giving rise to a minimum infection rate of 0.04% for this locality and 0.01% for the whole study site  differences were found between the fragments of the S or L segments analyzed . A single amino acid difference was found between the M fragment (255 aa) of the 5 viral strains analyzed. Results of phylogenetic analyses of the nucleotide sequences of amplified fragments from 3 segments belonging to 2 representative strains (H1MAU03 [SHM169867] and H2MAU03 [SHM169868]) isolated during this epidemic and previously described nucleotide sequences of RVFV are shown in The combination of ELISA, RT-PCR, and isolation assays has permitted the rapid and efficient identification of RVFV as the cause of the extended hemorrhagic fever outbreak reported in Mauritania during the last quarter of 2003. Of the 24 RVF cases diagnosed in the laboratory, 13 were diagnosed by IgM only; 8 were diagnosed by IgM, RT-PCR, isolation, or >1 method; and 3 were diagnosed by RT-PCR, isolation, or both. These data and those obtained during the epidemics of RVF in Kenya , who represent a large part of the population living in these areas.During this investigation, a high infection rate was found in sheep and goats that lived in close proximity to the patients (46.3% of IgM positive compared with 25% during the 1998 outbreak)  (Cx. poicilipes was found to carry RVFV during the outbreak. Spatial analyses of the results show that Cx. poicilipes was in fact predominant only in the village of Guimi, where the RVFV was isolated. This observation indicates that the levels of the different species vary according to the local environment. Mosquitoes from the Aedes genus, known for their role in RVFV maintenance and transmission, were scarce, likely due to their early appearance at the onset of the rainy season, whereas our investigation took place at the end of the rainy season. Indeed, in 2003, the last rainfall event was recorded at the beginning of October.Among the mosquitoes collected, several species known as RVFV vectors (,Genetic analyses of the 3 segments of RVFV isolated during this epidemic showed a low level of variation between isolates from the different provinces. This finding supports the hypothesis that the same strain was circulating in the different affected areas. The nucleotide sequences of the strains isolated during this epidemic compared with those isolated elsewhere in Africa and Saudi Arabia showed that they belong to the East/Central African cluster for the 3 segments. Previous reports have shown that some strains isolated in West Africa share 1 or 2 segments with strains belonging to the East/Central African cluster (This finding confirms the existence of RVFV strain exchanges between geographic areas. In fact, the spread of RVFV from East Africa to other regions has already been observed during the RVF outbreak in Saudi Arabia and Yemen in 2000\u20132001 ("}
+{"text": "To the Editor: Crimean-Congo hemorrhagic fever (CCHF) is a severe viral disease transmitted to humans by tick bite or contact with blood, excreta, or tissues of infected patients or livestock. The disease is endemic in many African, Asian, and European countries. Sporadic cases or outbreaks have been observed in the Balkan Peninsula  per reaction.One set of primers and 1 probe were designed to amplify an 84-bp genome region of the S RNA segment of CCHF virus on the basis of European sequences : primers CCEuS 5\u2032-TGACAGCATTTCTTTAACAGACATCA-3\u2032 and CCEuAs 5\u2032-AAACACGGCAGCCTTAAGCA-3\u2032, and probe 5\u2032-TCGCCAGGGACTTTATATTCTGCAAGG-3\u2032. A 25-\u03bcL reaction was conducted in a LightCycler  with 10 mmol/L of each deoxynucleotide triphosphate, 600 nmol/L of each primer, 200 nmol/L of probe, and 3 \u03bcL of RNA. Cycling conditions were 50\u00b0C for 30 min and 95\u00b0C for 15 min, followed by 45 cycles at 95\u00b0C for 15 s and 58\u00b0C for 30 s. A quantification curve was constructed with 10-fold serial dilutions of in vitro\u2013transcribed CCHF virus RNA. Positive results were obtained up to a dilution of 10Twelve samples had positive results: all 11 samples in group A and 1 in group B . Results6 to 28.99 \u00d7 106 geqs/reaction. The highest level was observed in the patient who died (23/03). High loads were observed in all primary case-patients  except for patient 154/04, from whom a sample was obtained 18 days after onset of disease. All primary case-patients had severe disease with high fever and clinically apparent hemorrhage. All secondary case-patients, except patient 34/03, were contacts of the patient who died  and had symptoms of disease \u22481 week after the death of patient 23/03.Viral loads ranged from 14 \u00d7 10Viral load of secondary case-patients was <250 geqs/reaction, which was much lower than that of primary case-patients. This finding suggests that the disease is more severe in primary case-patients and becomes a milder form in secondary case-patients. Samples of secondary case-patients 24b/03 and 25b/03 were obtained on day 9 of illness, and patient 24b/03 had a 4\u00d7 higher viral load than patient 25b/03. A possible explanation might be that because patient 24b/03 had closer contact with the person who died, he received a higher dose of virus, which might affect severity of the disease. Other secondary case-patients had milder symptoms with no clinically apparent hemorrhage and were not hospitalized. All hospitalized patients had leukopenia, except for the patient whose sample was taken 18 days after the onset of disease. No correlation was observed between viral load and cytokine levels or platelet counts, which suggests that other factors are involved in pathogenicity and immune response.The real-time RT-PCR was rapid and more sensitive than the RT-nested PCR because 1 additional positive sample was detected. Samples with positive results from the first round of the conventional RT-nested PCR  had the highest viral loads when tested by real-time RT-PCR.In conclusion, a 1-step real-time RT-PCR for detection and quantification of CCHF virus was developed, used with clinical samples, and provided informative data on the severity, course, and outcome of CCHF. Further studies, preferably in serial samples of patients, should provide insights into the pathology of CCHF and the effectiveness of antiviral drugs."}
+{"text": "In June and July 2008, two office workers were admitted to a Dublin hospital with Legionnaires' disease. Investigations showed that cooling towers in the basement car park were the most likely source of infection. However, positive results from cooling tower samples by polymerase chain reaction (PCR) did not correlate with subsequent culture results. Also, many employees reported Pontiac fever-like morbidity following notification of the second case of Legionnaires' disease. In total, 54 employees attended their general practitioner or emergency department with symptoms of Legionnaires' disease or Pontiac fever. However, all laboratory tests for Legionnaires' disease or Pontiac fever were negative. In this investigation, email was used extensively for active case finding and provision of time information to employees and medical colleagues. We recommend clarification of the role of PCR in the diagnosis of legionellosis and also advocate for a specific laboratory test for the diagnosis of the milder form of legionellosis as in Pontiac fever. Legionellosis presents as two distinct clinical entities: Legionnaires' disease (LD) comprises pneumonia with severe multisystem disease and Pontiac fever (PF) which is a self-limiting flu-like illness . Annually, on average, 6 cases of LD were reported to the Health Protection Surveillance Centre by the Departments of Public Health in Ireland between 1994 and 2007. This reported rate of two per million is low compared with other European countries, such as Spain and France where rates of 24.8 per million and 22.8 per million were reported, respectively, in 2007. In 2007, the overall European rate of reported infection was 11.4 cases per million [Legionella for these cases is rarely determined. However, outbreaks may provide a better opportunity to identify common sources of legionellae. Such outbreaks of LD can be caused by communal sources such as hot tubs, spa pool, hospital, domestic or hotel showers, and wet cooling towers [LD is transmitted from the environment by inhalation of an infectious aerosol . In Eurog towers . Over a ten-day period in June-July 2008, two cases of LD were reported to the Department of Public Health, Health Services Executive East (HSE-E) in Dublin. Both cases worked in a large Dublin-based insurance firm and were based in a newly built office block in the city. The office building consisted of seven floors over a basement car park. Two cooling towers were located in the basement. The staff smoking area was located immediately adjacent to the cooling towers. Air conditioning within the building was confined to the printing/postal room (3rd floor) and the conference/meeting rooms (6th and 7th floor). This paper describes the cases with LD, the outbreak investigation, and subsequent management. A retrospective cohort study of cases with PF will be reported separately .L. pneumophila serogroup 1 urinary antigen test (UAT) on the day of admission. He was admitted to the Intensive Care Unit for the management of renal failure associated with LD and was treated with clarithromycin. He was too ill to provide a history on admission. He had no history of travel abroad. He was hospitalised for 14 days. This case was notified to the Public Health Department on June 27th. The first case of LD was a 58-year-old Southern-European male smoker who worked in the postal department on the 3rd floor of the company office, and became unwell in early June. He was admitted on June 25th to a Dublin hospital with a 3-week history of fever, night sweats, chills, diarrhoea, and confusion. His chest X-ray showed right upper and lower lobe consolidation. Liver function tests and renal indices were elevated. Diagnosis of LD was confirmed by On July 7th, a second case of LD (case 2) was reported to the Department of Public Health. Case 2 was a 48-year-old Irish male nonsmoker, who worked on the 5th floor of the company office. He had been ill and off work for 7 days preceding his presentation to the same Dublin hospital as case 1, with a history of fever, headache, cough, diarrhoea, confusion, and paraesthesia of arms and legs. On admission he was diagnosed with pneumonia and LD was confirmed by UAT on the day of admission. He was treated on the ward with clarithromycin and ciprofloxacin and was discharged home after 10 days. Case 2 lived in a different area of Dublin from case 1. Apart from place of employment there were no other common links such as travel or social activities between both cases. As a result of two linked cases of LD an outbreak control team was formed with representatives from Public Health, Infection Control, Environmental Health, Microbiology, the Health Protection Surveillance Centre, and the Health and Safety Authority.On July 8th, it was reported to the Department of Public Health HSE-E by company management that many employees were absent from work and had reported severe flu-like illness. Consequently, active case finding was carried out on the morning of July 8th in the company. A standardised questionnaire was emailed to all company and contract employees who had worked in the building at any time during the previous 14 days. Information was requested on symptoms , potential exposure , smoking, travel, and demographics . Legionella infection was diagnosed by culture, UAT, or serology (a single high titre or a four-fold rise in specific serum antibody to L. pneumophila serogroup 1 or serogroups 2\u20136 and 8).We defined a case of LD as an employee of, or visitor to, the company office who had pneumonia with onset since June 1st, andWe defined a case of PF in the initial investigation as an employee of, or visitor to, the company office with symptoms of fever, headache, myalgia, and nonproductive cough with onset since June 1st. Sickness records were reviewed by Human Resources staff and absent workers were contacted to inform them of the outbreak and to enquire about their health and whether they had sought medical attention. Respondents who met our case definition for LD or PF were promptly telephoned by Department of Public Health medical or nursing staff and advised on appropriate medical follow-up.Legionella serology three weeks after likely exposure. Employees who attended the emergency department (ED) or their general practitioner (GP) were followed up to determine their symptomatology, investigation, results, and treatment. Subsequently, a designated clinic was arranged for all employees and contract workers who (a) met our case definition for PF or (b) had attended the ED or their GP with concerns regarding LD. All were invited for Legionella serology. No employee tested showed serological evidence of infection with L. pneumophila serogroups 1\u20136 and 8, three weeks after likely exposure. The serological tests were performed by the legionella reference services at the Respiratory & Systemic Infection Laboratory, HPA Centre for Infections, Colindale, UK.In total, 54 employees attended EDs in the Republic of Ireland and Northern Ireland or their general practitioner with symptoms and/or concerns about LD or PF; each had a UAT, all of which were reported negative. Of these at least 38 were reported as having been treated with an antibiotic. Subsequently, 37 of these employees with possible symptoms of PF attended a special clinic for On July 8th, Public Health doctors met on site with the company's Health and Safety manager, Risk Assessment manager, and Human Resources manager. Public Health doctors, Environmental Health officers of the Health Services Executive, and Environmental Health officers of Dublin City Council conducted an inspection and environmental risk assessment of the building. On July 9th, the Health, and Safety Authority, the organisation responsible for ensuring safety, health and welfare at work, inspected the building. There were two separate water-containing systems supplying the office. The primary system consisted of two cooling towers located in the large basement carpark of the building. Both cooling towers were immediately adjacent to the staff smoking area. This system was used to chill incoming water. The secondary system was a hot and cold water system used to service sinks and showers. There was no physical connection between the primary and secondary systems. Both systems were supplied with mains water. Managers reported the use of an alarmed temperature control system for the water supply to the building. There was no fountain in the building. Legionella spp. Mains water was checked at points entering the building. Samples from tower 1 were positive by PCR for Legionella spp. but below detection level on culture . Levels of L. pneumophila detected by PCR from the showers/sinks tested were below the threshold level for clinical significance defined by the chemical company, and L. pneumophila was not cultured from any showers/sinks tested.As a precautionary measure, after case 1 was reported and prior to any treatment, multiple water samples were taken from both water systems and sent for PCR and culture for  culture . SamplesBoth cooling towers were immediately deactivated, cleaned, and disinfected as recommended . The cooL. pneumophila serogroup 1 UAT (Binax). Sputum samples from both patients, after they had commenced antibiotics, did not culture Legionella species. However sputum from case 2 sent to the legionella reference services at the Respiratory Systemic Infection Laboratory HPA was positive by PCR testing for L. pneumophila DNA. However, there was insufficient DNA to allow sequence typing of the strain. The environmental isolate from cooling tower 2 was identified as L. pneumophila serogroup 1, monoclonal antibody subgroup \u201cKnoxville\u201d.Both patients admitted to hospital were confirmed cases of LD by After the first case of LD was notified, an email alert was sent to 600 company employees informing them of the symptoms of LD and advising them to seek medical attention from their general practitioner if they had any concerns regarding illness. This alert was repeated to all employees three days later. In addition, every line manager endeavoured to contact their employees to confirm they had received relevant public health information of LD and were not symptomatic. Similar efforts were also made to check on contract employees and visitors to the company since June 1st. Following reporting of the second case of LD, an alert was sent to all general practitioners and ED consultants in the region by fax and email. In addition, due to the high level of concern among the employees in the company, two educational/information sessions on LD were held in the office by medical doctors.L. pneumophila cultured from cooling tower 2 was typed as serogroup 1 mAb2+ve which is a recognised cause of clinical infection and also a serogroup detected by UAT, we can reasonably conclude that cooling tower 2 was the most likely source of this cluster. At present, Ireland does not have legislation requiring the registration of cooling systems at national level [This paper describes a cluster of LD among office workers in Dublin. The only common link among the two cases was their place of employment. Consequently, the most likely source of this cluster was the cooling towers in the basement car park. Definitive microbiological confirmation of the cooling towers as the source of infection required an isolate from one of the patients to match the environmental isolate. As al level . A systeal level . Legionella pneumonia to other types of pneumonia indicate that LD is not \u201catypical\u201d and clinical features such as diarrhoea and confusion are not sufficiently distinctive to distinguish LD from other causes of community-acquired pneumonia. Consequently, in order to increase awareness of LD as a possible diagnosis, after the initial case of LD was confirmed we immediately corresponded with company employees, management, as well as GP and hospital colleagues. Several outbreaks have observed a relationship with the dose of inoculum. For example, an outbreak has been described where two workers in a cooling tower with different levels of exposure developed pneumonia and PF, respectively [Early identification and treatment of patients with LD have important implications for clinical management of patients and outcome . Studiesectively . Immunolectively , 9. SuchL. pneumophila as well as by nonpneumophila species [Legionella cells. In this cluster, while PCR provided time information on the number of Legionella in the environmental samples tested, equivalence with the number of CFU by culture was not found. Our results were similar to previous studies which showed that PCR results are usually higher than culture values [Legionella cells that may not necessarily be pathogenic and thus PCR is not an accurate measure of a real health risk. Nevertheless, in this outbreak, detection of Legionella species by PCR was a marker of environmental contamination by Legionella and led to preventive measures. As culture of Legionella remains the \u201cgold-standard\u201d in diagnosis of LD [Few studies have addressed the practical value of PCR in routine clinical and environmental microbiology laboratories. PCR offers theoretical advantages, such as a high sensitivity, rapid availability of results, and the potential to detect infections caused by various serogroups of  species , 10, 11.e values . It coulis of LD , we suggThe affected premises in this cluster ware a new purpose built \u201cgreen building\u201d which was occupied for three months prior to the reported onset of illness. A green building is defined as \u201cthe practice of increasing the efficiency with which buildings use resources\u2014energy, water, and material\u2014while reducing the building's impact on human health and the environment during the building's lifecycle, through better siting, design construction, operation, maintenance, and removal\u201d . HoweverL. pneumophila serogroup 1 antigens in a urine sample within one hour with a specificity of 95% [L. pneumophila serogoup 1 among PF patients [The nonspecific presentation of LD makes clinical diagnosis very difficult in most patients with community-acquired pneumonia of uncertain aetiology. Thus, the key to diagnosis is appropriate microbiological testing . Both pay of 95% . Sensitipatients \u201319. IndeThis investigation demonstrated that timely intervention is vital in control of a cluster of legionellosis. In order to control, and ideally prevent, future clusters of legionellosis we recommend the statutory notification of high risk sites and also improved diagnostic tests."}
+{"text": "Cryptococcus gattii infection during 1999\u20132006 to travel destinations of the general public on Vancouver Island, British Columbia, Canada. Findings validated and refined estimates of risk on the basis of place of residence and showed no spatial progression of risk areas on this island over time.We compared travel histories of case-patients with  Cryptococcus gattii is a fungus that infects the lungs and central nervous system of mostly immunocompetent humans and animals (C. gattii emerged on the east coast of Vancouver Island (VI), British Columbia (BC), Canada . EnvC. gattii\u2013infected case-patients with travel patterns of the general public to validate and refine these risk areas on VI. We also examined spatial progression of these areas over time to assess whether C. gattii spread from a single focal point since its emergence in 1999.This study compared travel histories of C. gattii\u2013infected case-patients were defined as BC residents with culture-confirmed C. gattii infection or HIV-negative residents of BC with Cryptococcus sp. infection diagnosed by antigen detection or histopathologic analysis. Analysis included all cases diagnosed from January 1999 through December 2006 in which the patient had documented travel history on VI. Case-patients were interviewed by using a standard questionnaire and asked about travel to any city outside their city of residence in the 12 months before symptom onset or diagnosis (www.hellobc.com/en-CA/default.htm) provided aggregated monthly visitor volume to 14 visitor centers in major tourist destinations . Maps were created by using ArcMap version 8.2 .Proportion of visits to each visitor center city was defined as number of visits to a visitor center city divided by total number of visits to all visitor center cities. For case-patients, the proportion was similarly defined. In both instances, visits to multiple cities by the same person were counted multiple times. Differences between proportion of case-patient visits and Tourism BC visits were evaluated by Fisher exact test and StatXact software . Analysis was conducted for all years combined and in 2 four-year increments (1999\u20132002 and 2003\u20132006) to assess potential spread of >1 visitor center city. Of these, 62 (75.6%) resided on VI and 20 (24.4%) lived on the BC mainland. A significantly greater proportion of visits to Parksville  and Nanaimo  were reported for patients than for Tourism BC visitors (Travel history data were available for 104 (60.1%) case-patients. Eighty-two (78.8%) had traveled to visitors . Similarvisitors .When analysis was restricted to data concerning mainland residents , a greater proportion of mainland case-patients visited Courtenay , Parksville , Nanaimo , and Qualicum Beach  than did Tourism BC visitors during 1999\u20132006; however, only Parksville reached statistical significance. Because of the small number of patients who resided on the mainland (n = 20), we could not further restrict this subset analysis to earlier (1999\u20132002) and later (2003\u20132006) periods.Residents of VI may be exposed in their place of residence, in addition to their travel destination. However, we could not accurately weight patient exposure in the home environment to exposure at the travel destination. Minor differences in results obtained for all case-patients compared with only mainland patients may be caused by this limitation or by differences in travel preferences between these groups.2 test). A higher proportion of included patients resided in central VI. However, travel patterns of central VI residents did not differ from travel patterns of other VI residents (data not shown). Our analysis assumes that travel patterns of Tourism BC visitors represent those of the general BC public. However, characteristics and activities of persons who use Tourism BC visitor centers may differ from those of persons who do not. Therefore, caution is necessary when generalizing results to the entire BC population. Our interpretation is limited by its inability to account for duration of time spent in each visitor center city and specific activities of persons while there, factors that may contribute to exposure risk.Although travel history data were unavailable for 39.9% of the case-patients, they were not significantly different in terms of mean age  or sex .C. gattii outbreak. Alternatively, results may indicate a true increase in travel-associated risk in areas near Parksville and Nanaimo. Some case-patients who resided in areas with high incidence rates may have acquired their infections by travel to these 2 areas. Although Parksville and Nanaimo may represent areas of higher risk, environmental sampling suggests fungal colonization in southern and central eastern VI, and travelers can be exposed to C. gattii in these regions (The refinement of geographic risk from our analysis may in part be the result of a reporting bias that produced larger than expected risk differences for certain areas. BC residents may be more likely to visit or travel through Nanaimo, a commercial center on VI and transportation gateway to the rest of the island (To determine travel-related risk for malaria ("}
+{"text": "In March 2007, we investigated a cluster of Nipah encephalitis to identify risk factors for Nipah infection in Bangladesh.We defined confirmed Nipah cases by the presence of IgM and IgG antibodies against Nipah virus in serum. Case-patients, who resided in the same village during the outbreak period but died before serum could be collected, were classified as probable cases.We identified three confirmed and five probable Nipah cases. There was a single index case. Five of the secondary cases came in close physical contact to the index case when she was ill. Case-patients were more likely to have physical contact with the index case . The index case, on her third day of illness, and all the subsequent cases attended the same religious gathering. For three probable cases including the index case, we could not identify any known risk factors for Nipah infection such as physical contact with Nipah case-patients, consumption of raw date palm juice, or contact with sick animals or fruit bats.Though person-to-person transmission remains an important mode of transmission for Nipah infection, we could not confirm the source of infection for three of the probable Nipah case-patients. Continued surveillance and outbreak investigations will help better understand the transmission of Nipah virus and develop preventive strategies. Pteropus fruit bats, in to humans are though drinking of raw date palm sap contaminated by bats, contact with infected animals and possibly through direct contact with bat secretionAmong the 122 Nipah cases identified between 2001 to 2007 in Bangladesh, 87 (71%) died and 62 (51%) developed illness following person to person transmission Nipah virus has been isolated from human saliva, urine, nasal and pharyngeal secretions In April 2007, a joint investigation team formed from the Institute of Epidemiology, Disease Control and Research (IEDCR) and the International Centre for Diarrhoeal Disease Research, Bangladesh  investigated a cluster of fatal encephalitis in a village of Sadar Upazila (sub-district) of Kushtia District. The objectives of the investigation were to identify the cause of the outbreak and the risk factors for development of illness.We defined suspect case patients as persons having fever with headache and/or cough or persons having fever with new onset of altered mental status or seizures residing in the outbreak area with an onset of illness during March and April, 2007. We identified suspect case-patients by collecting information from the local health workers in the community and by asking community residents if they were aware of anyone meeting the suspect case-definition in the affected community. We also investigated all deaths in that community in that time period. We asked family members of the decedent, if the decedent had symptoms compatible with the suspect case definition prior to death. We used structured questionnaires to record history of illness and general information about exposures for each suspect case-patient. The team requested the local health authority in the outbreak area to report to the IEDCR if any case-patient with fever and altered mental status came to the local health facility for treatment.The team collected blood samples from living suspect case-patients. The samples were centrifuged in the field then transported on wet ice to the laboratory at IEDCR where they were stored at \u221270\u00b0C. We tested the samples at IEDCR with an immunoglobulin M (IgM) capture enzyme immunoassay that detects Nipah IgM antibodiesA team of two Bangladeshi anthropologists conducted in-depth interviews with living confirmed and probable case-patients and with family members who cared for cases, including the deceased case-patients. They also conducted informal discussions with neighbors of case-patients with the objective of exploring possible modes of transmission of disease.The field team enrolled confirmed and probable case-patients in a case-control study to investigate exposures associated with Nipah infection, including person-to-person transmission. We selected three neighborhood controls for each case-patient, starting from the fourth closest courtyard to the case-patient to reduce over matching of demographic characteristics of neighboring household, confirming that no members in the households of that courtyard were ill during the outbreak period. Each courtyard had two to three households and we selected only one household from each courtyard which was closest to case-patient's household. The household resident closest in age to the case-patient was eligible to participate as a control. We selected controls closets in age to the cases not to control for confounding effect of age but to provide interviewers with easy guidelines to select controls. The field team selected only one control from each courtyard. This process was repeated at the next closest household until we enrolled three controls for each case.We used proxy respondents for each case-patient who was too sick to answer, or who had died. Proxy respondents included family members and friends who were aware of the case-patients activities and probable risk exposures in the 30 days preceding their illness. Multiple proxy respondents were used to ensure maximum reliability of the information as friends or colleagues might be more informed about possible exposures outside the home whereas family members were likely to be better informed about domestic exposures. The field investigation team used a standardized structured questionnaire to collect information on demographics, signs and symptoms of illness and possible risk factors for Nipah transmission such as: exposure to ill patients, including touching, staying in the same room, feeding, sharing a bed or cleaning body secretions; exposure to sick animals in the surrounding area; history of climbing trees; sighting bats near area of residence or working place, contact with bats, eating fruits picked from the ground; and drinking raw date palm sap.We used descriptive statistics to analyze the socio-demographic and clinical profiles of case-patients. For the case-control study, we used an unmatched analysis because neighbors were chosen as controls to ensure that controls and case-patients were representative of the same population, not to control for confounding factors. We enrolled persons closest in age as controls, not to control confounding by age, but rather to provide simple standardized guidelines to the interviewers for control selection. We used odds ratios (OR) to estimate the association of each exposure with disease and calculated 95% confidence limits (CI) around the odds ratio. To assess that observations were not only due to chance, we used the chi square test when expected cells were \u22655 and the Fisher exact test when expected cell sizes were <5. We considered any association to be statistically significant if the p value was <0.05. To test the hypothesis that Nipah virus was transmitted from the index case to other case- patients, we excluded the index case in all the analyses of person-to-person transmission.All human study participants gave informed verbal consent for participation in this investigation. The Ethical Review Committee at ICDDR, B reviewed and approved a protocol for encephalitis surveillance and outbreak investigation. As the study was part of an emergency outbreak investigation, approval from an Institutional Review Board was not required.The team collected 13 serum samples from 19 suspect case-patients. Seven of the serum samples were collected within less than seven days of illness onset. Three of the four serum samples that were collected after 10 days of illness onset had detectable IgM and IgG Nipah virus antibodies and were identified as confirmed cases. Five (26%) of the 19 suspected case-patients, who resided in the same outbreak village and died during the outbreak period but before samples could be collected, were categorized as probable cases. We also identified one additional death in the outbreak area but the decedent had chronic abdominal problem and had severe abdominal pain before death. He was not included into the study as the deceased did not have symptoms compatible with the case-definition.All the cases were clustered in time. The index case developed illness on March 17, 2007, followed by a single secondary wave of illness 12\u201316 days later . The meaProxies reported that the index case, Patient A , developed illness on March 17, 2007 and died on the sixth day of illness on March 22, 2007. On the first two days of illness Patient A developed fever, headache, cough, muscle pain and joint pain which progressed to restlessness, difficulty in breathing, vomiting, altered mental status, spitting of blood tinged saliva and loss of consciousness over the last four days of illness. Patient A reportedly did not have any exposure to identified risk factors for Nipah infection, including drinking raw date palm sap or partially bat eaten fruits, contact with other Nipah patients, climbing trees or contact with bats or sick animals. Five of Patient A's caregivers  subsequently developed Nipah compatible illness.Patient B (confirmed case), a friend, took care of Patient A during the entire illness episode; she slept with her at night, carried her to the doctor, cooked food for her, doused her head with water several times, cleaned her vomit, bathed her and shared rice with her from the same plate. Patient B developed Nipah illness 8 days after Patient A died (duration of exposure to illness onset 8\u201313 days). Patient C (probable case), another friend, also took care of Patient A and slept with her during the last three days of her illness; she developed illness within 11 days of contact with Patient A (duration of exposure to illness onset 9\u201311 days). Patient C died after seven days of illness. Patient D (confirmed case), a neighbor of Patient A, massaged the index case's head with oil and cleaned her oral secretions during her last two days of illness when she developed breathing difficulty, altered mental status and began spitting. Patient D became ill within 12 days of contact with Patient A. Another neighbor of Patient A, Patient E (confirmed case), reported that she only touched the hand of the index case once, to check for fever, and contracted the illness 12 days after illness onset and seven days after death of the index case. We do not know on which day of illness or how many times she visited Patient A during her illness episode. Patient F (probable case), Patient A's brother-in-law, only came in physical contact with Patient A on the third day of her illness. He became sick 11 days after contact with the index case and died after six days of illness. None of these five subsequent cases (Patients B-F) had exposure to any other Nipah patient or patient with fever and altered mental status other than Patient A. They also did not have any history of drinking raw date palm sap or coming in contact with any bat or other sick animal.The grave of a Muslim religious leader, who died in 2001, is in the outbreak village and his followers from different villages gather annually for an anniversary ceremony. On the third day of the index case's illness, on March 19, 2007, this religious ceremony was in the courtyard adjacent to the grave of the religious leader. Although the index case was quite ill at this time, with a severe cough and a varying level of consciousness, she still attended the ceremony. All the other seven case-patients (case-patients B to H) also took part in the ceremony. During the ceremony, the index case lost consciousness. A group of people, including Patient B, C, and D along with other unidentified women, carried her back to her home. We could not confirm if any other men apart from Patient D helped in carrying her home.Proxy respondents of the two probable case-patients, Patients G and H, who were present at the religious gathering, did not recall observing any physical contact between Patient G or H and the index case in the religious gathering. Proxy respondents also did not report any contact with other Nipah cases, contact with anyone with fever and altered mental status or seizures or contact with sick or dead animals prior to their illness onset. They also did not have any history of possible exposure to environmental risk factors for Nipah infection. Patient G and H both developed illness 11 and 14 days after attending the religious ceremony. Patient G first developed fever and headache with generalized weakness. On the third day of illness she developed severe respiratory distress and died on the same day. Patient H reportedly only developed fever, headache, restlessness and severe weakness. After one day of illness he was taken to a hospital where he died before admission. However, the manager of the place where Patient H worked reported that four days before his death he had borrowed some money saying that he was not feeling well and needed to seek medical care, but the manager did not know the details of his illness.We enrolled the three confirmed and five probable cases and three unmatched controls for each case-patient in a case-control study. We used proxy respondents for six case-patients (6/8). Case-patients and controls were similar in age  and sex .The only risk factor associated with acquiring Nipah infection was having physical contact  with the index case during illness  in the bivariate analysis. Though we identified two bat roosts within three and six kilometers from the outbreak area, there were no significant associations between illness and drinking raw date palm sap, climbing trees, sighting bat in the area of residence or working place, eating fruits picked from the ground or having contact with bats or sick animals .The findings of this outbreak investigation, specifically the epidemic curve with a single secondary peak of illness supported by a strong association between contact with index case and illness provide compelling evidence for person-to-person transmission of Nipah infection from the index case to the subsequent cases. In this outbreak, five of the subsequent case patients were directly involved in caring for the index case when she was sick. The five subsequent cases developed illness within 8\u201313 days of contact with the index case which is consistent with the incubation period of human Nipah virus infection observed in other settings A unique finding and a major limitation of this investigation is that the index cases and patient G and H did not have definite history of identified risk factors. These three case-patients did not have any history of consuming raw date palm sap within one month prior to death which has been implicated as a possible route of transmission of Nipah virus directly from its natural reservoir in Bangladesh Continued surveillance and outbreak investigations will help in better understanding of transmission of Nipah virus from bats to humans and then from humans to humans. Behavior change communication promoting feasible steps, to avoid unprotected contact with respiratory and oral secretions, such as adequate hand washing or the use of respiratory barriers, while caring for patients with respiratory symptoms both at family and hospital levels could minimize spread of the disease by limiting person-to-person transmission."}
+{"text": "Eight outbreaks of paralytic polio attributable to circulating vaccine-derived poliovirus (cVDPV) have highlighted the risks associated with oral poliovirus vaccine (OPV) use in areas of low vaccination coverage and poor hygiene. As the Polio Eradication Initiative enters its final stages, it is important to consider the extent to which these viruses spread under different conditions, so that appropriate strategies can be devised to prevent or respond to future cVDPV outbreaks.This paper examines epidemiological , and virological  parameters in order to infer the numbers of individuals likely to have been infected in each of these cVDPV outbreaks, and in association with single acute flaccid paralysis (AFP) cases attributable to VDPVs. Although only 114 virologically-confirmed paralytic cases were identified in the eight cVDPV outbreaks, it is likely that a minimum of hundreds of thousands, and more likely several million individuals were infected during these events, and that many thousands more have been infected by VDPV lineages within outbreaks which have escaped detection.Our estimates of the extent of cVDPV circulation suggest widespread transmission in some countries, as might be expected from endemic wild poliovirus transmission in these same settings. These methods for inferring extent of infection will be useful in the context of identifying future surveillance needs, planning for OPV cessation and preparing outbreak response plans. Much of the success of the Polio Eradication Initiative (PEI) to date can be attributed to massive use of oral polio vaccine (OPV), administered through routine immunisation services and supplementary immunisation activities in the form of National Immunisation Days (NIDs) and subnational immunisation days (sNIDs) in over 100 countries since 1988. Though it is a powerful tool for preventing poliomyelitis, OPV has two disadvantages. In addition to carrying a low risk of vaccine-associated paralytic poliomyelitis (VAPP) among vaccinees or their close contacts, it is now known that vaccine viruses can be serially transmitted through human hosts, and may revert genetically towards wild-type transmissibility and virulence.c is used to denote \u201ccirculating\u201d , and i for VDPV excreted by immunodeficient individualsa (for \u201cambiguous\u201d) is used to describe VDPV isolates from persons with no known immunodeficiency or environmental isolates whose ultimate source has not been identifiedThe degree of genetic change in vaccine-derived polioviruses (VDPV) is routinely assessed by determining the number of nucleotide substitutions in the VP1 gene, relative to the sequence of the Sabin vaccine viruses, and typically occurs at a rate of approximately 1% per annumEight outbreaks attributable to cVDPV have so far been fully documented: in Hispaniola, Indonesia, Egypt, Madagascar (\u00d72), Philippines, China and Cambodia, resulting in a total of 114 virologically-confirmed (and an unknown number of undiagnosed or unreported) casesSpread of VDPV infection is likely to be a function of virus transmissibility, population immunity levels, and other population characteristics such as density, socio-economic status, sanitation levels and hygiene-related behaviour, ascertainment efficiency of infection and/or cases, the nature and scope of response activities, and chance. Both the transmissibility and virulence (as measured by the case-to-infection ratio) of VDPVs are difficult to define, as they change over time. These properties take on particular importance as the PEI approaches its final stages.As part of the plan to discontinue OPV after wild poliovirus (WPV) eradication, there is discussion of vaccine stockpiles, and response strategies to manage any polio outbreaks in the \u201cpost-certification\u201d eraThere has been little discussion of the geographic spread of cVDPV outbreak strains, or of the numbers of infections associated with these and other reported VDPV episodes. In this paper, we develop lines of argument to infer the extent of VDPV spread in outbreak populations. We apply the reasoning to data from three of the largest reported cVDPV outbreaks  in order to give crude estimates of the potential numbers of individuals infected with VDPV in each of these settings. We then summarise findings for five smaller cVDPV outbreaks (Madagascar (\u00d72), Philippines, China and Cambodia), and discuss individual VDPV isolates, including iVDPV, and their implications for vaccine-derived virus spread.The data used here come from published reports of eight documented cVDPV outbreaks, shown in The further apart that cases or  isolates are identified in space and time, then the further and longer the implied extent and duration of virus circulation. The total duration of an outbreak, both before and after the initiation of response activities, is also indicative of the extent of infection spread.Given that virus excretion starts within hours or days after infection, and usually lasts 4\u20136 weeksA low prevalence of immunity to poliovirus infection (i.e. enteric immunity) is likely to encourage cVDPV emergence and spread. The degree of \u201cnaturally-induced\u201d immunity can be inferred from the time since the corresponding wild serotype was eliminated, while vaccine-induced immunity can be inferred from the type and extent of past vaccination coverage. Vaccine-induced immunity is complicated however, since not all vaccinated individuals become immune (or seropositive), responses differ by type, and some unvaccinated individuals are immunised by secondary transmission of vaccine virus. Prevalence of immunity to type 2 (at least as reflected by seropositivity) in any age cohort is likely to be equal to or higher than the proportion who have received three routine doses or two doses during campaigns. Immunity (seropositivity) to types 1 and 3 is likely to be lower than the uptake percentages, and to vary considerably between populations, as there is evidence for per dose efficacy as low as 10\u201320% in some parts of India The maximum number of individuals who could be infected during a cVDPV outbreak is the number susceptible during the period that the virus circulates.The age distribution of cases reflects the distribution of immunity in a population at the start of an outbreak, with a higher average age of cases being consistent with a lower crude prevalence of infection immunity, and thus with more extensive (and more rapid) virus spread.The relationship between the average age of cases (and infected contacts), and the extent of spread is complicated. The case-to-infection ratio of wild poliovirus is age-dependent, with a higher number of infections per case among younger personsThe greater the delay between illness onset in the index case, and the initiation of response activities, the more widespread virus circulation is likely to be. Similarly, extensive and high-quality responses are consistent with shorter transmission duration and reduced geographic spread compared to circumstances in which the outbreak responses are of poorer quality.In a polio-free area, the minimum annual expected AFP rate is at least 1 per 100,000 population aged under 15 years. Data on the completeness of AFP reporting and case investigation (including the proportion of AFP cases from whom two adequate stool specimens are obtained) are collected in many countries.Data on the likely efficiency of AFP case detection prior to and during an outbreak can be used to infer what proportion of cases may have been missed. The extent and results of additional surveillance activities conducted during VDPV circulation, including environmental sampling, community stool surveys, and (retrospective and active) case searches provide information on the extent of VDPV transmission in the community. Extensive sampling with only negative results suggests limited transmission, whereas poor surveillance is at least consistent with widespread infection transmission.The extent of VDPV spread will vary between populations. Cases from disparate social groups suggest wider circulation than those occurring within the same population subgroup. Population mobility will influence the geographic extent of virus spread.cVDPV isolates identified in crowded populations and/or those with poor hygiene suggest more extensive transmission compared with those found among socially privileged populations. Similarly, populations with a high annual growth rate  will be more prone to extensive VDPV circulation in the absence of high and continuous levels of vaccination coverage, due to the relatively high proportion of new susceptibles being introduced into the population.Polioviruses evolve at an annual rate of approximately 1% of nucleotide substitutions per site in the VP1 geneThe extent of genetic diversity among isolates provides information on the patterns of poliovirus transmission. In areas with sensitive surveillance, low genetic diversity among contemporary isolates is consistent with narrow chains of transmission with limited branching, whereas high diversity follows early and potentially extensive branching of the chains of transmission. High diversity implies prolonged circulation in populations with large immunity gaps.. In terms of virulence, the case-to-infection ratios for wild viruses appear to be greatest for type 1 (\u223c1\u2236200) and lowest for types 2 and 3 (\u223c1\u22361000)OPV viruses and their wild parent strains vary by type in their relative transmissibility and virulence. For OPV, transmissibility and virulence are in the order of 2>1,3The estimated occurrence of 0.36\u20131.92 cases of VAPP per million initial doses of OPV administered The first recognized cVDPV outbreak occurred on Hispaniola resulting in 21 VC and 21 polio-compatible (PC) cases reported in 2000\u20131Although fewer cases were reported in Haiti (8 VC and 4 PC) than in the Dominican Republic (DR) (13 VC and 17 PC), the Haiti cases occurred over a longer duration of time (1 year versus 6 months) suggesting that transmission there was more widespread. Cases were reported from a greater number of departments in the DR than in Haiti (5 versus 3), but this may reflect poor case ascertainment in Haiti, rather than more localized spread of infection.The last polio case associated with wild poliovirus was in 1985 in the DR and in 1989 in Haiti, suggesting that \u201cnaturally-induced\u201d immunity to infection was restricted to adults over 15 and 11 years of age in the two countries respectively.NIDs were conducted in DR between 1983 and 1995 and reported routine OPV3 coverage was \u223c80% nationally for over a decade prior to the outbreak . This implies that by 2000, susceptibles were concentrated among the under fives , of whom approximately (1\u22120.8)\u00d71,200,000\u200a=\u200a240,000 children <5 years old were unvaccinated when the outbreak occurred. It is likely that at least 200,000 were susceptible to infection.In Haiti, the prevalence of immunity was probably much lower, with reported national OPV3 coverage fluctuating between 30% and 58% between 1989 and 1999 (but as low as 7% in some communities), and no history of NIDs. This is consistent with up to 70% of children <11 years of age being susceptible to infection when the outbreak occurred, or around 1,600,000 children .The median age of the virologically-confirmed cases was higher in Haiti than in the DR (7 years [range: 2\u201312 years] versus 3 years [range:9 months\u201314 years]), reflecting differences in the age distribution of immunity to infection between the two countries. This age difference is consistent with more extensive infection spread in Haiti relative to the DR, both because a greater proportion of the population was susceptible to infection, and also because a broader age range of infected people is likely to correspond to a broader range in contact patterns between infected and susceptible individuals.In the DR, three OPV campaigns were initiated: 5, 7 and 9 months after paralysis onset in the index case see , resultiand that adequate stool samples were taken was therefore only around 0.1 in the DR (0.3\u00d70.3) and close to 0 in Haiti. As such, it is highly unlikely that the index cases were the first infected individuals in either country. However, the fact that the virus in the DR index case appears close to ancestral to all subsequent DR viruses is consistent with its being among the earlier infected individuals in that country. No AFP cases were identified retrospectively.AFP surveillance performance indicators declined prior to the outbreak. By 1999, the non-polio AFP rate per 100,000 population aged under 15 years was 0.3 in the DR and 0.12 in Haiti, with adequate stool samples taken from only 30% of AFP cases in the DR and from 0% in Haiti. The probability that a non-polio AFP case was reported From 12 July 2000 to 31 July 2001, 123 AFP cases were reported from the DR, from which 13 (11%) had type 1 cVDPV isolated. Only 33 AFP cases were investigated during approximately the same period in Haiti, of which 8 (24%) were associated with cVDPV. This corresponds to an annual non-polio AFP rate of 3.8/100,000 in the DR, and \u223c0.9/100,000 in Haiti. The difference in the AFP rate in each country and in the proportion attributed to cVDPV is further evidence that more cases went unreported in Haiti than in the DR. The poorer AFP ascertainment in Haiti compared to the DR is consistent with more widespread transmission on this part of the island.Sampling among healthy contacts of AFP cases was conducted in both countries, with 2/36 contacts positive for type 1 cVDPV in Haiti and 10/205 positive in the DR. Environmental sampling was conducted at the peak of the outbreak from sewage, canals, and public latrines in several sites, with 2/12 (17%) samples from Haiti and 5/43 (11%) from the DR positive for type 1 cVDPVUp to five million Haitians (55% of the population) live in abject poverty, over four million are without access to safe water and up to three million are slum-dwellersCharacterisation of isolates from the first two reported cases showed that the outbreak strain had 97.4% and 98.1% sequence identity to Sabin 1, suggesting that the initiating OPV dose occurred in late 1998 or early 1999, 18 months before illness onset in the index caseSequence analysis of the outbreak isolates suggests that the initiating dose occurred in Haiti, and was followed by repeated divergence of the virus lineagesThe outbreak strain was derived from Sabin 1, which is the least virulent of the Sabin strains, and is less transmissible than Sabin 2The first two outbreak isolates were shown to be highly neurovirulent in a transgenic mouse model, and replicated to high titres at 39.5\u00b0C in cultured cellsVDPV infection appears to have covered a broader area and infected many more people in Haiti than in the DR due to the lower levels of population immunity, poorer sanitation and hygiene, and a slower response to the outbreak on this part of the island. The virological evidence suggests that the virus circulated for over two years in Haiti, and for around nine months in the DR.In Haiti, response, surveillance and virological data suggest that case ascertainment may have been as low as 20%, which is also consistent with fewer reported cases over a longer time period. A case-to-infection ratio of 1\u2236200 would suggest that around 12,000 infections (i.e. 12\u00d75\u00d7200\u200a=\u200a12 cases\u00d7under-reporting factor\u00d7case-to-infection ratio), occurred in the outbreak period alone. A case-to-infection ratio of 1\u22361000 would imply 60,000 infections. Since the initiating dose was given in Haiti around 20 months prior to the onset of paralysis in the index case, in a population containing over one and a half million susceptibles under 10 years old, it is likely that the total number of infections was at least 100,000.Thirty polio cases were reported from the DR between July 2000 and January 2001, with surveillance indicators and virological data suggesting that case ascertainment was probably around 50%. If the cVDPV had regained a high degree of virulence (1\u2236200 case-to-infection ratio) for the entire duration of its circulation, then this would imply that around 12,000 infections occurred during this period. It is likely that the average case-to-infection ratio of the cVDPV was lower than 1\u2236200, and that case ascertainment was lower than 50% in the early stages of the outbreak (given the results of the retrospective case search and surveillance indicators for 1999). If case ascertainment had been 40% and the average case-to-infection ratio in the order of 1\u22361000, then the total number of infections in the DR would have been closer to 75,000.In June 2005, a type 1 cVDPV outbreak began in Indonesia, resulting in 46 VC cases45/46 virologically-confirmed cVDPV cases occurred between June and October 2005 on Madura island, while one case occurred on the neighbouring island of Java. VDPV cases occurred in all four of Madura's districts (and in 16/68 sub-districts), with several distinct clusters occurring in the rural northern areas of three of the districts , indicatPrior to the co-circulating outbreaks of WPV and VDPV in 2005, the last case associated with WPV was reported from Indonesia in 1995, suggesting that naturally-induced immunity was restricted to individuals >10 years old in 2005. NIDs were last conducted in 2001, following which polio immunisation was restricted to routine vaccination. Data on vaccination coverage from convenience samples and non-polio AFP cases suggest that immunisation levels were particularly low in Madura, with over 50% of those <5 years of age having received less than 3 doses of OPV.With approximately 1.2 million children under 10 years old on Madura island, it is likely that over half a million were susceptible to infection during the period of cVDPV emergence. Within Indonesia, the number of susceptible children under 10 years old is likely to have been over 10 million by 2005.The median age of the 45 Madura cVDPV cases was 2 years (range: 6 months to 14 years). 56% (25) of cases were under 3 years old and 20% (9) were 5 years or over. The broad age distribution of the cases is consistent with absence of WPV circulation for over a decade and variable coverage by vaccination services over the same period, giving rise to the potential for extensive geographic cVDPV spread.Improved surveillance in Indonesia following the detection of WPV in May 2005 is likely to have accelerated the confirmation of the index case as cVDPV in August 2005, within two months of virus isolation.In response to the detection of WPV cases, NIDs were conducted in August, September, and November 2005 , with reAFP performance surveillance indicators for Madura prior to the recognition of the cVDPV outbreak were low, consistent with poor case identification and the potential for widespread virus circulation.and had adequate stool samples collected in the year prior to recognition of the index case.The non-polio AFP rate per 100,000 children <15 years of age was 0.4 per year in 2003 and 0.7 in 2004. Adequate stool samples were collected from 69% of reported cases in 2004, suggesting that only about half (0.70 * 0.69\u200a=\u200a0.48) of non-polio AFP cases were reported The non-polio AFP rate increased to 3.3 per 100,000<15 year olds during the first half of 2005 when the cVDPV cases were detected (4/8 reported AFP cases were confirmed as cVDPV during this period), consistent with poor case ascertainment before the recognised outbreak period. AFP surveillance performance was lowest in Sumenep district, which reported the fewest VDPV cases.Active searches for AFP cases with paralysis onset in the three months prior to the index case were conducted in neighborhoods of confirmed VDPV or WPV cases, and resulted in no further cases being identified.2, and came from poor, rural households, with low utilisation of health services, and suboptimal hygiene and sanitation practices, providing ideal conditions for virus spread.The majority of the Madura cases occurred in sub-districts with a population density of 500\u20131000 persons/kmCharacterisation of outbreak isolates demonstrated a sequence difference between the outbreak strain and Sabin 1 of 1.1\u20132.2%, suggesting cVDPV circulation for up to two years prior to detection of the index case, and implying 26\u201352 \u201cgenerations\u201d of transmission during this period.A phylogenetic analysis of the isolates showed four distinct lineages were represented in separate geographic clusters, which were recombinant with other species C enteroviruses. The heterogeneity among the isolates is consistent with extensive transmission of the outbreak strain.Type 1 is the least virulent of the Sabin strains, consistent with a large number of infections prior to identification of the index case.If the outbreak strain had fully regained a case-to-infection ratio consistent with wild type 1 when the cases occurred, and if case ascertainment was complete (46 VC and 10 PC cases), then the minimum number of infected individuals during the outbreak might have been as low as 10,000. A lower case-to-infection ratio of 1\u22361,000 would imply in the region of 50,000 to 60,000 infections during the outbreak period.These arguments indicate that the spread of VDPV infection in the Indonesian outbreak was likely to have been extensive on Madura Island, and limited on Java. Four distinct lineages of the outbreak VDPV circulated for up to two years (26\u201352 \u201cgenerations\u201d) before transmission was terminated, and were present in all districts in Madura among subgroups of the population who were poorly immunised, had not been exposed to WPV circulation for over a decade and who lived in conditions conducive to poliovirus spread. Ascertainment is unlikely to have been 100% due to the reported logistical difficulties encountered during the response activities and limited retrospective searches for AFP cases. The apparently later introduction, and relatively limited circulation of type 1 WPV on the same island, also suggests that immunity levels to type 1 may have been recently boosted by the circulating VDPV strain, although vaccination response activities would have limited WPV spread. Taken together, it is not unreasonable to estimate that at least 100,000 individuals were infected in Indonesia during the full period in which the VDPV circulated, with the vast majority residing on Madura.stored poliovirus isolates revealed that type 2 cVDPV circulated in Egypt between 1988 and 1993A retrospective analysis of The recognised cVDPV cases were from 7 of the 27 governorates of Egypt, with onset dates between 1988 and 1993 . The preThough the last reported wild type 2 polio case in Egypt was in 1979, the exact date of eradication of this serotype is unknown since AFP surveillance began only in 1990There were approximately 20 million children under 15, and around 1.25 million births per year, in Egypt throughout the 1980sAge information is no longer available on the cases from whom the isolates were obtained.There was no specific response to the outbreak in Egypt as it was ascertained retrospectively. Transmission is likely to have ceased in the mid-1990s following an increase in OPV coverageThe 30 confirmed cases are likely to represent only a fraction of those attributable to the cVDPV as polio surveillance is known to have been poor during the outbreak period. There was no routine AFP surveillance. The stored isolates represent cases visiting particular health facilities after 1988.Data on the social characteristics of the cases have not been reported, but both the living and sanitary conditions, and the population density (among the highest in the world) and frequent mixing patterns in Egypt favour poliovirus spread, as shown by the persistence of wild type 1 until early 2005.The outbreak isolates had between 93% and 97% nucleotide sequence identity to the Sabin type 2 strain, and a regression analysis of the VP1 evolution rate suggests that the lineages resulted from an OPV infection that occurred in about 1983 (95% CI: 07/79\u201303/86), as shown in Sequence analyses show a high degree of nucleotide diversity between the outbreak isolates, consistent with multiple, extensive chains of transmission and incomplete case ascertainmentThe Egypt outbreak was associated with type 2 which is the most transmissible of the Sabin strainsThe neurovirulence of two isolates from the outbreak was measured in transgenic mice and found to be similar to the prototype wild type 2 straincVDPV infection appears to have been much more extensive in Egypt than in the other known cVDPV outbreaks, with continued circulation for approximately 10 years, the occurrence of recognised cases over a broad area that comprised at least 50% of the total population, and evidence of multiple chains of transmission. Very high population density, particularly in slum areas in \u201cmega-cities\u201d and a high birth rate are likely to have contributed to extensive spread of infection. Virological evidence, along with poor case reporting before the 1990s, suggest that many hundreds of cases were probably associated with the outbreak. Taken together, the evidence indicates that the cVDPV strains became endemic in the population completely filling the niche vacated by the disappearance of indigenous wild type 2 viruses. It is likely that an appreciable proportion  of the approximately 12 million children born in Egypt 1983\u20131993 were infected with these type 2 cVDPV viruses.In addition to these 3 large cVDPV outbreaks, 5 further outbreaks of cVDPV have been documented. In addition to these multi-case outbreaks, there have been at least 35 isolations of VDPV strains which appear, because of VP1 divergence ranging from around 1\u20133%, to have circulated in populations for periods of \u223c1\u20133 years The long-term excretion of VDPV by some immunodeficient individuals (iVDPV) has been recognized since 1962Person-to-person transmission of VDPV infection for prolonged periods has been recognised in several settings, and has resulted in eight fully documented multi-case outbreaks of polio. In addition, over forty VDPVs have been isolated from individual AFP cases, healthy individuals, immunodeficient individuals and in environmental samples in other settings. The important risk factors for cVDPV emergence-low prevalence of immunity to poliovirus infection, low OPV coverage and poor sanitation-are likely to exist in populations where case ascertainment is incomplete. It is thus likely that the number of cVDPV cases recognised in these eight outbreaks falls well below the true number of cases, particularly in Egypt and Haiti, with the actual incidence of VDPV-attributable paralytic disease likely to have numbered in the many hundreds.The spread of VDPV infection was most extensive in Egypt, where several million individuals may have been infected by type 2 cVDPV which became endemic following the disappearance, of indigenous wild type 2 virus. In Hispaniola and Indonesia, at least 100,000 individuals are likely to have been infected in each setting, while up to 10,000 infections may have occurred in each of the five smaller outbreaks. The isolates from single AFP cases, healthy contacts and environmental samples identified in other episodes, are together likely to correspond to several thousand infections. A large type 2 VDPV outbreak is currently ongoing, with roughly 130 cases attributed to the independent emergence of seven cVDPV lineages in northern Nigeria Each of the eight \u201coutbreaks\u201d ended because of OPV intervention. In Egypt, this intervention occurred as part of the scale-up of routine OPV during the 1990s . Special NIDs were organised to control the seven other outbreaks.Reports of cVPDV outbreaks and references to other VDPV isolates have so far focused on the numbers of clinical cases involved in these episodes. Their full implications come to light when the underlying infection incidence is considered. Insofar as eradication of all poliomyelitis is the PEI target, this will require total cessation of all poliovirus transmission. To describe the problem of vaccine-derived polio as 114 virologically-confirmed cases, worldwide, over some twenty years, gives a very different impression than a description which suggests a minimum of hundreds of thousands, and more likely several million infections by vaccine-derived viruses, some of which became endemic in large populations. It is also possible that other vaccine-derived virus lineages have circulated for limited time periods, but failed to cause any clinical cases and were thus unrecognizedAlthough the methods developed here give wide-ranging estimates of the numbers infected during each episode, they nevertheless highlight the need to focus on the extent of infection spread in the event of future VDPV episodes. As such, these methods will become increasingly important for planning surveillance, control and response activities in the current and future stages of the eradication programme, as attention focuses towards reducing cases, and ultimately infections, to zero."}
+{"text": "Underreporting of Human Alveolar Echinococcosis, Germany We estimated the total number of human alveolar echinococcosis cases in Germany from 2003 through 2005 using the multiple source capture-recapture method. We found a 3-fold higher incidence of the disease than that shown by national surveillance data. We propose a revision of the reporting system to increase case ascertainment. Echinococcus multilocularis, is a rare zoonosis in Germany, mainly occurring in the south , caused by the metacestode stage of the fox tapeworm In 2001, AE reporting became mandatory in Germany  and that the study population be closed. If only 2 sources are used, these should be independent  conducted in June 2006 among all registered pathology laboratories in Germany (\u2248525). Pathologists were requested to complete a questionnaire reporting all echinococcosis cases diagnosed from 2003 through 2005 to RKI.>1 source, the first 3 digits of the case-patient\u2019s postal code or the referring physician\u2019s postal code had to be identical in addition to the above criteria.We defined confirmed AE case-patients as persons with positive results of histopathologic examination or with liver lesion showing typical morphologic features, identified by imaging techniques. Only case-patients with a first diagnosis from 2003 through 2005 were included. Because reporting of AE is anonymous, we used 3 proxy matching identifiers. Matching criteria were identical: 1) year and month of birth, 2) sex, and 3) year and month of diagnosis . For case-patients for whom month of birth or month of diagnosis was missing in The distribution of matched and unmatched observed cases by source is displayed in a Venn diagram . To predWe selected the final model using Akaike\u2019s Information Criterion (AIC), which indicates how well a model fits the data, considering the number of variables included. Small values of AIC correspond to a better adapted model (N) from the selected model. Analysis was performed with Stata 9.0  . Pathologists reported 49 AE cases in the survey, of which 25 were the first diagnosis, 5 were previously diagnosed, and 19 had no date of first diagnosis. N ranged from 184 to 399 cases (Log-linear estimates for 99 cases . Model 5We estimated that the national surveillance system failed to detect 67% of AE cases in Germany over 3 years. Underreporting may occur for several reasons. Pathologists might be unaware of their obligation to report. Furthermore, reports almost exclusively come from microbiologic laboratories, and, consequently, case-patients who do not undergo serologic testing, or who have seronegative results, are likely to be missed. Finally, the reporting procedure is arduous because forms are detailed and must be first ordered from RKI.Capture\u2013recapture estimates can be biased if the underlying assumptions are violated. Because case identification was based on several variables, the potential for mismatching was considered small. However, the lenient criteria may have led to overmatching. Including more or fewer matching criteria had only a small effect on the estimate.In the final models, we excluded cases reported through PAS when first-diagnosis status was unknown. Log-linear analysis that included these cases resulted in a higher estimate; therefore, we are confident that the exclusion did not overestimate the number of cases. Varying catchability can be addressed by stratification. Although the sources differed with regard to geographic distribution, we considered stratified analysis inappropriate due to missing postal codes for several case-patients, zero values in 1 stratum, and small numbers in general, which would increase the uncertainty around our estimate.AE is not equally distributed in Germany, and the different geographic distribution of cases reported by PAS compared with RKI and EER indicated that PAS had missed case-patients mainly from the south. The number of histopathologically diagnosed cases was therefore likely underestimated. The importance of this for the estimated true number of AE case-patients presented here cannot be ascertained.The negative dependence between RKI and PAS can be explained by diagnostic practices. Unpublished data from EER suggest that histopathologically diagnosed cases are less likely to have serologic test results than those without histopathologic examination. If a case-patient has had a histopathologic examination with positive results early in the diagnostic decision-making process, additional serologic testing is unnecessary, which reduces the chance of these case-patients being reported to RKI. Reporting to EER is independent of serologic testing, which could explain the greater overlap between EER and PAS than between RKI and PAS.Despite the limitations, the study did demonstrate poor reporting of AE. To improve the national surveillance system, the focus of reporting should be shifted from microbiologic laboratories and pathologists to referring physicians, who usually collate the various diagnostic results.Sustaining a surveillance system for AE in Germany is a major challenge because the disease is rare. However, a recent report on increasing human AE in neighboring Switzerland ("}
+{"text": "To the Editor: On November 22, 2006, an infection control nurse notified the Marion County (Oregon) Health Department about acute gastroenteritis among persons who had attended a reception at a medical facility on November 16, 2006. With a holiday weekend only hours away, the county health department asked the state health department to join the outbreak investigation.>3 loose stools within a 24-hour period) within 18\u201372 hours of the event.After interviewing the caterer, organizers, and several attendees, we modified a questionnaire template to reflect potential exposures. Using this questionnaire, we conducted a retrospective cohort study by telephone among reception attendees identified from a ticket list. We defined a case of acute gastroenteritis as reported vomiting or diarrhea (www.cdc.gov/epiinfo); and assessed the significance of the association between acute gastroenteritis and consumption of implicated oysters by the \u03c72 or Fisher exact test.Sanitarians inspected the facility and the caterer\u2019s kitchen. We traced implicated oysters (the source of the outbreak) through distribution records; screened stool specimens for norovirus by RT-PCR; tested oysters from the implicated lot for norovirus by qRT-PCR; entered data into a custom outbreak database template; calculated relative risks (RRs) and 95% confidence intervals (CIs) using Epi Info  had minor symptoms. The median incubation period was 36 hours (range 31\u201363 hours). None of the 10 attendees with acute gastroenteritis sought medical attention; stool specimens from 2 of them tested positive for norovirus (1 positive for genogroup II and 1 positive for both I and II).Illness was associated with consumption of raw oysters on the half shell , which was reported by 8 of the 10 attendees with acute gastroenteritis. No other foods were associated with illness. No significant breaches in food-handling procedures were identified. The only food handler who reported illness had eaten several oysters at the event and became ill 36 hours later.The oysters had been individually quick frozen on the half shell and packed loosely in cartons after being harvested in South Korea by growers approved by the US Food and Drug Administration. For the reception, a single 6-kg box of oysters was thawed and served raw. The box was from a shipment of 2,200 boxes legally imported in October 2006. Boxes from the same shipment had been distributed to 5 states. Oysters from 4 other cartons were consumed (some cooked) at 2 other Oregon locations. Public health officials in other states were notified and asked to report any related illnesses; none were identified.Noroviruses (genogroups I and II) were detected in oysters from an intact carton of the implicated lot. Sequencing was not attempted. The implicated lot was voluntarily recalled by the national distributor; most of the lot was embargoed or recalled before the oysters were consumed.Oysters are a recurrent source of outbreaks and sporadic cases of norovirus infection, vibriosis, and other infections  facilitated a quick and efficient response to the outbreak described here. Questionnaire design, interviews, data entry, and analysis were completed within 6 hours of the initial report, and distributors and regulatory agencies worked quickly to recall other oysters from the same source, thus probably preventing additional illnesses. We believe that widespread use of such templates would increase the number of outbreaks that could be investigated thoroughly.Because thorough outbreak investigations are time-consuming and gastroenteritis outbreaks are common, resource issues often affect decisions about how intensively to pursue investigations. Our use of integrated questionnaire, data entry, and analysis templates ("}
+{"text": "A widespread outbreak of tularemia in Sweden in 2000 was investigated in a case-control study in which 270 reported cases of tularemia were compared with 438 controls. The outbreak affected parts of Sweden where tularemia had hitherto been rare, and these \u201cemergent\u201d areas were compared with the disease-endemic areas. Multivariate regression analysis showed mosquito bites to be the main risk factor, with an odds ratio (OR) of 8.8. Other risk factors were owning a cat (OR 2.5) and farm work (OR 3.2). Farming was a risk factor only in the disease-endemic area. Swollen lymph nodes and wound infections were more common in the emergent area, while pneumonia was more common in the disease-endemic area. Mosquito bites appear to be important in transmission of tularemia. The association between cat ownership and disease merits further investigation. Tularemia is caused by Francisella tularensis, a fastidious, gram-negative rod. F. tularensis subsp. tularensis, or type A, occurs mainly in North America and is more virulent than F. tularensis subsp. holarctica, or type B, which occurs throughout the Northern Hemisphere. Type A is usually transmitted to humans by tick bites or contact with rabbits; type B is associated with water and animals living near water, and its transmission seems more complex \u20136.In Sweden, >6,000 human cases of tularemia have been reported since the disease was first described in 1931. However, incidence varies greatly from year to year, ranging from a few cases in some years to >2,700 cases in 1967. The ulceroglandular form of tularemia is by far the most common in Sweden, except for an outbreak in the winter of 1966\u201367, when a large proportion of pulmonary tularemia cases occurred in farmers who processed hay contaminated by dead, infected voles Most cases occur within a relatively small area in the central part of Sweden, with only sporadic cases in other areas. In recent years, however, the disease seems to have spread to areas south of the disease-endemic area. This shift was apparent in the 2000 outbreak, when 187 (40%) of 464 cases were reported to have been transmitted south of the disease-endemic area. The reason for this spread is unknown.We studied the risk factors for acquiring tularemia in Sweden, as well as the prevalence of the risk factors in the disease-endemic and the new, \u201cemergent\u201d areas during the outbreak of 2000. We performed a matched case-control study, using a modified questionnaire designed by a Finnish group that was studying a concurrent tularemia outbreak in Finland.Tularemia has been a notifiable disease under the Communicable Diseases Act in Sweden since 1968. Physicians who diagnose a case, either clinically or by microbiologic means, report to the county medical officer and the Swedish Institute for Infectious Disease Control (SMI). Cases were defined as tularemia in all persons ages >18 whose illness was reported to SMI from August 1 (week number 31) to November 21, 2000 (week number 47) and who resided in any of seven counties in central Sweden, representing both disease-endemic and emergent areas . The casControls matched for age, sex, and place of residence were drawn from the computerized Swedish National Population Register, in which the name, date of birth, personal identifying number, and address of all citizens and residents are stored. Matching for place of residence was done on the first three digits of the five-digit postal code, since this three-digit area corresponds to a small town, village, or municipality. We chose controls whose date of birth was as close as possible to that of the patient, with a difference <12 months. Two controls were selected for each case. A questionnaire was mailed to the two controls; if neither responded within 2 weeks, a third control was chosen in the same way. If a control reported having had fever during the period of investigation and no diagnosis other than tularemia was made, he or she was excluded from the analysis.On the standardized questionnaire mailed to both case-patients and controls were questions on whether they had had an elevated temperature during the 4-week period 2 weeks before and 2 weeks after onset of symptoms in the patient. Additional data were collected on symptoms, medication, and referral to hospital. Participants were also asked about number of persons in the household and symptoms in other household members during the defined period.The second part of the questionnaire contained questions on exposure to presumed risk factors for acquiring tularemia during the 4-week period preceding the reported day of onset of illness. The following exposures were recorded: owning cats, dogs, or other animals; visiting golf courses and forests; participating in farming procedures of different kinds; having contact with dead animals, with or without wearing gloves; visiting or swimming in lakes or rivers; drinking water from lakes or wells; picking berries or mushrooms; and being bitten by mosquitoes, mites, ticks, deer flies, or other insects. Respondents were also asked if they had used insect repellents. Visits to areas other than place of residence during the defined period of exposure were also recorded.In the 2000 epidemic, cases occurred in places where tularemia had rarely been reported. To evaluate whether this spread was connected to any new risk factors, the seven counties in the study area were divided into three categories : the disThe endemic group consisted of 84 case-patients and 159 controls, a total of 243 persons, while 300 persons were in the emergent group (105 case-patients and 195 controls). We chose to analyze these two groups separately for symptoms and exposures as above, but to improve the discrimination between the old and new tularemia areas, we excluded the third group, consisting of 29 case-patients and 60 controls from the border area, from this part of the analysis.A matched univariate analysis, with calculation of odds ratios (OR) and 95% confidence intervals (CI) by the Mantel-Haenszel method, was done in EpiInfo 6.04  and the Stata program . Multivariable analysis was done by conditional logistic regression for matched data in the Stata program.Two hundred seventy cases fulfilled the above criteria. All the case-patients were ill during the summer or in the early autumn. No cases were reported during 2000 before the start of the study period on August 1. Of cases, 86% were confirmed with serologic testing or culture. The questionnaire was sent to all 270 case-patients and 670 controls. Replies were received from 243 (90%) of 270 case-patients and 438 (65%) of 670 controls, for a total of 681 (72%) of 940. Twenty-five cases and 21 controls were excluded because of confusion about the time periods used in the questionnaires. One control was excluded because of a documented episode of tularemia, one control was excluded because of an episode of febrile illness that could have been tularemia, and another control was excluded because the wrong person filled out the questionnaire. Thus, 218 cases and 414 controls, from which 202 matched pairs or triplets could be arranged, remained for further analysis.With regard to symptoms, 198 (95%) of 209 case-patients reported having had a fever during their episode of tularemia. Of these, 147 (86%) of 171 reported swollen lymph nodes, 143 (79%) of 180 wound infection, 26 (20%) of 132 sore throat, 39 (28%) of 138 cough, and 10 (5.2 %) of 193 pneumonia diagnosed by a physician. As a measure of severity of disease, 34 (16%) of 212 were hospitalized. The median duration of hospitalization was 4 days. No deaths were reported.The matched univariate analysis  gave staSex and age distributions were similar in the endemic and emergent groups. Having had swollen lymph nodes (47 [78%] of 60 vs. 85 [96%] of 89 [p=0.001]) or a wound infection (48 [72%] of 67 vs. 78 [89%] of 88 [p=0.007]) was significantly more common in the emergent group, while having had pneumonia (7 [9.3%] of 75 vs. 1 [1.1%] of 94 [p=0.02]), was significantly more common in the endemic group.The matched univariate analysis in the endemic group showed significant results for the following exposures: owning a cat, farming, and being bitten by mosquitoes. In the emergent group, being bitten by mosquitoes and visiting woods or forests appeared as risk factors . After mWe report results from a study of a tularemia outbreak in Sweden, with a spread of the disease into new geographic areas. The use of a mailed questionnaire with matched controls from the Population Register had a high response rate even among controls.Frequency of reported symptoms from case-patients are consistent with earlier data, showing ulceroglandular tularemia to be the dominant form in Sweden Statistically significant independent associations were found between acquiring tularemia and the following exposures: being bitten by a mosquito, doing farm work, and owning a cat. The results for mosquito bites could have been influenced by recall bias, since this transmission route has always been thought to be the most common in Sweden and many patients might thus have been told by their physician that mosquitoes caused the infection. However, the predominance of the ulceroglandular form of tularemia and the seasonal variation of the disease support the theory that mosquito bites are the major route of transmission in Sweden.Farming has not been connected with tularemia in Sweden since the outbreak in 1966\u201367. However, about pulmonary tularemia was reported among farmers in Finland in 1982 Transmission of tularemia from cat to humans, mainly caused by F. tularensis subsp. tularensis, but also by F. tularensis subsp. holarctica, has been described from North America, from both sick and healthy cats, and with or without the patient\u2019s being bitten by the cat \u201317. Furt The risk for acquiring tularemia, however, is relatively small even in the disease-endemic areas, where the overall incidence in this outbreak was approximately 66 per 100,000 population. On the basis of these findings, recommendations to the population in general about not owning a cat therefore seem unwarranted. However, informing the public about the risk of spread of tularemia from cats to humans seems reasonable.In the separate analysis of risk factors, owning a cat and being bitten by mosquitoes appeared as independent risk factors in both groups. Farming appeared as a risk factor only in the disease-endemic area. The reason for this difference remains unknown and merits investigation. One explanation could be different farming practices, since the disease-endemic areas are heavily forested with small plots of arable land interspersed, but the emergent areas have more open, continuous farmland. No new risk factors were found in the emergent group that could explain the spread of tularemia into new areas. However, mosquito bites and spending time in the forest play a relatively bigger role in the emergent areas, but farming and contact with cats were relatively more important in the disease-endemic area  and 2. TThis study has elucidated some of the basic epidemiology of human tularemia in Sweden. More research is needed on the epidemiology of the disease in animals. Important questions that remain unanswered are\u2014What is the reservoir (or reservoirs)? What is the interaction between infection in different wild species? And What triggers an outbreak in humans? A follow-up field study with collection of samples from mosquitoes, water, and rodents is planned."}
+{"text": "BAFF and APRIL share two receptors \u2013 TACI and BCMA \u2013 and BAFF binds to a third receptor, BAFF-R. Increased expression of BAFF and APRIL is noted in hematological malignancies. BAFF and APRIL are essential for the survival of normal and malignant B lymphocytes, and altered expression of BAFF or APRIL or of their receptors  have been reported in various B-cell malignancies including B-cell non-Hodgkin's lymphoma, chronic lymphocytic leukemia, Hodgkin's lymphoma, multiple myeloma, and Waldenstrom's macroglobulinemia.BAFF, APRIL, TACI and BAFF-R gene expression in 40 human tumor types \u2013 brain, epithelial, lymphoid, germ cells \u2013 to that of their normal tissue counterparts using publicly available gene expression data, including the Oncomine Cancer Microarray database.We compared the expression of TACI in multiple myeloma and thyroid carcinoma and an association between TACI expression and prognosis in lymphoma. Furthermore, BAFF and APRIL are overexpressed in many cancers and we show that APRIL expression is associated with tumor progression. We also found overexpression of at least one proteoglycan with heparan sulfate chains (HS), which are coreceptors for APRIL and TACI, in tumors where APRIL is either overexpressed or is a prognostic factor. APRIL could induce survival or proliferation directly through HS proteoglycans.We found significant overexpression of Taken together, these data suggest that APRIL is a potential prognostic factor for a large array of malignancies. APRIL and BAFF are two members of the TNF family. BAFF is a type II transmembrane protein that can be secreted after proteolytical cleavage from the cell membrane,2. APRILEvidence has been presented that BAFF/APRIL contribute to malignancies of B cells and plasma cells: non-Hodgkin's lymphoma -16, Hodget al demonstrated that TACI binds also HS proteoglycans like syndecan-1, syndecan-2 and syndecan-4 [Recombinant APRIL binds to several cell lines that do not express detectable mRNA for TACI and BCMA and proteoglycans were identified as APRIL-specific binding partners. This binding is mediated by heparan sulfate (HS) side chains and can be inhibited by heparin,27. Bindndecan-4 .BAFF, APRIL and of their receptors \u2013 BAFF-R, BCMA, and TACI \u2013 in various cancers, compared to their normal tissue or cell counterparts and in association with disease staging.These data demonstrate that BAFF/APRIL are potent growth factors in B cell malignancies. Furthermore, APRIL could be implicated in tumor emergence and/or progression due to its ability to bind HS proteoglycans -28. Therhttp://www.oncomine.org[http://amazonia.montp.inserm.fr/[BAFF, APRIL, BCMA, TACI, BAFF-R and HS proteoglycans genes in 40 human tumor types and their normal tissue counterparts as indicated in Table 1 , in Burkitt lymphoma compared to normal B cells (P = 5.4E-5), in diffuse large B cell lymphoma compared to normal B cells (P = .01) and in follicular lymphoma compared to normal B cells (P = 2E-6)[BAFF overexpression in glioblastoma compared to normal brain  [in vitro produce high amounts of BAFF. BAFF is expressed in astrocytes in the normal human central nervous system and is strongly upregulated in activated astrocytes in the demyelinated lesions of multiple sclerosis[BAFF expression is readily induced in the central nervous system by neurotrophic viruses and correlates with the recruitment of antibody-secreting cells[BAFF was found significantly overexpressed in breast carcinoma compared to normal breast , in esophageal adenocarcinoma compared to normal esophagus , in clear cell renal cell carcinoma compared to normal kidney  and in adult male germ cell tumor compared to normal testis .40 tumor types were investigated, including 36 solid tumors and 4 hematological tumors . Four in= 0.008) -37 [P = 3.7E-4 and P = 5.5E-4)[P = 3.5E-4 and P = 0.006)[P = 9.2E-10 and P = 0.009)[P = 7.6E-6)[P = 0.002) and in adult male germ cell tumor compared to normal testis  ,45, in e= 5.5E-4),46, in g = 0.006),36, in h = 0.009),48, in d= 7.6E-6),50, in p= 7.6E-6); P = 0.0ola et al; P = 1.6et al[APRIL expression in tumor lesions correlates with B-cell lymphoma aggressiveness. We found here that BAFF or APRIL expression could be also associated with tumor aggressiveness in other cancers  and in patients presenting glioma, dead at 3 years compared to patients alive at 3 years (P = 1.8E-4)[APRIL is overexpressed in patients dead at 3 years compared to patients alive at 3 years (P = .005)[APRIL expression in two independent studies (P = .003 and P = .004)[APRIL compared to stage I (P = 0.003)[APRIL in patients with glioma or bladder carcinomas who were dead at 3 years compared to patients alive at 3 years. An explanation could be that a part of APRIL protein is quickly secreted and no more detectable by immunostaining. APRIL protein could also be used by tumor cells as a growth factor or released in the blood circulation.Recently, Schwaller et al demonstrs Figure . APRIL i= 1.8E-4) (AdditioP = .005). High tuP = .004),55. Stag = 0.003) . APRIL p = 0.003) and norm = 0.003). Interes = 0.003),58. In a = 0.003) suggesti = 0.003). NeverthBCMA and TACI expressions in MM since microarray data were validated by real time RT-PCR and flow cytometry[BCMA or BAFF-R expression between tumor cells and their normal counterparts could be found in the 40 cancer types available from Oncomine database. By contrast, a significant TACI overexpression was found in smoldering myeloma compared to normal plasma cells  and in thyroid carcinomas compared to normal thyroid   . Heparin could be also useful in blocking APRIL binding to surface HS proteoglycan. Of note, we found here always one HS proteoglycan gene overexpressed in tumors that also highly expressed APRIL. A recent study reported that a brief course of subcutaneous low molecular weight heparin favorably influences the survival in patients with advanced malignancy and deserves additional clinical evaluation[The analysis reported here demonstrates that valuation. Thus, tThe authors declare that they have no competing interests.JM designed research, performed the analyses and wrote the paper. JLV and JDV have been involved in drafting and reviewing of the manuscript. BK is the senior investigator who designed research and wrote the paper. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/9/83/prepubBAFF, APRIL, TACI, BAFF-R and heparan sulfate proteoglycans in human tumor types to that of their normal tissue counterparts using publicly available gene expression data, including the Oncomine Cancer Microarray database.Expression of Click here for fileClassification of cancers included in the analysis.Click here for fileGene expression profile of Syndecan 1\u20134 and glypican 1\u20136 was determined using Oncomine Cancer Microarray database in bladder carcinoma, esophagus adenocarcinoma, brain tumors, breast cancer, head and neck tumors, lymphoma, pancreatic adenocarcinoma and adult male germ cell tumors presenting an overexpression of APRIL or an association with a bad prognosis or tumor progression.Click here for file"}
+{"text": "There was an error in the text in the \"Embryo micro-injection\" section of Materials and Methods. The correct lbx2-MO sequence is the reversed version of the published sequence in the text. It is 5'-CATGTCTTTAGAGCTGGAGGTCATC-3'."}
+{"text": "We found a statistically significant difference of CEB between the group with granulocyte pleocytosis and the control group. These results indicate a high degree of anaerobic metabolism caused by the oxidative burst of neutrophils. Similarly, we found a statistically significant difference of CEB between the control group and groups with tumorous oligocytosis plus pleocytosis and monocyte pleocytosis. This difference can be attributed to the oxidative burst of macrophages. Our findings suggest that CEB combined with CSF cytology has a great importance for diagnosis, differential diagnosis, and early therapy of CNS diseases.Urgent examination of cerebrospinal fluid (CSF) provides immediate important information about the character of central nervous system (CNS) impairment. Although this examination includes energy parameters such as glucose and lactate concentrations, it does not commonly use Coefficient of Energy Balance (CEB). In this study, we focused on CEB because it enables more exact assessment of actual energy state in the CSF compartment than glucose and lactate alone. CEB informs about the actual functioning condition of present cells, and it does not require any other analysis or costs. Using Kruskal-Wallis ANOVA, we examined a large CSF sample ( Unlike blood analysis, an examination of CSF allows the following of immune and metabolic processes in CNS without disturbing other organ systems as the CSF compartment is considerably autonomous. The urgent  examination of CSF should be accessible everywhere 24 hours a day to provide important information in the decision-making diagnostic and therapeutic algorithms , 2. The Qglu(Qglu = \u230aglucoseCSF\u230b/[glucoseblood]), and concentration of the product of anaerobic metabolism, that is, lactate in CSF [\u03c1 = \u22120.770) with CSF cellularity than lactate or glucose concentrations in purulent meningitis [Biochemical parameters of energy metabolism in the CSF compartment typically comprise a concentration of the energetic substrate, that is, glucose in CSF and blood, glucose quotient e in CSF , 4. Gluce in CSF . An impoe in CSF \u201311, as we in CSF . Becausee in CSF ; therefoe in CSF \u20137, 13\u201316e in CSF \u20137, 13\u201316ningitis , and CEBningitis .aerobic way. In aerobic glycolysis pyruvate is further metabolized by the Krebs cycle and the respiratory chain to CO2 and H2O producing 38 molecules of adenosine triphosphate (ATP) from 1 molecule of glucose. If the amount of oxygen in the CSF compartment is low, the extent of anaerobic glycolysis increases. In anaerobic glycolysis pyruvate is converted to lactate producing only 2 ATP molecules from 1 molecule of glucose [To calculate CEB, it is important to understand the processes of energetic metabolism of glucose. Glucose as an energy substrate is changed by glycolysis to pyruvate in CNS. If a sufficient amount of oxygen is dissolved in CSF compartment, the glycolysis takes place primarily in an  glucose , 13. Obv glucose  is a mattabolism \u20137, 13\u201316Qglu more than 0.55), concentration of lactate is low, and value of CEB within aerobic metabolism is in the reference range, that is, 28.00\u201338.00 [Under the physiological conditions in CSF, the considerable amount of oxygen is dissolved there and a natural consumption of glucose is continuously supplied from blood. As a result, concentration of glucose is quite high  or noninfectious etiology . When serous CNS inflammation occurs, a permeability of blood-CSF barrier increases, humoral immune components and cells penetrate CSF, and activated immune system raises energy demands. Limited tissue reserves of glucose [Qglu under 0.55), concentration of lactate slightly increases, and value of CEB ranges from 10.00 to 28.00 [Serous inflammations can be either of infectious  burst of professional phagocytes\u201d when purulent CNS inflammation or another kind of pathologies occurs. See Qglu), concentration of lactate significantly increases, and value of CEB distinctly falls, ranging between 10.00 and highly negative values [Mycobacterium tuberculosis, Listeria monocytogenes, rarely Borrelia sp., Treponema pallidum, Cryptococcus neoformans, and Candida albicans) or at the malignant meningeal infiltration, MMI [During this process a consumption of oxygen is substantial, so an intensive anaerobic metabolism of glucose is developed, and as a result, concentration of glucose in CSF significantly decreases  CSF examination, without the necessity of any other analysis or costs. We examined a large CSF sample from patients with variant neurological symptoms and compared CEB values among eight cytological groups to match the findings with the above-mentioned studies. We hypothesized that values of CEB would be significantly different in groups accompanied by the oxidative burst of professional phagocytes. This means that significantly different CEB would be in the group with granulocyte pleocytosis or tumorous pleocytosis/oligocytosis, possibly monocyte pleocytosis. Our results confirm this hypothesis. They suggest that CEB, commonly assessed with CSF cytology, can differentiate between variant pathologies in CNS specify and accelerate differential diagnosis and early targeted therapy of CNS diseases.http://www.likvor.cz). In all cases, physicians requested this examination because of a suspicion of some neurological disease . Firstly we examined a total cell number in the Fuchs-Rosenthal chambre, basic biochemistry, secondly we made a qualitative cytological examination, and thirdly we assumed levels of IgG, IgA, IgM, and their CSF/S quotients, and \u03b22-m in CSF and serum and its CSF/S quotient. In the cytological examination we used standard  and special staining techniques in an effort to detect malignant elements  and the presence of CNS tissue destruction (Oil Red 0) by detecting of lipophagic macrophages. Cytological samples were obtained from CSF using a gentle sedimentation technique. With immunoglobulins, their intrathecal oligoclonal synthesis, if existent, was determined numerically (Reiber).We analyzed 8183 CSF samples in the Laboratory for CSF and Neuroimmunology , granulocyte oligocytosis , granulocyte pleocytosis , monocyte oligocytosis , monocyte pleocytosis , lymphocyte oligocytosis , lymphocyte pleocytosis , and tumorous oligocytosis plus pleocytosis . We included TO in the same group as TP because of small sample size and the same etiology. Pleocytosis is determined as the number of leukocytes in the Fuchs-Rosenthal chamber greater than 4/1\u2009\u03bcL; oligocytosis as the number of leukocytes in the Fuchs-Rosenthal chamber in reference range (to 4/1\u2009\u03bcL), but with a pathological cell composition\u2014the attributive adjective according to the predominant cells .According to the findings in cytology we divided the sample into 8 groups: control group ; Qglu and CEB were calculated.Concentrations of glucose in CSF and serum, total protein in CSF, and lactate in CSF were analyzed photometrically ; concentration of beta-2-microglobulinDescriptive methods, Kolmogorov-Smirnov test, box-and-whiskers plots, and Kruskal-Wallis ANOVA with pairwise posthoc multiple comparisons were used for statistical analysis.Descriptive statistics of CEB with individual cytological syndromes in cerebrospinal fluid and results of testing of concordance among 8 groups are summarized in Tables transf(CEBtransf = log\u2061(40 \u2212 CEB)) for creation of box-and-whiskers plots\u2014see Figures Because the distribution of CEB in our data set is not normal and CEB values are often strongly negative, we performed the transformation to CEBP < 0.001), respectively, significantly decreased values of CEB, as well as in comparison with all other cytological groups, that is, LO, LP, MO, MP, TO + TP and GO (P < 0.001 or P < 0.01). Similarly, the group with TO + TP presents the statistically significant difference of CEB from controls (P < 0.001) and other cytological groups (P < 0.001 or P < 0.01), except for MP . Consequently, our results support the above-mentioned theory of the oxidative burst of professional phagocytes and probability of simultaneous occurrence of anaerobic scope of CEB and certain cytological patterns.As can be seen from Tables P < 0.001). But the same difference is between MP and GP, LP and GP (P < 0.001): this means that the values of CEB in GP are even significantly lower than in MP and LP . It can be seen in P < 0.01) and no difference between GO and MO, LP. It may be caused by the fact that finding of GO is not usually caused by purulent neuroinfection. This cytological pattern appears in the initial stages of nonpurulent inflammations and in early stage of cerebral ischemia [In contrast to these results, the group with GO shows less significant difference of CEB in comparison with controls  is present. An activation of brain capillaries endothelium occurs, and then the permeability of blood-brain barrier increases\u2014an influx of humoral immune components and immunocompetent cells to CSF raises to prepare this initially immune-privileged space for prospective invasion of pathogen and fully fledged inflammation process [\u03bcL. As can be seen in  process , 16, 36. process , 6, 14. secondary infection after trauma or neurosurgical performance may bring a slight modification of results to our data set. In these cases the input of pathogens to the immune unprepared space causes slow acceleration of inflammation, and in addition there are often many other complications like production of glucocorticoids due to stress, a primary disease of CNS itself, a noninfectious inflammation after neurosurgical performance, a contamination of CNS by nonpathogenic microbes, usually a protective antibiotic therapy, polymorbidity, and so forth. These patients do not often develop an adequate inflammatory response, including cellularity of cerebrospinal fluid. A detection of metabolic manifestation of the oxidative burst by CEB significantly contributes to the solution of this difficult situation [Inclusion of data from patients with ituation , 37, 38.To sum up, the \u201canaerobic shift\u201d of CEB towards the values below the lower reference limit always reliably reveals the presence of pathology in CNS.Advances in laboratory technologies affect an investigation of cerebrospinal fluid; processes occurring in CNS can be more accurately characterized by still new biomarkers measured at specialized centers. But we target the most fundamental energy parameters in CSF because they provide the first information about pathological processes in CNS to the physician, and they are generally available, low-cost, and irreplaceable. The Coefficient of Energy Balance enables more exact assessment of actual energy state in the CSF compartment than glucose and lactate alone, and not only does it specify and accelerate diagnostic and therapeutic decision, but it also determines the direction of further examinations. n = 8183) we found statistically significant difference of CEB values in cytological groups accompanied by the oxidative burst of professional phagocytes. Our results confirm the ability of CEB to distinguish between variant pathologies in CNS, and thus support the benefit of CEB as an instrument for early differential diagnosis and early targeted therapy of CNS diseases. We believe that the combination of CEB and cytology in CSF examinations is crucial because cytology describes the presence of concrete cells, and CEB adds information about the functioning condition of these cells. Of course, it is also necessary to build diagnosis on all clinical and laboratory findings comprehensively, not only on one or several laboratory findings. Using a large data set  analysis of cerebrospinal fluid. We are convinced that a calculation of CEB should be included into an urgent CSF analysis and automatically set in a Laboratory Information System. Further studies of CEB in relation to specific clinical diagnosis are needed to clarify sensitivity and specificity of CEB. Results from CSF open the possibility of using CEB for examinations of other body fluids."}
+{"text": "Case 2 had VPA for schizoaffective psychosis with 10 VPA concentrations during an 8-week admission. To maintain a VPA level \u226550\u2009\u03bcg/mL, VPA doses increased from 1500 to 4000\u2009mg/day. Case 3 had tuberous sclerosis and epilepsy and was followed up for >4 years with 137 VPA concentrations. To maintain VPA concentrations \u226550\u2009\u03bcg/mL, VPA doses increased from 3,375 to 10,500\u2009mg/day. In Cases 2 and 3, the duration of admission and the VPA dose were strongly correlated  with almost no change after controlling for VPA concentrations, indicating progressive autoinduction that increased with time.Valproic acid (VPA) can autoinduce its own metabolism. Cases requiring VPA doses >4000\u2009mg/day to obtain therapeutic plasma concentrations, such as these 3 cases, have never been published. Case 1 received VPA for seizures and schizophrenia and had >50 VPA concentrations in 4 years. A high dose of 5,250\u2009mg/day of VPA concentrate was prescribed for years but this dose led to an intoxication when switched to the enterocoated divalproex sodium formulation, requiring a normal dose of 2000\u2009mg/day. VPA metabolic capacity was significantly higher ( It is repilepsy . The theor mania .\u03b2-oxidation as a fatty acid (30%), with the cytochrome P450  as a minor component in the metabolic process. At low doses, \u03b2-oxidation may be the most important pathway, while at therapeutic doses glucuronidation may be more important [In spite of decades of use, VPA metabolism is not completely understood \u20138. VPA ptty acid 0%, with \u03b2-oxidation (prospective study) [Regarding the potential of causing drug-drug interactions (DDIs), VPA was traditionally considered to be a moderate inhibitor of several enzymes including CYP2C9, epoxide hydroxylase, several UGTs, and the N-glucosidation pathway of phenobarbital . First, e study) ; (2) CYPe study) ; (3) pose study) ; (4) arie study) ; (5) olae study) , TDM [18e study) , and a pe study) ; (6) cloe study) , 22, a pe study) , and stae study) , 25); ane study) . In summ\u03bcg/mL. Recent advances in pharmacokinetic knowledge have allowed us to offer a hypothesis about these patients' needs for such high VPA doses to reach therapeutic concentrations. On February 19, 2015, we conducted a PubMed search using the words \u201cvalproic acid and (high dose or high dosage)\u201d limiting them to title or abstract, human participants, and case reports, which provided 33 articles. None of these 33 articles described the therapeutic use of VPA doses >4000\u2009mg/day. We decided to publish these cases because it is very likely that other clinicians have seen similar patients but did not write about them.The following 3 cases, accumulated by the senior author in the last 20 years of his clinical practice, describe 3 patients needing high doses of VPA to reach and maintain therapeutic concentrations \u226550\u2009The senior author collected these 3 cases during the last 20 years while working as a clinician and/or consultant in the public mental health system in Kentucky, USA. During 8 of those years, he managed a 30-bed treatment-refractory unit for psychotic patients in a state hospital with approximately 1600 admissions/year. He acquired Cases 1 and 3 in that setting. For 14 years he has also acted as a consultant for difficult cases, including those needing high doses of psychiatric medications in 4 state hospitals for severe mentally ill patients, 4 hospitals for adults with intellectual disabilities, and 2 nursing homes. Case 2 was collected as a consultant for another psychiatrist (third author). In the senior author's experience, VPA is probably the most frequently prescribed psychotropic drug in the public mental health system facilities of Kentucky, with more than 1000 patients every year treated with VPA at state facilities. The 3 patients in this paper participated in pharmacogenetic studies after written informed consent forms were signed by them and/or their guardians.\u03bcg/mL, which is the same as mg/L.These 3 cases contain TDM information that was collected for clinical purposes many years ago. The VPA TDM was done by immunoassay in the same psychiatric hospital using the same clinical laboratory at the hospital; levels were taken as trough concentrations (early AM before taking medications) and after reaching steady state. The VPA concentrations were measured in In the last 10 years, the senior author has increasingly used the pharmacological concept of concentration-to-dose ratio (C/D ratio) to study TDM in order to personalize the prescription of psychiatric medications in psychopharmacology. The C/D ratio is a measure of the ability to eliminate the drug and is influenced by genetic, personal, and environmental factors. Inducers decrease the C/D ratio and inhibitors increase the C/D ratio. In comparing individuals taking the same drug, a very low C/D ratio indicates an individual with very fast metabolism, while a very high C/D ratio indicates one with very slow metabolism. Each drug is different and unique and has its own normal ranges for C/D ratios determined by its own bioavailability and elimination from the body.The senior author has used the C/D ratio to interpret TDM of clobazam , clozapiVPA TDM, unfortunately, is a little more complicated since it does not follow linear kinetics. The relationship between VPA dose and total concentration is nonlinear; the concentration does not increase proportionally with the dose but increases to a lesser extent due to saturable plasma-protein binding . As the \u03bcg/mL. Therefore the C/D ratio in this patient is 100/2000 or 0.05. The C/D ratio multiplied by 1000 would be 50, an easier number to grasp.The senior author has started using VPA C/D ratios in his teaching and clinical practice in the last 3 years and is not aware of any published article using them. Therefore, as the reader may not be familiar with the use of C/D ratios for valproate VPA, this section provides a short introduction. Based on experience as a lecturer, the senior author has figured out that VPA C/D ratios have values too low to be easily understood by physicians, who do not tend to be particularly strong in mathematics. To more easily understand the VPA C/D values, they are also presented with a value obtained by multiplying by 1000. To understand this, let us assume that a VPA dose of 2000\u2009mg/day provides a total VPA concentration of 100\u2009Next, we can use a published case of VPA toxicity  to expla\u03bcg/mL at a dose of 1500\u2009mg/day . The VPA C/D ratio multiplied by 1000 was 520. In a study of VPA TDM [\u03bcg/mL with an average maintenance dose of 629\u2009mg/day  and a VPA C/D ratio multiplied by 1000 of 121. In the same study, 23 males had an average VPA concentration of 70\u2009\u03bcg/mL with an average maintenance dose of 617\u2009mg/day  with a VPA C/D ratio multiplied by 1000 of 113.Other examples of VPA C/D ratio are calculated using published data , 31. A cThese 3 cases needing VPA doses >4000\u2009mg/day to obtain VPA therapeutic concentrations had very low mean C/D ratios multiplied by 1000: in the 20s or lower, possibly due to VPA autoinduction.For this paper, we have completed the Drug Interaction Probability Scale (DIPS)  for eacht-test compared mean VPA C/D ratios from the two VPA formulations, one probably accompanied with autoinduction and the other not. Pearson correlations between VPA dose and day of admission were used in Cases 2 and 3 to demonstrate that higher doses were needed to keep the VPA concentrations therapeutic over time, which is consistent with a pattern of autoinduction. Partial correlations were used to demonstrate that VPA concentrations did not explain the correlation between VPA dose and day of admission.Available data for each of the 3 cases was dictated by clinical care; many years later we tried to use statistical techniques to accommodate the available data and the questions asked in each case. The Statistical Package for the Social Sciences (SPSS 22) was used to analyze the data. In Case 1 an independent sample A Caucasian male was followed up for more than 4 years between the ages of 30 and 34 years. His initial weight was 85\u2009Kg. He smoked 10 cigarettes per day. His psychosis started when he was 12 years old and had been refractory to treatment for many years at the time of admission. The patient had three seizures of unknown origin in the 3 months before coming under the care of the senior author. Academic neurologists had examined him at least twice. All tests, including a CT scan of the head, were negative. After a careful review of all records, it was the impression of the senior author that at least 1 or 2 of the 3 seizures might have been associated with rapid withdrawal of high doses of benzodiazepines, particularly temazepam, used as PRNs. He arrived at the unit with four antiepileptic medications in his regimen: carbamazepine, phenytoin, diazepam, and VPA. These medications had been progressively added by the neurology consultants. The blood levels of all four of these medications were subtherapeutic. After several months the senior author was able to change the regimen to VPA only, without the patient having any seizures during his long admission. The VPA treatment is described in detail in It is likely that this case presentation reflects the result of VPA autoinduction only present during VPA concentrate versus no autoinduction during divalproex sodium use. The DIPS score was 6, which corresponds to a probable drug interaction in this case .\u03bcg/mL, progressively higher doses from 1500 to 4000\u2009mg/day were needed to the point that the last VPA C/D ratio multiplied by 1000 had a value remarkably low (at 17) while being on a high dose of 4000\u2009mg/day and with a VPA concentration of only 67\u2009\u03bcg/mL. The mean C/D ratio multiplied by 1000 was 25 with a range from 17 to 33  between the number of days of admission and the dose, indicating that, with an increasing length of admission, the dose increased, and this high value remained after correcting for VPA concentrations using a partial correlation  . His initial weight was 90\u2009Kg. He had 7 prior psychiatric admissions. On presentation the patient met the criteria for hypomanic episode with psychotic features. He had a positive viral panel for hepatitis C and his Hep C RNA PCR was positive, but his liver function profile and metabolic panel were unremarkable. Sublingual asenapine 20\u2009mg/day, one of his outpatient medications, was restarted by the third author on hospital day 2 in addition to VPA 1000\u2009mg/day, which the patient had not received as an outpatient. Other medications are described in 17 to 33 . As a maConsidering the need for higher doses to maintain the same VPA concentration, the DIPS score was 5 , indicating a \u201cprobable\u201d relationship between VPA and autoinduction activity.Patient 3 was a Caucasian male nonsmoker with a history of tuberous sclerosis, which manifested as seizure disorder and mental retardation. His initial weight was 71\u2009Kg. He had been an inpatient at a state psychiatric hospital for 22 years before the senior author started to manage his treatment (day 1) when the patient was 48 years old. Two years later he developed a right renal mass, which was highly suspicious for renal cell carcinoma on biopsy. It was followed by a right-sided nephrectomy and cholecystectomy (day 860). However, the final pathology suggested that the mass was actually angiomyolipoma, which is common if there is renal involvement in patients with tuberous sclerosis. Thorough investigation for left-sided pathology indicated that his left kidney was functioning well with stable masses present on CT. From day 1322, the patient progressively worsened with new-onset episodes of ataxia and confusion. High clinical suspicion for a brain mass led the senior author to investigate with MRI. The results indicated that the patient had a mass in the right lateral ventricle \u201csuggestive of giant cell astrocytoma\u201d and that \u201cfoci of enhancement at the gray-white junction and numerous signal abnormalities in the skull suggest the possibility of metastatic disease.\u201d Giant cell astrocytomas occur in 6\u201314% of patients with tuberous sclerosis and should be suspected when there is a new focal deficit, signs of increased intracranial pressure, or unexplained behavioral changes. After day 1420, when the brain tumor was diagnosed, we started trying to reduce the VPA dose and manage it on the basis of the patient's comfort rather than the VPA concentration level.\u03bcg/mL. On day 1 the VPA dose was 3375\u2009mg/day, on day 400 it was 5000\u2009mg/day, on day 943 it was 9000\u2009mg/day, and on day 1029 it was 10500\u2009mg/day, which was maintained until it was clear that his physical deterioration was due to a brain tumor. At that time other medications were discontinued, only leaving phenytoin and a lower VPA dose  described in VPA dose .r = 0.97; p < 0.001) between the number of days past admission and the dose, indicating that with an increasing length of admission, the dose increased, and this significance remained after correcting for VPA concentrations using a partial correlation  , indicating a \u201cprobable\u201d relationship between VPA and the autoinduction of its metabolism.These 3 cases reflect 3 extremely challenging patients who required very high doses of VPA >4000\u2009mg/day to maintain VPA therapeutic levels. As usual in these challenging cases, the pharmacological data is somewhat limited since patients were treated with polypharmacy and went through complex changes in medications. In spite of the data limitations, we think that these cases need to be published to acknowledge that while these patients are very rare, they exist. In support of the above findings, however, the senior author was closely involved in the treatment of the 3 patients and all VPA concentrations were measured in the same lab. It is rare to see publications describe so many large measures of VPA concentrations.The data is limited but undeniable and should indicate to any clinician that these 3 patients needed daily doses of VPA to get therapeutic VPA concentrations. The second and greater limitation in analyzing these cases is our interpretation of the data many years after they were collected\u2014in the third case, almost 20 years. It has required that much time to accumulate evidence in the literature to understand what happened and to realize that VPA may be an inducer; in fact, it can induce its own metabolism. Therefore, although these VPA concentrations were collected prospectively and used to modify VPA doses, they were reviewed retrospectively to provide the unified hypothesis that it is possible that these high VPA doses were required because these 3 patients were unusually sensitive to VPA autoinduction. In retrospect, after having a possible pharmacological explanation now after many years, it would have been better to collect more frequent TDM data, but what is described is what is available. Although we acknowledge that the data is limited and our interpretation is even more limited, we have tried to support our interpretation through the use of statistical tests and a validated scale.We think these cases contribute to the scarce literature by indicating that clinicians must be aware that VPA may behave as an inducer. In Cases 1 and 3, patients with likely VPA autoinduction presented significant clinical challenges to the senior author, who had no idea why the patients required much higher VPA dosages to obtain therapeutic VPA concentrations. Case 2 was identified only 4 years ago when the senior author realized that VPA autoinduction was clinically relevant for some peculiar patients. As a matter of fact, Case 2 was identified by the third author, who had rarely seen similar cases. The senior author suggested to her that these cases were probably explained by autoinduction and she was able to identify this patient as a candidate for VPA autoinduction shortly after admission.p = 0.001) in VPA plasma concentrations upon switching dosage forms from divalproex sodium to VPA [\u03bcg/mL to 145\u2009\u03bcg/mL and required halving the dose to 2000\u2009mg/day of divalproex sodium to get VPA concentrations in the 80s\u2009\u03bcg/mL. In retrospect, during treatment with VPA concentrate, the VPA C/D ratio multiplied by 1000 was extremely low, at a mean of 15 and a range of 10\u201321, indicating an extraordinarily high capacity to metabolize VPA. This is compatible with VPA concentrate inducing its own metabolism. We propose that after 4 weeks on divalproex sodium the autoinduction was lost and the patient's metabolism normalized, with a mean VPA C/D ratio multiplied by 1000 of 39 and a range of 28 to 48. We cannot explain why this patient's VPA concentrate appeared to induce its own metabolism while divalproex sodium did not; we assume this patient had some rare genetic variation that explains his peculiar response.The first patient was very challenging, probably the most challenging patient in the senior author's career, even with his more than 25-year history of treating treatment-refractory patients. The patient metabolized the CYP1A2 drugs clozapine and olanzapine normally but got intoxicated on clozapine during an infection . He appem to VPA . TherefoThe second patient had a relatively short admission (<60 days) but demonstrated a very high and significant correlation between length of admission and VPA dose. The final C/D ratio was 17, extremely low. This type of patient is probably more common than Case 1; his treating psychiatrist, the third author, has seen several similar cases in her 25-year practice in patients with severe mental illness including 20 years at this psychiatric hospital.\u03bcg/mL. It is possible that phenytoin treatment contributed in some way to the facilitation of VPA autoinduction, since phenytoin is a major inducer and can displace VPA from plasmatic proteins. It is remarkable that although increases in VPA dosage were needed to get therapeutic VPA concentrations, the phenytoin dosage needed to obtain therapeutic phenytoin concentrations was relatively stable throughout this time period. Phenytoin doses ranged from 500\u2009mg/day at the beginning to 400\u2009mg/day at the end. This minor reduction in dosage may be explained by the greater VPA concentrations at the end. VPA is an inhibitor of phenytoin metabolism. Unfortunately, 20 years ago, free VPA and phenytoin concentrations were not available at this psychiatric hospital. It would have been important to measure them [The third patient probably deserved a Guinness record since he was treated with 10500\u2009mg/day for 400 days and had a VPA C/D ratio multiplied by 1000 which yielded a value less than 10 most of the time. We have the impression that progressive VPA autoinduction explained the need to go from 3450 to 10500\u2009mg/day to achieve and maintain VPA concentrations >50\u2009ure them .The induction of metabolic enzymes such as UGTs implies that the amount of these proteins increases when they are induced; this is almost always explained by increasing protein synthesis mediated by the so-called nuclear receptors  or other transcription factors such as aryl hydrocarbon receptors. The potent AED inducers  bind to the pregnane X receptors. Phenytoin and phenobarbital appear to also bind to the constitutive androstane receptors. VPA is a less potent inducer but probably works in a similar way. An in vitro study suggested that VPA might also activate constitutive androstane receptor and pregnane X receptor pathways .We think it is possible that these unusual patients needing very high VPA dosages have unusual genetic variants at the nuclear receptors, which made them very sensitive to VPA autoinduction."}
+{"text": "Alu elements, the most abundant human repeats. The methylation of Alu elements is an important mechanism to suppress Alu transcription and subsequent retrotransposition. Decades of studies revealed that Alu methylation is highly dynamic during early development and aging. Recently, many environmental factors were shown to have a great impact on Alu methylation. In addition, aberrant Alu methylation has been documented to be an early event in many tumors and Alu methylation levels have been associated with tumor aggressiveness. The assessment of the Alu methylation has become an important approach for early diagnosis and/or prognosis of cancer. This review focuses on the dynamic Alu methylation during development, aging, and tumor genesis. The cause and consequence of Alu methylation changes will be discussed.DNA methylation primarily occurs on CpG dinucleotides and plays an important role in transcriptional regulations during tissue development and cell differentiation. Over 25% of CpG dinucleotides in the human genome reside within  Alu repeats of short interspersed repetitive elements (SINEs) which comprise 13.7% of the human genome. A full-length Alu element is approximately 300 nucleotides in length and contains two almost identical monomers separated by an A-rich region. Most of the Alu repeats share extensive homology over the 300\u2009nt sequence with a recognition site for the restriction enzyme AluI  Alu methadiation , 89, andadiation , 91. Intger Alus . A recenrticular . The racd groups . Alu methylation status and carcinogenesis has been established, many studies demonstrated the strong correlations between the global Alu methylation level and the clinical outcomes of patients with cancer [ Alu methylation from the adjacent normal salivary gland to the intermediate, mucous, and squamous cells [ Alu elements [ Alu elements gain or lose methylation in tumors. The Alu elements proximal to CpG islands tend to be hypermethylated in ependymomas, whereas those in intergenic regions are more likely to be hypomethylated. In addition, the methylation levels of some specific Alu elements, either hypermethylated or hypomethylated in tumors, were confirmed to be associated with tumor aggressiveness. Since some Alu elements become hypermethylated while some others become hypomethylated, the methylation analysis on a panel of specific Alu loci, rather than global Alu methylation, could greatly improve the specificity and sensitivity for cancer detection and prognosis. For instance, a study of the MLH1 gene in gastrointestinal cancer found that the methylation of an Alu element in the first intron spread to its promoter. In normal cells, the spread of Alu methylation is limited and a clear boundary is present between that Alu element and upstream promoter [ Alu elements remains to be addressed, in which methylation levels could be used as common diagnostic or prognostic indicators for a variety of tumors.Although no direct connection betweenh cancer , 96. A sus cells . Our recelements . Comparepromoter . The abepromoter . Whether Alu elements play important roles in the genome and transcriptome, the consequences of aberrant Alu methylation in tumors could be multifaceted  to initiate Pol III-dependent transcription. The binding activity of TFIIIC is the key of the transcription activity of Alu. Jang and Latchman found that ICP27 is responsible for the increase in TFIIIC binding and in turn produced the increase in Alu transcription [ Alu corepressor 1 (ACR1) [ Alu transcription in vitro. Sperm Alu binding protein (SABP) binding is sufficiently selective to protect Alu from in vitro methylation without significantly altering the methylation of adjacent CpGs and protect a region (positions 25\u201333) downstream from the A box promoter element for Pol III [ Alu-RNA containing nuclear RNP particles [ Alu and might participate in Alu expression regulation.cription . The intcription . Alu cor1 (ACR1)  and nucl1 (ACR1)  were dem Pol III . Lukyanoarticles . The p68 Alu RNAs may affect nearby gene expression, distal gene expression, and global translation. For instance, the expression of an Alu in the promoter of the epsilon-globin gene was found to negatively regulate globin gene expression by transcriptional interference [ Alu RNA was found to be a modular transacting repressor of mRNA transcription [ Alu RNAs also affect translational initiation and are found to form stable, discrete complexes with double-stranded RNA-activated kinase PKR and antagonized PKR activation [ Alu RNA processing causes the degradation of a subset of critical stem cell mRNAs  and modulates the exit from the proliferative stem cell state [ Alu RNA accumulation induces retinal pigmented epithelium cytotoxicity, thus leading to geographic atrophy and blindness [Being more than a dramatic source of insertional mutagens, the expression of freerference . Recentlcription . Importativation . DICER-dll state . Meanwhilindness , 117. Alu Pol III transcripts have also been associated with the miRNA transcription. Recognized as an important component of the transcriptome, miRNAs are approximately 22 nt noncoding RNAs mediating posttranscriptional regulation [ Alu transcripts are often comprised of 3\u2032-flanking sequence prior to the Pol III transcription terminator \u201cTTTT.\u201d If located within the Alu or immediate 3\u2032-flanking regions, miRNAs may be expressed with Alu Pol III promoters. Chromosome 19 miRNA clusters (C19MC) were first shown to be transcribed by Pol III with upstream Alu promoters. Based on computational prediction and high-throughput small RNA sequencing, an additional twenty-three miRNAs at the downstream of the Alu elements were suggested to be transcribed by Pol III with Alu promoters [ AluSx is located in between the spliced MIR1975 but in the antisense orientation with respect to MIR1975 transcription. Thus, the following remains an open question: how frequently and under what condition would miRNA be transcribed with Alu promoters?Recently,gulation . Alu traromoters . Howeverromoters \u2013122. Recromoters , 123 andAlu elements may serve as cis-regulatory elements in the control of neighboring gene expression. Based on the analysis of the Alu consensus sequence, Alu elements contain transcriptional factor binding sites for the zinc-finger Pol II transcription factors SP1 and YY1, activator and repressor, respectively, in a sequence-specific binding manner [Alu elements [Alu subfamilies, the methylation and deamination resulting from the CG to TG transition could generate the CATG motifs attractive to p53 for binding in vivo [Alu elements contain negative calcium response elements [Alu elements with conserved noncoding elements in mammalian genomes were found to be the enhancers, such as that of FGF8 and SATB2 genes, and are involved in brain development [Alu element in the last intron of T-cell-specific CD8\u03b1 gene was found to serve as an enhancer with GATA3, bHLH protein, and LYF1 binding motifs [In addition to being part of the transcriptome, many g manner   ( Alu element containing the vitamin D receptor binding element in the promoter of CAMP gene mediates the vitamin D-cathelicidin pathway and becomes a key component of a novel innate immune response of human to infection [ Alu-DR (direct repeat) elements provide the binding sites for hepatocyte nuclear factor 4 alpha (HNF4\u03b1) which activates the transcription of a list of target genes [ Alu repeats, in particular AluS. Importantly, consensus Alu-DR2 elements arose predominantly via deamination of a methylated CpG dinucleotide [ Alus gain the palindromic inserted repeats separated by three base pairs and acquired the ability to function as estrogen receptor-dependent enhancers [Experimentally, somes (HREs) , includinfection . Alu-DR et genes . Laperricleotide . By acqunhancers , 145. Alu repeats, and, additionally, most RNA Pol IITF binding motifs cluster in the RNA Pol III promoter sequences, which provoke the thinking of the competition and cooperation among these binding proteins. On the other hand, how RNA Pol II and Pol III transcription machineries utilize Alu repeats in different tissues (such as SABP in sperm versus p68 in somatic cells) and under different microenvironments to recruit different transcription factors remains a mystery. Shankar found that a progressive loss of the Pol III transcriptional potential was observed with concomitant accumulation of Pol II regulatory sites [ Alu sequences is involved in the balanced activity between Pol II and Pol III. The methylation statuses of Alu elements would certainly determine the accessibility of Alu containing cis-elements and have a great impact on the binding of transcriptional factors. However, no genome-wide data has been generated so far to explore the Alu methylation patterns and the binding profiles of various factors on Alu elements.As shown in ry sites , while ary sites , and Polry sites . All of  Alu elements play important roles in the genome, from their contribution to the epigenome to their occurrence in a significant fraction of the human transcriptome. The time when they were considered to be nothing but junk is certainly long gone. DNA methylation is one of the key mechanisms controlling Alu expression. The dynamic Alu methylation during development and aging suggests Alu elements and their transcripts may be involved in the cell differentiation and tissue development. The methylation levels of a subset of Alu elements have been associated with tumor malignancy and aggressiveness. This indicates a potential biological and clinical relevance of the methylation of some Alu elements during tumorigenesis and progression. Since the interactions between genes and Alus are multidimensional, the impact of such epigenetic alterations on Alu elements may be multifold. Future integrative Alu epigenomic and transcriptomic studies will further elucidate the functional aspects of Alu elements and their transcripts.Decades of studies have demonstrated that"}
+{"text": "AbstractMetriocnemusvolitans Goetghebuer, 1940 revealed that these specimens belong to the genus Chaetocladius and are not con-specific with Gymnometriocnemusvolitans  sensu Gymnometriocnemuskamimegavirgus Sasa & Hirabayashi, 1993 fits well with the species figured and diagnosed by Chaetocladiusvolitans (Goetghebuer) comb. n. and for the use of Gymnometriocnemuskamimegavirgus for Gymnometriocnemusvolitans sensu Brundin. In addition, we provide DNA barcode data that indicate the presence of at least seven Gymnometriocnemus species in Norway of which six are collected as male adults. Two of these, Gymnometriocnemus (Gymnometriocnemus) pallidussp. n. and Gymnometriocnemus (Raphidocladius) autumnalissp. n. are regarded as new to science and diagnosed based on adult male morphology and DNA barcodes. The species Gymnometriocnemus (Gymnometriocnemus) marionensis S\u00e6ther, 1969 is re-established and a key to all Holarctic species is provided.Examination of the syntypes of  Gymnometriocnemus was suggested by Gymnometriocnemussubnudus  as type species and made the name available according to the International Code of Zoological Nomenclature. Edwards is thus credited authorship of the genus (Gymnometriocnemus species recognized (Raphidocladius from Norway (own data).The orthoclad genus he genus . The gencognized . Larvae cognized , but theMetriocnemusvolitans was described by Gymnometriocnemus by Gymnometriocnemus relied on the characters presented by Raphidocladius for Gymnometriocnemus species possessing an extremely long virga with needle-like sclerotization in the adult males. The species Gymnometriocnemusbrumalis  and Gymnometriocnemusacigus S\u00e6ther, 1983 were listed as members of the group, while Gymnometriocnemusvolitans was considered a possible member since immatures were unknown and virga had not been examined  acigus, with Gymnometriocnemus (Raphidocladius) brumalis claiming that the characters used by Raphidocladius in the World Catalogue of Chironomidae: Gymnometriocnemus (Raphidocladius) brumalis, Gymnometriocnemus (Raphidocladius) kamimegavirgus, Gymnometriocnemus (Raphidocladius) tairaprimus and Gymnometriocnemus (Raphidocladius) volitans  ancudensis , Gymnometriocnemus (Gymnometriocnemus) benoiti , Gymnometriocnemus (Gymnometriocnemus) brevitarsis , Gymnometriocnemus (Gymnometriocnemus) johanasecundus Sasa & Okazawa, 1994, Gymnometriocnemus (Gymnometriocnemus) lobifer , Gymnometriocnemus (Gymnometriocnemus) longicostalis , Gymnometriocnemus (Gymnometriocnemus) subnudus, Gymnometriocnemus (Gymnometriocnemus) terrestris Kr\u00fcger, Thienemann & Goetghebuer, 1941, Gymnometriocnemus (Gymnometriocnemus) mahensis  Gymnometriocnemus (Gymnometriocnemus) nitidulus  and Gymnometriocnemus (Gymnometriocnemus) wilsoni Freeman, 1961   has beene groups . We haveGymnometriocnemusvolitans (Goetghebuer), describe hitherto unknown species of Gymnometriocnemus and to present the DNA barcodes of Norwegian Gymnometriocnemus as a resource for future studies of this genus.The motivation for this study was to clarify the identity of Metriocnemusvolitans Goetghebuer from the Royal Belgian Institute of Natural Sciences (RBINS), five male and three female adults mounted between cellophane strips on two separate pins. Both pins bear the label \u201cEnv. d. Abisko, Aout 1939, Dr. Thienemann\u201d and \u201cMetriocnemusvolitans n sp\u201d  acigus S\u00e6ther, 1983 ) and two male syntypes and a female syntype of Gymnometriocnemus (Gymnometriocnemus) terrestris (RBINS).We also examined the male holotype and a male paratype of Gymnometriocnemus was collected using a variety of methods in different biosurveillance projects: Malaise traps, sweep netting and fogging of oak canopies (Supplementary file 1). This material is deposited in the NTNU University Museum insect collection (NTNU-VM). One to three legs were dissected off the specimens and submitted to the Canadian Centre for DNA Barcoding. Metadata, photos, sequences and trace-files are available in the Barcode of Life Data Systems  through the dataset DS-GYMNO with doi: 10.5883/DS-GYMNO. GenBank accessions are given in Supplementary file 1.Additional material of DNA extracts and partial COI gene sequences were generated using standard primers and bi-directional Sanger sequencing with BigDye 3.1 termination at the Canadian Centre for DNA Barcoding in Guelph. Protocols and original trace-files are available through the dataset DS-GYMNO in BOLD. Alignments were done on amino acid sequences and was trivial as indels were absent; only sequences > 300bp were used in the final alignment. The taxon ID-tree was generated using neighbour joining analysis and 1000 bootstrap replicates on Kimura 2-parameter (K2P) genetic distances in MEGA 6 .PageBreakGymnometriocnemus are given in Table Morphological terminology and abbreviations follow S\u00e6ther 1980). Antennal and fore leg ratios of Norwegian 980. AnteTaxon classificationAnimaliaDipteraChironomidaecomb. n.Metriocnemusvolitans Goetghebuer, 1940: 59.Metriocnemusvolitans Goetghebuer in Gymnometriocnemus, and the better preserved male specimens show characters that fits the diagnosis of Chaetocladius: wings with coarse punctuation and without macrotrichia on the membrane  marionensis in having very slightly larger megasetae on the gonostyli than specimens of Gymnometriocnemus (Gymnometriocnemus) subnudus  marionensis (c. 38 \u00b5m long) from Gymnometriocnemussubnudus (c. 17 \u00b5m long). Gymnometriocnemus (Gymnometriocnemus) marionensis with Gymnometriocnemus (Gymnometriocnemus) subnudus doubting the diagnostic value of the size of the megaseta and position of the end of R2+3  marionensis, but specimens from Michigan and North Carolina (ZMBN) identified by Ole S\u00e6ther as belonging to this species. This record of the species is the first from Norway and Europe.Only one specimen of this species from Norway has been available to us. It fits S\u00e6ther\u2019s description of dus Figs , but thi2+3 Fig. . We havePageBreakTaxon classificationAnimaliaDipteraChironomidaehttp://zoobank.org/8C19C165-6923-4A17-9C9D-380DD9413E4160.30921\u00b0N; 6.16453\u00b0E, 23.vi.2011, leg. Karl H. Thunes [BOLD ID: CH-eik131]. 5 Paratypes: 2 male adults as holotype except tree #1 60.314\u00b0N; 6.167\u00b0E, 21.vi.2011; 1 female adult as holotype except tree #18, 59.201\u00b0N; 9.920\u00b0E, 5.vii.2012; 2 male adults, Norway, Telemark, Porsgrunn, Brevik, Frierflauene, 59.0579\u00b0N; 9.66485\u00b0E, Malaise trap, 30.vi\u201327.vii.2010, leg. Geir S\u00f8li.Holotype: Male adult (NTNU-VM slide 143840), Norway, Hordaland, Kvam, Berge, oak canopy fogging, tree #3, Gymnometriocnemus.The species is named \u201cpallidus\u201d, Latin adjective meaning pale, referring to the conspicuous pale body colour compared to other Holarctic Gymnometriocnemus (Gymnometriocnemus) pallidus can be separated from other Gymnometriocnemus species by the following combination of characters in the adults: body pale yellow-green; male with short inconspicuous virga, gonostylus with convex outer margin and weakly developed crista dorsalis. Female with setae on most of wing surface, including numerous in cell m; antenna with apical flagellomere pointed and longer than flagellomere 4; genitalia with long rami, about the same length as notum.PageBreakPageBreakMale adult (n = 5 unless otherwise stated). Wing length 1.21\u20131.30, 1.28 mm. Colouration pale yellow-green body, legs and antennae; slightly darker bands on scutum; postnotum, dorsal side of head, ventral part of preepisternum pale brown; eyes dark brown.Head. Antennal ratio in Table Thorax. Antepronotum with 2-6, 3 setae. Dorsocentrals 10-17, 13; acrostichals 7-12, 10, minute and difficult to discern; prealars 3-4; scutellars 6-7.1+2; R2+3 approaching costa at 1/3 distance between R1 and R4+5. Macrotrichia present on membrane in apical half of wing, 0-2 setae in cell m. Veins Sc, R2+3, M and pseudovein without setae.Wing Fig. . Costa mLegs. Fore tibia with one spur, 35 \u00b5m long; mid tibia with two spurs ca. 20-25 \u00b5m long; hind tibia with well-developed comb and 2 spurs, ca. 20 and 35 \u00b5m long. Fore leg ratios in Table Hypopygium Fig. . Ninth tFemale adult (n = 1). Wing length 1.23 mm. Colouration as male.Head. Antenna Fig.  with fivThorax. Antepronotum with 6 setae. Dorsocentrals 18; acrostichals 11; prealars 3; scutellars 6.1+2; R2+3 approaching costa at 1/3 distance between R1 and R4+5. Macrotrichia present on membrane in whole wing. Veins M, Sc and R2+3 without setae.Wing Fig. . Costa w1 not measurable).Legs. Fore tibia with one spur, 20 \u00b5m long; mid tibia lost; hind tibia with well-developed comb and 2 spines, ca. 35\u201340 \u00b5m long. Tarsus of fore leg lost  subnudus and Gymnometriocnemus (Gymnometriocnemus) johanasecundus, but paler (see whole specimen figures in BOLD dataset DS-GYMNO). Males and females are almost completely yellow-green with pale brown posterior side of head and postnotum; pale brown scutal bands and ventral side of preepisternum. Gymnometriocnemus (Gymnometriocnemus) pallidus is also similar to these species in having a short, triangular anal point and a small virga, but the hypopygium of Gymnometriocnemus (Gymnometriocnemus) pallidus has a more prominent inferior volsella than Gymnometriocnemus (Gymnometriocnemus) johanasecundus and considerably stronger anal tergite setae than Gymnometriocnemus (Gymnometriocnemus) subnudus Gymnometriocnemus (Gymnometriocnemus) marionensis and Gymnometriocnemus (Gymnometriocnemus) pallidus sp. n. and is fairly widely distributed throughout Europe.We have seen specimens from eastern and central Norway that fit well with the original and later descriptions of the species, except for having a lower AR (1.0-1.1) compared to what Taxon classificationAnimaliaDipteraChironomidaehttp://zoobank.org/CAB0F99B-1A0A-4078-A68D-D8DB31D0DEB169.8306\u00b0N; 25.1856\u00b0E, 107 m a.s.l., 03.ix.2010, leg. Alyssa Anderson [BOLD ID: Finnmark201]. 3 Paratypes, male adults: 1 Norway, Finnmark, Vard\u00f8, Nedre Domen, lake and pond at road E75, PageBreak31.0341\u00b0E70.3215\u00b0N; , 120 m a.s.l., 05.ix.2010, leg. Alyssa Anderson; 1 Norway, Finnmark, Nordkapp, Nordkapp-Plateau, 71.1446\u00b0N; 25.7641\u00b0E, 220 m a.s.l., 01-ix-2010, leg. Trond Andersen; 1 Norway, Oppland, Dovre, Rondane National Park, Vidjedalsbekken (upper), Malaise trap, 61.9717\u00b0N; 9.83606\u00b0E, 1280 m a.s.l., 15.ix.2008, leg. Terje Hoffstad.Holotype: Male adult (NTNU-VM slide no. 136299), Norway, Finnmark, Porsanger, small pond near Gaggavann, The species is named \u201cautumnalis\u201d, Latin adjective meaning belonging to autumn, referring to the time of the year when the type material was collected.Gymnometriocnemus (Raphidocladius) autumnalis can be separated from other Gymnometriocnemus species by the following combination of characters in the adult male: body brown, dark brown; virga long and conspicuous with strong lateral sclerotization, anal tergite without dorsal anal point or ridge, gonostylus with convex outer margin and well-defined median crista dorsalis.Male adult (n = 4 unless otherwise stated). Wing length 1.30\u20131.52, 1.43 mm. Colouration completely brown, dark brown except for pale transverse bands posteriorly on abdominal tergites V\u2013VIII, narrower on tergite V.Head. Antennal ratio in Table Thorax. Antepronotum with 2 setae. Dorsocentrals 10-11; acrostichals 8-9, minute and difficult to discern; prealars 4-6; scutellars 2-5.1+2; R2+3 approaching costa at \u00bd distance between R1 and R4+5. Macrotrichia frequent on membrane in apical 1/3 of wing, absent from cell m, few (0-6) in cells cu+an. Veins Sc, R2+3, M, Cu, PCu and pseudovein without setae.Wing Fig. . Costa mLegs. Fore tibia with one spur, 40 \u00b5m long; mid tibia with two spurs ca. 20 \u00b5m long; hind tibia with well-developed comb and 2 spines, ca. 50 \u00b5m long. Fore leg ratios in Table Hypopygium Fig. . Ninth tFemale and immature stages unknown.PageBreakGymnometriocnemus (Raphidocladius) brumalis and Gymnometriocnemus (Raphidocladius) kamimegavirgus, but different in lacking an anal point or ridge on the anal tergite. This character is similar to characters reported for Gymnometriocnemusterrestris and Gymnometriocnemustairaprimus, but these two species can according to original descriptions be separated by having a higher AR (1.4 in Gymnometriocnemusterrestris) and a different shape of the superior volsella  autumnalis has so far only been recorded from the very north of mainland Norway and the Rondane mountains (1280 m a.s.l.) in Central Norway.The species is morphologically similar to volsella . The vironomidae . We haveTaxon classificationAnimaliaDipteraChironomidaePageBreakPageBreakGymnometriocnemus (Raphidocladius) brumalis cluster and all our specimens fit the description by Gymnometriocnemus (Raphidocladius) acigus from Gymnometriocnemus (Raphidocladius) brumalis (Gymnometriocnemus (Raphidocladius) brumalis can be regarded as separate species. We have examined the male holotype and a male paratype of Gymnometriocnemus (Raphidocladius) acigus and can confirm that the species fits our and Gymnometriocnemus (Raphidocladius) brumalis. The species has a Holarctic distribution.We have barcoded specimens from eastern, central and northern Norway that fall within the same genetic cluster although with quite large intraspecific divergence  , misidentifications e.g. .Gymnometriocnemus (Raphidocladius?) volitans (Goetghebuer) sensu Gymnometriocnemus (Raphidocladius) volitans (Goetghebuer) sensu Gymnometriocnemus (Raphidocladius) kamimegavirgus can be separated from other species of the genus Gymnometriocnemus by having well-developed, long virga (about the length of the gonocoxite); AR 0.9-1.1 (n=5); LR1 about 0.53-0.56 (n=3); wing membrane with setae at the apex only, occasionally with 1-2 setae proximally in cell an; R2+3 situated in the middle between R1 and R4+5; dark brown almost blackish thorax and head, slightly paler abdomen and legs.Gymnometriocnemusvolitans, and Sasa & Hirabayashi\u2019s description of Gymnometriocnemuskamimegavirgus except for slightly fewer setae on the abdominal tergites (Our examined material is from eastern, central and northern Norway, frequently collected near streams, rivers and moors. Male adults fit well with Brundin\u2019s description of tergites . The speGymnometriocnemusbrevitarsis is only known as female and therefore not included in the key.The species Gymnometriocnemus registered worldwide and the genus is present in all major biogeographical regions except Antarctica. Our findings through moderate sampling in Norway indicate that the number of species could be considerably higher also on a global scale and show that molecular data can be a great advantage in diversity assessments of targeted groups. Moreover, our study also highlights the importance of consulting type material for correct identification of Chironomidae if we are to avoid long term misconceptions of species.As a result of this study, there are now 17 species of"}
+{"text": "Cyclovirus to be discovered. This virus poses a significant potential threat to humans and poultry due to its global dissemination and infectiousness. We used three overlapping polymerase chain reactions (PCRs) to map the whole genome of AGV2. We then modelled the evolutionary history of these novel sequence data in the context of related sequences from GenBank. We analysed the viral protein characteristics of the different phylogenetic groups and explored differences in evolutionary trends between Chinese strains and strains from other countries. We obtained 17 avian-sourced AGV2 whole genomes from different regions of China from 2015 to 2016. Phylogenetic analyses of these Chinese AGV2 sequences and related sequences produced four distinct groups (A\u2013D) with significant bootstrap values. We also built phylogenies using predicted viral protein sequences. We found a potential hypervariable region in VP1 at sites 288\u2013314, and we identified the amino acid changes responsible for the distinct VP2 and VP3 groups. Three new motifs in the AGV2 5\u2032-UTR direct repeat (DR) region were discovered and grouped. The novel characteristics and diverse research on the AGV2 genome provide a valuable framework for additional research.Avian gyrovirus 2 (AGV2) was the second member of the viral genus  The AGV2 genome consists of ~2.38\u2009kb, with three partially overlapping open reading frames (ORFs): VP1 from nucleotides 953\u20132335; VP2 from nucleotides 450\u20131145; and VP3 from nucleotides 577\u2013951. These ORFs encode homologues of the chicken anaemia virus (CAV) viral protein, and these sequences have only ~40% nucleotide similarity with those of CAV34567Avian gyrovirus 2 (AGV2) was first reported by Rijsewijk In 2015, the virus was detected for the first time in a chicken flock and healthy humans in mainland ChinaWe obtained 17 avian-sourced AGV2 complete genomes, each of which had a length of 2376\u2009bp. These sequences had ~40.54% nucleotide similarity with CAV strain Cux-1 (M55918). However, they had very high levels, 93.8\u201399.3%, of nucleotide similarity with all six AGV2 whole genomes from GenBank  and much higher similarities with each other (95.7\u2013100%). Sequences of the virus were named using the format PPXXXX, where PP is the area and XXXX is the time of origin. We appended a suffix sequence number if further naming resolution was necessary. Sequences were deposited in GenBank under accession numbers KX708506\u2013KX708522 for whole genomes.Hind III and Bam HI endonuclease sites, suggesting evolution within the population. Eight sequences  had one Hind III site and two Bam HI sites; eleven sequences  had two Hind III sites and two Bam HI sites; two sequences (HLJ1506-1 and KJ452213) had no Hind III and two Bam HI sites; and two sequences (FR823283 and KU168250) had two Hind III sites and one Bam HI site.There were 179 mutations in the AGV2 whole genomes, of which 143 were found in more than one sequence (data not shown); the change rate was 7.53%. These mutations changed the number of important Phylogenetic analysis at the nucleotide level of the 17 avian-sourced AGV2 genomes sequenced in this study, together with that of all six (de-duplication) AGV2 and HGyV strains from different sources including the chicken , human  and ferret , revealed four distinct sequence groups , with bootstrap values of 100, 68, 100 and 87, respectively. The sequences in this study were the majority of the members in Groups A, B, and C . Group AAll VP1 sequences reported presently and obtained in this study are 1383\u2009bp in length. Based on the multiple alignments of these DNA sequences, the maximum divergence is 7.3%. An alignment of the predicted amino acid sequences yielded a maximum divergence of 2.7%. Three major groups were present in the VP1 protein sequence phylogenetic tree. Most of the virus sequences in this study were clustered in Group I, with three European sequences  and an avian-sourced sequence from Chile (JQ308212). Group II contained HLJ1508 and JL1511, one reference sequence from China (JQ690763), and one from Hungary (KJ452213). Group III contained two sequences: the first-discovered prototypic AGV2 from Brazil (HM590588) and another avian-sourced sequence from South Africa (KF436510) . Some prSubstitutions at certain positions determined the phylogenetic groupings of this virus protein. Minority characters at VP1 sites 154 (A to S), 288 (V to Q), and 242 (G to Q) belonged to Group II sequences. Substitutions at sites 212 (K to R), 242 (R to G), 270 (S to A), 310 (E to Q), 383 (P to Q), 401 (V to M), and 416 (L to I) were specific to sequences HM590588 and KF436510 of Group III.There were 15 amino acid substitutions in the VP1 protein , which on average is one substitution for approximately every 31 amino acids. We suspect that a hypervariable region is present in the middle and posterior of the VP1 protein, from sites 288 to 314. This region has 5 substitutions over its 26 amino acid length, which may be comparable to the CAV hypervariable region (sites 131\u2013165)The VP2 sequences reported presently and obtained in this study are 696\u2009bp in length, except for the ferret-sourced sequence KJ452213, which has a serine (S) insertion at site 162. The phylogenetic tree of VP2 at the amino acid level has four major groups. Unlike VP1, the VP2 sequences are well conserved within their major phylogenetic clusters; however, the inter-phylum branch lengths are relatively high . The VP2The majority of the same strains  constitute Group I. Substitutions at AGV2 VP2 sites 141 (R to Q), 156\u2013158 (GKR to RRG), 161 (Y to H), 174\u2013175 (EE to DD), and 179 (A to V) characterized Group III, which contained 2 new sequences (HLJ1508 and JL1511) and 1 reference sequence (JQ690763). Group IV contained 5 new sequences  and 2 reference sequences (KF436510 and HM590588) and had nearly the same substitutions as Group III, with additional substitutions at sites 14 (N to T) and 165 (A to T). Group II contained a unique strain (KJ452213) that had a T to S substitution at site 167 and a serine (S) insertion at site 162 .Almost all the novel VP3 predicted protein sequences were 124 amino acids in length, except for the unique sequence KJ452213, which has an arginine (R) insertion at site 122. Based on their bootstrap values, the VP3 sequences fell into five groups . The VP3The amino acid variations observed in the sequences occurred at 9 significant sites  at both the N- and C-termini, and two amino acid orders at these sites were the basis of the grouping. All fourteen VP3 members of Group I contained the homologous sequence R-Q-S-V-L-A-K-Q-N . Group V contained two reference sequences and five new VP3 proteins with the sequence Q-R-C-A-S-S-R-R-E , but other sequences did not follow this pattern.th Type-a base (C to T) as follows: 5\u2032-GTACAGGGGGGTACGTCATCAT-3\u2032. The Ending DR was the last DR in the string, and it differed at its 3\u2032 terminus from the other DR types: 5\u2032-GTACAGGGGGGTACGTCACAGC-3\u2032. The last four bases of the Ending DR form the first half of a 7-bp linker: 5\u2032-AGCCAAT-3\u2032 (The 5\u2032-untranscribed region (5\u2032-UTR) of AGV2 is by definition located between the canonical polyadenylation site (AATAAA) and the start of transcription1314CCAAT-3\u2032 .Based on the structural characterization of the AGV2 repeat units, six groups of DR region were classified. The combination of Type-b DR occupies the third and the first places and is defined as Group A and Group B, respectively. Group E is the combination of two Type-b DRs occupying the third and fourth places. Groups C, D, and F were three novel groups discovered in this research. The newfound Group C had two Type-b DRs as the third and fifth components, yet Group D had no Type-b DR and Group F had two Type-b DRs occupying the first and fifth places .Group A  and Group B  made up more than half of all DR regions, while Group E  and the three novel groups  made up lower percentages . The numThis report is the first to characterize AGV2 genomes originating from diverse geographic locales around the world and different hosts, and it provides significant additional AGV2 genomic information for the six unique complete genomes, one complete coding sequence (CDS) and one partial CDS previously retrievable from GenBank. This study provides a basis for further research on the relationships between the genetic element structure and virus phenotype.The complete genomes that we report here are each 2376\u2009bp in length, while all of the other sequences in GenBank are approximately 2380\u2009bp in length. The length of the poly C and G regions upstream of the 5\u2032-UTR account for this discrepancy. The phylogenetic analysis of the CDS at the amino acid level did not perfectly match the groupings based on the nucleotide sequences. This mismatch may be due to synonymous mutations; AGV2 isolates should be almost identical at the amino acid level, despite differences at the nucleotide level.th amino acid of VP3 (threonine in NX1506-1 and isoleucine in NX1506-2). The other mutation at nucleotide 157 of the 5\u2032-UTR in HLJ1603-1 and HLJ1603-2 was between a cytosine and a thymine. These observations indicate that these two couples are the same strains with a few mutations, and the consequences of these relationships merit further investigation.All the sequences that we obtained were highly similar. The pairs NX1506-2 and BJ1509 and HLJ1506-2 and GS1512 had particularly high pairwise similarity, but entirely different backgrounds. The same sequences were detected at different times and places, meaning that they may be epidemic strains in China or that some transmission carried them from one place to another during this time. The vaccine may be a reason for the observed similarities, in light of the role of AGV2 as a contaminant in poultry vaccinesThe epidemic situation of AGV2 varies throughout the world. The sequences in this study had similar substitutions as variants from southern Brazil and the NetherlandsThe study of the virus capsid protein VP1 may yield virus information related to the receptors and neutralization of the antigenic epitope, as occurred for CAVAGV2 VP2 may have a function similar to CAV VP2. The protein phosphatase activity of CAV VP2 is important but is not absolutely required for replicationSome important functional domains were predicted in AGV2 VP3, such as apoptin, which is known to translocate to the nucleus in transformed cells; this domain plays a role in the induction of apoptosis in these cells20The 5\u2032-UTR must be important for gene expression and regulation in AGV2. Although the detailed function of the DR region in 5\u2032-UTR is unknown, the disruption of the relative spacing of the DR region with other promoter elements and the start of transcription by the insertion of a 7-bp linker decreases the rate of virus replication in cultures of CAVIn this study, a high homology of AGV2 was observed within different hosts, including chicken, human and ferret, on the nucleic acid and protein levels. One possibility is that not enough various-sourced sequences could be referred; another potential reason is that AGV2 might infect different hosts.Cyclovirus.This study provides a basis for future research on the pathogenicity and genomic function of AGV2. We are dedicated to further unveiling the virology of AGV2 and All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The animal experiments were performed in strict compliance with the Guideline for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People\u2019s Republic of China. The Committee of the Ethics of Animal Experiments at the HVRI, CAAS, also approved the animal experiment protocols.via specific PCRIn the epidemiologic investigation from April 2015 to April 2016 by our group, 55 of 448 clinical specimens from suspected sick fowl or fowl embryos  tested positive \u00ae). Total DNA was extracted from the tissue homogenates as follows. One hundred microliters of tissue homogenates was lysed in 200\u2009\u03bcL of tissue lysis buffer . The DNA was precipitated with absolute ethanol, washed with 70% ethanol, and dried at room temperature. Subsequently, the DNA was resuspended in nuclease-free water and stored at \u221270\u2009\u00b0C.Clinical samples were oscillated and broken to obtain tissue homogenates as follows. Then, 300\u2009mg of tissues was put into a 2-mL Ep tube with 500\u2009\u03bcL of PBS and two high-pressure steam sterilization small steel balls were added, and then the solution was oscillated for 3\u2009min twice to break the tissues at a frequency of 29\u2009Hz  in a 50-\u03bcL total reaction volume, using an automated thermal cycler . These reactions ran under the following program: initial denaturation of 98\u2009\u00b0C for 5\u2009min, followed by 30 cycles of denaturation at 98\u2009\u00b0C for 10\u2009s, annealing at 60\u2009\u00b0C for 15\u2009s, and extension at 72\u2009\u00b0C for 10\u2009s; the final extension was carried out at 72\u2009\u00b0C for 7\u2009min. The PCR products were analysed by 1.5% agarose gel electrophoresis, and imaged using a BIO-RAD Universal Hood II system. In all PCR reactions, an AGV2 isolate previously identified in our laboratory was used as a positive control, and the PCR mixture was used as a negative control. The 3 regions were purified using an agarose gel with the AxyPrep\u2122 DNA Gel Extraction Kit (AXYGEN). DNA extraction and PCR were performed at least twice for each sample.\u00ae-T Easy vector , and the ligated products were transformed into Escherichia coli subcloning efficiency DH5a competent cells . Colonies that contained DNA inserts of the correct size were picked and grown overnight in 3\u2009mL of ampicillin-containing lysogeny broth (LB) liquid medium. The mini-preparation of plasmid DNAs was performed with a plasmid extraction kit , following the manufacturer\u2019s instructions. The plasmid DNAs were sequenced by Comate Bioscience Co., Ltd. .In the case of sequencing failure, the specific PCR products of the 3 regions were subcloned into the pMD18Contig assembly was performed using Seqman in Lasegene . Sequences were aligned using ClustalWHow to cite this article: Yao, S. et al. Novel characteristics of the avian gyrovirus 2 genome. Sci. Rep.7, 41068; doi: 10.1038/srep41068 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."}
+{"text": "This study seeks to better understand the human-nature interface and to measure the variability of plant use knowledge among cultures, through inter- and intracultural analyses. We compared plant collection, use, and management of two culturally distinct groups (Baitadi and Darchula) of the Nepal Himalaya. They inhabit different physiographic regions, yet share the same ecological landscape, environmental resources, and livelihood challenges. We hypothesized that the elderly, native, and traditional healers living in remote and rural places possess more diverse and detailed knowledge of plant use and conservation than young, non-native, and non-healers.A total of 106 people were contacted for interviews, and 100 (68 men and 32 women) agreed to share ethnobotanical, demographic, and socioeconomic information. They were asked about the three most important plants for their socioeconomic benefit, culture, primary health care, and livelihood.p\u2009<\u20090.001) attributed to the cultural heritage of the area. The low consensus of plant use  between Baitadi and Darchula district could be due to cultural divergence, varied accessibility, physiographic heterogeneity, and biodiversity uniqueness.The knowledge of plant collection, use, and its transfer was strongly associated with the cultural heritage whereas the ecogeographical condition influences the ways in which plants are collected and used. The divergent knowledge of plant collection, use, and transfer between the participants of Baitadi and Darchula was significantly  contains supplementary material, which is available to authorized users. Human communities that inhabit remote and rugged ecosystems use diverse livelihood strategies such as utilizing different ethnoecological environments , 2 definThe knowledge and practice associated with the collection and uses of plants vary within any culture, because of the abundance and quality of species, geography of the region, origin of the plants, residence of the people, social status, and relationships within the community , 19\u201322. Here, we compared the knowledge of plant\u00a0collection and use of two subculturally distinct groups inhabiting different physiographic regions within the same ecological landscape with access to similar environmental resources. Cross-cultural studies were parsimoniously studied before 1998 , but nowThe Kailash Sacred Landscape (KSL) is a trans-boundary landscape comprised of parts of the southwestern Tibetan Autonomous Region of China and adjacent parts of northern India and northwestern Nepal . At its Baitadi and Darchula, the study districts (29\u00b0 22\u2032 N to 30\u00b0 15\u2032 N/80\u00b0 15\u2032 E to 81\u00b0 45\u2032 E) located at the westernmost end of the country, represent far-western Nepal bordering India and China , and others (10%). Chhetri and Brahmin are relatively privileged groups with the highest well-being index [Dalit are receiving reserved access and opportunities provided by the Nepal Government, they are still disadvantaged due to the sociocultural and class system [Dalit. Brahmin, Chhetri, and Dalit predominate in Baitadi District. Agriculture, wage labor, medicinal plant collection and trade, and traditional healing are the major occupations in Baitadi [There are more than 30 ethnic and 10 minority groups in the study area including the indigenous groups Byanshi and Santhal in Darchula and Kusunda, Dom and Dhanuk in Baitadi . Chhetring index . Even ths system , 72. The Baitadi , 73\u201375;  Baitadi \u201379. Bait Baitadi . Sacred  Baitadi , and loc Baitadi . The leg Baitadi . As a re Baitadi , 84.Abies poles for mounting prayer flags in the yard. Abies pindrow Royle (Himisin) is a very good resource as prayer flag, fuelwood, furniture, butter churners, medicine,\u00a0and agricultural implements in mountain communities. Other temperate-alpine medicinal plant species Angelica archangelica L. (Gannanu), Dactylorhiza hatagirea (D.Don) Soo (Hathajadi), Neopicrorhiza scrophulariflora (Pennell) Hong (Katuko), Ophiocordyceps sinensis (Berk.) G.H.Sung (Yartsagumba), etc. are used and conserved in Darchula District for a long period [The Byanshi is a Tibeto-Burman minority group with a population of about 4000 in the country. About 500 live in Chyanrung and Byansh villages and about 500 in Rapla, Shitola, and Khalanga villages . They arg period .O. sinensis), Jimbu (Allium hypsistum Stearn), and Satuwa (Paris polyphylla Sm.). This trans-boundary trade and transhumance are important livelihood strategies [The average landholding of a Byanshi family in Darchula District is small and of poor quality, feeding a family only 3 to 4\u00a0months in a year . Faminesrategies , 90.Forest resources and alpine pastures complement the mountain agricultural system in both study districts, meeting fuel, fodder, timber, and medicinal needs. Fuelwood is commonly used for heating and cooking. Agriculture, livestock, woolen products, and the medicinal plant trade are four major livelihoods maintaining household economies. Because of the remote location, reliance on traditional medicine is associated with wild medicinal plants and on the harmonious existence of spirit and matter. Many traditional healers worship and pray to plants before collecting them, acknowledging the spiritual powers of the vegetation , 92. TheInformed consent forms were obtained from all oral interview participants written in accordance with the FAU IRB and Nepal research protocols. Inventory-based interviews  were carDalit caste group were interviewed. There were 57 participants including 47 Chhetri, 6 Brahmin, and 4 Dalit from 9 villages of Baitadi District and 43 participants including 11 Chhetri, 8 Brahmin, and 24 Byanshi from 12 villages from Darchula district. Informal discussions were held during the evenings while staying with local communities, and sometimes with tea vendors. Tea shops are excellent arenas for observing interactions between communities and discussion of open-ended questions [A total of 106 people were contacted for interviews, and 100 (68 men and 32 women) agreed to share ethnobotanical, demographic, and socioeconomic information. A two-page semi-structured questionnaire was developed in Nepali script prior to the start of fieldwork and administered for interviews. The interviews were carried out during three field visits, the duration of each trip lasting about a month between February and September 2017. A total of 100 participants including 58 from Chhetri, 14 from Brahmin, 24 from Byanshi, and 4 from uestions .The Plant List (Retrieved from www.theplantlist.org).In the interview, the participants were asked to list the three most important plants for each category, e.g., socioeconomic benefit, culture, primary health care, and livelihood. Demographic information was collected for each participant including socioeconomic status, age, occupation, education, family size, livestock, land ownership, where they migrated from, languages they speak, length of residence, distance of home from district center, nearest health post, and forest. Interviews were supplemented with other investigative techniques, such as participant observation, walk-in-the-woods interviews, and informal meetings . InterviMatching information (use reports) from at least three respondents was considered a common response for quantitative analysis . To dete species . IASc va species .The frequency of citation of a specific use, that is, the number of individual use reports (nur) for a type of use category, serves to establish the consensus across the respondents . The culies used . After aDalit).We grouped the socioeconomic and demographic data into nominal/categorical variable: (1) gender, (2) education, (3) occupation, (4) livelihood type, (5) access to opportunity, (6) food availability, (7) languages spoken and continuous variable: (1) age, (2) household size, (3) livestock size, (4) land size, (5) length of residence, (6) years of healing practice, (7) distance from home to forest, and (8) distance from home to health post. Cross-cultural analysis was made by (1) gender: male and female, (2) education: literate and non-literate, (3) occupation: traditional healers and non-healers, (4) livelihood type: suburban, hill, and sedentary (Baitadi District); rural, mountain and semi-nomadic (Darchula District), (5) food availability: <\u20096 and >\u20096\u00a0months, (6) language spoken: two languages spoken and more, and (7) access to opportunity: privileged group (Chhetri and Brahmin) and under-privileged  whereas that of Darchula District was 517 (12 person\u22121) . In order to investigate the relationship between plant knowledge and consensus at the group  level, plant species were ranked according to their IASc value. In Baitadi, an IASc value >\u20090.5 was obtained for five species: Prunus cerasoides, A. pindrow, Chrysopogon aciculatus (Retz.) Trin., Bergenia ciliata (Haw.) Sternb., and Quercus lanata Sm. whereas only three species: Angelica archangelica, N. scrophulariflora, and P. polyphylla were recorded as the highest IASc value >\u20090.5 species in Darchula  in Darchula District  with high IASc value (>\u20090.5) were important in Baitadi District. Of the five >\u20090.5 IASc species in Baitadi, only one species B. ciliata was used as medicinal, and the rest four were used for sociocultural purposes. A. pindrow was valued as a timber/wood species, Q. lanata as a fire-wood and fodder, C. aciculatus as forage for livestock feeding, and P. cerasoides for ritual ceremonies. The species appeared in quadrant \u201cA\u201d were highly consented in both districts whereas those that appeared in quadrant \u201cC\u201d were insignificant in uses and consensus. None of the species was highly consented in both district indicated that the use value of plant species was specific to the district groups and culturally divergent.Analysis of use value of plants in two different cultures revealed that three species ict Fig.\u00a0, right. p\u2009<\u20090.001\u20130.03) for both MUR and OUR   from participants who moved there more recently. Higher plant use knowledge was also associated with the participants who had a larger number of livestock (p\u2009<\u20090.001) and greater land size (p\u2009<\u20090.001) and family member (p\u2009=\u20090.001); however, the latter was insignificant (p\u2009=\u20090.148) to OUR. The settlement: distance from home to a health post (p\u2009<\u20090.001) and home to forest (p\u2009=\u20090.002) was positively associated to MUR; however, the OUR knowledge was insignificant to the distance of home to the forest. Unlike other studies, age of the participants did not show any statistically significant variation (p\u2009=\u20090.55) on means of the knowledge of plant use at all (Table\u00a0p\u2009=\u20090.028). Mixed result was obtained when the participants were categorized into healers and non-healers by their occupation and assessed their knowledge of MUR (p\u2009=\u20090.17) and OUR (p\u2009<\u20090.001). Though healers were more knowledgeable on MUR (7.79), it was statistically insignificant (p\u2009=\u20090.17). They also provided less knowledge about OUR (5.83) and UUR (1.99) than the non-healer respondents.The participants living in the study area for generations possessed the highest knowledge of plant use, which was significantly different (ll Table\u00a0. Contrarp\u2009<\u20090.001) for both MUR and OUR. The latter four were significant for OUR and UUR. Even though participants responded differently about the plant recognition, identification, collection, and uses [In the study area, the variation of plant use knowledge was closely related to the categorical variables: livelihood, access to opportunity, gender, occupation, and language spoken by the participants, among which livelihood was significant  among the participants of Darchula. All three species were used as medicine. The use of N. scrophulariflora against fever and headache was folkloric with higher IASc in KSL, Nepal, supported by the earlier findings [P. polyphylla was considered as a common antidoting plant and A. angelica as effective herbal for indigestion in traditional medicine. Species A. pindrow, B. ciliata, C. aciculatus, P. cerasoides, and Q. lanata received the highest IASc (>\u20090.5) among the participants of Baitadi. All species except B. ciliata were used for livelihood and ritual uses. P. cerasoides emerged as important for participants for ritual uses. Other ritual species with less IASc (<\u20090.5) reported in this study were Ficus religiosa\u00a0L., Mangifera indica\u00a0L., Butea monosperma (Lam.) Kuntze, and Selinum wallichianum (DC.) Raizada & H.O. Saxena. All these species have been previously reported as ritual and culturally valuable in KSL, India [Aegle marmelos (L.) Correa, F. religiosa, P. cerasoides, M. indica, and P. emblica L.\u00a0are cultural and religious plants of Nepal [Panch pallav  and used them ritually during three major events of life: birth, marriage, and funeral ceremonies. Besides these, Artemisia indica Willd. (Kurjo), Ocimum tenuiflorum L. (Tulsi), and Syzygium cumini (L.) Skeels (Jamun) were also reported as ritual plants in this study.The species with high consensus (>\u20090.5) were greatly varied at use types and district level. Out of 122 useful botanical taxa documented in this study, only three species findings , 116. P.L, India , 115. Aeof Nepal . ParticiA. pindrow, C. aciculatus, and Q. lanata from Baitadi District were regarded as useful for livelihood as wood, fuel, and fodder. Since we considered the Baitadi District as relatively more accessible than Darchula, all the useful plants with high consensus of the district are associated with the accessibility and availability. Of the five high IASc (>\u20090.5) species, F. religiosa, P. cerasoides, and Q. lanata are abundant at nearby settlements. The high consensus value species were not only being frequently used in the study area, they were also reported as highly useful in other villages of study districts [Other high IASc species such as istricts , 118. Ofistricts  and otheistricts , 120, whThe result of IASc was supported by informant consensus factor FiC. Naturally, both assess the consensus; however, the first assesses the consensus at species level whereas the latter evaluates at use types/category. The highest FiC (0.87) was reported for sociocultural livelihood use  followed by 0.85 for cultural/ritual uses in Baitadi District. In contrast, the highest FiC value (0.83) was recorded for medicinal use in the treatment of gallstone and endocrine aliments in Darchula district. Higher FiC values indicate the consent of informants on the specific use of a plant in a traditional use system . The ave\u22121, the tradition of wild plant collection, use, and management was a common strategy to combat poverty paired with geo-ecological constraints and sacredness of the landscape, resulting in insignificant difference between the groups of people with food deficiencies. The role of plant collection in Darchula and Baitadi districts in complementing food availability was appreciated [The frequent use of medicinal plants to cure ailments could be attributed to the high preponderance of digestive and infectious disorders in the area. This account aligns with the government report , 122 stareciated . The lowWe found a cultural distinction in collection, use, and conservation of plants. Mountainous pastoral communities of Darchula people often collect plants from remote areas for medical ethnobotany. They reported that their household economy was complemented by 8.13\u2009\u00b1\u20094.75% from the sales of medicinal plants of rural undisturbed areas whereas less (5.96\u2009\u00b1\u20095.46%) was reported in Baitadi District. The sedentary hilly farmers of Baitadi District value plants more for social, ritual, and livelihood, and often generalist collectors from nearby areas of settlement forage there. Conversely, the forage from distant forest areas in Darchula was associated with quality products, traditional medicine, and elder healers. Since the Darchula people are occupational traders of medicinal plants, they have long been foraging medicinal plant products from remote and relatively undisturbed areas and selling them to lowland groups and to India and China for food grains. While collecting medicinal plants, many collectors worship and pray to plants and acknowledge the spiritual powers for quality products  because Allium wallichii Kunth (Ban lasun), A. hypsistum (Jimbu), B. ciliata (Vedaite), Delphinium denudatum Wall. ex Hook.f. (Nirmasi), P. polyphylla (Satuwa), Nardostachys grandiflora DC. (Jatamansi), N. scrophulariflora (Katuko), O. sinensis (Yartsagumbu), Zanthoxylum armatum DC. (Timur), etc. The pursuit of collection, bartering, and trade of medicinal plants was dated back to the 1960s [B. ciliata, S. chirayita, and Valeriana jatamansii Jones were collected for trading. Nepal Government records (2000\u20132016) show that S. chirayita was traded 3.6\u00a0tons in 1999, 4\u00a0tons in 2008, and 2.7\u00a0tons in 2016 from both districts [Asparagus racemosus Willd., Berberis asiatica Roxb. ex. DC., and Cinnamomum tamala (Buch.-Ham) Nees & Eberm. were also collected from both districts in 1999 for marketing purpose [O. sinensis from remote alpine pasture in Darchula District gathered 140\u00a0kg in 1998 to 1440\u00a0kg in 2004 [People often trade medicinal plant products such as he 1960s , 10, 126he 1960s , and thehe 1960s , 73, 84.istricts . Other l purpose . The hou in 2004 , 4500\u00a0kg in 2004 , and 500Such directed and culture mediated collection and use of plants for personal gain  over communal incentives  yield a direct impact on resource conservation. Persistence of intercultural divergences occurred in specialized medicinal and ritual uses of plants or trade of certain species. The distinction of knowledge of cultural groups substantiates the hypothesis that cultural differences play an important role in the transfer and maintenance of indigenous knowledge. Despite the persistence of different strategies for collecting and utilizing plants, the geographic adversities have strengthened a homogenous strategy of employing locally available plants for livelihood and culture. The way of life in rural hills/mountains involves adjusting to the difficult environment, food deficits, and limited accessibility, with appropriate strategies . Thus, tp\u2009\u2264\u20090.001) were positively associated with the ethnomedicinal knowledge of plant use. The greater length of residence in an area helps in accumulating greater vast knowledge required to use the resources wisely [\u22121). This supports the tenet of a positive association between longer residence and greater knowledge.An intracultural difference of plant use knowledge at the individual level was evident with the cultural heritage. Participants\u2019 years of residence in the study area, the size of their livestock herd and land, family size, and the time required to access a health post . O. sinensis that grows well in alpine pastures is folkloric as a tonic and antipyretic in Darchula. This could be one reason that local communities forage the wild and distance sites for quality products regardless of the distance and geo-ecological constraints. While herding, summer grazing, and ascending for the collection of medicinal plants, local people share knowledge of plant identification, collection, uses, and management. Horizontal transmission of knowledge among shepherds and herders while herding and transhumance contributes to a high level of knowledge sharing, thus aiding conservation knowledge. Cultural values often aid the knowledge of plant use and collection in high altitude areas [Limited access to health centers compels people to use local resources for their primary health care, inferring the role of geography. IASc >\u20090.. O. sinede areas . In our de areas . The sizde areas , 133 whide areas . Househode areas , 135. Hode areas .p\u2009=\u20090.46\u20130.95) among them; however, the age group 40\u201359\u00a0years possessed the higher MUR 8.19\u2009\u00b1\u20090.53 comparable to MUR 7.2\u2009\u00b1\u20090.79 of the >\u200980\u00a0years age group. A non-existent relation (p\u2009=\u20090.32) was already reported from adjoining villages of our study sites [p\u2009=\u20090.34) [p\u2009=\u20090.023) and was higher (6.72\u2009\u00b1\u20095.2) among the male participants. This result was supported by the gendered division of labor where males are often engaged in summer grazing, livestock herding, and extraction of plants from inaccessible sites that would take them to relatively remote and distant sites. Pfeiffer and Butz [OUR and UUR were insignificant along age whereas the MUR was slightly significant indicating that the knowledge of medicinal plant use insignificantly decreased as age increases. Our results indicated that plant use knowledge was less varied among the participants because in situ transmission is underway when needed as described by Phillips and Gentry  and Panidy sites  and semi\u2009=\u20090.34) . Earlier\u2009=\u20090.34) . Guimbo \u2009=\u20090.34)  found thand Butz  reportedand Butz  and are Baidhya tradition\u2014a local healing tradition in far-western Nepal [A weak association of age and plant use knowledge could be attributed to the (non-random) selective samples and the unwillingness or inability to share information explicitly by the elderly groups. The secrecy of medicinal plant knowledge is a common practice in different parts of the world , and it rn Nepal , 75, 91\u2014rn Nepal , whereasrn Nepal .p =\u00a00.06) whereas the specialty knowledge like use of medicinal plants for ailments  and unique use reports  were transmitted through closed and vertical sharing with directed and dedicated apprenticeships under the tutelage of senior practitioners, resulting in constrained transfer. There were 75% (57 out of 77) traditional healers that learned medicinal plant use knowledge from their parents and grandparents , whereas 25% healers learned themselves or from peer healers . Vertical knowledge transmission is often associated with family members and the sharing of secretive knowledge [General knowledge such as OUR was commonly shared among communities through cultural learning . MUR was also influenced by the literacy level of participants . Higher knowledge of MUR among the non-literate participants could be attributed to their direct association with forest and natural resources and frequent and first choice of traditional and home-based medicines for ailments. Thorsen and Pouliot [p\u2009<\u20090.001).The participants who are non-literate had also significant UUR  between the participants of different livelihood type: semi-nomadic communities from rural mountains of Darchula (MUR 8.69\u2009\u00b1\u20093.01) and sedentary communities with the suburban setting of Baitadi (MUR 6.75\u2009\u00b1\u20092.67). Most of the people from Darchula District inhabiting in remote areas are occupational medicinal plant collectors and traders and do summer grazing, travelling during transhumance, and often seek high-value medicinal plants for trading purposes. The underprivileged groups  were knowledgeable on MUR (8.07\u2009\u00b1\u20093.2) however insignificant (p\u2009=\u20090.27). They were less knowledgeable on general use reports  and unique use report 1.60\u2009\u00b1\u20091.37, p =\u00a00.045). The Dalit are disadvantaged groups of the country and have limited access to the natural resources in Darchula District because of the sociocultural and caste system [p\u2009<\u20090.001). OUR was significantly different (p\u2009<\u20090.001\u20130.023) for five out of seven categorical variables: access to opportunity, gender, occupation, language spoken, and livelihood type. The higher mean values of OUR 7.38 and 8.92 compared to 3.60 and 2.88 respectively of privileged groups and from sedentary communities of Baitadi District indicated that the communities living with more amenities and better privilege were more knowledgeable about general non-medicinal uses (OUR) such as uses for livelihood and rituals and divergent from Darchula people.Among three response variables, MUR and OUR were significantly different  and unique use reports different from peers (UUR) was transmitted through closed and vertical sharing with directed and dedicated apprenticeships under the tutelage of senior practitioners. The regional particularities\u2014geographic  and cultural \u2014seem relevant in explaining the differences in plant use knowledge in Baitadi and Darchula districts, Nepal. These results contribute to a growing body of literature that expands our understanding of patterns of knowledge of useful plants across culture and geography.Additional file 1:Species use reports and IASc at district level. (XLSX 31\u00a0kb)Additional file 2:Socioeconomic variables and their analyses. (CSV 13\u00a0kb)"}
+{"text": "Autologous airway epithelial cells have been used in clinical tissue\u2010engineered airway transplantation procedures with a view to assisting mucosal regeneration and restoring mucociliary escalator function. However, limited time is available for epithelial cell expansion due to the urgent nature of these interventions and slow epithelial regeneration has been observed in patients. Human airway epithelial cells can be expanded from small biopsies or brushings taken during bronchoscopy procedures, but the optimal mode of tissue acquisition from patients has not been investigated. Here, we compared endobronchial brushing and endobronchial biopsy samples in terms of their cell number and their ability to initiate basal epithelial stem cell cultures. We found that direct co\u2010culture of samples with 3T3\u2010J2 feeder cells in culture medium containing a Rho\u2010associated protein kinase inhibitor, Y\u201027632, led to the selective expansion of greater numbers of basal epithelial stem cells during the critical early stages of culture than traditional techniques. Additionally, we established the benefit of initiating cell cultures from cell suspensions, either using brushing samples or through enzymatic digestion of biopsies, over explant culture. Primary epithelial cell cultures were initiated from endobronchial biopsy samples that had been cryopreserved before the initiation of cell cultures, suggesting that cryopreservation could eliminate the requirement for close proximity between the clinical facility in which biopsy samples are taken and the specialist laboratory in which epithelial cells are cultured. Overall, our results suggest ways to expedite epithelial cell preparation in future airway cell therapy or bioengineered airway transplantation procedures. Fewer epithelial colonies were generated in CM+Y than in direct 3T3+Y co\u2010culture but separation of epithelial cells from feeder cells by a transwell membrane allowed colony formation that was comparable with direct co\u2010culture Figure\u00a0d. This iFinally, as organoid derivation was recently reported from cryopreserved primary tumours  or by collecting cells by brush biopsy . We estimate that isolation of cells from a cell suspension in 3T3+Y could reduce the time required for epithelial cell expansion to 3\u20134 weeks in a case where only basal cells were to be transplanted on an adult tracheal scaffold. Colony formation assays suggested that airway epithelial cells required a continual supply of feeder products that was not recreated by feeding three times per week with 3T3\u2010J2\u2010conditioned medium: when cell\u2013cell contact was prevented by adding 3T3\u2010J2 feeder cells on a transwell membrane, epithelial colony growth was comparable with growth in direct co\u2010culture. Notably, our study revealed that patient tissue can be cryopreserved prior to the initiation of cell cultures. Although cryopreservation did slow the initiation of epithelial cell cultures, this finding could remove the necessity for specialist clinical and research facilities to be in close proximity in future airway and mucosal tissue\u2010engineering clinical procedures. Current protocols limit the number of sites capable of performing autologous airway cell therapies to those with both appropriate clinical and research facilities, but samples could be cryopreserved for transportation, expanding the potential application of these techniques. Overall, our findings suggest methods to facilitate cell preparation in future bioengineered airway transplantation procedures.To conclude, direct explant expansion of human airway epithelial cells in 3T3+Y culture conditions improves the early stages of culture compared with BEGM (88% culture success, approximately 400 000 cells after 12 days vs. 50% culture success, <250 000 cells after 2 weeks; Butler et al., The authors declare no conflicts of interest.Data S1. Supplementary MethodsThe following supporting information may be found in the online version of this article:Click here for additional data file."}
+{"text": "In this work, a silicone oligomer involving amino groups is employed to crosslink a non-crystallized poly(vinylidene fluoride-chlorotrifluoroethylene) matrix bearing double bonds (P(VDF-CTFE-DB)) via addition reaction. Thanks to the flexible silicone molecules, the modulus of the hybrids is reduced over 30% when compared with the pristine matrix. Most interestingly, the \u03b5r of the hybrids is improved to nearly 100% higher than that of the matrix when the silicone content reaches 30 wt %. This may be due to the dilution effect of silicone molecules, which favors macromolecular chain rearrangement and dipole orientation of the hybrids under an applied electric field. As a result, electric-field activated displacements of the above hybrid increases to 0.73 mm from 0.48 mm of the matrix under 60 MV/m. The maximum electric field-induced thickness strain increases from 1% of the matrix to nearly 3% of the crosslinked hybrid. This work may provide a facile strategy to fabricate PVDF-based hybrids with enhanced electromechanical performance under low activating voltage.Poly(vinylidene fluoride)-based ferroelectric polymers have large and tunable dielectric permittivity ( Sz) could be determined by polarization (P) under the electric field and Young\u2019s modulus (Y) of the elastomer, e.g., \u03b50 and \u03b5r are the vacuum permittivity (8.85 \u00d7 10\u221212 F/m) and dielectric permittivity, respectively, and E is the electric field applied on the film sample [\u03b5r/Y is defined as an electromechanical efficiency, which characterizes the energy conversation efficiency. Apparently, the higher the \u03b5r/Y, the larger the Sz may be realized under the given electric field, which means the DEs with high \u03b5r and low Y values are favorable to obtain large electromechanical strain. Meanwhile, a low activation electric field (Ea) is favorable for elastomers, especially for the wearable devices and medical instruments applications, where the high voltage is prohibited due to intimate contact with the human body.As an important branch of electro-active polymers (EAPs), dielectric elastomer (DE) has the merits of high electromechanical efficiency, large deformation, and fast response times, which has potential application in the fields of artificially intelligent and wearable devices, such as actuators, nano-generators, and biosensors ,5,6,7,8.m sample ,10,11. A\u03b5r ranging from 10 to 100, along with rather high breakdown strength (Eb) (100~300 MV/m). Meanwhile, their excellent comprehensive properties, including electrical activity, chemical and weather resistance, as well as biocompatibility, assure their applications for actuators dealing with high power  and sensors working under severe circumstances (high pressure or high irradiation) [Y of 0.3~0.4 GPa for their semi-crystalline structure, thus delivering a relatively low electromechanical efficiency (\u03b5r/Y). The reported study is mostly focused on improving \u03b5r to achieve high electromechanical performance [\u03b5r fillers, such as graphite, carbon black, or ferroelectric ceramics  are introduced into well-known elastomers, such as poly(acrylates), silicone elastomers, and polyurethane (PU) [\u03b5r particles inevitably causes the increase of Y and the reduced Eb, thus, the undesirable reduction of electromechanical efficiency. Therefore, how to adjust the \u03b5r/Y value of FPs reasonably is crucial to realize high electromechanical performance.Fluoropolymer (FP) plays an increasingly important role in the building, automobile, and other industries because of its excellent performance of self-flame-retardancy, self-cleaning, low friction coefficient, high corrosion resistance, and high temperature resistance. Poly(vinylidene fluoride) (PVDF)-based fluoropolymers have been well studied as electrostrictive EAPs for their ferroelectric properties and multiple crystalline morphologies, which results in a tunable diation) ,13,14,15formance ,18,19,20ane (PU) ,27,28,29Y of high-\u03b5r FPs is the other option to improve \u03b5r/Y. In the present work, an amorphous fluoropolymer matrix (P(VDF-CTFE):poly(vinylidene fluoride-chlorotrifluoroethylene)) with relatively low modulus is chosen as the pristine matrix, where the desired -CH=CF- unsaturation is inserted by a dehydrochlorination process catalyzed with triethylamine (TEA) as active sites. A silicone oligomer capped with NH2 groups (NH2-PDMS-NH2) is introduced by the addition reaction between amine and -CH=CF- units, which results in not only the anchored silicone oligomer, but also the cured matrix. As desired, the modulus of the hybrid polymer is significantly reduced thanks to the addition of flexible silicone cross-linkers. Most interestingly, the dielectric permittivity of the hybrid is enhanced nearly 100% compared to the pristine matrix. Both the reduced Y and the improved \u03b5r are responsible for the tripled strain of the optimized hybrid DEs. This work may provide a facile strategy to fabricate PVDF-based dielectrics with large electromechanical strain and low actuating voltage.In addition to increasing the permittivity of the elastomers, the decreasing Mn = 120,000 g/mol, polydispersity index (PDI) = 2.20) was provided by Zhonghao Chenguang Research Institute of Chemical Industry . Low molecular weight silane oligomer with amino groups (NH2-PDMS-NH2) was purchased from Silok Chemicals Co. Ltd.  and used as received. Triethylamine (TEA), ethyl acetate (EAc), N-methyl pyrrolidone (NMP) and anhydrous ethanol were provided by Tianjin Chemical Reagent Co. Ltd. .P(VDF-CTFE)  bearing double bonds (P(VDF-CTFE-DB)) (VDF/CTFE/DB = 68/8/12 in molar ratio), was synthesized from P(VDF-CTFE) (VDF/CTFE = 80/20 in molar ratio) by a controlled dehydrochlorination process as follows : Into a Silicone oligomer crosslinked P(VDF-CTFE-DB) hybrid films with a thickness of ~50 \u03bcm (+/\u2212 5 \u03bcm) were prepared via a typical solution cast process ,32 on glFor expression convenience, the materials and hybrids used in the present work are indicated by abbreviations as listed in \u22121 and 16 scans. X-ray diffraction (XRD) measurement was conducted on a Rigaku D/max 2400 diffractometer  with the X-ray wavelength of 1.542 \u00c5 , the 2\u03b8 diffraction angle was from 20\u00b0 to 80\u00b0, at a rate of 15\u00b0/min, and a step of 0.02\u00b0. Scanning electron microscopy  observation was performed on a JSM-2400, and the samples were fractured with liquid nitrogen and coated with a thin layer of gold prior to the observation.Fourier-transform infrared (FTIR) spectra were recorded on an IR spectrophotometer  with a resolution of 2 cm2 flow. The elastic modulus was determined by a commercial DMA-Q800 analyzer . Specimens were fabricated into sheets with a size of 35 mm \u00d7 10 mm \u00d7 0.1 mm. Proton nuclear magnetic resonance (1H-NMR) spectra were carried out on a Bruker  400 MHz spectrometer with acetone-d6 as the solvent and tetramethylsilane as an internal standard.Differential scanning calorimetry (DSC) measurements were carried out on a Netzsh PC-200 DSC , and the calorimeter had been calibrated with indium before use. All the measurements were carried out under nitrogen atmosphere from room temperature to 180 \u00b0C at a heating rate of 10 \u00b0C/min. Thermogravimetric analysis (TGA) was tested on a STA 449F3  at a heating rate of 10 \u00b0C/min under NThe dielectric properties and AC electrical conductivity of all hybrid films were obtained via a HP4284A LCR meter  at frequencies ranging from 100 Hz to 1 MHz with 1 V voltage bias at room temperature. Gold electrodes with a diameter of 3 mm and a thickness around 30 nm were sputtered on both sides of the hybrid films by JEOL JFC-1600 auto fine coater  for all electrical properties\u2019 characterizations. The electric field-induced electromechanical response, including the thickness strain and tip displacement of the films, was measured by using a laser distance-detecting instrument  and high voltage amplifier.1H-NMR as shown in 2-CH2-CH2-CF2-) and head-tail (-CF2-CH2-CF2-CH2-) connections of VDF units. The signal at 3.2\u20133.6 ppm is identified as protons on VDF units adjacent to CTFE units (-CF2-CH2-CFCl-CF2-). Compared to P(VDF-CTFE), new multiple signals at 6.2\u20136.7 ppm are assigned to protons on double bonds (-CF2-CF=CH-CF2-) after the removal of HCl from (-CF2-CFCl-CH2-CF2-) [The chemical composition of P(VDF-CTFE-DB) is confirmed with H2-CF2-) .2-CF=CH-CF2-), as illustrated in \u22121 and out-plane bending vibration signal of C-H bonds in -CF=CH- at 704 cm\u22121 through the FT-IR results of P(VDF-CTFE-DB), as presented in Based on the integral calculation, the chemical composition is estimated to be 68 mol % VDF, 8 mol % CTFE, and 12 mol % DB  is almost doubled with respect to eight of the pristine matrices at 100 Hz. However, when PDMS content is over 30 wt %, \u03b5r of the hybrids starts to decrease, which may contribute to the dilution effect of low-\u03b5r PDMS. The frequency dependence of the dielectric loss (or tan\u03b4) of the hybrids is plotted in The frequency dependence of dielectric permittivity of the hybrids measured on a HP4284A LCR instrument at room temperature is shown in To evaluate the effects of PDMS on the mechanical modulus of the matrix, DMA measurements are performed and the results are shown in \u03b5r/Y of hybrids bearing varied PDMS are listed in \u03b5r/Y) of the hybrids reaches a maximum value of 2.40 at PDMS content of 30 wt %, which is nearly 200% higher than that of the P(VDF-CTFE-DB) matrix. As suggested by the above-mentioned equation, the thickness strain of the hybrid should be improved accordingly.The dielectric constant, Young\u2019s modulus, and \u03b5r, but low Y at the same time, or, a large \u03b5r/Y value. In addition, enough high Eb is also necessary for the dielectrics to work stably under a high electric field.Apparently, in order to obtain materials with large thickness strains, the dielectrics should possess high Sz for the pristine matrix and the hybrids against the increasing electric field are shown in Sz of all the samples increases with the elevation of the electric field, which agrees well with equation mentioned in introduction part. Meanwhile, when the silicone content is less than 30 wt %, Sz under the consistent electric field increases with the increasing silicone contents. For instance, Sz values of neat matrix and hybrids containing 10 wt %, 20 wt %, and 30 wt % silicone are 0.5%, 0.6%, 0.7%, and 1.3%, respectively, under 50 MV/m. This suggests the addition of silicone molecules could effectively weaken the intermolecular interactions, thus increasing the mobility of molecular dipoles and decrease the modulus of the hybrids . The significantly enhanced strain can be attributed to the following factors: On one hand, the introduction of flexible silicone oligomers (no more than 30 wt %) can improve thw electromechanical efficiency (\u03b5r/Y) values of the hybrids, as indicated in 14 \u03a9\u00b7cm) can help improve the electric resistance of the hybrid, thus leading to higher breakdown strength (Eb). It is worth pointing out that all the above tests were performed without any pre-stretching.Most interestingly, a maximum Ea) need to produce a 3% thickness strain are compared with other reported PVDF-based EAPs, including stretched P(VDF-CTFE) [Ea values for the five above-mentioned PVDF-based EAPs produce a 3% thickness strain are 150 MV/m, 118 MV/m, 112 MV/m, 95 MV/m, and 71 MV/m, respectively. The result suggests that c-P(VDF-CTFE)-PDMS hybrids possess the advantage of low actuating voltage as well.In order to evaluate the electromechanical conversion ability of c-P(VDF-CTFE)-PDMS hybrids prepared in this work, actuating fields (DF-CTFE) , e-beam DF-CTFE) , copolymDF-CTFE)  and P(VDDF-CTFE) ,38, and In this study, a novel hybrid fluoropolymer is prepared by using a flexible silicone oligomer as a chemical cross-linker. The optimized hybrid dielectric indicates a simultaneously-improved dielectric constant and reduced modulus, which is 100% higher and 30% less than that of the pristine matrix, respectively. Therefore, the hybrid may produce a significantly enhanced electromechanical strain under the applied electric field, e.g., 3% thickness strain under 70 MV/m, which is three times that of the pristine fluoropolymer. This work may provide a simple strategy to fabricate PVDF-based EAPs with significantly enhanced electromechanical strain."}
+{"text": "This article was republished on October 10, 2018, to correct a figure error that was introduced by PLOS staff during the typesetting process. Fig 3B contained an image that was not available for publication under the Creative Commons Attribution License (creativecommons.org/licenses/by/4.0/). Fig 3B has therefore been replaced with an image in the public domain. The caption remains the same.The publisher wishes to apologize to the authors and readers for this error. Please download this article again to view the correct version."}
+{"text": "Demographic data such as gender, age, marital status, education level, and income were collected and analyzed. The results indicated the following: (a) Residents\u2019 top three favorite activities were internet surfing, drinking tea and chatting, and traveling; (b) The Leisure Motivation Scale had a high reliability, with a Cronbach\u2019s alpha of 0.93; (c) In general, residents\u2019 intellectual, social, competence mastery, and stimulus avoidance scores all reached high levels; and (d) Significant differences among gender, marital status, and education level were found in leisure motivation. The findings are discussed in regard to the status quo of leisure behaviors and leisure motivations of Chinese residents. Leisure motivation can be defined as a requirement, reason, or satisfaction that stimulates involvement in a leisure activity ; it is aWalker and his colleagues found that the type of self-construal each person has affects his or her emotions, cognitions, and motivations . The resCulture significantly influences leisure motivation, as individual beliefs and attitudes partially rely on the social circumstances to which they belong. Social behavior is primarily guided by personal goals in individualistic cultures, while the goals of the collective have a dominant influence on forming behavior in collectivistic cultures . Walker Additionally, the psychological values that motivate leisure and recreational cyclists were explored, and the major intrinsic motivational factors of cyclists were found to include competence mastery, solitude, exploration, physical challenge, adventure experiences, stimulus seeking, social encounters, and relaxation/escapism . The leiLeisure plays an important role in Chinese society. In recent years, substantial attention has been paid to the People\u2019s Republic of China in the sphere of leisure studies. Therefore, leisure motivation has already become one of the important leisure studies in the Western world, but it is only slightly focused on in China, and its importance is often overlooked or ignored. Thus, this study aims to examine the relationship between leisure activities and leisure motivations of the residents in China, and the reported results will add to the limited research in the area of leisure motivation in collectivism nations.An ethics approval was not required for this research as per Zhejiang Agriculture and Forestry University\u2019s guidelines as well as regulations. However, an ethical approach is expected. For this research the oral consent of the participants was obtained after principles expressed in the Declaration of Helsinki. The study has no harm to participants, as the names of the residents have not been used in order to preserve anonymity, and the data were analyzed anonymously.This questionnaire comprises four items, namely, leisure activities engaged in, weekly leisure time, leisure frequency, and weekly leisure spending. The respondents entered information related to the leisure activities they enjoy participating in frequently, the number of hours they spend on leisure each week, the number of occasions they participate in leisure each week, and their weekly leisure expenses.To study leisure motivation, the Leisure Motivation Scale has been used in all kinds of settings. Beard and Ragheb developed a comprehensive list of 48 leisure motivations, which can be categorized into four subscales: intellectual, social, competence mastery, and stimulus avoidance. The shortened 32-item instrument was recommended for use in a research setting in which the time given for administration was a major consideration, and data could be combined across individuals . As a reData for the current study were collected between 8 and 23 October 2016 in Hangzhou. A convenience sample of residents was selected and recruited to pilot in Zhejiang Library, West Lake, Shopping Mall (Intimes) and Zhejiang University; 25 residents refused to participate. After excluding the participants who did not meet the criteria, 453 residents remained.The majority of respondents (56.1%) were female. Half the residents were 25\u201335 years old. With regard to marital status, 47.2% of the responding residents were single and had relatively more time for leisure than did married residents. For education level, most of the residents had attained a college degree or above because Hangzhou is one of the immigrant cities in China, and its economic development level is very high. Regarding monthly income, more than half the residents have an income of over 3000 RMB.The statistics software package SPSS version 24.0 was used for all data entry and analyses. Data analysis consisted of three stages. First, frequencies were used to examine the residents\u2019 current leisure situation. Second, the reliability of the Chinese version of the Leisure Motivation Scale (LMS) was examined. Third, independent samples t-tests and analysis of variance (ANOVA) were conducted on leisure motivation using personal background as the independent variable.To examine leisure situations in Hangzhou, residents were asked what leisure activities they enjoy participating in frequently. Cronbach\u2019s alpha was used to estimate the reliability of the LMS in this research. Data were collected by using the LMS, which is divided into four subscales, namely, Intellectual, Social, Competence Mastery and Stimulus Avoidance. The mean scores and standard deviations (SD) are shown in Independent sample t-tests were used to examine the differences in the four leisure motivations by gender. p < .01). The results showed that both males and females reported the highest scores in competence mastery and stimulus avoidance, but females had a much stronger desire to pursue peaceful and calm leisure elements than did males.There were significant differences in both competence mastery and stimulus avoidance between males and females  were also examined.The top three favorite activities of residents are internet surfing (56.1%), drinking tea and chatting (46.1%), and traveling (44.4%). The results show that with the rapid technology development as well as the social and economic progress over the past twenty years in Hangzhou, the residents\u2019 lives in terms of leisure have changed substantially. Internet surfing and other high-tech leisure activities account for a much higher proportion of residents\u2019 leisure participation, while traditional leisure such as playing poker and mahjong (7.7%) and hobbies  (10.2%) are less frequent in residents currently. These results are consistent with Su\u2019s conclusion , in whicHangzhou is a famous city for its natural beauty and is always crowned as the \u201cCapital of Tea\u201d, \u201cHome of Silk\u201d, and \u201cParadise on Earth\u201d. Hangzhou is best known as the origin of the Tea of Longjing, a notable green tea. Teahouses can be seen everywhere in Hangzhou. Therefore, it is not surprising that drinking tea and chatting (46.1%) are some of the residents\u2019 favorite leisure activities. In addition to internet surfing (56.1%) and drinking tea and chatting (46.1%), traveling (44.4%) is also a favorite leisure activity of residents. Hangzhou has rich tourism resources, including West Lake, Lingyin Temple, and Xixi National Wetland Park. Traveling is essential for the residents of Hangzhou. Among the top five activities, traveling was the strongest predictor for social motivation, while drinking tea and chatting was the the strongest predictor for competence mastery.As shown in There were significant differences in intellectual motivation by marital status. Residents who were separated or divorced had the highest scores on intellectual motivation. The findings indicated that single residents were more likely to show intellectual motivation. Thus, partnership played an important role in intellectual motivation. In addition, education seemed to affect residents\u2019 leisure motivations. Residents who attained higher education levels may be better able to financially maintain their daily lives. The results showed that residents with high education levels had the lowest mean scores on social motivation.This research makes several significant contributions to the literature on leisure. First, by focusing on leisure activities and leisure motivations of the residents of China, the researchers determined the reasons why residents choose to engage or disengage in different activities and how residents\u2019 gender, age, marital status, education level and income relate to their leisure motivations. Second, the current findings provide a foundation for further in-depth study of leisure motivation. The study of residents\u2019 leisure should involve their own history and culture. Finally, these findings provide beneficial information for investigating Chinese leisure motivations.Because of an oversight, career, which should be another important factor that affects leisure behavior, was not measured in this research. Many existing researches on leisure motivation involved the career factor. The shortened version of the Leisure Motivation Scale was used in a sample of 1,127 British vacationers . StarzykS1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S3 Appendix(DOCX)Click here for additional data file."}
+{"text": "Microglia-mediated neuroinflammation is important in Alzheimer\u2019s disease (AD) pathogenesis. Extracellular deposition of \u03b2-amyloid (A\u03b2), a major pathological hallmark of AD, can induce microglia activation. Adiponectin (APN), an adipocyte-derived adipokine, exerts anti-inflammatory effects in the periphery and brain. Chronic APN deficiency leads to cognitive impairment and AD-like pathologies in aged mice. Here, we aim to study the role of APN in regulating microglia-mediated neuroinflammation in AD.\u2212/\u22125xFAD) by crossing APN-knockout mice with 5xFAD mice to determine the effects of APN deficiency on microglia-mediated neuroinflammation in AD.Inflammatory response of cultured microglia (BV2 cells) to A\u03b2O and effects of APN were studied by measuring levels of proinflammatory cytokines (tumor necrosis factor \u03b1 [TNF\u03b1] and interleukin-1\u03b2 [IL-1\u03b2]) in cultured medium before and after exposure to A\u03b2O, with and without APN pretreatment. Adiponectin receptor 1 (AdipoR1) and receptor 2 (AdipoR2) were targeted by small interference RNA. To study the neuroprotective effect of APN, cultured HT-22 hippocampal cells were treated with conditioned medium of A\u03b2O-exposed BV2 cells or were co-cultured with BV2 cells in transwells. The cytotoxicity of HT-22 hippocampal cells was assessed by MTT reduction. We generated APN-deficient AD mice  induced by A\u03b2O. Compound C, an inhibitor of AMPK, abolished these effects of APN. Knockdown of AdipoR1, but not AdipoR2 in BV2 cells, inhibited the ability of APN to suppress proinflammatory cytokine release induced by A\u03b2O. Moreover, pretreatment with APN inhibited the cytotoxicity of HT-22 cells co-cultured with A\u03b2O-exposed BV2 cells. Lastly, APN deficiency exacerbated microglia activation in 9-month-old APNOur findings demonstrate that APN inhibits inflammatory response of microglia to A\u03b2O via AdipoR1-AMPK-NF-\u03baB signaling, and APN deficiency aggravates microglia activation and neuroinflammation in AD mice. APN may be a novel therapeutic agent for inhibiting neuroinflammation in AD.The online version of this article (10.1186/s12974-019-1492-6) contains supplementary material, which is available to authorized users. Alzheimer\u2019s disease (AD), the most common cause of dementia in the elderly, is clinically characterized by progressive cognitive impairment typically memory decline . While tMicroglia, the resident phagocyte of the central nervous system (CNS), is pivotal for surveillance in the CNS. They respond rapidly to pathological triggers, such as neuronal death or protein aggregates, and remove them via phagocytosis and degradation . It is wAdiponectin (APN) is an adipokine secreted predominantly from adipocytes and circulates as oligomers, including full-length trimers, hexamers, high molecular weight (HMW) multimers, and globular adiponectin (gAPN) in the blood . The bioIn the present study, we studied whether APN can modulate neuroinflammatory response of microglia in AD in vitro and in vivo models. We found that APN suppressed microglial neuroinflammatory response induced by A\u03b2O via AdipoR1-AMPK-NF-\u03baB signaling pathway in BV2 cells. Importantly, APN inhibited the cytotoxicity of HT-22 hippocampal cells co-cultured with A\u03b2O-treated BV2 cells. Moreover, we showed that APN deficiency increased microglia activation associated with upregulation of TNF\u03b1 and IL-1\u03b2 in the cortex and hippocampus of 5xFAD mice. Taken together, our findings suggest that APN inhibits microglia-mediated neuroinflammation in AD.\u2212/\u2212 mice were previously described [\u2212/\u2212 mice to generate APN\u2212/\u22125xFAD mice. Genotyping was performed by polymerase chain reaction (PCR) of tail DNA as described previously [5xFAD transgenic mice  were a gescribed . 5xFAD teviously . All malMurine BV2 microglia cells were a generous gift from Dr. Nicolai Savaskan (Universit\u00e4tsklinikum Erlangen (UKER), Germany), and HT-22 hippocampal cells were obtained from Dr Kiren Rockenstein . BV2 cells and HT-22 cells were cultured in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) (Gibco) with 10% fetal bovine serum (FBS)  and 1% penicillin/streptomycin (Gibco). The cells were grown in a humidified incubator at 37\u2009\u00b0C with 5% CO2. BV2 cells were pretreated with APN (10\u2009\u03bcg/ml)  or Compound C (10\u2009\u03bcM)  for 2\u2009h and then treated with A\u03b2O (10\u2009\u03bcM) for 24\u2009h in serum-free culture medium. The cellular morphology of BV2 cells was observed by light microscopy with phase contrast .42 peptide  was dissolved in 221.7\u2009\u03bcl cold HFIP  (Sigma-Aldrich) to a concentration of 1\u2009mM. The solution was incubated at room temperature (RT) for 1\u2009h, and then placed on ice for 10\u2009min. After incubation, the solution was aliquoted into non-siliconized microcentrifuge tubes (100\u2009\u03bcl solution containing 0.45\u2009mg A\u03b242) and then dried overnight at RT. The residues were dissolved in 20\u2009\u03bcl dimethyl sulfoxide (DMSO) and then added with F12 medium to obtain a 100\u2009\u03bcM stock solution. The solution was incubated at 4\u2009\u00b0C overnight and then centrifuged at 14,000\u00d7g for 10\u2009min at 4\u2009\u00b0C. Then, the A\u03b2 oligomers were presented in the supernatant. The presence of A\u03b2 oligomers was confirmed by immunoblot using anti-A\u03b2 antibody .A\u03b2O were prepared as previously described . In brieMouse AdipoR1 and AdipoR2 siRNAs and non-targeting control siRNA were purchased from Santa Cruz Biotechnology. BV2 cells were seeded in a six-well tissue culture plate until 80% confluency. Then, BV2 cells were transfected with siRNA using lipofectamine 3000 reagent . The mixture of siRNA duplex and reagent was diluted in Opti-MEM medium (Gibco) and incubated at RT for 45\u2009min. Then, the siRNA duplex and reagent mixture were added to BV2 cell. After 6-h incubation, medium containing siRNA was removed and cells were further cultured for 18\u2009h before using in experiments and analysis.The concentrations of TNF\u03b1 and IL-1\u03b2 in culture medium were examined by Mouse Quantikine ELISA Kits according to the manufacturer\u2019s protocol (R&D Systems). The optical density of each well at 450\u2009nm was determined by a CLARIO star microplate reader .g at 4\u2009\u00b0C for 20\u2009min to remove any insoluble materials, including nuclei and large debris, and the cytosolic protein concentration in supernatants was determined by Bradford test . Samples were then assessed in duplicate via RayBio\u00ae Mouse TNF\u03b1 ELISA Kit  and IL-1\u03b2 Mouse Quantikine ELISA Kits (R&D Systems) according to the manufacturer\u2019s protocol. The concentration (pg/ml) of cytokines was normalized to total protein content (pg/mg of protein).Production of TNF\u03b1 and IL-1\u03b2 were assessed in tissue homogenates. Briefly, frozen cortex and hippocampus were incubated in ice-cold lysis buffer  with PMSF for 30\u2009min and then sonicated 3\u2009\u00d7\u200915\u2009s with a 2-min interval between each sonication in ice-cold lysis buffer. Samples were centrifuged at 14,000\u00d7Total RNA was extracted from BV2 cells using Trizol reagent  with a DNase  treatment according to the manufacturer\u2019s instructions. cDNA synthesis from 1\u2009\u03bcg of total RNA in a reaction volume of 20\u2009\u03bcl was performed with ImProm-IITM Reverse Transcription System  according to the manufacturer\u2019s instructions. PCR amplification with specific primers was utilized following thermal cycling: 95\u2009\u00b0C for 10\u2009min, 35\u2009cycles of denaturing at 95\u2009\u00b0C for 1\u2009min, annealing at 64\u2009\u00b0C for 1\u2009min, elongation at 72\u2009\u00b0C for 1\u2009min, final extension at 72\u2009\u00b0C for 7\u2009min and holding at 4\u2009\u00b0C. PCR products were electrophoresed in 1% agarose gels. All samples were normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The following primers were used: AdipoR1: forward (5\u2032-AACTGGACTATTCAGGGATTGC-3\u2032), reverse (5\u2032-ACCATAGAAGTGGACGAAAGC-3\u2032); AdipoR2: forward (5\u2032-CCACCATAGGGCAGATAGG-3\u2032), reverse (5\u2032-TGAACAAAGGCACCAGCAA-3\u2032); GAPDH: forward (5\u2032-AAGCCCATCACCATCTTCCAG-3\u2032), reverse (5\u2032-AGAAGACTGTGGATGGCCCCT-3).g for 10\u2009min at 4\u2009\u00b0C, followed by collecting supernatant within cytosolic protein fraction. The protein concentration was quantified using the Bradford assay .Cytosolic protein from BV2 cells was isolated as described previously . Brieflyg for 3\u2009min at 4\u2009\u00b0C. After removing the supernatant, Buffer B working reagent containing DTT, protease inhibitor, and phosphate inhibitor was added to the pellet and the microcentrifuge tubes were vortexed at the highest setting for 10\u2009s. The pellet was detached from the microcentrifuge tube wall and incubated on ice for 1\u2009h. The nuclear extraction was collected as supernatant. The protein concentration was quantified using the DC assay .Nuclear protein isolation protocol was performed with a nuclear extraction kit  according to the manufacturer\u2019s protocol as described earlier . BrieflyT172 , rabbit anti-\u03b1-Tubluin , rabbit anti-p-NF-\u03baB p65S536 , rabbit anti-NF-\u03baB p65 , rabbit anti-p-I\u03baB\u03b1 (Ser32) , mouse anti-I\u03baB\u03b1  antibody were incubated at 4\u2009\u00b0C overnight, followed by HRP-conjugated secondary antibodies  at RT for 1\u2009h. The immunoblot signals were visualized by Westernbright Quantum HRP substrate .Cell homogenates (20\u2009\u03bcg/well) were loaded onto 10% SDS polyacrylamide gels in denaturing conditions at 80\u2009mA for 90\u2009min and transferred electrophoretically  to polyvinylidene fluoride (PVDF) membrane. Immunoblotting was performed as described previously . Nonspec4 cells/well overnight, and then washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 20\u2009min at RT. The slides were rinsed with PBS and incubated with 0.05% Triton X-100 in PBS for 15\u2009min at RT, followed with blocking solution  at RT for 1\u2009h. Slides were then incubated with mouse anti-Iba-1  and rabbit anti-AdipoR1  or goat anti-AdipoR2  at 4\u2009\u00b0C overnight. After incubation, the cells were washed with 0.05% Triton X-100 in PBS three times and incubated with appropriate Alexa-Fluor-conjugated secondary antibody  at RT for 1\u2009h. The coverslips were mounted with by slow fade\u00ae anti-fade DAPI reagent . The fluorescent images were captured with a Nikon Eclipse Ecliose NiU microscope  and digitized with SPOT software 5.0 .BV2 cells were seeded on glass chamber slides at a concentration of 1\u2009\u00d7\u200910The 9-month-old mice were anesthetized by ketamine/xylazine , and transcardially perfused with ice-cold PBS, followed by 4% paraformaldehyde in PBS. Dissected brains were harvested and post fixed with 4% paraformaldehyde at 4\u2009\u00b0C for 24\u2009h, and then dehydrated in 30% sucrose in PBS and stored at 4\u2009\u00b0C. Fixed brains were sectioned in 10\u2009\u03bcm with a cryostat.n\u00a0=\u20094) were acquired with a Nikon Eclipse NiU microscope  and digitized with SPOT software 5.0  in identical settings to determine the degree of overlap between AdipoR1, AdipoR2, TNF\u03b1 or IL-1\u03b2 immunoreactive cells, and Iba1-expressing cells in the cortex and hippocampus. Analysis of the fluorescent intensities and the number of labeled TNF\u03b1 or IL-1\u03b2-positive microglia cells were quantified with ImageJ software .For immunofluorescent staining as described previously , sectionT172, 1:100, Cell Signaling Tech. Inc., USA) at 4\u2009\u00b0C overnight, followed by incubation with HRP-conjugated secondary antibody  at RT for 1\u2009h. Sections were developed by brown color staining and counterstained with hematoxylin.For immunohistochemistry, mice at 9\u2009months old were anesthetized with ketamine/xylazine . After transcardially perfused with PBS and 4% paraformaldehyde, dissected brains were post fixed in 4% paraformaldehyde for 24\u2009h at 4\u2009\u00b0C. Fixed brains were incubated in gradient ethanol for dehydration followed by xylene before embedding in paraffin wax. Tissue sections (10\u2009\u03bcm thick) were obtained by using a rotary microtome. Tissue sections were rehydrated by graded ethanol to water before antigen retrieval. Hydrogen peroxide solution was used to inactivate endogenous peroxidase. Sections were incubated with a primary antibody (rabbit anti-p-AMPKn\u00a0=\u20092) and acquired with a water-immersion objective lens (\u00d7\u200910 or \u00d7\u200920) of Nikon Eclipse NiU microscope . The number of amyloid plaques was counted with ImageJ cell counter plugin. To determine the number of microglia around plaques, the quantitative images of amyloid plaque were randomly selected for their similar size in the cortex (n\u00a0=\u200980 plaques from 2 mice per genotype) using a water-immersion objective lens (\u00d7\u200920 or \u00d7\u200940) of Nikon Eclipse NiU microscope . The cell bodies of microglia within a 25-\u03bcm radius circular area from the plaque edge were quantified with ImageJ software .Each cryosection (40\u2009\u03bcm thick) was stained with 0.01% thioflavin-S (Sigma-Aldrich) followed by immunostaining with anti-Iba1 . The quantitative images of amyloid plaque were selected in the similar location in the cortex  in serum-free culture medium and then treated with BV2 CM for 24\u2009h.BV2 cells were seeded in a 6-well plate to 80% confluent. The cells were pretreated with APN or Compound C (10\u2009\u03bcM) for 2\u2009h followed by A\u03b2O treatment for 24\u2009h in serum-free culture medium. After incubation, the supernatant (CM) was collected and filtered with a 0.22-\u03bcm sterile filter  to remove cells and cell debris. For the control group, BV2 cells were incubated for with medium only for 24\u2009h. For activation of HT-22 cells, the cells were seeded in 96-well plates (1.5\u2009\u00d7\u2009105/well) were seeded in 24-well plate in serum-free culture medium. BV2 cells (1.5\u2009\u00d7\u2009104/well) were seeded in the inserts  Membrane, Corning Costar Corp, USA) and directly placed above the HT-22 culture. BV2 cells were pretreated with APN or Compound C for 2\u2009h followed by A\u03b2O treatment for 24\u2009h. Then, the viability of HT-22 cells in the lower compartment was determined by MTT assay.HT-22 cells -2, 5-diphenyltetrazolium bromide (MTT). HT-22 cells were incubated with MTT solution at a concentration of 5\u2009mg/ml in a humidified incubator at 37\u2009\u00b0C for 1\u2009h. Afterwards, the supernatants were removed and then 200\u2009\u03bcl of dimethyl sulfoxide (DMSO) was added to detect formazan crystal developed in the viable cells. The absorbance of the solution in each well was determined at 570\u2009nm using a CLARIO star microplate reader  as described by the manufacturer. Cell viability was expressed as a percentage of viable cells obtained relative to that of controls.P\u00a0<\u20090.05.Statistical analyses were performed by Prism 6 . Quantitative data were expressed as mean\u2009\u00b1\u2009standard errors of the mean (S.E.M.) and analyzed using one- and two-way ANOVA followed by post hoc comparison using Tukey\u2019s test. The difference was considered to be statistically significant when Previous studies have shown that biological effects of APN were mediated through AdipoR1 and AdipoR2 . In ordeNext, we investigated the proinflammatory effect of A\u03b2O on BV2 cells. We found that exposure of A\u03b2O for 24\u2009h induced the release of TNF\u03b1  is a known energy metabolic sensor that is activated by phosphorylated AMPKTP ratio . AccumulTP ratio . AMPK, dTP ratio . Therefoent Fig.\u00a0a. We nexTo investigate the roles of AdipoR1 and AdipoR2 in regulating the anti-inflammatory effect of APN on A\u03b2O-treated BV2 cells, we used siRNA to knock down AdipoR1 or AdipoR2. Western blot analyses showed that AdipoR1 and AdipoR2 expression were all significantly inhibited by siRNA at the dose of 100\u2009nM Fig.\u00a0a, b. TheIt has been demonstrated that activated microglia release toxic agents with neurotoxic effects which correlate with the onset and progression of neurodegenerative disease , 57. Sin\u2212/\u22125xFAD) by crossing APN knock-out mice (APN\u2212/\u2212) with 5xFAD mice (APN\u2212/\u22125xFAD). We measured levels of the proinflammatory cytokines TNF\u03b1 and IL-1\u03b2 in the cortical and hippocampal fractions of wild type mice (WT), APN\u2212/\u2212 mice, 5xFAD mice, and APN\u2212/\u22125xFAD mice at 9\u2009months old. We found that TNF\u03b1 and IL-1\u03b2 levels were both increased in the cortex  of APN\u2212/\u22125xFAD mice, compared with that of the 5xFAD  in the cortex. We found that APN\u2212/\u22125xFAD mice showed a lower proportion of small amyloid plaques, but a higher proportion of large amyloid plaques, compared with those in 5xFAD mice. We also found that the number of microglia within a predefined area of 25-\u03bcm radius around small plaques and medium plaques was decreased in the APN\u2212/\u22125xFAD mice, compared with that in the 5xFAD mice -\u03b3 signaling . Nicolas42 impaired AMPK phosphorylation to activate GSK3\u03b2 and induce tau hyperphosphorylation in a fly model of AD [AMPK acts as an energy sensor and plays an important role in the regulation of energy metabolic homeostasis. In response to low AMP concentration, AMPK is activated by phosphorylation of \u03b1 subunit at Thr172 via upstream kinases to increase cellular ADP to ATP and AMP to ATP ratios . AMPK acel of AD . Our resel of AD . In thisel of AD . It had el of AD . We beliS276 was required for the interaction of p65 with target gene and thereby promoted p65 transcriptional activity, while phosphorylation of p65S536 increased proteasomal degradation and nuclear export of p65 [Activation of NF-\u03baB is critical in the transcription of genes that are involved in the inflammatory response . Brieflyt of p65 . Our rest of p65 . AMPK ret of p65 , Forkheat of p65 , and pert of p65 , 71, whit of p65 . Our pret of p65 .AdipoR1 and AdipoR2 are two major receptors for APN. It has been reported that AdipoR1 and AdipoR2 had distinct binding affinities. AdipoR1 bound to gAd with high affinity, while AdipoR2 had high binding affinity to both globular and full-length APN . Besides\u2212/\u2212 mice [It was notable that APN inhibited the release of TNF\u03b1 and IL-1\u03b2 levels from A\u03b2O-exposed BV2 cells. Moreover, APN prevented cytotoxicity of HT-22 neuronal cells that were co-cultured with A\u03b2O-exposed BV2 cells. TNF\u03b1 was an important mediator of neuroinflammation that was widely studied in AD. TNF\u03b1 antagonist prevented inhibition of long-term potentiation (LTP) induced by A\u03b2 . Also, T\u2212/\u2212 mice . Further\u2212/\u2212 mice . A prosp\u2212/\u2212 mice . IL-1\u03b2 w\u2212/\u2212 mice . In addi\u2212/\u2212 mice . Our fin\u2212/\u22125xFAD mice. Abundant IL-1\u03b2-expressing microglia cells and non-microglia cells were found in APN\u2212/\u22125xFAD mice. In the brain, IL-1\u03b2 was synthesized and released mainly by the microglia and astrocytes [\u2212/\u22125xFAD mice. APN\u2212/\u22125xFAD mice had much more expression of TNF\u03b1 than 5xFAD mice. We could not find direct evidence to show whether TNF\u03b1 originated from neurons themselves or glia cells, but abundant studies have shown that the expression of TNF\u03b1 was increased in neurons, microglia, reactive astrocytes, and epithelia cells upon brain injuries and chronic disorders [\u2212/\u2212mice at 9\u2009months old compared to wild-type mice, but we found cerebral IL-1\u03b2 and TNF\u03b1 levels were significantly increased at 18\u2009months old in our previous study [We previously reported that microglia activation and proinflammatory cytokines TNF\u03b1 and IL-1\u03b2 were increased in the cortex and hippocampus of aged APN-KO mice . In the trocytes , 89, andtrocytes . We specisorders . In addiisorders . Moreoveisorders , 93. It isorders . Acrp30 isorders . We specus study .\u2212/\u22125xFAD mice. One possibility was that APN deficiency in 5xFAD mice induced microglia into a dysfunctional state. We observed a large number of microglia cell display abnormal morphological features with shortened or gnarled processes and dystrophic spheroid formation, which might affect motility of microglia process contacting with the plaque and phagocytic ability of microglia [Microglial activation is associated with amyloidosis not only in transgenic mouse models of AD , but alsicroglia , 101. ThSome limitations should be noted in our study. Firstly, it was reported that adiponectin receptors were expressed in the hippocampus in mice . Using tTo conclude, we found that APN attenuated the inflammatory response of A\u03b2O-activated microglia via AdipoR1-AMPK-NF-\u03baB signaling pathway, and APN deficiency enhanced neuroinflammation in the 5xFAD mouse model of AD. This anti-inflammatory effect of APN on A\u03b2O-exposed microglia might protect neurons against cytotoxicity in AD. The study provides evidence that APN may be a potential novel therapeutic agent in AD.Additional file 1:APN treatment induced morphological changes of A\u03b2O-treated BV2 cells. Representative images were depicting morphology of BV2 cells pretreated with APN (10\u2009\u03bcg/ml) for 2\u2009h prior to incubation with A\u03b2O (10\u2009\u03bcM) for an additional 24\u2009h. Photomicrographs were taken directly from culture plates by phase-contrast microscopy. At the quiescent state, the BV2 microglia showed the typical ramified shape. Incubation with A\u03b2O of BV2 cells revealed an amoeboid shape which cell body was enlarged and extended processes were lost. Pretreatment with APN converted the amoeboid morphology of A\u03b2O-stimulated BV2 cells to a ramified morphology. Three independent experiments were performed. Scale bar 200\u2009\u03bcm. (TIF 1630 kb)Additional file 2:n\u2009=\u20093). One-way ANOVA with Tukey\u2019s multiple comparison test revealed a difference between groups. *p\u2009<\u20090.05. (TIF 199 kb)BV2 microglia in co-culture system exacerbated neuronal loss under A\u03b2O exposure. HT-22 cells were treated with A\u03b2O with or without pre-treatment of APN, compared with HT-22 cells co-cultured with A\u03b2O-exposed BV2 cells in a transwell system. Data were presented as the mean\u2009\u00b1\u2009SEM for at least three independent experiments, and each performed in triplicates  Quantification of GFAP fluorescence intensity WT mice, APN\u2212/\u2212mice, 5xFAD mice, and APN\u2212/\u22125xFAD mice (n\u2009=\u20094). One-way ANOVA with Tukey\u2019s multiple comparison test revealed the difference between groups. **p\u2009<\u20090.01, ***p\u2009<\u20090.001; ns, statistically not significant. (TIF 3235 kb)APN deficiency exacerbated astrogliosis in 5xFAD mice. (a) Representative images of GFAP immunoreactivity of astrocytes in the cortex and hippocampus of WT mice, APNAdditional file 4:T172 (black arrows) in the cortex of 5xFAD mice and APN\u2212/\u22125xFAD mice at 9\u2009months old. Scale bar 200\u2009\u03bcm. (TIF 2716 kb)APN deficiency reduced the level of phosphorylated AMPK in 5xFAD mice. (a) Representative image of immunohistochemistry staining of p-AMPKAdditional file 5:\u2212/\u22125xFAD mice. Scale bar 30\u2009\u03bcm. (b) Quantification of the percentage of thioflavin-S-labeled amyloid plaques according to their plaque size  in the cortex . (c) Representative images of Iba1-labeled microglia (red) surrounding different size of thioflavin-S-labeled amyloid plaque (green) in the 5xFAD mice and APN\u2212/\u22125xFAD mice. Images on the right represented magnified portion of microglia clustering around amyloid plaque (\u00d7\u2009400). Scale bar 30\u2009\u03bcm. (d) Quantification of the number of Iba1-labeled microglia within a 25-\u03bcm radius from different size of plaque edge (n\u00a0=\u200980 plaques from 2 mice per genotype). Data were presented as the mean\u2009\u00b1\u2009SEM. Two-way ANOVA with Tukey\u2019s multiple comparison test revealed a difference between groups. **p\u2009<\u20090.01, ***p\u2009<\u20090.001; ns, statistically not significant. (JPG 768 kb)APN deficiency increased the amyloid plaque size and decreased microglia clustering around amyloid deposits in 5xFAD mice. (a) Representative images of thioflavin-S-labeled amyloid plaque (green) in the 5xFAD mice and APN"}
+{"text": "Using gold nanoparticles (AuNPs), modified by aptamers it was possible improving the limit of detection (LOD) that reached 550 cells/mL, while without amplification the sensitivity of the detection of SK-BR-3 cells was 1574 cells/mL. HER2 negative cell line MDA-MB-231 did not resulted in significant changes of fs. The viability studies demonstrated that cells are stable at experimental conditions used during at least 8 h. AuNPs were not toxic on the cells up to concentration of 1 \u03bcg/mL.Detection of the breast cancer cells is important for early diagnosis of the cancer. We applied thickness shear mode acoustics method (TSM) for detection of SK-BR-3 breast cancer cells using DNA aptamers specific to HER2 positive membrane receptors. The biotinylated aptamers were immobilized at the neutravidin layer chemisorbed at gold surface of TSM transducer. Addition of the cells resulted in decrease of resonant frequency, f HER2 positive breast cancer is among most aggressive type of disease with low percentage of survival. However, substantial progress in early stage diagnostics together with progressive methods of therapy substantially improved the situation. Nevertheless, despite of the current achievements, the diagnostics of the breast cancer is rather difficult considering that at the first stage it is usually without significant symptoms. Most of the patients are in general diagnosed in late stage of this disease. The main reason is lack of the effective early stage diagnostics tests. For the purpose of diagnostics of HER2 positive breast cancer various methods have been developed. However, approx. 20% of the tests can be casual.The epidermal growth factor receptor (EGFR) is at the front of the signaling cascade, that affect the cell proliferation and differentiation . This inApplication of the gold nanoparticles is rather useful for cell and tissue imaging as well as for visualization of the targeted drug delivery by means of aptamers . For theAmong biosensors those based on acoustics principles are of special interest. They allowing study in real time the processes connected with cell adhesion, cell morphological changes, apoptosis as well as detection of the cells based on receptors specific to the cancer markers in their membranes . They al2 atmosphere. All experiments were performed on cells in the exponential growth phase. Cell lines were obtained from the Cancer Research Institute . All cell lines were screened for contamination by mycoplasma.Human breast cancer cells SK-BR-3 (HER2 positive) and MDA-MB-231  were incubated in Dulbecco\u2018s modified Eagle\u2019s medium (DMEM) , supplemented with 10% fetal bovine serum  and 1% penicillin-streptomycin . Cell cultures were maintained at 37 \u00b0C (human body temperature) in humidity within a 5% CO2PO4, 1.8 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl, pH 7.4). The deionised water was prepared by PureLab Classic UV .Neutravidin (NA) was purchased from Thermo Fischer Scientific GmbH . Inorganic salts, ethanol, ammonia, hydrogen peroxide (p.a.) were from Slavus . Trypan blue for microscopy and trypsin were purchased from Sigma Aldrich. The DNA aptamers modified at its 5\u2019-end by biotin were purchased from Eurogentec . The aptamer sequences  were takThe breast cancer cells SK-BR-3 (HER2 positive) and control MDA-MB-231 cells (HER2 negative) were three times washed with 2 mL of PBS, and then incubated with 1 mL of trypsin at 37 \u00b0C for 5\u201310 min. FBS was then added to quench the proteinases. After washing with PBS buffer, the trypsin treated cells were used for experiments. Before the experiments it was necessary to prepare samples with known concentration of the cells.5 cells/mL.For this purpose we used the method based on direct cell counting in a B\u00fcrker cell . In further experiments the undamaged cells were counted and diluted for desired concentration in the range of 50\u20135 \u00d7 104 cells/well. Subsequently the cells were incubated during 72 h in an incubator . Then the growth medium was removed and the cells were washed twice in a PBS. Cells in PBS were added into the wells. The concentration of the cells has been determined after 1, 2, 4, 6, 8 and 10 h in PBS following their incubation at various temperatures: 37 \u00b0C in the incubator, at ambient temperature 20 \u00b0C and at 4 \u00b0C in refrigerator [The cells were added into the 24 wells plate with a concentration of 5 \u00d7 10igerator ,23.The cell viability was based on a trypan blue test. The trypan blue does not penetrate through the plasmatic membrane of undamaged cells, while the damaged cells are blue colored ,23. The 4 cells/mL have been added into the 24 well plate and incubated during 72 h. Then the cultivation medium has been removed, and after cell washing replaced by fresh DMEM medium without FBS, or alternatively instead of DMEM the PBS has been used. Subsequently the AuNPs have been added in certain concentration.In one series of experiments we used gold nanoparticles (AuNPs) for amplification of the cell detection. For this purpose it has been necessary to analyze the cytotoxicity of the nanoparticles. We used AuNPs conjugated with streptavidin (StpA-AuNPs) in a concentration range 1\u201310 \u03bcg/mL. Cytotoxicity has been analyzed in the special incubator IncuCyte ZOOM\u2122 kinetic imaging system . The breast cancer cells in a concentration 10s, and motional resistance, Rm. While the fs value reflects the changes in mass or thickness of the sensing layer at the surface of AT-cut quartz crystal, the Rm value is responsible for the viscosity contribution [2. The crystal has been mounted into the acryl flow cell. The cell was the generous gift of Professor M. Thompson . The construction of the cell can be found in the paper by Tassew and Thompson [The thickness shear mode method (TSM) is thanks to its sensitivity one of the most used acoustics methods in biosensing and for surface characterization. It is based on the measurement of the series resonant frequency, fribution . The expThompson . The dig3), water and hydrogen peroxide (H2O2) in a volume ratio 1:5:1 for 25 min followed by rinsing in MilliQ water. This process was repeated three times. The crystal was subsequently washed by copious volumes of distilled water, then by 90% ethanol, and finally dried in the stream of nitrogen gas. The clean crystal has been then mounted into the flow cell and was ready for further modification. For this purpose the water solution of neutravidin (NA) in a concentration of 125 \u00b5g/mL has been allowed to flow to the surface of the gold layer of the quartz crystal.Prior to the preparation of the sensing layer at one side of the quartz crystal, it was necessary to accurately clean the gold layer. For this purpose we used chemical cleaning by basic Piranha solution. The crystal has been immersed into the hot mixture (70 \u00b0C) of basic Piranha, composed of ammonium (NHThanks to the chemisorption (presence of SH groups at the neutravidin molecules) a self-assembled NA layer was formed. The steady-state conditions, where both the resonant frequency and motional resistance become stable, has been reached after 35 min following starting of the NA injection. Because the DNA aptamers were diluted in the PBS, the NA surface was first cleaned by distilled water and then by PBS. Then the biotin-modified DNA aptamers in a concentration of 0.25 \u03bcM have been allowed to flow during 10\u201315 min until the resonant frequency and motional resistance reached steady-state values. Due to high affinity of biotin to NA a stable aptamer layer was formed. The sensing surface was then washed by PBS in order to remove physically adsorbed aptamers. Because the cells were incubated in a DMEM cultivation media, the surface of the sensing layer was washed by this solution. The sensing layer has been then ready for study of the interaction with the breast cancer cells. The scheme of the crystal modification is presented on the 12 NPs/mL have been used. The NPs were modified by biotinylated HB5 aptamers as follows: 2 \u00b5L of NPs in a concentration of 0.5 mg/mL have been mixed with 0.25 \u03bcM aptamers in a PBS. The solution has been incubated during 1 h. Then the solution of aptamer-modified NPs has been centrifuged at 13,400 rpm during 35 min. After centrifugation the supernatant with unbounded aptamers has been removed and the pellet was diluted in PBS.For amplification of the detection of the cancer cells we used gold nanoparticles modified by streptavidin (StpA-AuNPs). The NPs of 20 nm diameter in a concentration of 3.5 \u00d7 102), cell viability was tested under different conditions\u2014in incubator at 37 \u00b0C and 5% CO2, at room temperature at 20 \u00b0C, and in refrigerator at 4 \u00b0C [4 cells/mL in some wells in multiwell plates, which is related to the cell proliferation during settling. After incubation in PBS, the cells could no longer be divisioned.Viability is one of the basic parameters in cell culture handling. During measurements, the cells are exposed to adverse conditions . Therefore, the survival of the cells under these conditions had to be verified. We tested the effect of PBS buffer on the cell viability. To compare the effects of temperature and the concentration of carbon dioxide . We observed a decrease in the cell concentration by 16% after 8 h incubation, when testing the viability of the SK-BR-3 cells in PBS at 4 \u00b0C. After PBS administration we observed a decrease in the cell number by 18% in incubator at 37 \u00b0C. From our cell viability results in PBS, we can conclude that the SK-BR-3 cell line is stable under laboratory conditions. Our results are consistent with Han et al. [The lowest cell number decrease was observed at room temperature, but even at 37 \u00b0C and 4 \u00b0C, there was no significant reduction in the cell number over time compared to the original number . It was therefore important to analyze what concentration of AuNPs is not toxic and does not induce cell apoptosis. Verification of the cytotoxic effect of AuNPs is a major factor that predetermines them in biomedical applications. AuNPs have long been considered inert, non-toxic and biocompatible. Several studies were focused on the cytotoxicity of AuNPs under different conditions  and most of them confirmed the non-toxic properties of AuNPs. On the other hand, some studies demonstrated several adverse effects of AuNPs on the cell membranes, mitochondria or nuclei and caused DNA damage, oxidative stress, apoptosis, mutagenesis, etc. ,28. TherThe optimal concentration of StpA-AuNPs (gold nanoparticles conjugated to streptavidin) with a diameter of 20 nm for in vitro experiments was determined by monitoring the cells in the Incucyte incubator. Incucyte allows us to monitor the influence of various factors on the development of adherent tumor cells. We observed the cell confluence in DMEM culture medium or PBS buffer after addition of nanoparticles in the concentration range 1\u201310 \u00b5g/mL to SK-BR-3 (HER2+), MDA-MB-231 (HER2-) and healthy breast cancer cell line MCF10A. Cells were scanned with an Incucyte scanner during 90 h. The result of these measurements for the SK-BR-3 cell line in DMEM are shown in We observed minimal cytotoxic effect on the cells in both cases (DMEM and PBS) only at a AuNPs concentration of 1 \u00b5g/mL. In the case of cells in DMEM medium after 10 h of addition of nanoparticles, the cells began to proliferate again. An opposite effect was observed in PBS. After addition of PBS to the cells, the confluency was reduced by 2.5%, and then after 33 h caused a 1% decrease yet. Thus, we can conclude that for further experiments 1 \u00b5g/mL of AuNPs can be considered as an optimal concentration.Smolkova at al.  verifieds, on average by 157.8 \u00b1 27.3 Hz. However, the changes of motional resistance, Rm, were minimal. This is evidence of the formation of a rigid self-assembled layer of NA at the gold surface [s and Rm values become stable have been reached after 35 min following starting of the NA injection. Washing of the surface by deionised water resulted in slight increases of fs and Rm values. This corresponds to removal of physically adsorbed NA molecules. The NA forms very stable layers at gold surface. This has been demonstrated also by the study of the topography of these layers using atomic force microscopy (AFM). The results are presented in the s and Rm values, which agree well with the already discussed effect of the changed ionic conditions on the molecular slip between the sensing layer and surrounding water solution [s value in average by 57.10 \u00b1 16.03 Hz and in an increase of Rm by 4.80 \u00b1 0.76 \u03a9.In this part we present the study of the interaction of breast cancer cells with the surface of acoustics aptasensors. Three different aptamers were used for sensor preparation . Two of  surface . The stesolution . Additiom is caused by the increase of molecular slip between the sensing layer and the surrounding PBS. This phenomenon has been observed previously in our work [s and increase of Rm. The effect is similar to those caused by changed ionic strength. After this procedure the aptasensor was ready for analysis of the interaction with the cells. s and Rm values folowing addition of the breast cancer cells SK-BR-3 at concentration of 5 \u00d7 104 cells/mL. It can be seen that addition of the cells in a flow injection mode resulted in the decrease of the resonant frequency and in an increase of the motional resistance. This is evidence of specific interaction of the cells containing HER2 receptors with the aptamers.The decrease of resonant frequency is due to the strong binding of biotinylated HB5 aptamers to NA molecules, while the increase of Rour work . The was4 cells/mL) has been much lower in comparison with those based on HB5 aptamers (see s and Rm were not significant. The significant changes of fs and Rm were observed only when MDA-MB-231 cells were added at the TSM surface modified by sgc8c aptamers which are specific to PTK7 (s and Rm following addition of SK-BR-3 cells (concentration 5 \u00d7 104 cells/mL) to a surface of a TSM transducer which contained only an NA layer without aptamers. Addition of the cells resulted in decrease of the fs by 5.0 \u00b1 3.5 Hz and increase of Rm by 0.50 \u00b1 0.36 \u03a9 . The most sensitive response has been obtained for aptasensors prepared by 0.25 \u00b5M HB5 and 0.5 \u00b5M HeA2_3 aptamers. d values presented in We also compared response of aptasensors formed by different specific aptamers see  followin5 cells/mL. The changes of the fs and Rm values for each concentration were measured on a separate TSM transducer. It can be seen that for both aptasensors fs decreases and Rm increases with an increased concentration of the cells. Already at relatively low cell concentration (500 cells/mL) the fs decrease by 4.60 \u00b1 0.08 Hz for aptasensor based on HB5 aptamers. The changes of fs for HeA2_3 based aptasensors were 2 times higher. HB5 aptamer recognizes \u201cHER2 epitope peptide\u201d and HeA2_3 binds to the HER2 protein. It is possible that the HER2 proteins were not been fully available for HB5 aptamers, therefore we obtained less sensitivity of detection in this case. The prognostic concentration of HER2 positive circulating tumor cells (CTCs) in the blood is currently unknown. It seems that only 26.9% of patients with HER2 positive tumor have HER2+ CTCs in their blood. At the same time approx. 24.2% of patients with HER2- tumor has HER+ CTCs in their blood. Therefore the diagnostics of HER2+ CTCs is crucial for the establishment of correct targeted therapy [The response of HeA2_3 and HB5 based aptasensors as a function of various concentrations of HER2 positive breast cancer cells SK-BR-3 is summarized on  therapy . From th3 cells/mL) immobilized at the surface of TSM transducer resulted in decrease of resonant frequency, fs, by 22.4 \u00b1 3.54 Hz and in an increase of motional resistance, Rm, by 0.90 \u00b1 0.42 \u03a9 .Amplification of the detection is important step in the development of high sensitive TSM cell sensors. Gold nanoparticles are often used for this purpose due to their unique electrical and optical properties. They can also be easily modified by receptors such as aptamers or antibodies. Therefore we examined the possibility of amplification of the detection of breast cancer cells by the complexes of gold nanoparticles (AuNPs) conjugated with streptavidin (StpA) and modified by biotinylated HB5 aptamers (HB5-StpA-AuNPs). The interaction of HB5-StpA-AuNPs with the SK-BR-3 cells . In our case \u03b4 = 0.2 \u03bcm. This value is less then average thickness of the breast cancer cells, which is in the order of 5\u201310 \u03bcm [2 [This increase of the sensor sensitivity is not so remarkable. One of the possible reason is the limitation in the penetration depth of ultrasound wave. According to Glassford  the pene 5\u201310 \u03bcm . Therefo10 \u03bcm [2 . However0 is the fundamental frequency (8 MHz), \u0394m is the mass change (in grams) and A = 0.2 cm2 is the area of the piezocrystal. The formation of stable NA layer resulted in decrease of resonant frequency by 158 Hz. This corresponds to the mass changes by 218 ng. The number of NA molecules at the surface can be calculated as: (\u0394m/Mw)Na =  \u00d7 6.02 \u00d7 1023 = 2.25 \u00d7 1012 molecules, where Mw = 60,000 g/mol is molecular weight of NA and Na = 6.02 \u00d7 1023 is Avogadro\u2019s number. In analogy with this calculation it is possible to estimate mass changes and number of molecules after the immobilization of HB5 aptamers and the cells. In the case of aptamers the frequency decrease by 57 Hz corresponds to the mass changes of 78.7 ng. Considering that Mw of aptamer is 26 678.5 g/mol, the number of HB5 molecules at the surface is 1.8 \u00d7 1012, which is comparable with the number of NA molecules. We should, however, note that changes of resonant frequency following immobilization of the aptamers are accompanied also by increase of motional resistance and thus in increase of the contribution of viscous forces. Therefore the viscosity contribution into the mass changes can not be considered as negligible. Thus, the number of immobilized aptamers can be less in comparison with those determined based on the Sauerbrey equation.The changes in resonant frequency due to chemisorption of NA, formation of DNA layer as well as due to adsorption of the cells can be connected with the changes of the mass and shearing viscosity of the layers at TSM transducer. For relatively rigid layers like those formed by NA, from the changes of the resonant frequency it is possible to calculate the mass of the molecular layer using Sauerbrey equation :\u0394fs= \u22122.Taking into account that the NA molecule has 4 binding sites for biotin, but 2 are not available due to chemisorption, a maximum of 2 binding sites can be occupied by aptamers. From the above rough estimation it is, however, clear that the NA surface is not maximally saturated by aptamers. This result is not unexpected, considering that DNA aptamers form 3D structures in a water solution, have certain volume and due to negative charge cannot be too close to each other at the surface. Therefore they cannot occupy all NA binding sites.4 cells/mL at the sensing surface resulted in a frequency decrease by 46.7 Hz. This corresponds to the increase of the mass by 64.5 ng. The weight of one cell is approx. 1.5 to 3.5 ng [Addition of breast cancer cells at the concentration of 5 \u00d7 10o 3.5 ng . This meo 3.5 ng .3 cells/mL and the linear range was 104\u20131.5 \u00d7 105 cells/mL. Shan et al. [In the current literature there are only a few studies focused on the detection of cancer cells using the acoustics TSM method. The proposed cell sensors are characterized by different values of detection limits. In particularly, Pan et al.  developen et al.  developen et al. .\u22125 to 102 ng/mL.Only one paper has been so far reported on the development of TSM biosensor for detection breast cancer cells. Zhang et al.  reportedDespite the fact that our acoustics aptasensor is less sensitive, it represent good alternative to the electrochemical sensors. First, the acoustics response can be easily and directly monitored without any labels. This technique can be easily operated. Relatively low cost TSM devices are currently available at the market. In addition, by optimalization of the aptamer immobilization using nanostructures such as carbon nanotubes, dendrimers, etc. it is possible to increase of aptamer surface density which may improve the detection limit.In this work we demonstrated label-free detection of HER2 positive SK-BR-3 breast cancer cells using thickness shear mode acoustics method (TSM). The sensor based on immobilization of biotinylated DNA aptamers adsorbed at the neutravidin self-assembled monolayer at the gold surface of TSM transducer allowed specific detection of the cancer cells with limit of detection (LOD) of 1574 cells/mL (HB5 aptamer based sensor) or 1418 cells/mL (HeA2_3 aptamer based sensor). The amplification of detection using gold nanoparticles modified by aptamers allowed to improve detection limit only up to 550 cells/mL. This is due to the fact that the thickness of the cell layer is much higher (5\u201310 \u03bcm) in comparison with shear wave penetration depth (0.2 \u03bcm). Therefore the decrease in the frequency observed reflects only small part of the whole cell mass. Thus, further improvement of the sensitivity of detection by acoustics method can be obtained by optimization of aptamer immobilization using nanostructures such as carbon nanotubes, dendrimers or other nanomaterials. We believe that using this approach further progress in acoustics cell sensors will be possible toward practical application in the cancer diagnostics in particularly based on the detection of circulating tumor cells (CTC)."}
+{"text": "Cryoglobulinemic vasculitis is most commonly observed in the setting of hepatitis C infection. However, many other etiologies have been identified as well. We herein report a case of cryoglobulinemic vasculitis in a 56-year-old male secondary to infective endocarditis. The patient presented with a five-week history of a painful, purpuric rash and was found to have positive blood cultures, elevated cryoglobulins, and vasculitis on histology. He recovered after treatment with intravenous antibiotics and steroids. Although rarely identified as a cause of cryoglobulinemic vasculitis, infective endocarditis must be considered on the differential diagnosis as a delay in treatment can worsen the prognosis for the patient. Cryoglobulins are circulating immunoglobulins that can precipitate in cold environments and cause vasculitis, particularly affecting the skin, kidneys, and peripheral nerves. Although associated with hepatitis C infection in up to 90% of cases, cryoglobulinemia has been linked to many other infections, such as the hepatitis B virus and HIV, and autoimmune conditions like Sjogren\u2019s syndrome, systemic lupus erythematosus, and rheumatoid arthritis . Since mA 56-year-old male with no prior medical history except for chronic low back pain was admitted to the hospital for a painful rash over his legs, feet, and hands with associated malaise for five weeks. The patient stated his rash became more prominent when he was cold. The patient denied any history of drug use. He denied any fever or arthralgias. A punch biopsy performed by a dermatologist three weeks prior revealed only mild spongiosis with a superficial perivascular lymphocytic infiltrate. The dermatology service was consulted for the rash. His physical examination included a toxic-appearing man breathing room air with subtle palpable purpura and scattered 1-3 mm ulcers mainly on the acral surfaces of his hands and feet but also bilateral lower legs Figure . No septCryoglobulinemia entails the presence of abnormal antibodies that are water-soluble in warm environments yet precipitate in colder conditions. Immune complexes produced from precipitation cause inflammation that affects the small vessels, most notably in the skin and joints . The mosAlthough classically associated with hepatitis C, the presence of cryoglobulins in patients with IE has previously been reported -4. In thDespite not being commonly observed in this setting, cryoglobulinemic vasculitis has been documented as the presenting symptom of IE -7. In a As with the cases above, our patient presented with a purpuric rash on his legs and mild renal impairment. Unlike the previous cases, however, his skin lesions were also present on the upper extremity. He had growth of methicillin-sensitive Staphylococcus aureus on blood cultures and vegetation on the aortic valve seen on transthoracic echocardiogram, strengthening the case for IE as a diagnosis. The patient\u2019s IE exhibited a relatively indolent course of illness. We believe that this slow progression uniquely allowed for the development of cryoglobulinemia significant enough to be a prominent contributor to his overall IE disease course, rather than showcasing the more classical IE features: septic emboli, internal organ infarcts, and other vascular phenomena. In his case, the patient was administered antibiotics in conjunction with systemic and topical corticosteroids, which addressed both the infectious and inflammatory nature of his condition.Infective endocarditis may, on rare occasions, cause cryoglobulinemia, which typically features a purpuric lower extremity rash and renal dysfunction. Clinicians should appreciate the high mortality rate of IE, especially if not rapidly detected. A treatment regimen consisting of corticosteroids without antibiotics may worsen patients with IE due their immunosuppressive effects. Therefore, failure to consider IE in the differential diagnosis of cryoglobulinemia may seriously compromise a patient\u2019s prognosis."}
+{"text": "Cancer vaccines provide a complex source of neoantigens. Still, increasing evidence reveals that the neoantigen quality rather than the quantity is predictive for treatment outcome.\u2212/\u2212 tumor model, we performed a side-by side comparison of two autologous cell-line derived tumor lysates (namely 328 and A7450 T1 M1) harboring different tumor mutational burden . Mice received repetitive prophylactic or therapeutic applications of the vaccine. Tumor incidence, immune responses and tumor microenvironment was examined.Using the preclinical Mlh1\u2212/\u2212 tumor cells in IFN\u03b3 ELISpot, but likewise YAC-1 cells, indicative for stimulation of both arms of the immune system. By deciphering local effects, vaccines shaped the tumor microenvironment differently. While A7450 T1 M1 prophylactically vaccinated tumors harbored low numbers of myeloid-derived suppressor cells (MDSC) and elevated CD8-T cell infiltrates, vaccination with the 328 lysate evoked MDSC infiltration. Similar effects were seen in the therapeutic setting with stable disease induction only upon A7450 T1 M1 vaccination. Untangling individual response profiles revealed strong infiltration with LAG3+ and PD-L1+ immune cells when treatments failed, but almost complete exclusion of checkpoint-expressing lymphocytes in long-term survivors.Both tumor cell lysates delayed tumor formation in the prophylactic setting, with the A7450 T1 M1 lysate being more effective in decelerating tumor growth than the 328 lysate . Comparable results were achieved in therapeutic setting and could be traced back to antigen-driven immune stimulation. Reactive T cells isolated from A7450 T1 M1-treated mice recognized autologous Mlh1By applying two tumor cell lysates we demonstrate that neoantigen quality outranks quantity. This should be considered prior to designing cancer vaccine-based combination approaches. The idea of using whole tumor lysates as vaccines dates back to the late 1970ies and aims at the induction of a vigorous immune response against cancer \u20134. As suSeveral strategies were employed to prepare Ags from whole tumor cells and thus produce a highly immunogenic vaccine. Common strategies include chemical treatment, radiation as well as repetitive freeze/thaw cycles. With these methods, standardized, applicable sources of tumor-specific Ags can be generated. Besides, tumor cell lysates are also useful in high-risk, tumor-free patients\u2014especially for prophylactic approaches.Lynch syndrome (LS), the most common hereditary cancer syndrome, represents the paragon for cancer vaccination approaches \u201312. Affe\u2212/\u2212 mouse model that resembles features of the human LS counterpart [To move forward in developing vaccination strategies, we employed the MLH1nterpart , 18. Thenterpart , 20. Whinterpart . We hypo\u2212/\u2212 cells  cell lines: 328, A7450 T1 M1 and one lymphoma cell lines: 1351) were established in our lab. YAC-1 cells were originally cultured in DMEM medium, supplemented with 10% FCS , 6\u00a0mM Glutamine, and penicillin/streptomycin antibiotics . Tumor lysates were prepared from cell cultures in P15 as described [Mlh1escribed . Brieflyg . Cells were seeded in a 6-well plate (density: 3\u2009\u00d7\u2009105\u00a0cells/ml). GM-CSF was added  cells were harvested every third day. Therefore, non-adherent cells were gently pipetted up and down, transferred in a centrifuge tube, pelleted , the supernatant discarded and the pellet resuspended in freshly prepared medium. Cells were counted and re-cultured in DC medium containing GM-CSF with no other cytokines to generate highly pure DCs. On the 9th day, supernatant was collected and centrifuged. DCs were phenotyped using the following FITC-, PE-, APC-, and PE/Cy7-labeled antibodies (1\u00a0\u03bcg each): anti-CD11c , anti-CD83 (Biolegend), anti-CD11b (Immunotools), anti-CD40 (Biolegend), anti-CD80 (Immunotools), anti-CD86 (Immunotools), anti-MHC class I/II (Immunotools), and anti-CD19 (Immunotools). Afterwards, DCs were loaded with protein lysate (50\u00a0\u00b5g/tumor lysate). After 24\u00a0h of incubation, peripheral blood mononuclear cells were added in a ratio of 1:10 (DC:immune cell) [DCs were generated from murine femur and tibia as described . Brieflyne cell) , 24 and The cell lines 328 and A7450 T1 M1 were processed likewise , 21 for Furthermore, mapping the mutations and their statistics on a linear gene product (proteins of interest) was done with a \u2018lollipop\u2019 mutation diagram generator . Based oAll animal experiments were approved by the German local authority: Landesamt f\u00fcr Landwirtschaft, Lebensmittelsicherheit und Fischerei Mecklenburg\u2010Vorpommern (7221.3\u20101\u2010026/17), under the German animal protection law and the EU Guideline 2010/63/EU. Mice were bred in the animal facility of the University Medical Center in Rostock under specific pathogen\u2010free conditions. Mlh1 genotyping was done according to [A detailed treatment schedule is provided in Fig.\u00a0PET/CT imaging scans were performed on a small animal PET/CT scanner  according to a standard protocol as described before , 20.4Cl, 10\u00a0mM KHCO3 , and 0.1\u00a0mM EDTA . Negative controls consisted of lymphocytes stained with appropriate isotypes . Cells were washed, resuspended in PBS and analyzed by flow cytometry on a Flow Cytometer . Data analysis was performed using BD FACSuite software (BD Pharmingen).Blood samples were taken routinely from the retrobulbar venous plexus. Blood samples were stained with a panel of conjugated monoclonal antibodies  followed by lysis of erythrocytes  in combination with the Bio-Plex Manager Software.3 targets/well  were seeded in IFN\u03b3\u2013specific mAb -coated, 96-well microtiter plates. Peripheral blood leukocytes (5\u2009\u00d7\u2009104 cells/well) or splenocytes (1\u2009\u00d7\u2009104 cells/well) were added in triplicates and co-cultured overnight. Finally, bound antibody  was visualized by BCIP/NBT ; spots were counted using an ELISpot reader. Presented are the numbers of IFN\u03b3-secreting cells corrected for background levels counted in the absence of target cells, which was always\u2009\u2264\u20095 spots/well. Target cells without effector cells showed no background level.2.5\u2009\u00d7\u200910All values are expressed as mean\u2009\u00b1\u2009SD. In case of PET/CT data, raw tumor sizes are presented. After proving the assumption of normality (Kolmogorov\u2013Smirnov test), differences between vaccinated and control mice were determined using the unpaired Student\u2019s t-test or one-way ANOVA (Bonferroni or Dunnett\u2019s multiple comparison). Kaplan\u2013Meier survival analysis was done by applying the log rank (Mantel Cox) test. Statistical analyses were performed using GraphPad Prism 5 . The criterion for significance was set to p\u2009<\u20090.05.\u2212/\u2212 cell lines established from spontaneous GIT were used. The drug response of the cell lines 328 and A7450 T1 M1 was determined before and revealed no significant differences towards standard cytostatic drugs [In this study, two Mlh1ic drugs . By asse+CD8+ T cells, which were activated and additionally positive for IFN\u03b3. By contrast, the phenotypes of leukocytes from 328 lysate-loaded DCs changed faintly compared to the control , making these candidates unlikely to act as tumor rejection Ags.Then, the coding microsatellite (cMS) mutational profile was analyzed comparatively on a panel of putative MSI target genes  or moderate  TMB [To test the immunogenicity of whole cancer vaccines on a more global level, Mlh1/Mb) TMB  \u2212/\u2212 A7450 T1 M1-treated mice. Median survival time was 37\u00a0weeks, whereas it was only 22\u00a0weeks in control mice (p\u2009<\u20090.001). The Mlh1\u2212/\u2212 328 vaccine had a minor impact on survival, reaching a median survival of 25\u00a0weeks , skin malignancies (1 case) or died spontaneously (2 cases). Mice receiving the Mlh1\u2212/\u2212 328 tumor lysate showed a comparable tumor spectrum. Here, 70% suffered from GIT or generalized lymphomas in the spleen, one mouse developed a thymic lymphoma, and two mice died because of unknown malignancy (suspected lymphomagenesis).Prophylactic vaccination yielded significantly prolonged cancer-free survival in Mlh1\u2212/\u2212 A7450 T1 M1 lysate was reflected by immunological changes in the peripheral blood. While T cell numbers only gradually increased, we observed elevated levels of circulating NK cells . In line with the increased number of NK cells upon A7450 T1 M1 vaccination, leukocytes from vaccinated mice reacted against NK target cells YAC-1 (p\u2009<\u20090.01).Then, the reactivity of peripheral blood leukocytes was assessed upon co-incubation with different target cells by IFN\u03b3-ELISpot assay Fig.\u00a0a. Autolo\u2212/\u2212 A7450 T1 M1 vaccine largely prevented infiltration of CD11b+/Gr1+ myeloid-derived suppressor cells and F4/80+ tumor-associated macrophages (TAMs). While these cell types were barely detectable, we observed high numbers of infiltrating CD11c+ DCs as well as CD8+ cytotoxic T cells (CTL). By contrast, the Mlh1\u2212/\u2212 328 lysate triggered MDSC infiltration in the tumor, CTL were occasionally found. PD1 expression was not altered by any vaccination and, thus, expression levels were highly comparable with control tumors.Next, the tumor microenvironment was studied in detail to explore the quantity and quality of leukocyte infiltrates. Prophylactic vaccination leveraged the microenvironment Fig.\u00a0b. The Ml\u2212/\u2212 A7450 cells compared to the 328 lysate was shown before [Then, we moved to the therapeutic approach Fig.\u00a0. The surn before  and  and drug response [\u2212/\u2212 A7450 T1 M1 vaccinated mice compared to those receiving the 328 lysate was reflected by systemic immunological changes. T cell numbers gradually increased, and mice had elevated levels of circulating NK cells, recognizing autologous target but also YAC-1 cells in ELISpot IFN\u03b3 assays. NK cells are a subset of innate lymphocytes with great potential to kill cancer cells directly, and thus, the elevated number of NK cells detected here may have also prevented early tumor formation [\u2212/\u2212 A7450 T1 M1 vaccination. Here, numbers of tumor-infiltrating CD11c+ DCs were elevated; tumor-promoting MDSCs and TAMs low and provide a reasonable explanation for the delayed in vivo tumorigenesis compared to 328-vaccinated mice.By using cell lines for vaccination, this approach provides a virtually limitless source of neo-Ags, permits standardized, large-scale vaccine production, and is\u2014from the economic point of view\u2014very cost-effective . To get o growth . While tresponse , their cresponse , with knormation \u201335. Due ormation . Still, \u2212/\u2212 tumors. Other preclinical studies likewise described boosted Ag cross-presentation, increased immune-supportive M1 macrophages, as well as circulating T cells upon gemcitabine [. Several extrinsic as well as intrinsic factors foster resistance, especially after initial response. Treatment failure might be finally attributable to insufficient T-cell responses ; poor T-cell homing to Mlh1\u2212/\u2212 tumors, dysfunction or death of T cells within the tumor, and immune escape mediated by upregulation of immune-checkpoint molecules LAG-3 and PD-L1. Hence, the balance between immune-mediated tumor prevention/elimination and escape is a narrow ridge [+ lymphocytes.Therapeutic application prolonged progression-free survival, but again only when mice received the A7450 T1 M1 vaccine. The ultra-hypermutated-derived lysate 328 failed to provide a clear survival benefit. Adding chemotherapy to either vaccination improved the outcome by inducing long-term stable disease (\u2265\u20094\u00a0weeks). Once more, tumor growth control was more effective in the A7450 T1 M1 combination group, assuming that the coupled application of the tumor lysate and low-dose chemotherapy induced immunogenic cell death. This converted dying cancer cells into a vaccine vulnerable to be taken up by DC. These, in turn, activated T cells to kill tumor targets. In support of this, half of the mice received partial remission. Here, chemotherapy itself may play a supportive role in re-activating the immune system against Mlh1citabine , 38. Indcitabine , 40. Sticitabine , whereascitabine . While mow ridge , 44. A pIn humans, MMR-D tumors are often characterized by an increased density of intratumoral T cells and most patients are eligible to immunotherapy. Still, we here add evidence that the neo-Ag quality, rather than quantity defines response. These findings are supported by a recent study comparing pancreatic cancer and melanoma Ag load and T cell responses . While tTo improve vaccine efficacy prospectively, some additional aspects must be considered: (I) the choice of the right target Ags, whose mutation frequency is high and ideally shared among cancers; (II) the time-interval and dosing of vaccines; (III) the route of application; (IV) the choice of adjuvant and/or combinatorial agent as well as (V) a change of the standard of care in humans from the tumor to the host by treating patients with immunotherapy in first-line and thus before a history of previous anti-cancer chemotherapy.Finally, we would like to mention that there are some limitations to this study. Firstly, we only injected vaccines without additional adjuvants that might play a supporting role in immune stimulation. Secondly, mice were vaccinated with a lysate of only one cell line instead of different ones. Hence, there is a possibility that we have missed certain Ags that evoke immune responses when applied together and thus individual tumor clones may have been unrecognized.Prophylactic as well as therapeutic vaccination with whole tumor lysates delay tumor growth. Still, not only tumor mutational burden but also neoantigen quality predict vaccination efficacy. In addition to the number of mutations provided by a vaccine, the ability to evoke T cell responses and induce an inflamed tumor microenvironment is crucial for treatment responses."}
+{"text": "Differences in the way males and females look or behave are common in animals. However, discrete variation within sexes (sex-limited polymorphism) also occurs in several vertebrate and invertebrate lineages. In birds, female-limited polymorphism (FLP) in which some females resemble males in coloration is most prominent in hummingbirds, a group known for its morphological and behavioural sexual dimorphism. Yet, it remains unclear whether this intrasexual colour variation in hummingbirds arises through direct selection on females, or indirectly as a non-adaptive byproduct resulting from selection on males. Here, we analysed specimens from more than 300 hummingbird species to determine the extent, evolutionary history and function of FLP. We found that FLP evolved independently in every major clade and occurs in nearly 25% of hummingbird species. Using phylogenetically informed analyses, we rejected non-adaptive hypotheses that FLP is the result of indirect selection or pleiotropy across species. Instead, FLP is associated with ecology, migratory status, and marginally with social dominance, suggesting a socioecological benefit to females. Ultimately, we show that FLP is not only widespread in hummingbirds and likely adaptive, but may also be useful for understanding the evolution of female ornamentation in systems under strong sexual selection. Adaptive explanations for FLP are similar to those for the evolution of ornaments and weaponry in females ,18\u201321. Ton morph ,13,22,23e choice   ,28,29. Iochromes . Howeverochromes ,34, theysexually antagonistic intralocus conflict, we predict higher transition rates from sexual monochromatism to FLP and from FLP to sexual dichromatism than from sexual dichromatism to FLP or from FLP to sexual monochromatism. Second, we test the pleiotropy hypothesis by examining the relationship between the degree of androchromic plumage and bill length, a well-studied trait known to differ between sexes across species, underlying resource use and a sex's ecological niche  or socially  relevant. These socioecological traits relate to potential inter- and intraspecific social competition, and thus can act as a proxy in exploratory analyses on the relationship between social competition and FLP. Ultimately, this study provides the most species-rich comparative study to date of FLP in any taxonomic group, illuminating the pervasiveness and potential complex function of variable female ornaments in a family of birds that has become a model for the study of ornamentation and sexual selection.Here, we establish the pervasiveness and evolutionary origin of FLP in hummingbirds using museum specimens from nearly every species in the family, and then explore adaptive and non-adaptive hypotheses underlying this trait in a phylogenetically informed context. We begin by quantifying the prevalence of FLP within hummingbirds and performing ancestral state reconstruction to determine how many times the trait has evolved within the group. Next, we test the two non-adaptive hypotheses for FLP. First, since sexually antagonistic intralocus conflict should resolve through the eventual evolution of complete sexual dichromatism , if FLP he i.e. \u201337). Alt. Altsexuhe N = 16 542; electronic supplementary material, figure S1) across all recognized species for which there are skins (N = 307) at The American Museum of Natural History, the Louisiana State University Museum of Natural Science, National Museum of Natural History, The Field Museum of Natural History, and The Natural History Museum of Los Angeles.We assessed Trochilidae specimens ((b)We evaluated specimens clearly labelled as \u2018male\u2019 or \u2018female\u2019 on the tag, including those in which sexing methods were unknown (60% of samples) or for which gonad data were present on the tag (38% of samples). We did not evaluate specimens that were labelled as \u2018sexed by plumage\u2019 (2% of samples). Analyses comparing specimens without gonad data to those sexed reliably suggested that individuals sexed with unknown methods were done so reliably . Nonetheless, we took the uncertainty of sexing reliability into account in our analyses by using thresholds as a form of sensitivity analysis when classifying species as exhibiting or not exhibiting FLP (see below for details).(c)All assessed specimens were qualitatively scored on an ordinal scale of androchomic plumage by one researcher (E.S.D.) following Bleiweiss ,40. EachN = 532 individuals from N = 39 species) were examined at the Moore Lab of Zoology by a second investigator. These specimens were only classified as \u2018heterochromes\u2019 (Class 1 or 2) or \u2018androchromes\u2019 (Class 3 or 4) and not used in frequency histograms or unimodality tests; they were only used to classify FLP for phylogenetic analysis.Additional specimens from species already present in the dataset , and if that species has not been classified previously as exhibiting FLP. Those species that were categorized as lacking FLP were further classified as being either sexually dichromic or sexually monochromic based upon qualitative assessments of specimens and field guides [Here, we classified a species as d guides . PhylogeWe chose to present a more conservative analysis of FLP presence in the text because it better takes into account risks associated with mis-sexing. Additionally, we chose a moderate absence threshold to balance minimizing excessive branch pruning while also minimizing the risk of falsely classifying a species as lacking FLP. Although this absence threshold could have incorrectly included more species that were mislabelled as lacking FLP, the chances of this are relatively low given that the majority of hummingbird species did not exhibit FLP. Such a threshold also decreased excessive pruning of the tree, which is known to bias transition rates and node states . Importa(d)To determine the number of independent evolutionary origins of FLP in hummingbirds and whether or not FLP is an intermediate plumage stage between sexual monochromatism and dichromatism, we reconstructed ancestral character states and transition rates. Three discrete trait classes  were mapped onto a previously published molecular phylogeny , which wape [geiger [corHMM [c). For each dataset, we used the transition rates model that had the lowest AICc score and considered all where \u0394AICc is less than 2. We then used the results found in corHMM to reconstruct trait evolution on trees in ape and estimated the likelihoods of root states. Three plumage states were estimated at each node in the model: sexual monochromatism, sexual dichromatism, and FLP. Eight ancestral state reconstructions were conducted using the eight matrices that varied in their thresholds for identifying FLP. All species that were not present in our datasets were pruned from a given tree, resulting in final trees ranging from 152 to 198 species, depending on which dataset was used . We then used the most-supported transition rate model to test for non-adaptive sexually antagonistic intralocus conflict. We estimated the standard deviation of the likelihood of root states by randomly pruning 10% of species over 1000 simulations to determine if tree topology strongly affected ancestral state reconstruction.We estimated the root state without predicting the root state likelihoods. Since we did not fix a root state, we instead conducted an ancestral state reconstruction and estimation using a continuous-time Markov chain model to determine the root state likelihoods using the R  packagesape , geiger  [geiger  and corH [corHMM . We used(e)To test the non-adaptive pleiotropy hypothesis, we analysed photographs of male and female specimens that ranged from heterochrome to androchrome in 22 species  across all hummingbird clades. We also measured the wing and bill lengths (see below for details) of each specimen. We constructed a light box and photographed individual specimens with standardized settings from five angles: dorsal, ventral, each side, and with the bill perpendicular to the camera lens. A few species had additional photographs taken if iridescence was not visible from these angles. A white balance card was placed in the field of view and used for white balance normalization in RawTherapee . On avermageJ [Florisuga mellivora), UV was highly correlated (greater than 97%) with visible blue in plumages with high UV reflectance .To quantify the degree of androchromic coloration and bill length, each species' specimen's photographs were analysed in ImageJ  by a sinmageJ . HowevermageJ , and in MASS [N = 6), which were then excluded from subsequent analyses. We fit a linear model of relative bill length (bill length minus mean bill length of the species) with LD colour values to determine if androchromic plumage is associated with andromorphic bills and the slope of this relationship for each species. Accounting for multiple tests, we established an \u03b1 = 0.0031. Finally, we calculated Blomberg K [Next, we trained a linear discriminant analysis (LDA) within each species using the R package MASS  to extraomberg K  to deter(e)N = 167 species). Each species was categorized as migratory or not based on classifications from [N = 290 species) . Using caper [We chose three socially relevant traits for which species-level data were available: migratory status, social dominance, and body size  by cons from  We found that 209 of 307 hummingbird species met either the presence or the absence threshold. Of these, 47  exhibited FLP. The two species not included in the ancestral state reconstruction and that exhibited FLP were not present in the phylogenetic tree . FLP was(b)c score for the ER model (AICc = 360.47) was lower than that of the all rates different matrix (AICc = 367.15) and the symmetrical rates matrix (AICc = 362.95), indicating that transition rates between plumage dichromatism, monochromatism, and FLP were similar . Furthermore, none of our sensitivity analyses resulted in an ARD with higher transitions from sexual monochromatism to sexual dichromatism and from sexual dichromatism to FLP . Some of our sensitivity analyses resulted in the symmetric rates matrix being more supported when there were higher transition rates from monochromic to FLP and from FLP to dichromic than from monochromic directly to FLP. However, these models demonstrate higher transition rates from FLP to dichromic and dichromic to FLP than other transitions . The only sensitivity analysis in which the all rates different matrix was supported found the highest transition rates from dichromic to FLP ; though, for this analysis, AICc scores did not distinguish between the all rates different, the symmetrical rates, and the equal rates matrices . Together, these results do not support the non-adaptive hypothesis that FLP is an intermediate state and that dichromatism is a resolved state of intralocus conflict.Estimating transition rates allowed us to determine whether FLP is an intermediate state between sexual monochromatism and dichromatism, as predicted by sexually antagonistic intralocus conflict and the evolution of sexually dimorphic male ornaments  ,27. The (c)Heliangelus amethysticolis exhibited a significant negative correlation  between relative bill length and LD1  between bill length and LD1 . There was also no general pattern of association between other morphological traits and androchromic variation in females across most species, though some species did exhibit significant trait associations .For 16 species, we assigned LD scores to each individual male and female, where higher LD scores were associated with more androchromic plumage . Linear  and LD1 , whereas and LD1 . Heliang(d)\u03b2 = 0.088, p = 0.47), social dominance  or migratory status . However, when excluding monochromic species, species were less likely to have FLP if they were migrants , and there was a trend for behaviourally dominant species to exhibit FLP over non-dominant species . There was no relationship with body size and FLP . Finally, we found a slight but significant negative association with mean precipitation , but no other significant climate associations, when excluding monochromic species .When comparing the binary character \u2018FLP\u2019 to \u2018no FLP\u2019 for all species, we found no significant effect of body size (. 4Previous work has demonstrated that multiple hummingbird species exhibit FLP where some females look like males ,28,29. WTo determine why FLP is so common in hummingbirds, we began by exploring two non-adaptive hypotheses. First, we tested if FLP was a non-adaptive byproduct of sexually antagonistic intralocus conflict where females resemble males due to strong selection on males and a shared genetic architecture between the sexes ,27. AccoNext, we tested the non-adaptive pleiotropy hypothesis whereby androchromic plumage is a consequence of indirect selection on other andromorphic traits through pleiotropy . In hummAlthough non-adaptive pleiotropy may play a limited role in the evolution of FLP within individual species, non-adaptive hypotheses do not fully explain the evolution and maintenance of FLP in hummingbirds generally. We explored potential socioecological factors that might provide preliminary evidence for an adaptive function of male-like coloration in females. We found significant associations with migratory status, lower mean precipitation, and marginal association of FLP with social dominance, all of which are linked to interspecific interactions and competition over resources \u201371. Soci. 5In summary, our results indicate that FLP is widespread across the hummingbird lineage and has evolved independently multiple times. We rejected non-adaptive hypotheses for FLP by analysing evolutionary transition rates, confirming the bimodal distribution of androchromic plumage within hummingbird species, and finding no association between androchromic plumage and andromorphic morphology. Instead, we found preliminary support for the idea that socioecological traits best predict the evolution of FLP across the phylogeny, making social selection a potential explanation for the evolution and maintenance of this trait. Hummingbird plumage ornaments are classic examples of sexual selection, but these traits may have a more complex function than previously considered. Gaining a better understanding of female signalling traits in a group that exhibits widespread variation in female plumage within and across species will enable us to test hypotheses explaining female ornamentation, which are quite distinct from those underlying ideas of classic sexual selection on male ornaments ,8,20. ByClick here for additional data file."}
+{"text": "Globodera rostochiensis and Globodera pallida, are quarantine restricted pests of potato causing major yield and financial losses to farmers. G. rostochiensis was first reported from Kenya\u2019s key potato growing area in 2015. We sought to determine the diversity, prevalence and distribution of PCN species across the country by conducting a country-wide survey between 2016 and 2018, which included a more focused, follow-up assessment in three key potato growing counties. A total of 1,348 soil samples were collected from 20 potato growing counties. Information regarding local potato farming practices, potato cultivar use, their diversity and availability was also recorded. PCN cysts were obtained from 968 samples (71.8%) in all the counties surveyed, with Nyandarua County recording the highest PCN field-incidence at 47.6%. The majority of PCN populations, 99.9%, were identified as G. rostochiensis, while G. pallida was recovered from just one field, in a mixed population with G. rostochiensis. Inconsistencies in PCR amplification efficiency was observed for G. rostochiensis using the recommended EPPO primers, compared with ITS primers AB28/TW81, indicating that this protocol cannot be entirely relied upon to effectively detect PCN. Egg density in Nyandarua County varied between 30.6 and 158.5 viable eggs/g soil, with an average egg viability of 78.9 \u00b1 2.8% . The PCN-susceptible potato cultivar named Shangi was the most preferred and used by 65% of farmers due to its shorter dormancy and cooking time, while imported cultivars  with resistance to G. rostochiensis were used by 7.5% of farmers due to unavailability and/or limited access to seeds. Thus, most farmers preferred using their own farm-saved seeds as opposed to purchasing certified seeds. Establishing the distribution and prevalence of PCN and elucidating the local farming practices that could promote the spread of PCN is a necessary precursor to the implementation of any containment or management strategy in the country and ultimately across the region.Potato cyst nematodes (PCN), such as  Potato is a valuable and nutritious staple crop, driving both food security and Growth Domestic Product (GDP) growth globally . ApproxiGlobodera rostochiensis and G. pallida , where they were air-dried, thoroughly mixed, sieved to remove stones and debris before extraction of cysts from a 200 g sub-sample using the Fenwick can floatation method . BrieflyTM Base thermocycler (Applied Biosystems Life Technologies) with multiplex primers , which are species-specific for G. pallida (265 bp amplicon) and/or G. rostochiensis (434 bp amplicon) targeting the 18S rRNA gene and the internal transcribed spacer ITS1 region and ITS primers (TW81 5\u2019-GTT TCC GTA GGT GAA CCT GC-3\u2019 and AB28 5\u2019-ATA TGC TTA AGT TCA GCG GGT-3\u2019) targeting the ITS1-5.8S-ITS2 regions . Each PCR reaction mixture  contained 12.5 \u03bcl PCR master mix (One Taq\u00ae 2X Master Mix with standard buffer), 1 \u03bcl of each primer (forward and reverse), 8.5 \u03bcl nuclease-free water and 2 \u03bcl of DNA (template) totaling 25 \u03bcl volume per reaction. PCR amplicons were electrophoresed through 2% agarose gel at 100V for 1 h to confirm successful amplification and size of the amplicons against a 100 bp ladder . For sequencing, species-specific PCR reactions were conducted using a singleplex approach with ITS5/PITSp4 primers for G. pallida and ITS5/PITSr3 for G. rostochiensis; PCR-amplicons were purified using the QIAquick PCR Purification Kit  and sequenced by Sanger sequencing. DNA sequences were manually edited using BioEdit Sequence Alignment Editor  taxonomic guide, under a Leica MZ12.5 dissection microscope. The number of cysts (empty or containing eggs) positively identified as PCN were counted and recorded. About 1\u201310 cysts recovered from samples were subjected to molecular identification using modified EPPO multiplex-PCR protocols  and ITS t Editor . The edit Editor  and otheon 4.5.0 .X < 70%), mid PCN-incidence (70% \u2264 X < 90%) and high PCN-incidence (X \u2265 90%). Further classification for the in-depth study in Nyandarua County was undertaken at the sub-county level, where infestation levels were classified according to: 1 < X < 25 cysts, 26 < X < 110 cysts, 111 < X < 230 cysts, 231 < X < 495 cysts, and 496 < X < 985 cysts per 200 g of soil.A focused, more in-depth study was undertaken in Nyandarua County, which has the largest area of cultivated potato in Kenya. Data related to incidence and infestation level of PCN across surveyed farms were collected from five sub-counties . For each sub-county, the average PCN-infestation level was calculated as the mean number of cysts per 200 g soil recovered from all the farms visited. Incidence (%) at the county level was further determined as the number of fields where cysts were isolated from the total number of sampled farms and expressed as a percentage. Based on these results, counties were classified as low PCN-incidence (50% \u2264 n = 14)], cluster 2 (between 100 and 200 cysts (n = 11)], cluster 3 (between 50 and 100 cysts (n = 5)], and cluster 4 (between 20 and 50 cysts (n = 16)]. The samples were assessed for egg viability using Nile Blue stain, which stains dead eggs and/or juveniles (J2)  with 1 ml of 0.01% Nile Blue stain and incubated in the dark at room temperature for 48 h. After incubation, the stain was carefully rinsed and replaced with 1 ml sterile distilled water. Cysts were then individually removed, placed on a glass slide and gently cut open to expose the contents (The impact of PCN on potato production was estimated using the les (J2) , allowincontents . The totFinally, in each field, soil infectivity (SI) was calculated as the mean number of viable eggs per cyst, multiplied by the total number of cysts extracted in 200 g soil and expressed as viable eggs/g soil. For each sub-county, the mean SI was calculated from all the studied fields. In each sub-county, potato yield losses (t/ha) directly attributable to PCN were estimated based on the SI damage threshold determined by H1 gene, which confers resistance to G. rostochiensis. The cv. Jelly, which is resistant to G. rostochiensis with H1 gene  during field sampling. A semi-structured questionnaire was used to gather agronomic and socioeconomic data to complement the phytosanitary data obtained from the epidemiological survey. Data on farm size (acres), potato cultivars grown, potato seed source, and crop management strategies, e.g., rotation periods, use of alternate crops, etc. were collected. Enumerators were trained prior to conducting the interviews. Data were collected directly onto tablets and collated using Epicollect\u00ae Excel\u00ae Version 1911, mean and standard error of the means were calculated using the \u201cpsych\u201d package, while the model of significance was detected by analysis of variance with a chi-square test and the pairwise comparison of the treatments undertaken using Tukey\u2019s multiple comparison test using the \u201clsmeans\u201d on R version 3.6.1.The data was analyzed using Microsoftn = 18/22) farms were infested with PCN.Cysts were detected from farms in all the counties prospected and were recovered from a total of 902 field samples (82.8%) during the initial survey (2016\u20132017) and in 66 fields (25.6%) in the follow-up survey in 2018. Nyeri County presented the lowest incidence (53%), while counties such as Trans Nzoia, Taita Taveta and West Pokot had 100% incidence . Of the G. rostochiensis  from a combination of both protocols. A relatively lower PCR amplification efficiency was observed with EPPO primers in PCN-species identification. However, cysts from 61 fields were amplified using ITS primers AB28/TW81  were identified as B28/TW81 , which pin Kenya .H1 gene in cv.s Manitou, Markies, Destiny and Jelly (positive control) but not for cv. Shangi, indicating its susceptibility to G. rostochiensis , 26 < X < 110 cysts (22.1%), 111 < X < 230 cysts (14.0%), 231 < X < 495 cysts (5.8%), and 496 < X < 985 cysts (5.8%) respectively per 200 g soil. On average, higher infestation levels were found in Kinangop, with the largest number (66.7%) of highly infested fields or \u201chotspots\u201d with cyst counts \u2265231 cyst/200 g soil , followed by Kipipiri (80.0%), Ol\u2019Joro Orok (78.6%), Ol\u2019Kalou (52.9%) and Ndaragua (33.3%). Among these, infestation levels varied: between 1 < 0 g soil , 4.p \u2264 0.05) in Kipipiri and Ol\u2019Joro Orok; Kinangop and Kipipiri presented the highest SI levels (viable eggs/g soil) with 158.5 and 90.7 viable eggs/g soil, respectively, while the lowest SI levels were observed in Ol\u2019Kalou (30.6 viable eggs/g soil). Yield losses attributed to PCN, considering the damage threshold of 2.75 t/ha per 20 viable eggs/g soil  identified farm-saved seed, which is recycled over several seasons, and ware-potatoes bought from local markets  as their principal source of planting material. Government and private institutional sources of seed, such as Agricultural Training College-Farms (ATCs) (2.3%), seed aid from the Ministry of Agriculture (2.1%), National Research Organizations (KALRO) (1.7%) or private certified seed multipliers (1.9%) accounted for just 8.9% of seed supply. Noteworthy, the diversity on the sources of potato seed used for planting also varied among counties, although the two informal sources for seed (\u201cFarm-saved\u201d and \u201cMarkets\u201d) were predominant across counties (n = 7) was recorded in Nyandarua and Taita Taveta. Only eight counties reported seed multipliers as an accessible source of planting material, and the highest percentage of farmers in Meru County (11.4%) accessed quality declared certified seeds  indicated that they employed crop rotation to manage potato pests and diseases . Thus, iG. rostochiensis in Nyandarua County in Kenya  from the current study and separately documented as a new disease report  under the following accession numbers: MG309873.1, MG309920.1, MN382342.1 \u2013 MN382349.1, MN378550.1, MN378566.1, and MN378644.1.Reference sequences used in this study for phylogenetic analysis are freely available at the NCBI database (HM, SH, LC, WR, MN, and ZK designed the study. LC and HM analyzed the data. DC, SH, MN, LC, JB, and HM wrote the manuscript. All authors improved and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Papio ursinus individuals from southern Africa\u2014indicating a high degree of continuity in the southern African baboon population. This level of population continuity is rare in recent human populations but may provide a good model for the evolution of Homo and other large primates over similar timespans in structured populations throughout Africa.Baboons are one of the most abundant large nonhuman primates and are widely studied in biomedical, behavioral, and anthropological research. Despite this, our knowledge of their evolutionary and demographic history remains incomplete. Here, we report a 0.9-fold coverage genome sequence from a 5800-year-old baboon from the site of Ha Makotoko in Lesotho. The ancient baboon is closely related to present-day  Papio) are Old World Monkeys, widely distributed throughout Africa and the Arabian Peninsula. The six extant species of baboon occupy largely independent geographic ranges  and Southern  clades (P.\u00a0ursinus) has two deeply diverged subspecies (ursinus and grisepes), whose history and distribution may have been shaped by historical changes in range driven by aridification cycles  clades . The soun cycles . Thus, tP.\u00a0anubis) nuclear genome, and to the P.\u00a0ursinus mitochondrial genome. For these libraries, we estimated an average endogenous DNA content of 8.5% and obtained a total mean mapped autosomal coverage of 0.93\u00d7 and mitochondrial coverage of 36.6\u00d7. The Panu_2.0 reference genome does not contain a Y chromosome, but comparison of coverage on the X chromosome (0.47) to the autosomes  indicates that the phalanx belonged to a male. Fragment lengths , olive (P.\u00a0anubis), hamadryas (P.\u00a0hamadryas), Kinda (P.\u00a0kindae), yellow , and chacma (P.\u00a0ursinus) baboons. We included geladas (Theropithecus gelada) as an outgroup. These publicly available data include the complete coding sequence (CDS) of CYTB, part of the CDS of NADH5 and the complete tRNA-His, tRNA-Ser, and tRNA-Leu sequences. The resulting tree . Ha Makotoko is at the eastern end of the range of this subspecies, which extends to the south and west of the Kalahari Desert  to present-day P.\u00a0ursinus (8.7%), suggesting a relatively constant level of genetic diversity. In principal component analysis (PCA), the ancient baboon falls closest to the two P.\u00a0ursinus individuals  has a Z score of 12.1 suggesting that the ancient baboon shares significant drift with P.\u00a0anubis to the exclusion of P.\u00a0ursinus. However, this is also consistent with differential attraction to the reference genome \u2014a common source of bias in ancient DNA studies  that has a Z score of 18.6 indicating that the ancient baboon shares more drift with the reference than other P.\u00a0anubis individuals.Next, we analyzed the autosomal genome together with 14 present-day baboons sequenced as part of the baboon genome project diversity panel . Heterozividuals A. D statividuals  suggest  studies . This inD statistics can be explained with small amounts of gene flow from the reference into other samples  of ancestry related to P.\u00a0cynocephalus. However, we do not detect this signal using ADMIXTURE ; Z\u2009=\u200915.9) so it may be an artefact of the graph fitting or reflect structure within the P.\u00a0ursinus population rather than admixture.To further investigate the effect of reference bias, we constructed admixture graphs using TreeMix  and qpGr samples C. The qpDMIXTURE  (supplemDMIXTURE  online oatistics  (f3(P.\u00a0uTSPY locus can distinguish between subspecies (T.\u00a0gelada TSPY sequence (TSPY copies) confirms the male sex determination. The ancient baboon haplotype appears consistent with that reported for P. ursinus griseipes in the ursinus/kindae hybrid zone in Zambia   and a larger sample of n\u2009=\u200916 males represented by one third phalanx per individual  (13C\u2009=\u2009\u201316.99\u2009\u00b1\u20090.19\u2030 and \u03b415N\u2009=\u20094.46\u2009\u00b1\u20090.18\u2030 and a C:N ratio of 3.3) reflect a predominantly C3 diet, typical of P. ursinus, although the low value may reflect a diet more heavily focused on browse or fleshy fruits. The nitrogen values reflect low trophic level species and a largely vegetarian diet.We investigated the metrics of the phalanx  online.  0.7\u2009mm) . However 0.7\u2009mm) . Carbon P. ursinus ursinus persisted in the foothills of the Maloti-Drakensberg Mountains throughout the past 6,000\u2009years. This is despite the fact that paleoenvironmental proxies predict drier conditions across the summer rainfall zone which may, for example, have limited human occupation across all of southeastern Southern Africa from 6.0 to 3.5\u2009ka  reference sequence  from the bammds package  by restricting to sites that were polymorphic in a single TSPY we aligned reads from the ancient baboon that had not aligned to the reference to the T. gelada TSPY sequence; GenBank: AF284278.2 (P.\u00a0hamadryas sequence from the same reference, and four other partial sequences (T.\u00a0gelada sequence to identify differences.To analyze 284278.2 . We alsoequences  which weGenome Biology and Evolution online.evaa019_Supplementary_DataClick here for additional data file."}
+{"text": "The cardinal feature of adaptive immunity is its ability to form memory responses that can be rapidly recalled to contain pathogens upon reencountering. Conferring a robust memory immune response to an infection is a key feature for a successful vaccination program. The plasmablasts are cells that not only can secret non-neutralizing antibodies but also can secrete the specific antibodies essential to neutralize and inactivate the invading pathogens. Dengue has been recognized as one of the most important vector-borne human viral diseases globally. Currently, supportive care with vigilant monitoring is the standard practice since there is as yet no approved therapeutic modality to treat dengue. Even though the approved vaccine has become available, its low efficacy with the potential to cause harm is the major hurdle to promote the widespread usage of the vaccine. Despite the decades of research on dengue, the major challenge in dengue vaccine development is the absence of suitable experimental animal models that reflect the pathological features and clinical symptoms, as seen in humans. Dengue is transmitted by the bite of mosquitoes carrying infectious dengue virus (DENV), which has four distinct serotypes. Recently, cases resulting from unconventional transmission routes, such as blood transfusion, organs as well as stem cells and bone marrow transplantations, and mother-to-infant vertical transmission, have been reported, suggesting an alternate route of DENV transmission exists in nature. This review discusses issues and challenges needing to be resolved to develop an effective dengue vaccine. Development of a robust and reliable dengue animal model that can reflect not only dynamic human clinical symptoms but also can answer around why preexisting neutralizing antibodies do not confer protection upon re-infection and immune protection marker for dengue vaccine efficacy evaluation. Historically, dengue has been registered and associated with human beings for more than a thousand years . ConsequAedes spp. mosquitoes carrying infectious dengue virus (DENV). Upon infection, various clinical symptoms including asymptomatic, mild undifferentiated fever, classical dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS) have been well-established [Dengue is mainly transmitted by ablished . The ratablished ,6,7,8,9.ablished ,11. If tFor symptomatic dengue, the majority of DF patients can return to normal conditions within a week, while only a low percentage of the afflicted persons may develop severe dengue, DHF/DSS. DF and DHF presentations bear similarities to other acute febrile illnesses, resulting in difficulty for the diagnosis of severe DHF by medical professionals. One of the salient clinical features that can differentiate DHF from DF is the plasma leakage due to an increase in vascular permeability . The criExperimental human studies suggest that pre-exposure to DENV can induce protection as a result of the development of immunity, which was first observed in humans by Siler et al. and Simmons et al. ,16. The Dynamic clinical features observed in dengue patients may result from intrinsic and extrinsic factors. Age, ethnic or genetic background, and immune and nutritional status of the individual are intrinsic factors. Country locations, circulated virus strains and multiple infections, population density, and competency of the vector mosquitoes are the extrinsic factors. Generally speaking, people of all ages are susceptible to DENV infection, independent of other factors. In clinical presentations, age has been a major factor for the differential presentations of the dengue patients ,28,29. YIn order to get a much more comprehensive understanding of dengue, studying the pathogenic parameters involved with the infected host is necessary. Since dynamic clinical course occurs in affected patients and the clinical manifestations change with time, it is very difficult to obtain one specimen at a time point that can completely demonstrate the whole course of pathological features induced by the virus in humans . An atteHigh viremia is one of the major clinical features in acute dengue patients, and yet, the morphology of dengue viral particles in the circulation of the infected patients has been more elusive and difficult to convincingly demonstrate , despiteBecause there are very scanty autopsy data available, the pathogenesis of DHF/DSS has not yet been addressed clearly. Salient features on abnormalities include vascular congestions and dilation with edema and multiple focal hemorrhages in most organs, mild to moderate pleural effusion and ascites, mononuclear cell infiltration of interstitial tissues and alveolar walls of lungs, focal myocardial congestion, and a decrease in mature lymphocytes with the proliferation of mononuclear forms in the germinal centers of lymph follicles . InteresThe vascular congestion, dilation, and increased permeability could lead to extensive edema and hemorrhage that were observed in the gastrointestinal tract, the skin, as well as other tissues, leading to loss of plasma volume associated with electrolyte disturbances . In addiAs of today, despite robust and intensive investigations on dengue, the immunological response and pathological features of DENV infection have not yet been fully delineated, resulting in a major hurdle to develop effective vaccines and therapeutic modalities for dengue. One of the most important tasks in the understanding of the pathogenesis of dengue and dengue vaccine development is the development of an animal model that can capture the salient features of human dengue. These include the ability to support virus replication, induction of clinical signs, and immune responses that are comparable to human dengue virus infection. An attempt to reproduce the key features of the human dengue disease in animals was undertaken during the early 1920s. All in all, more than 500 different animal species were investigated, but the disease was not replicated in these tested animals ,59,60,61Recent advancements in technology and knowledge on animal biology and the scientific understanding gained on the pathogenic causes of dengue in suitable animal models have been improved ,65. Howe\u2212/\u2212 mouse model transplanted with human cells [\u2212/\u2212 mice are permissive to DENV2 infection and present viruses in plasma, the liver, spleen, and nervous system as well as bleeding and blood vessel leakage [\u2212/\u2212/STAT2\u2212/\u2212 and type1 IFN receptor (STAT1\u2212/\u2212/AR\u2212/\u2212) mice are susceptible to DENV infections [Immuno-deficient or knockout mouse models reconstituted with human cells or cell lines are available for studies of DENV infection. For example, SCID mice reconstituted with human peripheral blood lymphocytes (SCID-PBL), SCID mice transplanted with human cell lines (SCID cell lines), and NOD/SCID-hu mouse models ,68,69. Ian cells ,73. NOD/an cells ,75. STAT leakage ,77. Howe leakage . STAT1\u2212/fections . Better fections ,79. In gfections . Consequfections ,82. Howefections , the uti9 RNA copies/mL of blood in healthy subjects who donated the blood [5 PFU/mL of blood in a healthy individual [Asymptomatic DENV infection accounts for a large portion of affected dengue patients. Transmission of DENV to a recipient infused with blood collected from dengue inapparent subject has been demonstrated since 1939 . Recentlhe blood . It is ehe blood . Blood sdividual . Since adividual ; we submAs there is no perfect animal model available, it is not an easy task to systematically investigate the immunological factors effectively. Studies have found that the majority of DENV infected subjects would only seek professional help on the fourth day after the onset of fever A. By thiAlthough dengue has been viewed to be a self-limited disease, the complexity of clinical presentations in acute patients has been known and well-documented . During The salient biological aspect of the adaptive immunity is its recalled capability, existing as a form of memory response that is very rapid and timely to contain the pathogens upon reencountering. Developing a robust memory response to an infection is a crucial feature of the success of the vaccination program in preventive medicine. Scientifically, samples obtained from healthy subjects who have been exposed to DENV infections do demonstrate the recall immunity upon re-stimulation with the viral components ,102. Des\u00ae do not appear to be protected, especially in those who were seronegative at baseline, even though mediocre protection has been demonstrated in those who had evidence of previous infection [\u00ae show that people whose sera are negative for DENV infection are not suitable for Dengvaxia\u00ae vaccination. It remains unknown why three doses of Dengvaxia\u00ae do not confer a protected effect in these seronegative individuals. One of the major challenging tasks in dengue vaccine trials is that there is no surrogate indicator of immune protection for the evaluation of vaccine efficacy.The best strategy to reduce the dengue burden would be a widespread administration of an effective vaccine. Development of an effective dengue vaccine has been attempted for more than 60 years . Numerounfection ,116. ThiAnother enigma is around immune non-responding in affected subjects. People living in dengue-endemic regions are more likely to have been exposed to the DENV infection multiple times, and yet, some certain subjects do not have detectable antibodies in their body. This could be because human is one of the natural hosts for the DENV, suggesting that some individuals may not elicit a vigorous immune response to the infection and therefore can cope with DENV, the so-called immune non-responders . InteresAlthough recall ability in the immune response is very critical in the development of effective vaccines against many pathogens, the scenario seems not to be that favorable in DENV infection ,125. TheOne of the key clinical aspects of DENV infection is that high viremia is observed in patients during the acute febrile stage. Oddly, a limited antibody response occurs during the high viremic period in re-infected subjects that have been well-documented ,128. IndA schematic illustration demonstrating the gap that requires researchers\u2019 attention is shown in To better understand the roles of antibodies and their secreting cells, it is imperative to have appropriate animal models to work with. One of the major challenges in dengue research is to have an animal model that can recapitulate the cardinal features of human dengue. Even though there are many approaches to develop the aforementioned animal models, the major obstacle remains in to define the outcomes of the disease and the immune response reproducible in the animal model, and whether it resembles that observed in humans.With the advancement of technology and knowledge in dengue, the new concept in system biology and epigenetic research, the much more comprehensive picture on the development of a perfect animal model mimicking human dengue would unravel soon. This would lead to the knowledge necessary for vaccine and therapeutic development.Ethics approval and consent to participate: This studies in Conceptualization, K.C.; writing\u2014original draft preparation, Y.-W.C. and G.C.P.; writing\u2014review and editing, S.A. and G.C.P.; visualization, K.P.; funding acquisition, G.C.P. All authors have read and agreed to the published version of the manuscript."}
+{"text": "The buzz about hyaluronan (HA) is real. Whether found in face cream to increase water volume loss and viscoelasticity or injected into the knee to restore the properties of synovial fluid, the impact of HA can be recognized in many disciplines from dermatology to orthopedics. HA is the most abundant polysaccharide of the extracellular matrix of connective tissues. HA can impact cell behavior in specific ways by binding cellular HA receptors, which can influence signals that facilitate cell survival, proliferation, adhesion, as well as migration. Characteristics of HA, such as its abundance in a variety of tissues and its responsiveness to chemical, mechanical and hormonal modifications, has made HA an attractive molecule for a wide range of applications. Despite being discovered over 80 years ago, its properties within the world of fascia have only recently received attention. Our fascial system penetrates and envelopes all organs, muscles, bones and nerve fibers, providing the body with a functional structure and an environment that enables all bodily systems to operate in an integrated manner. Recognized interactions between cells and their HA-rich extracellular microenvironment support the importance of studying the relationship between HA and the body\u2019s fascial system. From fasciacytes to chronic pain, this review aims to highlight the connections between HA and fascial health. The polysaccharide hyaluronan, built from repeating disaccharide units, first entered the scientific world in 1934 when it was discovered by Meyer and Palmer ,3. GlycoHA is synthesized by integral membrane proteins through the addition of monosaccharides to the reducing terminal. Unlike other glycosaminoglycans, the primary structure for HA contains no peptide, which is consistent with its synthesis in the plasma membrane rather than the Golgi . HA prodIn both vertebrates and bacteria, the HA structure is identical . HA can The high molecular weight and the semi-flexible polymer of HA are key factors leading to the high viscosity of dilute solutions. HA solutions can display increased viscosity due to mutual macromolecular crowding . AdditioWhen the carboxyl groups of HA are fully ionized at extracellular pH, HA\u2019s osmotic activity is disproportionately high in relation to its molecular weight. This molecular characteristic means that it is capable of intense impact on the movement and distribution of water, and thus, water homeostasis . HA viscAs early as 1986, a team of researchers reported that HA can influence cell migration by cooperating with the intercellular matrix in cell detachment from the ECM . We haveTissue HA enters the bloodstream in significant amounts via the lymphatic system of ducts where it is rapidly absorbed by liver endothelial cells and subsequently degraded ,3. In huThere are many excellent reviews on the roles of HA in different fields such as in angiogenesis, reactive oxygen species, HA digestion, cancer, cancer therapeutics, cancer metastasis, chondrocytes, lung injury, wound healing, diabetes, leukocyte trafficking, and immune regulation . This reIt is understood that most vertebrate cells synthesize HA at some point in their natural history ,32,33. IFibroblasts reside in close proximity to collagen fibers . Their tSmooth muscle cells have organelles typical of secretory cells. Smooth muscle cells synthesize various types of collagens, as well as proteoglycans and adhesive glycoproteins . HA syntSynoviocytes are found within the architecture of diarthrodial joints, specifically, the delicate joint lining. The joint lining is divided into two functionally and anatomically distinct layers: the intimal lining layer and the sublining layer . SynovioOne of the biggest breakthroughs to date in the discipline of HA research was reported in 2018 by C. Stecco\u2019s team . A new cOur knowledge of the ECM composition and function has increased considerably over the last decade. Numerous studies have demonstrated that the ECM is a source of signals affecting cell adhesion, shape, migration, proliferation, survival, and differentiation . HA is tWhen examining the components of the ECM, a network of fibers (mostly collagen and or elastic microfibrils) can be observed; these are responsible for the three-dimensional scaffold that functions as the structural framework of organs . Due to Considerable study over the past two decades has established that ECM elasticity or, in contrast, stiffness, affects fundamental cellular activities, including cellular spreading, growth, proliferation, migration, and differentiation . ModificA variety of extracellular signals regulate the expression of HA and its receptors, concluding that HA-receptor signaling is a tightly controlled protocol. \u201cBinding of the HA ligand to its receptors triggers signal transduction events which, in concert with other ECM and cytoskeletal components, can direct cell trafficking during physiological and pathological events.\u201d . For exaAlthough fascia may not be the most glamourous organ system, it is one of the most influential. The fascial system is now being recognized with roles in pathology, fluid movement and proprioception . The fasBy general definition, fascia is the ECM plus cells, organized as a three-dimensional network that surrounds, supports, suspends, protects, connects, and divides muscular, skeletal and visceral components of the body ,50. It iThe diversity of existing categories of fascia reflects not only the complex architecture of fascia itself, but also the diverse group of professionals working in different fields. Schleip et al. (2012) describe three separate fascia terminologies, which if appropriately applied, allow one to discuss discipline distinctions as well as tissue continuities .There are two emerging definitions of fascia: one that anatomically defines fascia as \u201ca sheath, a sheet or any other dissectible aggregations of connective tissue that forms beneath the skin to attach, enclose and separate muscles and internal organs\u201d and the \u201cfascial system\u201d that highlights the more functional properties of the three-dimensional fascia which infiltrates all body systems so that they operate in an integrated manner . The anaThe fascia incorporates a spectrum of anatomical objectives such as adipose tissue, visceral adventitia and neurovascular sheaths, muscle aponeuroses, deep and superficial fascia, joint capsules, ligaments, tendons, membranes, meninges, myofascial expansions, periosteum, retinacula, septa, tendons, and all the intramuscular and intermuscular connective tissues including endo-/peri-/epimysium ,49,53,54The deep fascia that surrounds skeletal muscle cells (endomysium) and skeletal muscle fascicles (perimysium) and named muscle bellies (epimysium) is essential for translating muscle movement at a joint . This muDeep fascia is more than a tough barrier structure of collagen and elastin. It is a metabolically active tissue layer which provides protective functions as well as the recently revealed contributions to gliding . From a The deep fascial layer of densely packed collagen bundles and elastin fibers has HA concentrated on its inner surface, which is in contact with the underlying muscle epimysium. Solutions of HA can be highly viscous, thus affecting the movement of HA-containing fluid layers within and underlying the deep fascia . HoweverA study from Fede (2018) quantified, for the first time, the HA content of human fascial samples obtained from a variety of anatomic sites. The authors demonstrated that the average amount of HA varies across anatomic sites, specifically sites that allow various degrees of fascial plane sliding and gliding . For exaA study looking at the lubrication of fascia and HA was reported by Roman (2013). In this study, the authors utilized the squeeze film lubrication theory of fluid mechanics for flow between two plates, as well as the Navier\u2013Stokes equations to quantify the frictionless movement of connective tissue and HA . They aiThe influence of HA in fascial gliding can also be observed with the histochemical distribution of HA in microscopically thin layers of loose connective tissue between individual layers of deep fascia. In the study by the C. Stecco (2011), samples of deep fascia along with the underlying muscles were taken from the neck, abdomen and thigh of unembalmed cadavers. Samples were stained with hematoxylin\u2013eosin, Azan\u2013Mallory, Alcian blue and a biotinylated HA-binding protein specific for HA. The stained deep fascia revealed a layer of HA between fascia and the muscle within the loose connective tissue dividing the dense irregular layers . It was In summary, HA found between neighboring fascial layers promotes fascial gliding which is facilitated by the presence and activity of fasciacytes. Changes in the concentration, molecular weight, or even covalent modification of HA, as well as changes in binding interactions with other macromolecules, can have dramatic effects on the sliding movement of fascia .Understanding the connective tissue matrix of fascia, specifically HA, together with the mechanical forces allowed and limited by fascial planes can help uncover specific treatment modalities to relieve chronic pain syndromes . CompletHA is essential for two fascial structures gliding across each other; therefore, the regulation of HA could affect the functions of fascia implicated in myofascial pain . MyofascMovement stimulates HA production and turnover, whereas immobility can increase the concentration of HA without effective HA recycling, increase the viscosity, and reduce the lubrication and gliding of the layers of connective tissue and muscle . HA-mediAn increase in the production of HA is the body\u2019s initial effort to increase the gliding efficiency between two surfaces, much like that of a synovial joint . If HA iPlacing this information into the bigger picture of fascial relationships reinforces the concept that modifying HA at the interface between loose connective tissue and layers of deep fascia may play an essential role in the etiology of pain associated with movement ,55.Once thought to be just a structural molecule in the ECM, HA is now being considered a chief regulator of a variety of inflammatory responses . IncreasHigh molecular weight HA displays anti-inflammatory and immunosuppressive properties, whereas low molecular weight HA is a potent proinflammatory molecule . Recall In conclusion, an important component of pain therapy is to reverse these changes in HA. When the HA becomes adhesive rather than lubricating, the distribution of lines of force within the fascia become distorted. This is referred to as the densification of fascia . ThickenStudies suggest that fascia reorganizes itself along the lines of tension imposed or expressed in the body. This may potentially create restriction between all structures enveloped by fascia itself with mechanical and physiological effects as a consequence of reduced gliding facilitated by HA .From an osteopathic perspective, fascial techniques aiming to release such tensions, decrease pain and restore function are based on various studies that have explored the viscoelastic properties of HA in the connective tissue . More reDue to different anatomical locations and to the qualities of the deep fascial tissue, it is important to recognize that different modalities of approach have to be taken into consideration when considering treatment options . Based oThe biocompatibility and negligible negative side effects of HA makes it one of the more readily available compounds used throughout many fields of medicine in the 21st century . DespiteThe most significant cause of intervertebral disc (IVD) degeneration is the deprivation of HA-linked aggrecans and subsequent water loss present in the ECM of the annulus fibrosus. A recently published manuscript by Bhattacharya (2020) reports that compressive loading causes intensive coiling of HA, which traps more water and aids in bearing compressive loads. The authors indicate that the hydration level of the IVD is strongly influenced by the HA molecules and water molecules in the ECM, which in turn impact the nanoscale mechanics of the annulus fibrosus. An avenue for exploration exists between HA treatment and IVD degeneration .Plantar fasciopathy is the most common cause of plantar heel pain and is considered to be a type of enthesopathy. In 2018, Kumai et al. published a report stating that high molecular weight HA was an effective treatment with no serious adverse drug reactions for plantar fasciopathy. This treatment contributed to the alleviation of pain in patients with plantar fasciopathy and improvement in their activities of daily living .A research study by Chin et al. in 2011 demonstrated that HA augmentation can alter a host\u2019s response to an ECM . In thisFinally, in vocal fold scaring when the lamina propria fascial layer is insulted or deficient, this negatively impacts the viscoelastic properties of the ECM. Use of a polymer such as HA appears most promising for lamina propria replacement therapy because it has the optimal viscoelasticity and also plays a role in the maturation and maintenance of vocal fold fascia .HA is an essential molecule in the ECM and therefore the body\u2019s fascia. The numerous functions of HA depend on its size, location, and interactions with its binding proteins. Although significant progress has been made in translating the biochemical properties of HA in human tissue, researchers must continue to investigate strategies of targeting the HA network for human myofascial dysfunction. This knowledge requires current research on inflammation, fibrosis, and cancers using both high molecular weight HA and chemically modified HA preparations, HA inhibitors, HA blocking peptides, and hyaluronidases to be extrapolated to the discipline of fascia. It is the author\u2019s hope that this brief review will serve as a foundation for communication and conversation across the various research fields exploring the highly dynamic and critical roles of HA and body health."}
+{"text": "Migraine is a primary neurological disorder associated with complex brain activity. Recently, mounting evidence has suggested that migraine is underpinned by aberrant dynamic brain activity characterized by linear and non-linear changes across a variety of time scales. However, the abnormal dynamic brain activity at different time scales is still unknown in patients with migraine without aura (MWoA). This study aimed to assess the altered patterns of brain activity dynamics over different time scales and the potential pathophysiological mechanisms of alterations in patients with MWoA.Multiscale entropy in 50 patients and 20 healthy controls (HCs) was calculated to investigate the patterns and altered brain complexity (BC) across five different time scales. Spearman rank correlation analysis between BC in regions showing significant intergroup differences and clinical scores  was conducted in patients with MWoA.The spatial distribution of BC varied across different time scales. At time scale1, BC was higher in the posterior default mode network (DMN) across participants. Compared with HCs, patients with MWoA had higher BC in the DMN and sensorimotor network. At time scale2, BC was mainly higher in the anterior DMN across participants. Patients with MWoA had higher BC in the sensorimotor network. At time scale3, BC was mainly higher in the frontoparietal network across participants. Patients with MWoA had increased BC in the parietal gyrus. At time scale4, BC is mainly higher in the sensorimotor network. Patients with MWoA had higher BC in the postcentral gyrus. At time scale5, BC was mainly higher in the DMN. Patients with MWoA had lower BC in the posterior DMN. In particular, BC values in the precuneus and paracentral lobule significantly correlated with clinical symptoms.Migraine is associated with alterations in dynamic brain activity in the sensorimotor network and DMN over multiple time scales. Time-varying BC within these regions could be linked to instability in pain transmission and modulation. Our findings provide new evidence for the hypothesis of abnormal dynamic brain activity in migraine. Migraine is a debilitating neurological disorder associated with brain excitability dysfunction characterized by attacks of moderate or severe unilateral throbbing and pulsating headache . The sugSome fMRI studies have captured the differences between migraine and healthy controls (HC) across different spatial scales. For example, Traditionally, brain dynamic activity has been mainly explored based on measuring the mean or variance of the sequence  in a sint-test within different groups. We then utilized two sample t-tests between the migraine without aura (MWoA) and healthy control (HC) groups over different time scales to explore the altered brain activity dynamics. Finally, we performed a correlation analysis to explore the relationship between aberrant and clinical symptoms to test the contributions of BC alterations to the clinicopathology of migraine.Based on previous evidence, we hypothesized that patients with migraine may show different patterns of abnormal brain activity dynamics over different time scales. To test our hypothesis, we first identified time scale-specific spatial distributions of BC using a one-sample Fifty patients with MWoA were enrolled from the outpatient clinic of the Departments of Neurology in two clinical centers: (1) the Hospital of Chengdu University of Traditional Chinese Medicine; and (2) Sichuan Provincial People\u2019s Hospital. Patients were enrolled in the study from August 2018 to April 2020. Twenty healthy controls were recruited from Chengdu University of Traditional Chinese Medicine and communities in Chengdu. All participants were scanned in the Huaxi MR Research Center, Sichuan University, China.The diagnosis of MWoA was established according to the ICHD III-beta criteria . Participants who met all the following inclusion criteria were included in the study: (1) female, 18\u201350 years old, right-handed; (2) fulfilling criteria for migraine without aura; (3) history of migraine without aura for 12 months or more; (4) did not receive acupuncture treatment or other preventive treatment within the last 3 months; and (5) no long-term use of analgesics. Patients and healthy controls with any of the following conditions were excluded: (1) macroscopic T2-visible brain lesions on magnetic resonance imaging (MRI) scans; (2) existence of additional psychiatric or neurological disorders; (3) taking any drugs affecting the central nervous system; (4) no current or previous antipsychotic medication, immunodeficiency, bleeding disorders, or allergies; (5) MRI contraindications or claustrophobia; and (6) alcohol or drug abuse. For the HC, they should either have no personal or family history of migraine or other headaches. This study was approved by the Ethics Committee of the Hospital of Chengdu University of Traditional Chinese Medicine and was conducted following the Declaration of Helsinki . Voluntary written informed consent was obtained from each subject after verbal and written explanation of the study.The clinical outcome measures were the change in the frequency of migraine attacks, duration, and headache impact test (HIT-6). In addition, migraine-specific quality-of-life questionnaires (MSQ) were assessed.2, interslice gap = 0 mm, and voxel size = 3.75 \u00d7 3.75 \u00d7 5 mm3. For each subject, 180 functional volumes were obtained.All subjects were instructed to rest with their eyes closed, not to think of anything in particular, and not to fall asleep during the scan. fMRI data were acquired with a Siemens Trio 3.0 Tesla MRI system equipped with a high-speed, eight-channel, phased-array head coil. The functional images were collected transversely using an echo-planar imaging (EPI) sequence with the following settings: TR/TE = 2,000 ms/30 ms, flip angle = 90\u00b0, 30 slices, 64 \u00d7 64 matrix, field of view = 240 \u00d7 240 mmAll patients had been free from a typical migraine attack for at least 1 week before the MRI scan. The scan would be postponed to ensure that the migraine patient is in the interval of migraine attack at the time of the scan. Further, after scanning, all participants reported that they did not experience any headaches or migraines and remained awake during the measurement.1) (p = 0.43). \u201cBad\u201d time points (FD > 0.5 mm), as well as their one-back and two-forward time points, were then scrubbed and interpolated by spline interpolation  . The fir1) . Afterwapolation .BC is calculated from the MSE method. MSE analyses were developed as a biologically meaningful measure of complexity . Sample m is the pattern length, r (similarity factor) represents a proportion of the standard deviation (SD) of the signal series is a distance threshold, and N is the length of the signal sequence. Cm(r) (Bi) is the sum which measures the average likelihood of m-length patterns in a signal series; Cm + 1(r) (Ai) is the sum which measures the average likelihood of m+1-length patterns in a signal series. Two patterns match if the distance is less than the tolerance of r. (3) comparing the sample entropy over a range of scales. Here, we used the previous parameters for MSE calculation that m = 1 and r = 0.35 and scale factors up to 5 (where e. Cm(r) , \u2211 Bi ism + 1(r) , \u2211 Ai is up to 5 . To furt up to 5 . Resultst-tests were utilized to assess the time-specific abnormal spatial distribution of BC in each group. Regional differences between migraines and HCs were examined using two-sample t-test at each scale. Gaussian random field (GRF) corrections  were conducted for the comparisons of five scales  for the following analysis. ROIs were defined as 4-mm spheres with a center at the peak position of statistical difference. Correlation analysis was then performed between the mean BC in the ROIs and the clinical symptoms of the MWoA patients.Statistical analysis of the BC was conducted using MATLAB. One-sample e scales . GRF corFifty patients were well matched with twenty HCs. The demographic characteristics of the participants are given in p < 0.005, cluster significant p < 0.05).The spatial distribution of BC at each group is shown in p < 0.005, cluster significant p < 0.05). In time scale2, patients with MWoA showed increased BC in the right precuneus, left postcentral gyrus, and left paracentral lobule . In time scale2, patients with MWoA showed increased left superior parietal gyrus and right parietal gyrus . In time scale4, patients with EOS showed increased BC in the left postcentral gyrus . Finally, In time scale5, patients with MWoA showed reduced BC in the left precuneus   in the precuneus, frontal cortex, and sensorimotor cortex over time scales 1\u20134 and decreased BC in the precuneus over time scale5. Differences in the paracentral lobule and precuneus were significantly correlated with clinical symptoms. Our findings shed light on multiscale abnormalities in brain activity dynamics in migraine and its relevance to clinical symptoms.Different and non-uniform distributions of BC topography were found in the HC and MWoA groups. Across all time scales, the precuneus and frontal gyrus had significantly higher BC than the HC group. These brain regions are consistent with those found by linear and single-time scale methods . Higher Time-scale specific differences between the MWoA and HC groups were detected. Over time scale1, patients with MWoA showed increased BC in the left precuneus, left medial frontal orbital, left postcentral gyrus, and left paracentral lobule. Over time scale2 and time scale4, the abnormalities in BC were mainly found in the sensorimotor network. Over time scale3, the abnormalities in BC were mainly found in the frontoparietal joint area. Over time scale5, the abnormalities in BC were mainly found in the DMN. The precuneus is considered the center of a wide spectrum of highly integrated tasks , such asTo better explore the possible physiological mechanisms underlying altered BC in migraine, a correlation analysis was also performed. We noted that clinical symptoms were associated with BC values in the paracentral lobule and precuneus. The paracentral lobule is an important part of the pain matrix and directly accepts pain signals . AbnormaSome limitations should be considered in the present study. Firstly, the data length used in this study is only 175 time points. Although BC is considered independent of data length , the effOur findings reveal altered brain activity dynamics in patients with migraine across multiple time scales measured using a novel method. These findings support the hypothesis of abnormal brain dynamics and provide details of non-linear anomalies of brain activity dynamics in patients with migraine. Moreover, the altered BC in the precuneus and paracentral lobule was associated with clinical symptoms, suggesting that the symptoms were related to abnormal brain dynamics. Our observations may provide novel insights into the pathophysiological mechanisms underlying migraine.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of the Hospital of Chengdu University of Traditional Chinese Medicine. The patients/participants provided their written informed consent to participate in this study.LZ, DL, and YW designed the study and conceptualized the protocol for healthy subjects. YZ, TX, and ZW adapted this protocol for patients with migraines without aura and evaluated them. WQ, HL, JL, and YY managed the literature searches and analyses. ZH, SG, and GZ undertook the statistical analyses. XW wrote the first draft of the manuscript. All authors contributed to and have approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "H)-yl)-N--yl)phenyl)-N-phenylaniline (TPA-2ACR) and 10,10\u2032-bis (PhCAR-2ACR), were designed and synthesized using a single-step Buchwald\u2013Hartwig amination between the dimethyl acridine and triphenylamine or carbazole moieties. Both materials showed high thermal decomposition temperatures of 402 and 422 \u00b0C at 5% weight reduction for PhCAR-2ACR and TPA-2ACR, respectively. TPA-2ACR as hole-transporting material exhibited excellent current, power, and external quantum efficiencies of 55.74 cd/A, 29.28 lm/W and 21.59%, respectively. The achieved device efficiencies are much better than that of the referenced similar, 1,1-Bis[(di-4-tolylamino)phenyl]cyclohexane (TAPC)-based device . Moreover, phenyl carbazole-based PhCAR-2ACR showed good device characteristics when applied for host material in phosphorescent OLEDs. Two small molecular hole-transporting type materials, namely 4-(9,9-dimethylacridin-10(9 Organic light emitting diodes (OLEDs) have drawn attention from research and commercial communities due to their advantages, such as wide view angle, thin films, high brightness, high contrast and less power consumption compared to conventional displays. Interestingly, OLED technology revealed the possibility of achieving 100% internal quantum efficiencies (IQE) by utilizing second-generation phosphorescence and third-generation thermally activated delayed fluorescence dopants ,3,4. At N-(1-naphthyl)anilino]-9,9\u2032-spirobi [9H-fluorene] (Spiro-NPB), N,N\u2032-Di(1-naphthyl)-N,N\u2032-diphenyl--4,4\u2032-diamine (NPB), and N,N\u2032-Bis(3methylphenyl)-N,N\u2032-diphenylbenzidine (TPD). This type of well-known materials grab solid attention in the development of small molecular type HTMs with certain modification in their molecular skeleton  658.33 for C48H39N3; (HRMS) MALDI-TOF: found [(M + H)+] 657.3132 molecular formula C48H39N3 requires [(M + H)+] 657.3144.Yield: 92%; white solid; N-(4-bromophenyl)-N-phenylaniline 2BrTPA , 9,9-dimethyl-9,10-dihydroacridine DMAC , palladium (II) acetate , tri-tert-butylphosphine  and sodium tert-butoxide  were added in a two-neck round-bottom flask equipped with condenser. Anhydrous toluene of 40 mL added, then subjected to inert conditions using nitrogen gas. The mixture stirred at 110 \u00b0C for 8 h under inert conditions, and reaction progress was observed using thin-layer chromatography. After completion of the reaction, the reaction mixture was filtered through a silica plug and worked up using water and chloroform several times. The organic layer was dried under anhydrous magnesium sulphate and concentrated under reduced pressure. Crude mixture was recrystallized from n-Hexane to obtain the pure target material TPA-2ACR.A mixture of 4-bromo-1H NMR  \u03b4 7.46 , 7.40\u20137.43 , 7.34 , 7.24\u20137.25 , 7.17 , 7.02\u20137.05 , 6.93\u20139.96 , 6.43 , 1.69 ; 13C NMR  \u03b4 147.3, 141.1, 135.4, 132.1, 130.1, 129.8, 126.4, 125.5, 125.2, 124.2, 120.6, 114.1, 31.3; MS (APCI) m/z: 660.22 for C48H41N3 [(M + H)+]; (HRMS) MALDI-TOF: Found [(M + H)+] 659.3294 molecular formula C48H41N3 requires [(M + H)+] 659.3300.Yield: 87%; white solid; \u22127 Torr using a Sunic organic evaporator inside a glove box under inert conditions. The fabricated device structure was as follows: indium tin oxide ITO (150 nm)/1,4,5,8,9,11-Hexaazatriphenylenehexacarbonitrile HATCN (7 nm)/HTL (43 nm)/4,4\u2032-Bis(N-carbazolyl)-1,1\u2032-biphenyl CBP:10% Iridium(III) bis(4-(4-tert-butylphenyl) thienopyridinato-N,C2\u2032) acetylacetonate PO-01 (20 nm)/1,3,5-Tri(m-pyridin-3-ylphenyl)benzene TmPyPB (35 nm)/8-Quinolinolato lithium Liq (1.5 nm)/Aluminum Al (100 nm). ITO and Al were used as anode and cathode, respectively. HATCN used as hole injecting layer (HIL), Liq applied as electron injecting layer (EIL) and TmPyPB used as electron transport layer (ETL). Yellow phosphorescent dopant of PO-O1 used for all devices in this study.The OLED device fabrication process is as follows: indium\u2013tin\u2013oxide (ITO)-coated glass substrates were cleaned and washed in isopropyl alcohol ultrasonic bath, and further washing conducted with deionized water for 20 min. Then, the cleaned substrates underwent UV\u2013ozone treatment for about 10 min. All the organic layers along with the metal cathode were fabricated on the pre-washed ITO coated glass substrate. The deposition rate of layers was applied around 5 \u00d7 10In this work, we designed and synthesized two small molecular hole-transporting-type materials. TPA-2ACR and PhCAR-2ACR were designed and synthesized between dimethyl acridine and triphenylamine or carbazole core. We utilized our synthesized materials for hole transporting and host materials. TPA-2ACR as a hole-transporting material exhibited excellent current, power, and external quantum efficiencies of 55.74 cd/A, 29.28 lm/W, and 21.59%, respectively when compared to that of the referenced similar TAPC-based device . The triplet energy of our materials was more than 3 eV, and they both showed excellent device performances. The phenyl carbazole-based PhCAR-2ACR showed current and external quantum efficiencies of 56.90 cd/A and 20.57%, respectively and the referenced CBP-based device revealed 47.83 cd/A and 18.16%. We believe that our acridine-based small molecular hole transport type materials can replace the TAPC hole-transporting and CBP host material in future OLED applications."}
+{"text": "In the Netherlands, the 2019 coronavirus (COVID-19) pandemic had a significant impact on daily life, with two extensive lockdowns enforced to combat the spread of the SARS-CoV-2 virus. These measures included the closure of bars and restaurants, and the transition from face-to-face to online education. A survey was conducted among Dutch pharmacy students and PhD-candidates to investigate the impact of COVID-19 lockdown on alcohol consumption, hangovers, and academic functioning. The analysis revealed a significant reduction in both quantity and frequency of alcohol consumption during the COVID-19 lockdown periods. This was accompanied with a significant reduction in hangover frequency and lower hangover severity during COVID-19 lockdown periods. The distribution of scores on academic performance showed great variability between respondents: while some participants reported impairment, others reported improved performance during the COVID-19 pandemic, or no change. Women reported that significantly more time investment was associated with maintaining these performance levels. Consistent among participants was the notion of reduced interactions with teachers and other students. Participants who reported more hangovers and most severe hangovers before COVID-19 benefited from the lockdown periods in terms of improved academic performance. Positive correlations were found between study grades/output and both the frequency and severity of hangovers experienced before COVID-19, suggesting that heavier drinkers, in particular, improved academic performance during the lockdown periods. In conclusion, COVID-19 lockdowns were associated with a significant reduction in both alcohol consumption and experiencing hangovers, which was, among heavier drinkers particularly, associated with significantly improved academic functioning. In The Netherlands, the 2019 coronavirus (COVID-19) pandemic had a significant impact on daily life, with two extensive lockdowns enforced to combat the spread of the SARS-CoV-2 virus. In March 2020, the number of people testing positive for SARS-CoV-2 grew exponentially, and the first lockdown period was enforced on 15 March 2020. The first lockdown lasted until May, 2020, and comprised working from home, the closure of universities, bars, restaurants and all non-essential stores. People were instructed to stay at home as much as possible, and for students\u2019 face-to-face education changed to online education. In May 2020, the number of SARS-CoV-2 infections decreased rapidly. After a summer period without lockdown measures, a second lockdown was implemented in November 2020 when the number of positive SARS-CoV-2 cases again rose exponentially. COVID-19 measures were the same as during the first lockdown, but during the second lockdown in addition mandatory wearing face masks at indoor venues and a night curfew were implemented. The lockdown lasted until April 2021 See .Although in the absence of vaccines, lockdowns were considered an effective way to reduce the spread of the coronavirus, there is a growing body of scientific literature pointing to the negative effects of lockdowns ,3. StudiVarious coping strategies have been adopted to adapt to the lockdown situation. Amongst others, such as being physically active or adopting a healthy diet, these also included substance abuse. For example, Prowse et al.  reportedAn increasing number of studies evaluated alcohol consumption during the COVID-19 pandemic, in particular the effects of lockdown. These studies show that alcohol consumption either increased, decreased, or remained the same during the COVID-19 pandemic ,19,20,21Previous research conducted before the COVID-19 pandemic that specifically focused on academic performance during alcohol hangover is very limited. In several studies, a negative impact of excessive alcohol consumption on academic functioning has been reported by part of the participants, and this has been related to alcohol hangover. For example, Hallett et al.  revealedTeaching at the department of pharmaceutical sciences of Utrecht University is mainly centered around small-scale student-centered and context-rich teaching formats with a strong focus on collaborative learning, such as problem-based learning (case-based learning) and project-and inquiry-based learning. These types of classes are supplemented with lectures and practical course work. Before the start of the pandemic, most teaching was done on campus, although some courses had already implemented some form of computer-supported learning for e.g., self-study and peer-feedback assignments. In line with other universities around the world, soon after the start of the COVID-19 pandemic, an overnight change had to be made from in-person teaching activities to online teaching.On the 11th of May 2020, the lockdown ended and universities partially reopened. However, social distancing measures were still enforced and as a result most teaching activities remained online. Throughout the summer of 2020 there were no major COVID-19 outbreaks in The Netherlands. Academic activities were minimal during the summer and most students enjoyed the holiday season. After summer (September 2020) limited on campus education was re-instated, where universities aimed at welcoming students on campus for practical course work, research internships and once a week for regular course work. From September onward the number of positive COVID-19 cases rose again, and a second lockdown was installed in November (2020). This lockdown lasted until April 2021. During the second lockdown regular classes switched to online education again, whereas practical course work and research internships continued on campus.Given the current lack of knowledge, the aim of the current study was to investigate the relationship of alcohol consumption and having hangovers with academic performance during COVID-19 lockdowns among Dutch pharmacy students and PhD-candidates. In addition, the impact of the first COVID-19 lockdown on social interactions with teachers and other students was also examined. It is hypothesized that changes in alcohol consumption (quantity and frequency) and the frequency and severity of hangovers during lockdowns will also be associated with impairments in academic performance.An online survey was conducted in the first week of June 2021 among students and PhD-candidates of the department of pharmaceutical sciences of Utrecht University, The Netherlands. The study was reviewed and approved by the Science-Geo Ethics Review Board of Utrecht University , and all participants gave electronic informed consent. The survey was designed via SurveyMonkey and took about ten minutes to complete. As an incentive participants could enter a prize draw to wine one of two Euro 100 vouchers. A thorough description of the study methodology and the forthcoming dataset have been published elsewhere .Participants of the study were students, PhD-candidates, and postdocs from the department of Pharmaceutical Sciences of Utrecht University, the Netherlands. The department of Pharmaceutical Sciences is responsible for one of the three Pharmacy programs in the Netherlands. Pharmacy students first follow a three-year Bachelor program, most-often followed by a Master program comprising three more years of education, resulting in a PharmD degree. Within the Bachelor of Pharmaceutical Sciences, the department hosts a research-oriented international program, the College of Pharmaceutical Sciences (CPS). In addition, the Utrecht Institute of Pharmaceutical Sciences (UIPS), the research institute of the department, offers a PhD program in Drug Innovation and is responsible for the further development of postdoctoral researchers. In the academic year 2020\u20132021, 662 students followed the bachelor pharmaceutical sciences and 142 students the College of Pharmaceutical Sciences. A total of 458 students were registered for the master, and 190 PhD-candidates and 30 postdocs were affiliated with UIPS. Since the department comprises a considerable number of international students, participants could choose to complete the survey in English or Dutch language.Retrospective assessments of alcohol consumption were made for (1) the year 2019 (the period before COVID-19), (2) the first lockdown period, (3) summer 2020 (no lockdown), and (4) the second lockdown (November 2020 -March 2021). Changes in academic functioning were assessed for the COVID-19 pandemic as a whole, in comparison to the situation before the start of the COVID-19 pandemic.Questions assessing demographics includes age, sex, weight, and height. Participants indicated whether they were a student, PhD-candidate or postdoc, and whether they lived alone, or together with family or others  during the COVID-19 pandemic.Questions about alcohol consumption included the number of alcoholic drinks participants consumed on average per week, and the number of days per week they consumed alcohol. Guidance was provided on serving sizes and how to convert these alcoholic drinks into units. Participants further reported how many hangovers they experienced and the average severity of their hangovers. Hangover severity was assessed on an 11-point scale ranging from 0 (absent) to 10 (extreme) . The queThe academic functioning scale was specifically designed for this study. Participants rated, compared to before COVID-19, their study/work performance during the COVID-19 pandemic on scales ranging from -5 (extremely worse) to +5 (extremely better), around a midpoint of 0 (unchanged). Six items relate to academic performance and include \u2018Quality\u2019 , \u2018Time\u2019 (\u201camount of time invested in study/PhD-/postdoc project\u201d), \u2018Grades/Output\u2019 (\u201cstudy grades/output\u201d), \u2018Knowledge\u2019 (\u201cacademic achievement/amount of knowledge gained\u201d), \u2018Reading\u2019 (\u201creading articles/text books\u201d), and \u2018Writing\u2019 (\u201cwriting assignments/ articles\u201d)\u201c. Two items relate to academic interactions and include \u2018Contact with teachers\u2019 (\u201ccontact with teachers or supervisors\u201d) and \u2018Interactions with students\u2019 (\u201cinteractions with other students/PhD-candidates/postdocs\u201d). Finally, two other items relate to satisfaction with academic life and include \u2018Balance study-private life\u2019  and \u2018Role-satisfaction\u2019 (\u201cthe extent you enjoy being a student/PhD-candidate/postdoc\u201d).Data were analyzed with SPSS . Mean and standard deviation (SD) were computed for all variables and distributions were checked for normality with the Kolmogorov\u2013Smirnov test and by visual inspection. Data were not normally distributed, and therefore nonparametric tests were conducted for the statistical analysis. For the purpose of the current study, only data from participants that consume alcohol was considered for the statistical analysis.p-value for multiple comparisons. Differences between the assessments were considered significant if the adjusted p value was <0.05. Between-group comparisons to evaluate sex differences were conducted with the Independent Samples Mann-Whitney U test. Differences between the groups were considered significant if p < 0.05, and a Bonferroni correction was applied for multiple related comparisons. Finally, Spearman\u2019s correlations were computed between the \u0394 scores for alcohol consumption outcomes (the average score during the two COVID-19 lockdowns\u2013before COVID-19) and academic functioning outcomes. Correlations were considered significant if p < 0.05, and a Bonferroni correction was applied to account for multiple comparisons.Within-subject comparisons to compare assessments that are made for four timepoints  were conducted with the Related-Samples Friedman\u2019s Two Way Analysis of Variance by Ranks test. A Bonferroni\u2019s correct was applied to adjust the n = 1452 students that were invited, n = 341 started the survey (response rate: 23.5%), and n = 250 completed the survey. Of these, n = 156 participants reported consuming alcohol and were included in the statistical analyses. n = 94 served as a control group (see Of the roup see . Demograp = 0.037). No significant sex differences were found for student status or living situation. Female students were slightly older than male students . For women, the number of drinking days per week was also lower than in men for each time period, but after Bonferroni\u2019s correction this difference reached statistical significance only for the first lockdown period (p = 0.012). In line, the average hangover severity reported by women was significantly lower than that reported by men for both the first lockdown period (p < 0.001) and second lockdown period (p = 0.009). Hangover frequency was also lower in women compared to men, with a significant difference found for the first lockdown period (p = 0.002).For all time periods, women consumed significantly less alcohol per week than men  and hangover severity  (See p = 0.068). Similarly, reduction in alcoholic drinks consumed and number of drinking days per week during COVID-19 lockdowns was largest among those who reported the highest levels of alcohol consumption before COVID-19. The correlations between assessments made for \u2018before COVID-19\u2032 and \u2018during COVID-19 lockdowns\u2019 were highly significant for both the amount of alcohol consumed  and the number of drinking days per week   See A,B. For 001) See C,D. In op < 0.001) and interactions with other (PhD-) students (p < 0.001). The distorted balance between work/study and private life (p = 0.009) did not reach statistical significance after a Bonferroni\u2019s correction was applied. In women the effects were of the same magnitude and in the same direction as observed in men. However, due to the larger sample size, in women more items reached statistical significance . None of the direct comparisons between men and women reached statistical significance.Academic functioning outcomes for men and women are summarized in p < 0.007 after Bonferroni\u2019s correction) was found between study grades/output and hangover frequency (p = 0.005).p = 0.005). The correlations between time and hangover frequency (p = 0.008) approached significance. In men, none of the correlations reached statistical significance.In women, a significant correlation was found between grades/output and hangover frequency (p = 0.011) and severity  of hangovers experienced before COVID-19, suggesting that in particular the heavier drinkers improved academic performance during the lockdown periods  revealing that those who consume no alcohol significantly more frequently live together with family, whereas those that consume alcohol most frequently live to together with friends.A total of Academic functioning outcomes of both groups are summarized in The analysis revealed a significant reduction in both quantity and frequency of alcohol consumption during the COVID-19 lockdown periods. This was accompanied by a significant reduction in hangover frequency and lower hangover severity. These effects were especially robust among female participants. Due to the relatively small sample size the differences did not always reach statistical significance among male participants, although they did point in the same direction.Overall, academic performance was not affected during the COVID-19 pandemic. However, the distribution of scores on academic performance show great variability between respondents: while part of participants reported impairment, others reported improved performance during the COVID-19 pandemic, or no change see . Women rBoth male and female participants reported a significant reduction in contacts with teachers and interactions with other students. Further, female participants reported a significant reduction in the balance between study and private life, and an overall reduction of role satisfaction. In contrast to differential distribution of lockdown effects on academic performance, there were only few participants that reported that social interactions improved during the COVID-19 lockdowns. Instead, the vast majority reported a reduction in these interactions See .The largest reduction in alcohol consumption and experiencing hangovers was observed among participants who reported the greatest alcohol intake before COVID-19 see . The datOur findings are in line with other studies in that subsamples exist that either increased or decreased alcohol consumption during COVID-19 lockdowns ,19,20,21The outcomes should be interpreted by taking into account the study\u2019s limitations and strengths. Firstly, the data were collected retrospectively. Therefore, recall bias may have influenced the study outcomes. Secondly, the questionnaire assessing academic functioning was developed in-house. We choose to use this questionnaire as it was short and to our opinion captured the most important aspects of academic performance and social interactions. To avoid having a lengthy survey, we decided not to use traditional more elaborate questionnaires. Third, the survey was conducted among Dutch pharmacy students. It is therefore unclear to what extent the findings can be generalized to other students in the Netherlands, and internationally. It was decided not to recruit participants from other departments, as the educational methodologies between the departments differ, and this could interfere with the interpretation of study outcomes. Fourth, much more women took part in the study than men. The obtained male/female ratio is however in correspondence with the male/female ratio of the department of pharmaceutical sciences as a whole, as well as most other departments at Utrecht University. It will also be interesting to compare our findings with prospective studies. This will also allow to determine the possible impact of retrospective reporting on the study outcomes. Finally, the current data suggest that, in addition to alcohol consumption, it may be even more important to consider the impact of frequency and severity of alcohol hangover on academic functioning. Therefore, we advocate for more research on the impact of alcohol consumption and hangovers on academic functioning.Although a proportion of students have reported increased alcohol consumption and poorer academic functioning, overall, the COVID-19 lockdowns were associated with a significant reduction in both alcohol consumption and experiencing hangovers, which was, in particular among heavier drinkers, associated with significantly improved academic functioning."}
+{"text": "Triticum aestivum ssp. aestivum L.) were used to investigate early vigour in a rain-fed field environment for 3 years, and additionally assessed under controlled conditions in a greenhouse experiment. The normalised difference vegetation index (NDVI) calculated from red/infrared light reflectance was used to quantify early vigour in the field, revealing a correlation  between the spectral measurement and the length of the second leaf. Under controlled environmental conditions, the measured projected leaf area, using a green-pixel counter, was also correlated to the leaf area of the second leaf , as well as to the recorded biomass . Subsequently, genetic determination of early vigour was tested by conducting a genome-wide association study (GWAS) for the proxy traits, revealing 42 markers associated with vegetation index and two markers associated with projected leaf area. There are several quantitative trait loci that are collocated with loci for plant developmental traits including plant height on chromosome 2D (log10 (P) = 3.19; PVE = 0.035), coleoptile length on chromosome 1B (\u2013log10 (P) = 3.24; PVE = 0.112), as well as stay-green and vernalisation on chromosome 5A (\u2013log10 (P) = 3.14; PVE = 0.115).Due to the climate change and an increased frequency of drought, it is of enormous importance to identify and to develop traits that result in adaptation and in improvement of crop yield stability in drought-prone regions with low rainfall. Early vigour, defined as the rapid development of leaf area in early developmental stages, is reported to contribute to stronger plant vitality, which, in turn, can enhance resilience to erratic drought periods. Furthermore, early vigour improves weed competitiveness and nutrient uptake. Here, two sets of a multi-reference nested association mapping (MR-NAM) population of bread wheat ( Global climate change is considered one of the biggest and most complex challenges the mankind has faced. One effect that has been observed since 1970, which leads to severe yield losses, is the increased occurrence of erratic drought phenomena . Specifi2 concentration  to investigate the physiological characteristics that drive EV in wheat in the field and under controlled conditions, (ii) to assess the efficiency of phenotyping methodologies under greenhouse and field conditions, and (iii) to identify genomic regions influencing EV in wheat.1 lines were generated. Subsequently, these 15 F1 lines were used for population development through inbreeding, which produces1474 F4-derived lines and were then segmented into 15 genetically diverse families. These 15 families comprised four Mace-derived families, forming a conventional NAM population which was denoted as the Mace-NAM (Ma-NAM) component of the MR-NAM, five Scout-derived families (Sc-NAM), and six Suntop-derived families (Su-NAM). The NAM population was genotyped using the DArT-seq genotype-by-sequencing platform, producing over 25,000 polymorphic markers  was randomly selected from a multi-reference nested association mapping population (MR-NAM). The MR-NAM population was developed based on 11 diverse founder lines which were crossed with the commercially used wheat varieties Suntop (AGT), Scout (LPB), and Mace (AGT), then consequently adapted to the environmental conditions of the western, northern, and southern cropping regions of Australia, respectively . The fou markers . The fir2. To precisely reach the target crop density, thousand seed weight and germination rate were determined for seed of each genotype, while the sowing rates were calculated for each genotype. To avoid any artefacts in seed size, the seeds of the lines used in each trial were sourced from a common site and the year of the seed propagation. Seeds were generated from fully irrigated seed-increase rows sown at 0.5 m row spacing and fertilizer was applied to provide for the non-limiting conditions for both water and nutrients. Furthermore, diseases and weeds were controlled as necessary. This allowed for the full potential seed size of each genotype to be expressed during seed production. In all trials, no specific selection for seed size were performed. In each year the trials received 120 kg/ha\u20131 of urea prior to sowing, and 40 kg/ha\u20131 of Starter Z\u00ae  was applied at sowing. Plant protection measures were applied as necessary. All field trials were designed as a partially replicated (p-rep) block design with percentage of partial replication of 34, 29, and 62% in 2015, 2016, and 2017, respectively.Field trials were conducted over 3 years from 2015 to 2017. All field trials were carried out under rain-fed conditions at the Hermitage Research Facility (HRF), Warwick, Queensland, Australia . The HRF site is characterized by alkaline, cracking, and heavy clay soils with high water-holding capacity. Cropping season is from May to October, with an average rainfall of 211 mm and an average temperature of 14\u00b0C. Further information regarding the environmental conditions is given in \u00ae model 505, manufactured by NTech Industries, Ukiah, CA, United States. By attaching the device to the body by using a harness, the measurement could be carried out constantly at a height of 1 m. The NDVI has been reported as a very useful index for studying the dynamics of canopy development and of senescence patterns of wheat  was used as a quantitative measure for EV. The NDVI measurements were collected using a hand-held NTech Greenseekerof wheat . This veof wheat . In thisIn 2017, detailed measures were captured to fully understand the physiological properties. A core set consisting of 30 genotypes was compiled from Set 2 and was intensively examined at the time of the NDVI measurement. The core set included all founder and reference lines from the MR-NAM population, along with selected good-performing lines from other unpublished experiments. From each of the 30 genotypes, ten plants per plot were randomly sampled for each genotype, while the leaf characteristics were recorded by measuring the length and width of the first (L1) and the second leaf (L2). Subsequently, the approximate leaf area for L1 and L2 was calculated by multiplying the measured length by the width. The sum of the calculated leaf areas of L1 and L2 was then expressed as the total leaf area (TLA).\u00ae , containing 19.4% N, 16% P, and 5% K, was added to the potting media to guarantee sufficient nutrients during the experiment. The trial was designed as a fully replicated, randomized, and complete block design with six replications per genotype.The GH experiment was conducted using 210 genotypes from Set 2. The greenhouse chamber temperature was set on 22\u00b0C during daytime and 17\u00b0C during night time, while the lighting conditions were set to provide a 12-h photoperiod. Furthermore, the plants were irrigated on a daily basis. A single plant was grown per pot using 250 ml pots with 70 mm diameter. The potting media had a pH of 5.5\u20136.5 and was a composition of 70% pine bark (0\u20135 mm) and 30% coco peat, as well as fertilizers. Furthermore, Osmocote\u00ae  programming language. The pixel counter identifies green pixels in an image to calculate a projected leaf area (PLA) and is a low-cost approach for image analysis , the linear mixed model (LMM) described in Eq. 1 was used. For the calculation of the LMM, the R-language-based packages lme4Pijkl is the phenotypic value of the ith genotype, in the kth replication, \u03bc stands for the overall mean, gi describes the fixed effects of the ith genotype. The random effects are Wk, which is the kth replication, Cj, which represents the jth column, and Rl representing the lth row. The error term is represented by eijkl.where Pearson correlation matrices were created using the R package psych. Principal component analysis was conducted using the R package stats. For the Pearson correlation, as well as for the principal component analysis, we used the BLUEs described in Eq. 1.10 (P) > 3 which corresponds to a p cut-off of 0.001 significance level. The phenotypic variation explained by a given QTL (PVE) was calculated separately, according to Association mapping was performed using the R package GenABEL . Genome-H2) and narrow sense (h2) heritability by using the R package sommer  difference between the genotypes was observed at 21 DAS and 29 DAS, with a smaller phenotypical variation at 21 DAS compared to the measurement at 29 DAS (p < 0.05) phenotypical variation at both time points, with a smaller variation at 21 DAS compared to 29 DAS (p < 0.05) phenotypical variation could be observed (p < 0.001) correlation between TLA at 21 DAS and leaf area L2 at 21 DAS (r = 0.89), than between TLA and leaf area L1 at 21 DAS (p < 0.05 r = 0.42). A similar correlation can be observed for L1 and L2 at 21 DAS, and L2 parameters at 21 DAS. Notably, a significant (p < 0.05) correlation was observed between NDVI 17 21 DAS and leaf length L2 at 21 DAS (r = 0.39). Furthermore, the relationship between the leaf area of L1 and L2 at 29 DAS and leaf parameters at 29 DAS shows similarities to the calculation made at 21 DAS. Leaf area L1 at 29 DAS shows stronger correlations with leaf TLA at 29 DAS (r = 0.79), compared to 21 DAS. However, the L2 area at 29 DAS (r = 0.93) shows a stronger correlation to TLA at 29 DAS. Interestingly, the correlation between area L1 and L2 at 21 DAS, and the L1 and L2 at 29 DAS (r = 0.47) is slightly smaller than the correlation with L2 area at 29 DAS (r = 0.51) and leaf length L2 at 29 DAS.Basic descriptive statistical indicators , variation (Var), standard deviation (SD), coefficient of variation (CoV) for NDVI, and leaf parameters of Set 1, Set 2, and the core set are all given in t 29 DAS . For leao 29 DAS . The laro 29 DAS . Regardiobserved . By compobserved . For allobserved . In ordeobserved . Person p < 0.05) differences can be observed. However, the recorded phenotypical differences show a lower variation compared to the field conditions (p < 0.05) phenotypical difference, particularly the L2, as well as the L1&L2 values  and with PLA 21 DAS . Furthermore, the PLA at 21 DAS shows a positive correlation to leaf width L2 (r = 0.34), as well as to area L2 (r = 0.38) and TLA (r = 0.39). As under field conditions, the TLA of L1 and L2 is more affected by area L2 (r = 0.96). However, area L1 (r = 0.85) seems to have a greater impact on TLA in the GH compared to the field. Furthermore, it is noteworthy that dry mass measured at harvest at 21 DAS shows a positive correlation to all captured leaf characteristics. Moreover, seed weight (SW) showed no significant correlation to any other trait  analysis was performed. The analysis was conducted for the core set data and included parameters such as the leaf measurements at 21 DAS and at 29 DAS in the field, or at 21 DAS in the GH, as well as dry matter content, grain weight, and the respective NDVI values from 2015 to 2017 . For the10 (P) = 3) for NDVI and PLA across all years in the genome-wide association study. A total of 41 QTL were associated [\u2013log10 (P) = 3] with either NDVI or PLA. The majority of trait-associated SNP markers (21) were associated with NDVI at 21 DAS in 2017 in Set 2  and in 2016 (NDVI\u201916), along with NDVI values from 2015 to 2017, and the PLA data from the GH trial in 2017 in the 221 genotypes of Set 2. Several SNP markers exceeded the arbitrary threshold of association (\u2013login Set 2 . Chromosariation . Most QTariation . In acco.08\u20130.04 .The first objective of this study was to provide information on physiological components that contribute to EV of wheat, both in the field and under controlled conditions. Several studies observed a strong contribution of leaf width to a specific leaf area, and consequently to EV . Our resThe second objective of this study was to evaluate high-throughput methods to precisely record physiological leaf characteristics in field and greenhouse trials. Rht B1b on chromosome 4B and Rht D1db on chromosome 4D have been reported to reduce coleoptile length and, consequently, EV, since they decrease epidermal cell length in leaf tissue (Rht B1b and of Rht D1b. Comparable results were reported in Rht-B1b, and only one parental line contained Rht-D1b, while no QTL was identified on chromosome 4D. However, a significant correlation could be observed between Rht-D1 and NDVI and the ground cover in certain environments. The Rht 8, on the short arm of chromosome 2D, is a GA-responsive dwarfing gene reported to have a secondary effect of reducing epidermal cell length. Hence, it is more appropriate for achieving good canopy cover in combination with a semi-dwarf growth habit (TraesCS2D01G051500 as a possible candidate gene for Rht8. We identified QTL QSG.qwr-2D.1 in the vicinity of TraesCS2D01G051500, and found this QTL to be significantly associated with NDVI17 at 29 DAS. Several studies have identified QTL associated with coleoptile length on chromosome 1B, including markers XpGTG-mTCGA294 (wsnp_CAP11_c2596_1325540 (QSG.qwr-1B, which is located at the same region on the long arm region of chromosome 1B as wsnp_CAP11_c2596_1325540, as reported in Vrn1, frost resistance gene Fr1, as well as genes for ear emergence time and for the plant height (The third aim of this study was to achieve a better understanding of EV genetics in wheat. The GWAS for Set 1 and Set 2 revealed 41 SNP markers for NDVI and for PLA, which were linked to 60 protein-coding regions across 17 chromosomes. Consistent with previous studies, the present study shows that EV in wheat is a quantitative trait with numerous QTL located across several chromosomes . Numerouf tissue . In our th habit . Chai etmTCGA294  and wsnp_1325540 . This stt height .The results of the current phenotypic investigation extended our insights into the EV trait in wheat. The key characteristics of EV and the relationship with other traits, such as biomass, were successfully identified. In addition, this study presents effective methods that can be used to detect EV in the field, as well as under controlled conditions. In particular, the connection between the leaf length parameters and the NDVI highlights the great potential of NDVI, especially if given recent advances in unmanned aerial vehicle or drone phenotyping platforms . NeverthThe original contributions presented in the study are included in the article/JC and LTH conceived the study. SV, SA, and JC performed the experiments. SV and AS analysed the phenotypic data. SV wrote the manuscript with further input from AS, RJS, JC, SA, and LTH. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Bacillus and Pseudomonas were the dominant genera. On the solid lecithin plate, 35 of the 39 OPB strains produced visible phosphate halos, and 24 strains showed a high ratio of P halo diameter (HD)/colony diameter (CD). In the liquid lecithin medium, all 39 OPB strains demonstrated P-solubilizing ability, but with significant differences. The Pseudomonas strain demonstrated the strongest P-solubilizing ability, at 70.91 mg\u00b7L\u22121. There was no significant correlation between the amount of released phosphorus by OPB strains and pH. The P-solubilizing characteristics of OPB were affected by the interaction of dissolved inorganic phosphate and alkaline phosphatase. In the microcosm experiments, the added OPB strains significantly promoted the decomposition and release of organic phosphorus (OP) in the sediments. OPB in the sediments of Sancha Lake is rich in diversity and had a strong ability to release OP in the sediments.The organophosphate-mineralizing bacteria (OPB) convert environmental organic phosphorus (P) into soluble P that can be directly absorbed and utilized by organisms. OPB is an important group of microorganisms in lake sediments. The P decomposed and released from the sediments by OPB is an important P-source in eutrophic water bodies. In this study, the egg-yolk organophosphate medium was used to isolate and screen OPB strains from the sediments of Sancha Lake. Furthermore, the obtained OPB strains were classified based on their 16S rDNA sequence. Both the solid and liquid lecithin hydrolyzing experiments were conducted to investigate the P-solubilizing characteristics of the obtained OPB strains. Microcosm experimentsiwere performed to study the P-release ability of OPB strains from sediments. A total of 39 OPB strains were isolated from the sediments of Sancha Lake. They belonged to three phyla, five families, and five genera, and contained two potentially new species.  When external input of phosphorus (P) is under effective control, the release of endogenous phosphorus from lake sediments significantly affects the increase in its concentration in water bodies . The eleOPB exists widely in the environment. The current research reports on cultivable OPB primarily focus upon soil . Some scThe Sancha Lake is an important source of drinking water in the eastern new district of the Sichuan Province. It has a serious eutrophication problem . Our preWe hypothesized that: (1) There are abundant and diverse OPB in the sediments of Sancha Lake and (2) OPB have a strong ability to release OP from the sediments.2 area in the Sancha Lake, with a drainage area of 161.25 km2 above the dam. The average water depth is 8.3 m, and the deepest value is 32.5 m. The primary water source of the Sancha Lake is the Min River, which accounts for about 80% of the total water volume of the reservoir. The other 20% comes from rainfall and the two streams of the Tiaodeng River and the Longyun River. The Sancha Lake area has a humid subtropical monsoon climate, with an annual average temperature of 15.2~16.9 \u00b0C, no freezes in all seasons, and average annual precipitation of 786.5 mm.The Sancha Lake is located in the eastern new district, Sichuan Province, China, 104\u00b011\u203216\u2033 E~104\u00b017\u203216\u2033 E, 30\u00b013\u203208\u2033 N~30\u00b019\u203256\u2033 N, the eastern suburbs of Chengdu, and the upper reaches of Jiangxi River, a tributary of the Tuojiang River in the Yangtze River system. There is 27 kmMn) and biochemical oxygen demand (BOD5) indexes of the surface water of the Sancha Lake have met the Class III requirements of \u201cSurface Water Environmental Quality Standard\u201d (GB3838-2002) in China, but the high TN and TP contents failed the requirements [\u22121, with an average value of 1.670 mg\u00b7g\u22121, and the TN range was 1.325~2.615 mg\u00b7g\u22121, with an average value of 1.931 mg\u00b7g\u22121 [\u22121, with an average value of 7.1 mg\u00b7L\u22121 [Since 2015, the pH, ammonia nitrogen, chemical oxygen demand 2SO4 0.5 g, NaCl 0.3 g, MgSO4\u00b77H2O 0.3 g, CaCO3, 5 g, FeSO4\u00b77H2O 0.36 g, MnSO4\u00b7H2O 0.03 g, KCl 0.3 g, agar 20 g, and egg-yolk emulsion 50 mL with distilled water to 1000 mL, pH 7.0. For preparing egg yolk emulsion, the eggshell was cleaned with 75% alcohol wipes and broken at one end. The egg white was removed, the egg yolk was poured into a sterilized flask and mixed aseptically with an equal volume of sterile 0.85% saline [Ten grams of wet weight sediment was transferred into a 250 mL Erlenmeyer flask which contained 90 mL of 0.85% sterile saline, shaken at 25 \u00b0C and 160 rpm for 0.5 h to make a sediment suspension. The 10-fold dilution method was used to prepare suspension dilutions up to 10% saline . Three rThe total bacterial DNA was extracted with the bacterial genomic DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd., following the kit instructions. Then, the extracted bacterial DNA was taken as a template, the universal primers 27F (5\u2019-AGAGTTTGATCCTGGCTCAG-3\u2019) and 1492R (5\u2019-GGTTACCTTGTTACGACTT-3\u2019) were used to amplify the bacterial 16S ribosomal DNA (rDNA) sequences with the polymerase chain reaction (PCR) . The PCR\u00ae AxyPrep DNA Gel Extraction Kit  and followed by sequencing with a 3730xl DNA Analyzer conducted by Shanghai Majorbio Bio-pharm Technology Co., Ltd. .The PCR reaction system (20 \u00b5L) was composed of 10 \u00d7 Ex Taq buffer 2.0 \u00b5L, 5 U Ex Taq polymerase 0.2 \u00b5L, 2.5 mM dNTP mix 1.6 \u00b5L, 27 F 1 \u00b5L, 1492R 1 \u00b5, DNA 0.5 \u00b5L, and ddH2O 13.7 \u00b5L. PCR reaction conditions were as follows: denaturation at 95 \u00b0C for 30 s, annealing at 56 \u00b0C for 30 s, extension at 72 \u00b0C for 90 s, for a total of 25 cycles, with initial denaturation at 95 \u00b0C for 5 min before the cycle, and extension at 72 \u00b0C for 10 min after the cycle. Each treatment was repeated three times. The PCR products were detected by 0.8% agarose gel electrophoresis. Then, the PCR products were purified by Axygenhttps://www.ezbiocloud.net accessed on 31 October 2021) to determine the similarity and related species of the strain. Then, similar and effectively published sequences of typical strains were retrieved from the database. After multiple sequence alignment by clustalX, sequence editing was performed. MEGA 7.0 was used to estimate the evolutionary distance using the Kimura 2-parameter (K2P) model. The neighbor-joining (NJ) method was used to construct the phylogenetic tree of the 16S rRNA gene sequence of the strains with a bootstrap value of 1000 [The contaminated sequences from the raw bacterial 16S rDNA sequence data were removed using SeqMan II . Furthermore, the obtained data were curated to remove regions with low-quality nucleotide scores  and assembled into contigs. The obtained entire 16S rDNA sequence was sent for BLAST alignment at EzBioCloud ( of 1000 .4)2SO4 0.5 g, NaCl 0.3 g, MgSO4\u00b77H2O 0.3 g, CaCO3 5 g, FeSO4\u00b77H2O 0.36 g, MnSO4\u00b7H2O 0.03 g, KCl 0.3 g, agar 20 g, added with distilled water to 1000 mL, pH 7.0. After autoclaving, this was cooled to 50 \u00b0C, and 3 g phosphatidylcholine was added). The plates were cultured at 28 \u00b0C for 3 days. The phosphate halo diameter (HD) and colony diameter (CD) were measured, and the value of HD/CD was calculated. The P-solubilizing ability of a strain was preliminarily determined by whether it exhibited a phosphate halo. The criterion for selecting colonies is a value of HD/CD > 1. HD/CD > 1.5 indicated a strong P-solubilizing ability [The OPB obtained by isolation and purification was spot-inoculated on a solid lecithin medium . Then, 2 mL of the bacterial suspension was transferred into 200 mL of liquid lecithin medium (same as solid lecithin medium except the agar) and cultured at 28 \u00b0C, 160 rpm for 108 h. The culture was sampled every 12 h to determine the DIP concentration, ALP activity, and pH. The P concentration was determined by the malachite green-phosphomolybdenum heteropoly acid spectrophotometric method [\u22121 by drop plate method were determined in lecithin medium [Each of the isolated OPB strains was inoculated to LB medium and cultured at 28 \u00b0C and 160 rpm for 24 h. The bacteria were collected from LB liquid medium and then were washed with sterile water. The bacteria above were resuspended in sterile water, prepared in a bacterial suspension .8 CFU mL\u22121. A total of 15 parallel samples were set for each group. A total of five groups were set, including four groups of representative strains and one group of control with no bacteria. The treated samples were cultured at 13 \u00b0C (the temperature of the Sancha Lake mud\u2013water interface) and 80 rpm for 5 days, and manually shaken twice a day [4Cl was added to a final concentration of 0.8 mol\u00b7L\u22121, shaken for 30 min, and then centrifuged at 3500 rpm for 10 min. The supernatant was filtered using a 0.45 \u00b5m membrane filter. The Standards, Measurements, and Testing (SMT) protocol was followed to extract various forms of P in the sediments after centrifugation [The collected fresh sediments were subjected to continuous irradiation under Co-60 \u03b3-ray with an energy of 20 kG\u03b3/h for 12 h to kill all the microorganisms in the sediments. Then, 200 g of sterilized sediment was transferred into a 1-L ground stopper reagent bottle, mixed with 800 mL of the filtered overlying water, and 10 mL of the bacteria suspension of the representative OPB strain with an approximate value of 10fugation ,24. The fugation .p = 0.05 in all statistical analyses.Statistical analysis was performed using SPSS statistical software . The one-way analysis of variance (ANOVA) was used to analyze the differences of HD/CD, DIP, OP, ALP and pH among different OPB strains. Significance levels were set at Bacillaceae, Aeromonadaceae, and Comamonadaceae; five genera, including Bacillus, Pseudomonas, and Aeromonas; and 22 species including B. thuringiensis, B. altitudinis, and B. mobilis. The details are listed in Bacillus, Pseudomonas, Acinetobacter, and Aeromonas [Bacillus and Pseudomonas as the dominant OPB genera in the sediments of the Sancha Lake, whereas Acidovorax was rare. According to the established strain identification method [6 CFU g\u22121 (dry sediment). The number of isolated OPB species and their P-solubilizing activities in this study were both higher than those from the Dutang reservoir sediments reported by Xuejin You et al. [In this study, a total of 39 OPB strains with stable P-solubilizing activities were isolated from the sediments of the Sancha Lake. They belonged to three phyla, including Firmicutes, Proteobacteria, and Bacteroidetes; five families, including eromonas ,25,26. On method , strainsu et al.  from thep < 0.05). Among them, the highest value was from SWSO1719. The HD/CD values of SWWO1711, SWWO1713, SWWO1714, SWSO1718, and SWWO1721 were significantly lower than those of other strains (p < 0.05). Among them, the lowest value was from SWWO1711.The HD/CD values of the 39 OPB strains are shown in \u22121. The best P-solubilizing result was shown by SWSO178, with a DIP concentration of 72.26 mg\u00b7L\u22121, an increase of 70.91 mg\u00b7L\u22121 compared to that of the control. The increase in DIP concentrations of SWWO175, SWWO1716, and SWWO1717 was not significant (p > 0.05). SWWO1717 exhibited the weakest P-solubilizing effect, with a DIP concentration of 1.82 mg\u00b7L\u22121, only 0.47 mg\u00b7L\u22121 higher than that of the control.The P-solubilizing results of the 39 OPB strains by liquid lecithin experiment are mentioned in \u22121. As we see, the quantitative liquid lecithin experiment and the qualitative solid lecithin experiment gave similar results in the determination of the P-solubilizing ability, but there were certain differences as well. The P-solubilizing substance (such as ALP) produced by bacterial metabolism in the solid plate probably did not diffuse or did not diffuse enough.SWWO175, SWWO1718, SWWO1719, and SWWO1720 exhibited no phosphate halos on the solid lecithin medium, but they displayed P-solubilizing ability in the liquid lecithin experiment. Among them, the DIP concentration in SWWO1718 culture reached 49.21 mg\u00b7LAccording to Bacillus, Pseudomonas, and Aeromonas. In addition, one representative strain was selected from the genus Acidovorax which was rarely reported with P-solubilizing characteristics. The growth dynamics of these four representative OPB strains were investigated in the liquid lecithin medium. Furthermore, the impact of changes in DIP, ALP, and pH on the P-solubilizing ability of these strains was explored.To further explore the P-solubilizing mechanism of OPB, one representative OPB strain with the highest activity was selected from each of the dominant genera, including p < 0.05) than that in the control. Among these, SWSO178 and SWSO1719 exhibited strong P-solubilizing ability, while SWWO1713 and SWWO1711 showed weak P-solubilizing ability. The P-solubilizing activity of SWSO178 and SWSO1719 reached a peak at 48 h, while that of SWWO1713 and SWWO1711 reached a peak at 72 h. The DIP concentration changes of the four OPB cultures presented different curves, nevertheless, the curves all had a trough\u2013peak\u2013trough pattern.As shown in The curves of changes in ALP activity of the four OPB strains also showed different trends in the liquid lecithin culture. The ALP activity of SWSO178 and SWSO1719 reached a peak at 36 h, showing a stronger enzymatic activity, while that of SWWO1713 and SWWO1711 reached a peak at 72 h, indicating a weaker enzyme activity. Considering the relationship between the DIP and ALP curves, DIP concentration increased with the increase in the ALP activity, but the DIP changes and peaks lagged in the liquid lecithin culture.The P-solubilization of OPB was due to the enzymatic hydrolysis of ALP produced by bacteria . In the ALP is a non-specific phosphomonoesterase  and an iAs shown in p > 0.05). The drop in pH may be because the cells entered the exponential phase, with vigorous growth, reproduction and metabolism, and increased ALP production. ALP thus promoted the hydrolysis of lecithin into DIP and organic acids. On entering the stationary phase, microbial cell metabolism slows down, respiration becomes weak, and cells begin to lyse. The choline produced by ALP to decompose lecithin in this study could not be completely hydrolyzed, leading to an increase in pH. The decline in pH of the culture is due to the production of organic acids or lecithin degradation products of OPB [+, such as the NH4+/H+ exchange and respiration. However, the decrease in the culture pH is not a necessary condition for phosphorus solubilization [The culture pH of the four representative OPB strains all showed a \u201ctrough\u2013peak\u2013trough\u201d pattern. s of OPB , or therlization . In factlization .p < 0.05). The DIP concentration of the overlying water in the control was stabilized at approximately 0.17 mg\u00b7L\u22121 after three days. In the SWSO178 and SWSO1719 cultures, the DIP concentration of the overlying water changed in a trough\u2013peak\u2013trough manner. In detail, the overlying water DIP concentration of SWSO178 culture was at the trough on day 1, peaked on day 3 at 0.952 mg\u00b7L\u22121, and then decreased, but was still higher than the control at the end of the experiment. The overlying water DIP concentration of SWSO1719 culture was the lowest on day 2, increased the maximum to 0.714 mg\u00b7L\u22121 on day 3, which was 4.2 times higher than that of the control, followed by a decrease on days 4 and 5, but were still higher than that of the control. In SWWO1713 and SWWO1711 cultures, the overlying water DIP changed in a down\u2013up way. In detail, the overlying water DIP increased slowly on day 1 peaked on day 4, at 0.544 mg\u00b7L\u22121 and 0.425 mg\u00b7L\u22121, respectively, and the values were, respectively 2.6 and 2.4 times compared with the control, and then reduced on day 5, but both concentrations were still higher than that of the control.The above-mentioned four representative OPBs were used for the microcosmic sediment P-release experiment. As shown in p < 0.05). SWSO178, SESO1719, SWWO1711, and SWWO1713 can increase DIP content in overlying water and reduce OP content in sediment, which may be related to the ALP secretion by the strains. After adding the SWSO178, SWSO1719, SWWO1711, and SWWO1713 strains, the sediment OP contents reduced by 14.5%, 13.1%, 8.6%, and 7.3%, respectively. The decrease in sediment OP in SWWO1711 and SWWO1713 cultures was consistent with the increase in DIP concentration of the overlying water, whereas in the SWSO178 and SWSO1719 cultures, the sediment OP decreased; in fact, the overlying water DIP also decreased in the early stage, but in the middle and later stages, there was a consistent decrease in the sediment OP and increase in the overlying water DIP. This may be because SWSO178 and SWSO1719 grew too vigorously in the early stage, and the soluble P released from sediment OP was not sufficient, therefore, the cells supplemented from the soluble P in the overlying water to meet their biological P needs. Baiwen Hu et al. [As shown in u et al.  came to The sediments of Sancha Lake are rich in OP, which is an important source of P in the water body. Under favorable environmental factors, high-efficiency OPB can convert OP in sediments that are insoluble or not directly usable by organisms into DIP and released into water bodies. Therefore, the upper water body of the Sancha Lake is at a risk of more serious eutrophication.Acidovorax has been rarely reported as OPB, while SWSO1719 and SWWO1721 were potentially new taxa. Bacillus and Pseudomonas were the dominant genera. The results show that the cultivable OPB in the sediments of Sancha Lake have a rich diversity and some new taxa, expanding the source of P-solubilizing microorganisms.A total of 39 OPB strains were isolated from the sediments of the Sancha Lake. These strains were found to belong to three phyla, five families, and five genera. All the 39 OPB strains could dissolve lecithin, but the OPB strains showed significantly different P-solubilizing ability. The primary mechanism of P-solubilization by OPB may be through the production of ALP. The correlation between the amount of P-released and pH was not significant. The P-solubilization characteristics of OPB were affected by the interaction between the DIP concentration and the ALP activity in the culture medium.The OPB strains could significantly promote the mineralization and decomposition of organophosphorus compounds in the sediments of the Sancha Lake. OPB play an important role in the decomposition and release of OP in the sediments of Sancha Lake. The P decomposed and released from the sediments by OPB may be an important P-source in eutrophic water bodies."}
+{"text": "Human identification based on motion capture data has received signification attentions for its wide applications in authentication and surveillance systems. The optical motion capture system (OMCS) can dynamically capture the high-precision three-dimensional locations of optical trackers that are implemented on a human body, but its potential in applications on gait recognition has not been studied in existing works. On the other hand, a typical OMCS can only support one player one time, which limits its capability and efficiency. In this paper, our goals are investigating the performance of OMCS-based gait recognition performance, and realizing gait recognition in OMCS such that it can support multiple players at the same time. We develop a gait recognition method based on decision fusion, and it includes the following four steps: feature extraction, unreliable feature calibration, classification of single motion frame, and decision fusion of multiple motion frame. We use kernel extreme learning machine (KELM) for single motion classification, and in particular we propose a reliability weighted sum (RWS) decision fusion method to combine the fuzzy decisions of the motion frames. We demonstrate the performance of the proposed method by using walking gait data collected from 76 participants, and results show that KELM significantly outperforms support vector machine (SVM) and random forest in the single motion frame classification task, and demonstrate that the proposed RWS decision fusion rule can achieve better fusion accuracy compared with conventional fusion rules. Our results also show that, with 10 motion trackers that are implemented on lower body locations, the proposed method can achieve 100% validation accuracy with less than 50 gait motion frames. Human identification using motion capture data has attracted much attention for its wide applications in authentication, surveillance, and medical applications . Some coIn gait recognition, the system authenticates or classifies the target person by using her/his walking manner. The used gait data mainly include the following four modalities: camera image gait data , inertiaExisting gait recognition works mainly rely on sensor data or image data for classifying the object, and the recognition accuracy is not high enough for some applications with stringent accuracy requirements. The OMCSs can obtain high-precision gait motions, which may be helpful for improving the gait recognition performance, but its potentials in gait recognition has not been studied. Though it requires the implementation of optical motion trackers, the system does not require sensitive information like face image and fingerprint, thus it will be acceptable for some users with privacy concerns. In addition, OMCS is widely used in sports training, physical treatment, and visual effects production applications ,13,14,15As such, in this paper we attempt to use an OMCS for human identification, and our goal is to achieve state of art identification accuracy with 10 optical trackers that are planted on the lower body locations of the target person, including thigh, lower leg, and foot, and both left and right body sides. The proposed method only requires the collection of several gait cycles of the participants, and the tracked body locations are chosen as the lower body part, which reduces the intrusion degree compared with other body parts such as chest and head. We propose a decision fusion-based gait recognition method, and compared with existing works, the proposed method has the following distinguished differences:(1) We use an OMCS to record the gait trajectories of the persons and study the gait recognition problem with the obtained data, which has not been considered in existing works. Though OMCS requires the implementation of optical trackers, the system can obtain high-precision gait motion data, which is helpful to improve the classification accuracy when only using a relative smaller number of gait motions for human classification. In addition, the system does not require a large number of optical trackers, and using only 10 optical trackers can achieve very good classification accuracy, and even 100% accuracy when sample number is large enough.(2) We use a powerful classifier, namely kernel extreme learning machine (KELM) , to cond(3) Instead of focusing on improving the classification accuracy of the base classifier, in this paper we highlight the importance of decision fusion of multiple motion frames, and we develop a first-classification-then-fusion method to achieve better classification performance, and the design of the decision fusion rules plays a vital role in improving the fusion performance. More specifically, we first conduct the classification process for each single motion frame, and then propose a reliability weighted sum (RWS) rule for combining the classification decisions of multiple motion frames. In the proposed RWS rule, we first transform the outputs of the KELM classifier into fuzzy decisions by using a membership transformation function, then compute the consistency matrix of all the fuzzy decisions by using a consistency degree measurement. With the obtained consistency matrix, we then use the Eigenvalue decomposition method to obtain the weight vector of the fuzzy decisions. A fuzzy decision with larger average consistency value to other fuzzy decisions means that it is more reliable, and the corresponding reliability weight value will be relatively larger, otherwise it will be relatively smaller.(4) The experimental results on the collected gait motion data with 76 participants demonstrate that the proposed rule outperforms several decision rules, including sum rule, belief rule, weighted belief rule, product rule, and majority voting rule. The results also show that the proposed method can achieve 100% classification accuracy with 10 motion optical trackers, which shows its potentials for its applications in authentication and multiplayer motion tracking applications. The remainder of this paper is organized as follows: Gait recognition has been extensively studied in for its wide applications in authentication, surveillance, training, and medical treatment ,2,3. In In the feature extraction step, the system needs to extract expressive feature representations from the raw data. Although the quality of the extracted features has significant impacts on the classification accuracy of the gait recognition task, yet it is still not unified that which feature is the best choice that suits all situations. For image data, existing feature extraction methods can be categorized as model-based methods and model-free methods . In modeIn the classification step, the most commonly used classifiers include support vector machine (SVM) , random We emphasize that the gait recognition problem in this paper is different from the above existing works, in which the classifier usually requires a very large training data samples, and each sample is composed by several gait cycles to achieve good classification performance. In the proposed method, we only record several gait cycles for training the classifier for single motion classification, and 1\u20132 gait cycles for classification, which is efficient for data collection process. However, the available number of gait motion data is quite limited and cannot be solved by methods that require a larger number of training and test data. To fully use the decisions of each single motion frame, in this paper we first propose a method that first conducts frame-level classification, and then develop a decision fusion method to combine the decisions of the single frames, while in existing work the decision is directly made by the output of the classifier, and each classification requires a bunch of gait motion data frames. In the following section, we will illustrate the detailed steps of proposed gait recognition method using decision fusion.We use optical motion trackers to record high-precision body motion trace to identify the target person. Before identification, the participants are required to wear a set of optical trackers, and then naturally and straightly walk through a flat test field with a length less than 5 m, which can be done in a very short period of time. There are 76 participants in total, with 46 females and 30 males, and their ages range from 20 to 60. The heights of the participants range from 144 to 178 cm, and the weights ranges from 42 to 115 kg. The sampling frequency of the body locations is 5 Hz, and there are 10 lower-body locations are recorded in each frame, which include thigh, knees, shin, ankle, and tiptoe, and both left and right sides are covered. The obtained walking lengths of the participants are different, which range from 2.37 to 4.15 m. An example of the recorded gait data is shown in T consecutive motion frames, denote as In this paper, we consider a gait recognition problem using high-precision optical gait motion trackers. Before identifying the target, the system previously prepared a set of training dataset with Feature exaction: For each person, the input raw data includes 10 motion tracks, and they cannot be directly used to classify the target object. In this paper, we want to identify the target person with a short gait motion capture trace; extracting the features from a trace recorded from motion tracker time series is not practical in this situation since each person may only has several gait cycles, and the number of collected gait motion is quite limited. As such, a feature exaction process that only extracts relative location distance and speed features from single frame data is proposed to obtain an expressive feature representation of the input data, and then the extracted features will be the input of the following identification process.(1) Unreliable feature calibration: Though the OMCS can record high precise gait trajectory data, we observe that a recorded motion instance may be biased due to sensing failure or noise interference, and the corresponding the features of the biased motion data are also unreliable. Therefore, it is necessary to detect and calibrate the unreliable feature data, and relief their impact on the classification performance.(2) Classification: In this paper, we will use a kernel extreme learning machine (KELM) to deal with the classification task for the feature data of each single gait motion frame. We will provide performance comparison results to demonstrate the advantages on classification accuracy and efficiency of KELM for the gait classification in the experimental section.(3) Decision fusion: The motion frame number of different persons are also different due the variation of walking speed of the target person. With the obtained KELM outputs of all the frames, we then need to combine them into a unified one to obtain the final global decision. Compared with single frame classification, we expect a classification accuracy improvement after combining the decision of multiple frames, and the decision fusion rule will play a vital rule on the final fusion accuracy. (4) In the next subsections, we will give a detailed illustration of the above four steps, along with their mathematical models.t), for two trackers Given a gait motion data, we use the relative distance of the tracker locations as the feature of the input motion data. The reason is that the relative distance metric can depict both walking action and physical body shape characteristics, which can effectively distinguish the differences between gait motions of two persons. In frame  of each feature, then find the unreliable features that is larger or lower than the probability thresholds. Since the feature is irregularly distributed, and cannot be reasonably depicted by one specified distribution, thus we use the kernel density estimation method to estimate the PDFs of the features. Given a feature vector  follows (3)fz=1L(43L)0.2 . With thThe above criterion requires us to compute the CDF For a feature data With the obtained features, we then use a pattern recognition classifier to identify the target person. The used classifier can be any proper classifier with acceptable classification performance, such as the commonly used support vector machine (SVM), and random forest. In the proposed decision fusion based gait recognition method, the classification performance of each single gait motion frame has critical influences on the following fusion accuracy, thus it is necessary to find a classifier with powerful classification capacities. However, from the test results, we found that the classification performances of SVM and random forest are not satisfactory to us. In this paper, we use KELM as the base classifier for its outstanding classification performance in both accuracy and efficiency. Let puted by (8)\u03b2=K+1Note that Remark\u00a01.A relative larger value of a KELM output means the probability that the target belongs to the corresponding class will be higher. Apparently, a class with maximal output will be regarded the hard decision of the classifier. For a KELM classifier, if an output Since we want to combine the decisions of the multiple motion frames, the output will be transformed to fuzzy decisions, and details will be introduced in the following subsection.To combine the decisions of the consecutive motion frames, in this paper we propose a reliability-weighted sum rule (RWS) that adjusting the fuzzy decisions by considering the differences among the fuzzy decisions. In general, a decision is relatively more consistent to other decisions, it is more reliable, otherwise more unreliable. In RMS, the obtained output data of KELM classifier are first transformed to fuzzy decisions by using a fuzzy membership function. More specifically, for frame Remark\u00a02.The above fuzzy decision means that, for one classifier output vector, a relative larger output of one class will be transformed to a larger membership compared with other classes, otherwise it will be relatively smaller. In particular, in KELM, we set the class label that the person belongs to as +1, and other class labels as \u22121. For example, in a classification task with 5 classes, the decision label vector when the person belongs to class 2 is With the above membership transformation process, we can obtain the fuzzy decisions of all the motion frames, which are denoted as ing rule . HoweverAs such, in this paper we propose a reliability estimation method by using the consistency degrees among the fuzzy decisions. In belief function theory, the consistency degree between two basic belief assignments (BBAs)  follows (11)\u03c6m1,Following the above definition, we define the consistency degree between two fuzzy decisions Since we do not have compound classes in the above Equation, thus the consistency degree equals to the inner product of If the obtained consistency value Remark\u00a03.For a complex classification task with multiple classes, there may exist several outputs with relative larger fuzzy membership values. For example, for a fuzzy decision Remark\u00a04.According to the above definition of decision consistency degree, we can see that, for two fuzzy decisions With above consistency computation method, we can obtain a consistency matrix od (EDM)  to compuWe can see that one eigenvalue corresponds to a unique eigenvector. The above EDM problem can be properly solved by using the well-known Singular Value Decomposition (SVD) method . When alRemark\u00a05.It has shown that eigenvector matrix A ,37. MoreNext, we can combine all fuzzy decisions into a unified global one by using the obtained reliability weights, as given byAt last, the final decision is made by choosing the class with maximal global membership value, as given byT) is increased. In general, only several gait cycles . Note that, in the proposed method, only KELM is required to be trained, and RWS rule does not need to be trained and it can be directly used for combining the fuzzy decisions. Given a data with several new motion frames, we obtain the outputs of the KELM classifier, and transform the outputs into fuzzy decisions In this subsection we present a toy example to give a better understanding of the proposed RWS rule. Consider a gait motion recognition problem with 5 possible persons, and suppose that we have 10 consecutive motion frames, and the corresponding fuzzy decisions are shown in Next, we compute the corresponding decision consistency matrix of the fuzzy decisions in In this section, we test the performance of the proposed method by comparing it with some acknowledged baseline methods. The experiment mainly includes two parts: the first one is the classification performance comparison of the single motion frame, and the results of KELM, SVM, and random forest will be provided. The other one is the performance comparison results of the proposed RWS rule and other fusion rules, including sum rule, belief rule, weighted belief rule, product rule, and majority voting rule. As mentioned before, the system has 10 motion trackers, which are implemented on the lower body locations, and each body location has one or two trackers. For one body side, there are 5 trackers, and the trackers are implemented at the following three locations: thigh (one tracker), lower leg , foot . Since the system requires us to plant optical trackers on human bodies, it is better to reduce the number of trackers as much as possible. As such, we also want to clarify that whether it is possible for us to implement only one tracker on the lower leg and foot locations, thus we choose three trackers each body side, and six in total. With the above considerations, we will test the recognition performances of single motion frames and multiple motions with 10 and 6 motion trackers. All the experiments are conducted in a Windows 10 system with Intel i7 CPU, and 16 GB RAM, and the algorithm is developed in MATLAB 2020a platform.In this subsection, we test the classification performance of KELM on single motion frames. We first use 9595 motion frames as the training dataset, and the number of each of consecutive motion frames of each participant ranges from 82 to 179, and average number of each participant is about 122, and the corresponding number of gait cycles are about 3\u20135. We use another 4787 motion frames for the 76 participants, and the number of consecutive motion frames of each participant ranges from 45 to 89, or 1\u20133 gait cycles. We can see that test data size is about the 1/2 of the training data.Except KELM, we also test the performance of support vector machines (SVM) and random forest, which are used in  and 27]27], respFrom In this subsection, we demonstrate the classification performance of the proposed RWS rule by comparing it with several well-known decision fusion rules. The details of the compared rules as illustrated as follows:Majority voting rule [(1) ing rule : MajoritSum rule [(2) Sum rule : In thisProduct rule [(3) uct rule : similarBelief rule [(4) ief rule : DepmsteReliability-weighted belief rule [(5) ief rule : The obtNext, we test the classification accuracy of the fusion results of the proposed RWS rule and the baseline fusion rules with different KELM parameter settings. In addition, since the number of the test motion frames influences the classification, we also test the classification accuracies with increasing ratio of test motion frames, and ratio increases from 0.1 to 1. We test the fusion accuracies with the 10 trackers and 6 selected trackers, and the obtained results are shown in From the two figures, we can observe that the proposed RWS rule can achieve higher fusion accuracy compared with other rules. More specifically, we can see that, given the same ratio of the test frames, in most times the proposed RWS can achieve higher accuracies. However, it does not mean that the proposed rule will always be better than other rules. For example, in In this paper, we have studied the gait recognition problem by using optical motion capture data, and we proposed a first-classification-then-fusion method, which includes the following four steps: feature extraction, unreliable feature detection, classification of single motion frame, and decision fusion of multiple frames. In particular, we proposed an RWS decision fusion rule to combine the fuzzy decision of the gait motions. The experimental results on 76 participants show that KELM achieves much higher classification accuracy and training efficiency compared with SVM and random forest in the single motion frame classification task, and they demonstrate that the proposed method RWS achieves better fusion accuracy compared with several existing fusion rules. Particularly, our results show that, with high-precision 3-D gait motion tracking data, the recognition method can achieve 100% accuracy when the full data of 10 optical trackers are used. It has to admit that, although the proposed method can achieve 100% recognition accuracy, the relative high implementation cost on both optical trackers and the capture cameras limits its application scenarios. On the other hand, our results indicate that the performance of other gait recognition systems, such as video surveillance, can be further improved if range sensor and depth sensor is integrated to enable measuring the distances of the captured persons. It has to be recognized that, though the proposed method can achieve very high recognition accuracy, it requires the implementation of optical motion trackers, which limits its practical application scenarios. Our future work is applying the proposed system in multiplayer motion tracking scenarios and test the performance with more complex trajectories. In this way, the system can support several players in the tracking field at the same time, which will greatly enhance its capability and efficiency."}
+{"text": "Hippocampal neurogenesis occurs throughout life, but it declines with age. D-galactose  enhances cellular senescence through oxidative stress leading to neurodegeneration and memory impairment. Caffeic acid (CA) acts as an antioxidant via decreasing brain oxidative stress. This study aims to investigate the advantages of CA in alleviating the loss of memory and neurogenesis production in the hippocampus in aged rats activated by D-gal. Fifty-four male Sprague-Dawley rats were unpredictably arranged into six groups. In the D-gal group, rats were administered D-gal (50 mg/kg) by intraperitoneal (i.p.) injection. For the CA groups, rats received 20 or 40 mg/kg CA by oral gavage. In the co-treated groups, rats received D-gal (50 mg/kg) and CA (20 or 40 mg/kg) for eight weeks. The results of novel object location (NOL) and novel object recognition (NOR) tests showed memory deficits. Moreover, a decline of neurogenesis in the hippocampus was detected in rats that received D-gal by detecting rat endothelial cell antigen-1 (RECA-1)/Ki-67, 5-bromo-2\u2032-deoxyuridine (BrdU)/neuronal nuclear protein (NeuN), doublecortin (DCX) by means of staining to evaluate blood vessel associated proliferating cells, neuronal cell survival and premature neurons, respectively. By contrast, CA attenuated these effects. Our results postulate that CA attenuated the impairment of memory in D-gal-stimulated aging by up-regulating levels of hippocampal neurogenesis. Aging is reliant on a time-based system of life that is related to functional impairment, leading to a progressive decline in the organs for maintenance of their functions ,2. Free 2+/calmodulin-dependent protein kinase II [D-galactose  is an aldohexose that is enormously discovered in honey, butter, yoghurt, milk, cherry, and kiwi . In norminase II . Moreoveinase II ,11.Nowadays, drug therapy is widely used for management of cognitive impairment. Therefore, there is a great need for specific drugs that can be used to prevent the process involved in aging and impairments of memory and cognitive functions. Phenolic acids have earned exclusive consideration as they have steady antioxidant activity. Caffeic acid (CA), or 3,4-dihydroxycinnamic acid, is a non-flavonoid phenolic phytochemical detected in many plants, fruits, tea, and wine . CA has Fifty-four Sprague Dawley male rats, weighing 280-300 g, 8 weeks of age were acquired from Nomura Siam International Co., Ltd. Pathumwan, Bangkok. The rats were housed in an environment with a 12 h light-dark cycle. Three rats were allocated a plastic cage (width \u00d7 length \u00d7 high = 37.5 cm \u00d7 48 cm \u00d7 21 cm) at the standard room temperature in the range 23-25 \u00b0C. The rats  were randomly arranged into control, D-gal, CA20, CA40, D-gal + CA20, and D-gal + CA40 groups. The study design was accepted by the Khon Kean University Ethics Committee in Animal Research (IACUC-KKU-33/63).Saline solution (0.9%), 1 mL/kg and propylene glycol 1 mL/kg were administered to the control group by intraperitoneal (i.p.) injection and by oral gavage, respectively. For the D-gal group, rats were administered D-gal  melted in 0.9% saline solution by i.p. injection. Rats in the CA20 and CA40 groups were orally treated with CA  at 20 and 40 mg/kg melted in propylene glycol, respectively. In the D-gal + CA20 and D-gal + CA40 groups, rats were administered with D-gal at the same dose as the D-gal group and CA at the same dose as the CA20 and CA40 groups. Each rat received drug administration for 8 weeks. On the first day of the treatment, each rat was administered with BrdU  melted in 0.9% saline solution at 250 mg/kg, with 1 mL/kg by i.p. injection.The NOL and NOR tests were performed after seventy-two hours of drug treatment. For the NOL test, rats were habituated to an arena (50 \u00d7 50 \u00d7 50 cm) for 30 min without stimuli. 24 h later, two similar objects were set in different corners of the arena. Then, rats were permitted to explore the objects for 3 min . After exploring, rats were backed to their enclosures for 15 min. The arena and two objects were cleaned with 20% alcohol to wipe out odor cues. After that, one of the objects was placed at the familiar location (FL), and another one was changed to a new or novel location (NL) in the arena. Rats explored the objects in the arena for 3 min . One day later, in the NOR test, each rat was familiarized with the arena for 30 min similar to the NOL test. Then, 24 h later, two comparable objects were laid down in different areas in the arena. Rats explored the objects for 3 min . Next, rats were transferred to their enclosures for 15 min. Next, one familiar object (FO) and a new object (NO) were deposited into the arena at the familiar location. Rats explored the objects for 3 min .\u00ae, XT version 12, Noldus, Wageningen, Netherlands) was used to track and record the time of exploration and movement in both tests. Distance moved was noted in the habituation trial. The exploration time was counted when rats surveyed the objects by chewing, licking, sniffing, or pointing the nose towards the object from fewer than 2 cm distance [A digital camera linked to a computer program  compound  was used to fix each hemisphere. After that, each hemisphere was promptly immersed in liquid nitrogen-cooled isopentane . Finally, the hemispheres were kept at \u221280 \u00b0C for immunohistochemical study.To fix the frozen brain, 4% paraformaldehyde was used and then a freezing microtome  was used to serially section (20 \u00b5m), in coronal plane, through the length of the DG between Bregma point \u22122.3 to \u22126.3 mm. Cryoprotective buffer was used to store all sections at 4 \u00b0C. For cell proliferation investigation, every 15 sections from the complete DG were chosen using a procedure of standardized random selection to get 9 sections per brain ,22. The The immunohistochemistry of doublecortin (DCX) and bromodeoxyuridine (BrdU)/the neuronal nuclear protein (NeuN) were carried out to evaluate the number of immature neurons and cell survival correlated to mature neurons, respectively. Sections were cut at 40 \u00b5m thickness and every 8th section was selected from the entire DG to represent each brain. Finally, 9 sections per brain were used to investigate the DCX and BrdU/NeuN positive cells ,23,24. FA fluorescence microscope  was used to examine cells within a range of three cells at the internal edge of the DG to evaluate the numbers of positive cells of Ki-67/RECA-1, BrdU/NeuN, and DCX. The blood vessel-associated Ki-67 positive cells were considered when a positive cell was detected within a two-cell diameter interval of a RECA-1 positive blood vessel . In anotp < 0.05. One-sample t-test was utilized to analyze the PI. Total exploring time, distance moved, while number of Ki-67/RECA-1, BrdU/NeuN, and DCX positive cells were evaluated using One-way ANOVA.GraphPad prism  software and SPSS , along with demonstrated mean \u00b1 SEM, were used to analyze all statistical data. Statistical significance was revealed as p > 0.05, p > 0.05, p < 0.05, p > 0.05, In the examination of habituation, the result of distance moved of all groups revealed no significant diversities (p > 0.05, p > 0.05, p < 0.05, D-gal group: p > 0.05, p < 0.05, ** p < 0.01, *** p < 0.001, p > 0.05, In the habituation trial, result of distance moved revealed no significant diversities in all groups (#p < 0.05, ##p < 0.01, p < 0.05, ** p < 0.01, *** p < 0.001, p > 0.05, Ki-67 is a nuclear protein that is expressed during cell proliferation and present in all cell cycle phases, except the G0 phase. RECA-1 is a vascular endothelial cell marker that is related to blood vessels. This study used RECA-1/Ki-67 immunofluorescence double staining to investigate blood vessel associated and non-blood vessel associated cell proliferation in the hippocampus. We found that the number of blood vessel associated Ki-67, non-blood vessel associated Ki-67 and total Ki-67 positive cells in the D-gal group were significantly less than in the control group (##p < 0.01, p < 0.0001, ###p < 0.001, p < 0.0001, BrdU is a thymidine analog, which is used to identify DNA generation in the S phase of the cell cycle. NeuN is found during early embryogenesis in post-mitotic neuroblasts and persists in differentiating and differentiated neurons during the cell lifespan. This study used BrdU/NeuN double staining to identify cell survival related to mature neurons. The results demonstrated that BrdU/NeuN positive cells in the D-gal group were significantly lesser than in the control group (##p < 0.01, p < 0.05, *** p < 0.0001, ####p < 0.0001, p < 0.05, DCX is a microtubule associated protein, which is detected in the developing nervous system of mammalians and used as a marker of neuronal differentiation. This study used DCX immunofluorescence staining to investigate the immature neurons. The consequences of the staining showed that the D-gal group demonstrated significantly more DCX positive cells than the control group (This current study confirms that CA administration reversed memory deficits in rats treated with D-gal. Rats receiving D-gal exhibited cognitive deficits and reduction in hippocampal neurogenesis, but co-administration with CA reversed these deteriorations. Moreover, our results revealed no significant differences in distance moved in all groups, indicating that locomotor activities were not affected by D-gal and CA.Spatial memory is a memory related to spatial locations, routes or configurations. Spatial memory depends on the functions of the hippocampus . In humaRecognition memory is a memory related to recollection of experiences from the past, which relies on integration of the hippocampus and cortical regions ,34,35. TIt is widely accepted that adult neurogenesis is influential in learning and memory performance. The hippocampus and adjacent cortical areas located in the medial temporal lobe are essential for learning and memory processes . Some prIt was confirmed that hippocampal neurogenesis was reduced after receiving D-gal, as demonstrated by reduction in RECA-1/Ki-67, BrdU/NeuN, and DCX positive cells in this current study. Similarly, a previous study also confirmed that Ki-67, BrdU/NeuN, and DCX positive cells are decreased after D-gal administration ,29. PrevThis investigation postulates that receiving D-gal leads to memory loss and hippocampal neurogenesis depletions in aging rats. Nevertheless, CA improves these deteriorations in D-gal-induced aging rats via restoring hippocampal neurogenesis. Further assessment to evaluate the hippocampal synaptic protein and antioxidant enzyme levels may benefit explanation of the mechanism of CA on memory and hippocampal neurogenesis in aging rats activated by D-gal."}
+{"text": "The electronic band structures of CS\u2013MX2 and CS\u2013MXY MS vdWH seem to be simple sum of CS, MX2 and MXY monolayers. The projected electronic properties of the CS, MX2 and MXY layers are well preserved in CS\u2013MX2 and CS\u2013MXY MS vdWH. Their smaller effective mass (higher carrier mobility) render promising prospects of CS\u2013WS2 and CS\u2013MoSeTe as compared to other MS vdWH in nanoelectronic and optoelectronic devices, such as a high efficiency solar cell. In addition, we found that the effective mass of holes is higher than that of electrons, suggesting that these heterostructures can be utilized for hole/electron separation. Interestingly, the MS contact led to the formation of a Schottky contact or ohmic contact, therefore we have used the Schottky Mott rule to calculate the Schottky barrier height (SBH) of CS\u2013MX2  and CS\u2013MXY ((X \u2260 Y) = S, Se, Te) MS vdWH. It was found that CS\u2013MX2  and CS\u2013MXY ((X \u2260 Y) = S, Se, Te) (in both model-I and -II) MS vdWH form p-type Schottky contacts. These p-type Schottky contacts can be considered a promising building block for high-performance photoresponsive optoelectronic devices, p-type electronics, CS-based contacts, and for high-performance electronic devices.Using (hybrid) first principles calculations, the electronic band structure, type of Schottky contact and Schottky barrier height established at the interface of the most stable stacking patterns of the CS\u2013MX 2  and CS\u2013MXY ((X \u2260 Y) = S, Se, Te) MS vdWH.Electronic band structure, type of Schottky contact and Schottky barrier height established at the interface of the CS\u2013MX Another class of 2D materials, XY , which exhibit planar structures15 with sixteen  possible combinations, consisting of an equal number of two different atoms, have attracted much attention due to their stable configuration.16 For each of these 2D binary monolayers, there are three different possible geometrical configurations, the puckered, buckled and planar structures. The hexagonal planar structure supports sp2 hybridization, whereas the favorable hybridization in group V monolayers (phosphorene and arsenene) is sp3, which shows that the hybridization in group IV\u2013VI binary monolayers is similar to those of phosphorene and arsenene. It is observed that CS monolayers in the planar configuration are metallic due to the strong overlap of the conduction and valence bands.15After the successful synthesis of graphene,et al.17(18) have selenized (sulfurized) MoS2(MoSe2) through a chemical vapor deposition (CVD) technique and named these Janus transition metal dichalcogenides (JTMDCs) with the chemical formula MXY  = S, Se). These materials have been shown to be promising for spintronic devices due to the SOC-induced Rashba spin splitting.19 Using density functional theory (DFT) calculations, Xia et al.20 showed that the atomic radius and electronegativity differences of the X and Y chalcogen atoms in MXY  monolayers are associated with the direct to indirect bandgap transition and induced dipole moment. Furthermore, Idrees et al.21 have also used DFT and shown that MoSSe, WSSe, MoSeTe and WSeTe (MoSTe and WSTe) monolayers are direct (indirect) bandgap semiconductors. They transformed indirect MoSTe and WSTe to direct bandgap semiconductors by using external electric fields. They have also investigated the absorption spectra, absorption efficiency, and photocatalytic behavior of these materials.Lu (Zhang) via van der Waals forces in a precisely controlled sequence produces van der Waals heterostructures (vdWH).22 This provides a versatile platform for exploring the uses of new phenomena in designing novel nanoelectronic devices.23,24 In this regard, the stackings of semiconductors with semiconductors (SS contact) and metals with semiconductors (MS contact) are of crucial importance, with a wide range of device applications.25 To date, many of the vdWH in the form of SS contacts have been investigated both theoretically26\u201337 and experimentally38\u201341 for novel extraordinary applications in optoelectronic devices.42\u201347The stacking of isolated 2D materials 48 It reduces the contact resistance, modulates carrier polarity in the channel for transistors, and also enhances the selectivity of carrier extraction for photovoltaic cells,49,50 hence it plays a key role in device performance. In MS contacts, there is another important phenomena, the Fermi level pinning (FLP) caused by metal-induced gap states (MIGS) and interface dipoles or defects created at the interface.51 It refers to the insensitivity of the SB to the work function of the metal.52 TMDCs have been used in almost every MS contact in both experiments53,54 and theory.55,56 The contact of single layer MoS2 (semiconductor) has already been proposed with Ti 57 and other metals of varying work functions.58In the case of MS contacts, the Schottky barrier (SB) is an energy barrier across the junction for the transport of carriers.2  and MXY ((X \u2260 Y) = S, Se, Te) monolayers allow the creation of MS contacts in the form of CS\u2013MX2 and CS\u2013MXY vdWH. Alternative ordering of the chalcogen atoms allows the creation of two models of the CS\u2013MXY vdWH. Therefore, we have fabricated the possible stacking patterns in CS\u2013MX2 and in both (two) models of CS\u2013MXY MS vdWH. After making the possible stacking configurations, we have investigated the electronic band structure, type of Schottky contact and Schottky barrier height established at the interface of the most stable stacking patterns of the MS vdWH under investigation. These findings show the capability to control and modify the properties of the CS, MX2  and MXY ((X \u2260 Y) = S, Se, Te) monolayers, and provide guidelines for the designing of high-performance devices based on MS vdWH.Indeed, the small lattice mismatch and identical symmetry of CS, MX2.59 with the empirical dispersion correction of Grimme,60 and the functionals of Perdew\u2013Burke\u2013Ernzerhof (PBE)61 and Heyd\u2013Scuseria\u2013Ernzerhof (HSE06)62 in the Vienna ab initio simulation package (VASP).63,64We have used DFT\u0393-point centered 6 \u00d7 6 \u00d7 1 Monkhorst\u2013Pack k-point grids in the first Brillouin zone and a cutoff energy of 500 eV were used in the PBE functionals for the geometric relaxations until achieving the convergence criterion of 10\u22124 eV \u00c5\u22121 (10\u22125 eV) for forces (energy). The Monkhorst\u2013Pack k-point grids were refined to 12 \u00d7 12 \u00d7 1 for the electronic structure calculations. The converged PBE wave functions were further used for HSE06 calculations, while the k-mesh here was not refined due to the high computational costs. A 25 \u00c5 vacuum layer thickness was used to avoid interactions between adjacent layers.ab initio molecular dynamics (AIMD) simulations,65 through the Nose thermostat algorithm at a temperature of 300 K for a total of 6 ps with a time interval of 1 fs to investigate the thermal stabilities of CS\u2013MX2  and CS\u2013MXY ((X \u2260 Y) = S, Se, Te) MS vdWH.We have also performed 66 to investigate the optical spectra of the imaginary part of the dielectric functions (\u03b52(\u03c9)) of CS, the MX2  and MXY ((X \u2260 Y) = S, Se, Te) monolayers and the CS\u2013MX2  and CS\u2013MXY ((X \u2260 Y) = S, Se, Te) MS vdWH.67\u201369Using the Quantum ESPRESSO package, the Bethe\u2013Salpeter equation (BSE) was also solved using the GW method3.2 , and MXY ((X \u2260 Y) = S, Se, Te) monolayers are found to be in agreement with ref. 2  and CS\u2013MXY ((X \u2260 Y) = S, Se, Te) MS vdWH.The calculated lattice parameters , electronic structure , and the photocatalytic and optical response of the CS , and with MXY (2\u20137%) monolayers is experimentally achievable70 in the fabrication of CS\u2013MX2 and CS\u2013MXY MS vdWH. Furthermore, the same hexagonal symmetry of the CS monolayer, as shown in Fig. S1,2  and MXY ((X \u2260 Y) = S, Se, Te) monolayers also allows the formation of these MS vdWH. The electronic band structure and stability of vdWH are very sensitive to layer stacking,71 therefore, four possible stacking patterns for the CS\u2013MX2  and CS\u2013MXY ((X \u2260 Y) = S, Se, Te) MS vdWH are fabricated, see 2  MS vdWH (2 layer is placed on top of the S(C) atom of the CS layer, in stacking (b), the M atom of the MX2 layer is placed on the top of the S atom of the CS layer, while both X atoms of the MX2 layer and the C atom of the CS layer are on the hexagonal site, in stacking (c), the M atom of the MX2 layer and S atoms of the CS layer are placed on hexagonal sites, while the X atoms of the MX2 layer is placed on top of the C atom of the CS layer, and in stacking (d), the M(X) atom of the MX2 layer is placed on the top of the C(S) atom of the CS layer. In the case of the CS\u2013MXY ((X \u2260 Y) = S, Se, Te) MS vdWH, two different chalcogen atoms (X and Y) are attached to the transition metal atom (M), therefore eight possible high-symmetry stacking sequences of layers are fabricated, separated into two models, with each having four stacking patterns. In model-I, similar chalcogen atoms are placed at the interface of two layers i.e. CS\u2013MXY, see i.e. CS\u2013MYX, see Fig. S2(a)\u2013(d).\u2020The lattice mismatch of CS with MX MS vdWH : in stacEb = E(CS\u2013MX2(CS\u2013MXY)) \u2212 E(CS) \u2212 E(MX2(MXY)), where E(CS\u2013MX2(CS\u2013MXY)) is the total energy of the CS\u2013MX2(CS\u2013MXY) MS vdWH, E(CS) is the total energy of the isolated CS monolayer and E(MX2(MXY)) is the total energy of the isolated MX2(MXY) monolayer, and the interlayer distances are presented in 2  and stacking (d) of model-I of the CS\u2013MXY ((X \u2260 Y) = S, Se, Te) MS vdWH are the thermodynamically most stable stacking patterns. In the case of model-II of the CS\u2013MXY ((X \u2260 Y) = S, Se, Te) MS vdWH, stacking (b) for CS\u2013MoSeS, CS\u2013MoTeS and CS\u2013WTeSe, and stacking (c) for CS\u2013WSeS, CS\u2013WTeS and CS\u2013MoTeSe vdWH were found to be the thermodynamically most stable stacking patterns. The varying stable stacking in the case of model-II of the CS\u2013MXY ((X \u2260 Y) = S, Se, Te) MS vdWH is due to the induced strain on account of the different chalcogen atoms and also may be due to the unlike interface atoms compared to model-I. These thermodynamically most stable stacking patterns of the MS vdWH under investigation are considered for further investigations. The negative binding energies show that the formation of all MS vdWHs is exothermic, see 2 and CS\u2013MXY MS vdWH. These values are in the range of the binding energies for other vdWHs.21,72,73 The calculated interlayer distances  and CS\u2013MXY ((X \u2260 Y) = S, Se, Te) MS vdWH are presented in The binding energies, nces see  also con74,75 to verify the thermal stability of the MS vdWHs under investigation. There is no structural distortion in the CS\u2013MX2  and CS\u2013MXY ((X \u2260 Y) = S, Se, Te) vdWH after heating them for 6 ps. The fluctuation in the total energy at 0 ps and 6 ps is very small, indicating that these configuration are thermally stable at 300 K, making these systems feasible and they can be obtained easily in future experiments.70 From AIMD simulations, the geometrical structures before heating (first row), with fluctuating energy (second row) and after heating (third row) of CS\u2013MoS2, and CS\u2013MoSSe in both model-I and -II MS vdWH are presented in Furthermore, we have performed AIMD simulations2 and CS\u2013MXY in model-I and -II MS vdWH are calculated and are presented in \u0393\u2013K and M-points of the BZ, while the MX2  monolayers are direct bandgap semiconductors with the CBM (VBM) lying at the K point of the first BZ.76 Similarly, in MXY  = S, Se, Te) monolayers, MoSSe, WSSe, MoSeTe and WSeTe are direct bandgap semiconductors, while MoSTe and WSTe are \u0393\u2013K-point indirect bandgap semiconductors.21 The electronic band structures of the CS\u2013MX2(CS\u2013MXY) MS vdWH seem to be simple sums of the CS and MX2(MXY) monolayers, see 2(CS\u2013MXY) MS vdWH. Most interestingly, we notice that the CS layer has opened a tiny bandgap after stacking with MX2 and MXY layers in the form of the MS vdWH, which is comparable with graphene based vdWH, such as G-MoS2,77 G-GeTe,78 G-GeC79 and G-SnO.80 The opening of the bandgap of CS monolayer is due to the lattice symmetry breaking while making the CS\u2013MX2  and CS\u2013MXY ((X \u2260 Y) = S, Se, Te) MS vdWH. All these results demonstrate that the projected electronic properties of the CS, MX2 and MXY layers are well preserved in the CS\u2013MX2 and CS\u2013MXY MS vdWH.Using the PBE functional, the calculated electronic band structures of CS\u2013MX2(MXY) monolayers in the corresponding CS\u2013MX2 and CS\u2013MXY in model-I and -II MS vdWH are investigated by partial density of states (PDOS), see 2 and CS\u2013MXY vdWH, the CBM of the MX2 and MXY layers are shifted towards the Fermi level, which is due to the stacking on the CS monolayer, while the main contributions are due to the C-p and S-p orbitals of the CS monolayers (which cross the Fermi level) in the CS\u2013MX2 and CS\u2013MXY MS vdWH, respectively. An approach in DFT, that hybrid functionals lead to better agreement with experiments than semi-local functionals, is not general,81 but depends on the considered materials. Therefore, we have also used the HSE06 functional to investigate the electronic band structures of the CS\u2013MX2  and CS\u2013MXY ((X \u2260 Y) = S, Se, Te) vdWH, see Fig. S3.Furthermore, contributions of the orbitals of the CS and MX2  and CS\u2013MXY ((X \u2260 Y) = S, Se, Te) in model-I and -II MS vdWH, see V), presented in 2(MXY) monolayers have deeper electrostatic potentials than that of the CS monolayer in CS\u2013MX2(CS\u2013MXY) MS vdWH. This difference in the electrostatic potentials may have a crucial impact on the charge injection and carrier dynamics when these systems are used as electrodes.82 It should be noted that a large potential difference will significantly influence the charge transportation of the 2D MS vdWH. This electrostatic potential at the interface of CS\u2013MX2 and CS\u2013MXY MS vdWH can successfully reduce the charge carrier recombination and increase the transfer and separation of the induced charge carriers, which enhances the power conversion efficiency.83 The surface conditions of the material affect the work function due to altering the surface electric field induced by the distribution of electrons at the interface.84 The calculated values of the work functions for the CS\u2013MX2 and CS\u2013MXY MS vdWH lie in the range of 1.46 to 2.71 eV, see 85 Using the HSE06 functional, the calculated average electrostatic potential of the CS\u2013MX2  and CS\u2013MXY ((X \u2260 Y) = S, Se, Te) in model-I and -II MS vdWH are presented in Fig. S4 and Table S1.\u2020We have also calculated the electrostatic potentials of the CS\u2013MX\u03c1 = \u03c1(CS\u2013MX2(CS\u2013MXY)) \u2212 \u03c1(CS) \u2212 \u03c1(MX2(MXY)), where \u0394\u03c1 is the total charge density difference, \u03c1(CS\u2013MX2(CS\u2013MXY)) is the charge density of the CS\u2013MX2(CS\u2013MXY) vdWH, \u03c1(CS) is the charge density of the CS monolayer, and \u03c1(MX2(MXY)) is the charge density of the MX2 or MXY monolayer. In the case of the CS\u2013MX2 vdWH, about 0.0023, 0.005, 0.429, 0.0052, 0.175, and 0.0806 electrons are transferred from the CS to the MoS2, MoSe2, MoTe2, WS2, WSe2, and WTe2 layer, respectively, at the interface of the CS\u2013MX2  MS vdWH. Similarly, in the case of the CS\u2013MXY ((X \u2260 Y) = S, Se, Te) MS vdWH in model-I (-II), about 0.0106(0.0351), 0.005(0.0135), 0.0044(0.0089), 0.0038(0.0293), 0.004(0.0091), and 0.005 (0.029) electrons are transferred from the CS to the MoSSe, MoSTe, MoSeTe, WSSe, WSTe, and WSeTe layers at the interface, respectively.Charge redistribution and transfer (quantitatively) from one layer to the other layer are investigated by charge density difference and Bader charge analysis using \u03942  and CS\u2013MXY ((X \u2260 Y) = S, Se, Te) MS vdWH are calculated by using   as compared to those of the other vdWH, demonstrating that these heterostructures render promising prospects for nanoelectronic and optoelectronic devices, such as a high efficiency solar cell. In addition, we found that the effective mass of holes is higher than that of electrons, suggesting that these heterostructures can be utilized for hole/electron separation.87 Using the HSE06 functional, the calculated carrier effective mass of the CS\u2013MX2  and CS\u2013MXY ((X \u2260 Y) = S, Se, Te) in model-I and -II MS vdWH are presented in Table S1.\u2020The effective mass of the CS\u2013MXng  ref.  and are 2(CS\u2013MXY) MS vdWH lie between the CBM and VBM of the MX2  and MXY ((X \u2260 Y) = S, Se, Te) monolayers, thus forming a Schottky contact. Using the Schottky Mott rule,88 the Schottky barrier height (SBH) of n(p) type Schottky contacts is calculated as \u03a6B,n = ECBM \u2212 EF, and the computed values of \u03a6B,n48 are presented in \u03a6B,p have higher values than \u03a6B,n, thus, the CS\u2013MX2  and CS\u2013MXY ((X \u2260 Y) = S, Se, Te) (in both model-I and -II) vdWH form p-type Schottky contacts. These p-type Schottky contacts can be considered to be a promising building block for high-performance photoresponsive optoelectronic devices,89 p-type electronics,90 CS\u2013based contacts,91 and for high-performance electronic devices.92 While making the CS\u2013MX2  and CS\u2013MXY ((X \u2260 Y) = S, Se, Te) vdWH, there is no chemical bond among CS and MX2 (MXY) layers, which may create an interface dipole, which can be calculated via the potential step \u0394\u03c1, as presented in \u03a6B,n = WCS + \u0394V \u2212 \u03c7  \u2212 WCS + \u0394V), where W represents the calculated work function \u03c7 is the electron affinity and I is the ionization energies of the vdWH and corresponding monolayers. We have calculated the work function and \u0394V, presented in \u03a6B,n and \u03a6B,p with and without considering \u0394V are quite unchanged. Hence, the interface dipole at the CS\u2013MX2 and CS\u2013MXY vdWH is neglected within the vdW layers.93Interestingly, MS contact led to the formation of a Schottky contact or ohmic contact. We can see from the electronic band structures in \u03b52(\u03c9)) of the CS, MX2  and MXY ((X \u2260 Y) = S, Se, Te) monolayers, see Fig. S42 and CS\u2013MXY ((in both model-I and -II) MS vdWH, see \u03b52(\u03c9) spectra of the CS, MX2  and MXY ((X \u2260 Y) = S, Se, Te) monolayers,  MS vdWH (see \u03b52(\u03c9) spectrum of CS is very weak as compared to those of TMDCs and JTMDCs. Furthermore, a slight blueshift is found in the spectra of all MS vdWH compared to those of the isolated monolayers. \u03b52(\u03c9) spectra for the vdW heterostructures overlaps with those of TMDCs and JTMDCs but is higher than that of the CS monolayer. This indicates the good absorption capability of the constructed heterostructure.94For use in practical applications in optoelectronic and photocatalytic nano devices, we have further calculated the imaginary parts of the dielectric function  and MXY ((X \u2260 Y) = S, Se, Te) (semiconductor) monolayers also allow the formation of MS contacts in the form of vdWH. Therefore, using (hybrid) first principles calculations, we have investigated the electronic band structure, type of Schottky contact and Schottky barrier height established at the interface of the most stable stacking patterns of the CS\u2013MX2  and CS\u2013MXY ((X \u2260 Y) = S, Se, Te) MS vdWH. The calculated electronic band structures show that the projected electronic properties of the CS, MX2 and MXY monolayers are well preserved in the CS\u2013MX2 and CS\u2013MXY MS vdWH. The smaller effective mass (higher carrier mobility) of electrons and holes render promising prospects of CS\u2013WS2 and CS\u2013MoSeTe as compared to other MS vdWH in nanoelectronic and optoelectronic devices. Interestingly, the MS contact of the CS  and MX2 , and MXY ((X \u2260 Y) = S, Se, Te) (semiconductors) monolayers led to the formation of a Schottky contact or ohmic contact, therefore we have used the Schottky Mott rule to calculate the Schottky barrier height (SBH) of the CS\u2013MX2  and CS\u2013MXY ((X \u2260 Y) = S, Se, Te) MS vdWH. The CS\u2013MX2  and CS\u2013MXY ((X \u2260 Y) = S, Se, Te) (in both model-I and -II) MS vdWH form p-type Schottky contacts, a promising building block for high-performance photoresponsive optoelectronic devices, p-type electronics, CS-based contacts, and for high-performance electronic devices.Lattice mismatch and the same hexagonal symmetry of the CS  and the MXThere are no conflicts to declare.RA-012-D2RA00668E-s001"}
+{"text": "Clostridioides difficile infection (HO-CDI) were reexamined using 2012\u20132018 data from a new cohort of US acute-care hospitals. This analysis revealed significant positive associations between total, third-generation, and fourth-generation cephalosporin, fluoroquinolone, carbapenem, and piperacillin-tazobactam use and HO-CDI rates, confirming previous findings.Previously reported associations between hospital-level antibiotic use and hospital-onset  Clostridioides difficile infection (CDI) in the United States has decreased in recent years, disease burden remains high, and the Centers for Disease Control and Prevention has recently declared CDI an urgent public health threat. Decreases in hospital-onset CDI (HO-CDI) rates have occurred despite relatively stable national rates of overall inpatient antibiotic use. Previously, we reported the association between hospital-level antibiotic use rates and HO-CDI rates in acute-care hospitals (ACHs) from 2006 to 2012. In this analysis, we reexamined the cross-sectional and temporal associations between hospital-level antibiotic use and HO-CDI rates using more recent data from a separate cohort of ACHs.Although the number of cases of International Classification of Diseases (ICD) code specifying enterocolitis due to Clostridioides difficile  in any secondary diagnostic position and inpatient treatment with metronidazole , fidaxomicin, or vancomycin  initiated on hospital day 3 or later after admission. Facility-level monthly rates of HO-CDI were calculated per 10,000 patient days (PD). Hospital antibiotic use was measured by days of therapy (DOT) per 1,000 PD and examined through monthly rates of total antibiotic use and use of 7 antibiotic classes: fluoroquinolones, third- and fourth-generation cephalosporins , piperacillin-tazobactam, carbapenems, \u03b2-lactam/\u03b2-lactamase inhibitor combination (excluding piperacillin-tazobactam), clindamycin, and penicillins. In addition, we evaluated combined use of 3 high-risk antibiotic classes: fluoroquinolones, cephalosporins, and carbapenems.We used adult hospital discharge and inpatient charge records for antibiotic use, CDI tests and treatment from ACHs contributing to the Premier Healthcare Database from January 1, 2012, to December 31, 2018. A case of HO-CDI was defined as a hospital discharge with an  patient case-mix index, proportion of discharges with a surgical diagnosis-related group (DRG), and community-onset CDI (CO-CDI) cases per 100 discharges. A case of CO-CDI was defined as a discharge with a ICD-9 or CD-10 diagnosis code for CDI in the primary position, no prior hospitalization in the same facility in 28 days, and inpatient CDI treatment initiated at any time during hospitalization. NAAT testing was assessed using inpatient charges for C. difficile tests where each test was categorized as NAAT if its description contained \u201cNAAT.\u201d \u201cPCR,\u201d \u201camplified,\u201d or \u201cDNA.\u201d All other C. difficile tests were categorized as \u201cnon-NAAT.\u201d NAAT primary utilization was defined quarterly as a binary variable. If all C. difficile tests in a given quarter included an NAAT test, a hospital was considered to have primary NAAT utilization; otherwise, a hospital was considered not to have primary NAAT utilization.The cross-sectional relationships between facility-level rates of HO-CDI and antimicrobial use were examined through multivariable generalized estimating equation (GEE) models accounting for autocorrelation between monthly HO-CDI rates within a facility, negative binomial distribution, annual trend, month of discharge, and included a natural log of patient day as offset. All GEE models adjusted for hospital confounders: urban or rural location, bed size, teaching status, census division, primary nucleic acid amplification test (NAAT) utilization, proportion of patients aged \u226565 years, average Gagne comorbidity score,Temporal associations between HO-CDI rate and antibiotic use were assessed for subsets of ACHs that achieved the following targeted decreases in antibiotic use over 2 consecutive years: <10% decrease, \u226530% decrease, \u226520% decrease, and \u226510% decrease (groups are cumulative and inclusive). Temporal trends in HO-CDI rates were assessed using GEE models similar to the cross-sectional analysis with the addition of adjustment for seasonality.P < .001). In a class-specific model, 10 DOT per 1,000 PD increases in the use of carbapenems, cephalosporins, and piperacillin-tazobactam were each independently associated with 1.3%, 0.6%, and 1.1% increases in the HO-CDI rate, respectively (Supplementary Table 2 online). In all models, primary NAAT utilization was associated with a 16% higher HO-CDI rate compared with hospitals without primary NAAT utilization (Supplementary Table 2).During 2012\u20132018, among 921 participating hospitals, the median hospital-level HO-CDI rate was 6.7 per 10,000 PD ; the median CO-CDI rate was 0.26 per 100 discharges  (Supplementary Table 1 online). More than half of hospital quarters (52.5%) had primary NAAT utilizations , and primary NAAT utilization peaked at 57% in 2015 . In a cross-sectional multivariable analysis, overall antibiotic use was significantly associated with the facility-level HO-CDI rate. For every 50 DOT per 1,000 PD increase in antibiotic use, there was a 2.8% increase in the HO-CDI rate .In this ecologic analysis, we used 2012\u20132018 data from a large cohort of ACHs to identify significant associations between facility-level HO-CDI rates and antibiotic utilization. Specifically, higher levels of total antimicrobial use as well as use of third- and fourth-generation cephalosporins, carbapenems, and piperacillin-tazobactam were associated with higher rates of HO-CDI. Decreases in total antibiotic use, fluoroquinolones, carbapenems, and combined use of fluoroquinolones, third- and fourth-generation cephalosporins, and carbapenems corresponded with decreases in HO-CDI. This analysis builds upon our previous investigation by confirming associations identified in a different hospital cohort over a different period and by adjusting for a confounder not previously available, that is, hospital-level use of a more sensitive NAAT diagnostic test. Although NAAT utilization was derived from administrative data, which is subject to several previously described limitations, showing that a simulated 30% reduction in antibiotics prescribed across all inpatient and outpatient locations of a regional network was associated with a 17% decrease in HO-CDI. Given reports of widespread inappropriate use, reductions in total antibiotic use of this magnitude are potentially feasible, particularly among hospitals with high antibiotic use. Although decreasing total antibiotic use is an attractive goal, our findings suggest that important reductions in HO-CDI rates may be achieved with a more targeted approach that focuses on combined use of cephalosporins, fluoroquinolones, and carbapenems. Hospitals with a \u226530% decrease in the combined use of those antibiotics observed a 16% decrease in the HO-CDI. This approach may also help prevent substitution of fluoroquinolones by other broad-spectrum antibiotics such as cephalosporins, an occurrence evident in recent reports on antibiotic use trends. Although specific reasons for and factors driving the changes in antibiotic use in our hospital cohort were beyond the scope of this investigation, we found that decreased antibiotic use consistently corresponded with decreases in the HO-CDI rate. These findings should encourage ACHs to invest efforts into monitoring antibiotic use and targeting unnecessary and inappropriate use across all classes of antibiotics.Like our previous study, the largest reductions in HO-CDI rates were identified in a small set of ACHs that decreased their total antibiotic use by 30%. These findings are also similar to those of a study by Rhea et alOur study, utilizing a new cohort of hospitals during more recent years, continues to identify associations between hospital-level antibiotic use and the HO-CDI rate and suggests that reducing antibiotic use, in conjunction with other infection control and prevention measures, can reduce facility-level rates of HO-CDI."}
+{"text": "Dasycladus vermicularis (Scopoli) Krasser 1898 from the Adriatic Sea (Croatia) regarding volatilome\u2013volatile organic compounds  and less polar nonvolatile compounds for the first time. Headspace solid-phase microextraction (HS-SPME) and hydrodistillation (HD) were used showing the great volatilome variability among fresh (HS-FrDV and HD-FrDV) and dried (HS-DrDV and HD-DrDV) samples after GC\u2013MS analysis. Aromatic aldehydes were dominant in both fresh and air-dried HS samples with benzaldehyde as the most abundant in fresh samples and decreasing 2.7\u20133.7 times after drying together with 2-phenylbut-2-enal that was not present after drying. Aliphatic compounds  were also present. C11-hydrocarbons (dictyopterpene C\u2019 and dictyopterpene D\u2019) were detected in HS-FrDV. (E)-Phytol was the most dominant compound in HD-FrDV and HD-DrDV. Diterpene alcohols -falcarinol) and sesquiterpene alcohol, cubenol, were dominant in HD-FrDV, and their abundance decreased after drying. C13-norisoprenoides (\u03b1-ionone and \u03b2-ionone) increased after drying. Aliphatic compounds were present in both HD-FrDV and HD-DrDV samples. The less polar nonvolatile compounds in the obtained fractions F3 and F4 were analysed and identified by UHPLC-ESI(+)-HRMS. Identified compounds belonged to a group of pigments (7 compounds), fatty acid derivatives (13 compounds), as well as steroids and terpenes (10 compounds). Porphyrin-based compounds (C55H74N4O5\u20137), xanthophylls, sphingolipid compounds, fatty acid amides, and phytosterols represented the majority of identified compounds. By implementing both in vitro and in vivo assays for antioxidant activity determination, F3 showed a higher activity than F4. Inhibitory concentrations (IC50) for F3 and F4 were 498.00 \u00b1 0.01 \u00b5g/mL and 798.00 \u00b1 0.81 \u00b5g/mL, respectively, while a 1.5-fold reduction in the ROS level was observed after pre-treatment of zebrafish larvae with 45 \u00b5g/mL of F3.The present research is a comprehensive investigation of  The pharmaceutical industry is in a great rise at the global level, which has caused the necessity for new sources of pharmaceutical compounds such as the potential of marine algae. Cyanobacteria  are well known for their production of antibiotics and active pharmaceutical compounds (API) ,2. DespiDasycladus vermicularis (Scopoli) Krasser 1898 (fam. Dasycladaceae) is a green macroalga that inhabits rocky substrates of North Atlantic and Mediterranean littoral zones. To survive under increased UV exposure and temperatures, macroalgae developed antioxidant defence mechanisms that activate under the overproduction of reactive oxygen species (ROS), thus reducing/preventing the occurrence of oxidative stress [D. vermicularis mostly focused on phenolic compounds with antioxidant and UV-radiation absorption activities such as coumarins and their sulfated metabolites such as 7-hydroxycoumarin-3,6-disulfate [D. vermicularis.isulfate . There iD. vermicularis using gas chromatography\u2013mass spectrometry (GC\u2013MS). In addition to the VOCs analysis, less polar nonvolatile compounds and their in vitro and in vivo antioxidative activities were investigated for the first time in this research. Therefore, full chemical profiles of volatile and nonvolatile polar compounds of D. vermicularis were investigated. The present research, as a continuation of our project Bioprospecting of the Adriatic Sea, has the following key goals: (a) identify and compare VOCs of FrDV and DrDV retrieved by both headspace solid-phase microextraction (HS-SPME) and hydrodistillation (HD) followed by gas chromatography\u2013mass spectrometry analysis (GC\u2013MS); (b) identify the composition of FdDV (freeze-dried D. vermicularis) less polar fractions using high-performance liquid chromatography\u2013high-resolution mass spectrometry with electrospray ionisation (UHPLC-ESI\u2013HRMS); (c) evaluate detailed antioxidant activity of the less polar fractions by four in vitro assays  assay, Folin\u2013Ciocalteu method, and ferric reducing antioxidant power (FRAP)); (d) determine the protective effect of the less polar fractions against hydrogen peroxide-induced oxidative stress in the zebrafish model along with the embryotoxicity assessment.Our previous studies ,11,12,13To analyse the headspace composition, solid-phase microextraction was used (HS-SPME). A more complementary headspace profile was attained using two fibres of different polarities: divinylbenzene/carboxene/polydimethylsiloxane (DVB/CAR/PDMS) and polydimethylsiloxane/divinylbenzene (PDMS/DVB). In the FrDV headspace (HS-FrDV), 90.99% of VOCs were identified in total with DVB/CAR/PDMS fibre and 91.74% with PDMS/DVB fibre. In the DrDV headspace (HS-DrDV) with both fibres, 100% of VOCs were identified. Aromatic compounds were dominant in both fresh and air-dried HS samples\u201455.25% (DVB/CAR/PDMS fibre) and 55.74% (PDMS/DVB fibre) in HS-FrDV and 77.44% (DVB/CAR/PDMS fibre) and 69.32% (PDMS/DVB fibre) in HS-DrDV. Benzaldehyde was the most abundant in fresh samples  and decreased 2.7\u20133.7 times after the drying, and the second-most abundant compound was aromatic aldehyde 2-phenylbut-2-enal , which could not be found in HS-DrDV (it probably evaporated). On the other hand, benzyl alcohol was the most abundant in air-dried samples  , as was 11-hydrocarbons (well-known pheromones) dictyopterpene C\u2019  and dictyopterpene D\u2019  with the highest abundance in HS-FrDV decreased after the drying. Dictyopterene C\u2019 could not be detected in HS-DrDV and the abundance of dictyopterpene D\u2019 decreased 4.3\u20135.0 times after drying, which can be explained by the oxidative degradation [The second-most abundant group of compounds belonged to aliphatic compounds: unsaturated hydrocarbons in HS-FrDV  and saturated hydrocarbons in HS-DrDV . Cradation ,15,16,17E)-nonadec-9-ene (12.79%) as the most dominant. This could probably be the result of fatty acids\u2019 decarboxylation, which follows its 4.4 times abundance decrement in HD-DrDV. The increment in saturated (2.4 times) and unsaturated alcohols (7.5 times) in HD-DrDV could be an indicator of the hydrocarbons\u2019 oxidation during the drying. Among them, hexadecan-1-ol (10.37%) and (Z)-octadec-9-en-1-ol (8.13%) were the most abundant.In the hydrodistillate of FrDV (HD-FrDV) and of DrDV (HD-DrDV), 86.36% and 90.88% of TIC  areas were identified altogether, respectively. Aliphatic compounds were dominant in both fresh (43.00%) and air-dried (61.42%) HD samples. In HD-FrDV, the majority of aliphatic compounds were hydrocarbons (both saturated and unsaturated), with the long-chain hydrocarbon docosane (7.69%) as the most abundant . It decrDiterpene alcohols -falcarinol) as well as sesquiterpene alcohol, cubenol, were dominant among the group of terpenes (17.80%) in HD-FrDV. The abundance of terpenes dropped down 3.3 times in HD-DrDV.13-norisoprenoides (\u03b1-ionone and \u03b2-ionone), increased 7.3 times after the drying mostly because of the increment in \u03b2-ionone, most probable due to carotenoids degradation.The abundance of carotenoid degradation products, CThe FdDV sample was extracted and fractionated . F3 and 55H74N4O5\u20137), were abundant, especially in F4 (55H74N4O6). Two other compounds were less polar, so they were much more abundant in F4. It is well known that the porphyrin ring is an essential part of the chlorophyll structure responsible for the antioxidant activity [a were found in green alga Ulva prolifera (formerly known as Enteromorpha prolifera) [Sargassum fulvellum [a was also detected but in a small amount, more in F3 than in F4. It has also shown antioxidant activity in green alga Ulva prolifera [No chlorophyll was detected, but its derivatives, porphyrin-based compounds (Cly in F4 . Among tactivity . Antimutolifera)  and its ulvellum . Pheophorolifera .Nannochloropsis oculata [Scenedesmus almeriensis [Xanthophylls, both present in green algae, zeaxanthin , and the one more characteristic for brown algae, fucoxanthin, were detected and identified  with a g oculata  and luteeriensis . The estCodium fragile, oleamide also suppressed, among others, the secretion of TNF-\u03b1 (tumour necrosis factor-\u03b1) and interleukins IL-1\u03b2 (interleukin-1 beta) and IL-6 (interleukin 6), and prevented the translocation of NF-kappa B into the nucleus, which are main features in the anti-tumour process [Ericaria sp. [Prymnesium parvum [Ericaria sp. [Fatty acid derivatives in F3 and F4 originate from palmitic, stearic, oleic, and behenic acids . Sphingo process . It was aria sp. . Erucamim parvum  and Ericaria sp. . Fatty aD. vermicularis: cholesterol, 24-methylenecholesterol, brassicasterol, and clionasterol [Steroids and terpenes consisted of 10 identified compounds . Four conasterol .D. vermicularis fractions (F3 and F4), and to obtain better insight into their antioxidant behaviour, different assays were used. The results for four assays , 2,2-diphenyl-1-picrylhydrazyl (DPPH), and oxygen radical absorbance capacity (ORAC)) are depicted in p < 0.0001) different activity for F3 and F4 fractions, namely, a 50-fold higher activity was obtained for F3. As no polyphenols were found in these fractions (p > 0.001) activity for F3 than F4.To analyse the antioxidant potential of the obtained ractions , this re50 values  in a higher amount, which probably contributed to the antioxidant responses [Further on, the detailed reduction of radical cations by implementing ABTS assay was studied. Different concentrations of F3 and F4 fractions were prepared ranging from 0.005 to 5 mg/mL to obtain dose\u2013response curves  and calc0 values . As can esponses .Danio rerio exposed to H2O2-induced oxidative stress. F4 induced a concentration-dependent increase in mortality  = 17.13 \u00b1 1.27 \u00b5g/mL) and developmental abnormalities  = 29.52 \u00b1 4.27 \u00b5g/mL), as presented in Ericaria crinita and Ericaria amentacea fractions enriched with mentioned fatty acid amides negatively affected the survival of zebrafish embryos [2O2. However, pre-treatment with 45 \u00b5g/mL of F3 significantly protected zebrafish from H2O2-induced mortality . H2O2 increased ROS production in zebrafish up to 255.8 \u00b1 6.99% compared to the control treatment group on artificial water \u2013HRMS analysis. Both fractions contain the compounds that have antioxidative properties such as porphyrin-based compounds  and xantv/v) formic acid, were both hypergrade (HPLC-MS LiChrosolv\u00ae) and purchased from Supelco Co. .Water and acetonitrile, both containing 0.1% , TPTZ , L-ascorbic acid (\u226599%), DPPH , fluorescein, AAPH  dihydrochloride, 97%), and ABTS  sulfonic acid, >99.0%) were purchased from Sigma-Aldrich .Dasycladus vermicularis (Scopoli) Krasser 1898 was collected in July 2021 by a single-point collection from the Adriatic Sea  with the sampling geographical coordinates 44\u00b017\u203201\u2033 N; 15\u00b012\u203227\u2033 E. The sea depth was 3 m with the sea temperature at 25 \u00b0C. The macroalga was transferred to the laboratory in an air-tight plastic box containing both alga and seawater immediately after the collection. It was maintained at 4 \u00b0C in the dark for not more than 24 h until further analysis. A part of the collected D. vermicularis was air-dried and placed in the dark at room temperature for 10 days. Both fresh and air-dried samples were sliced into small pieces before further analysis. The identification of the collected alga was performed by marine biology experts Donat Ptericiolli and Dr Tatjana Bakran-Petricioli, professor at the Faculty of Science, University of Zagreb.D. vermicularis was freeze-dried for fractionation, as described in A part of The excess seawater from the fresh sample was removed by placing a part of the sample between two layers of filter paper for a few minutes. HS-SPME was performed using an autosampler, PAL Auto Sampler System , and two SPME fibres of different polarities. One fiber was covered with DVB/CAR/PDMS (divinylbenzene/carboxen/polydimethylsiloxane) and the other one with PDMS/DVB (poly-dimethylsiloxane/divinylbenzene). Both fibres were conditioned prior to the extraction according to the manufacturer and were purchased from Supelco Co. . Prepared samples, with the mass of 1 g, were placed into HS-20 mL glass vials sealed with a polytetrafluorethylene (PTFE)/silicon septa stainless-steel cap. Equilibration of the sample was performed at 60 \u00b0C for 15 min and then it was extracted for 45 min. The temperature of the injector was set to 250 \u00b0C. Thermal desorption of the sample from the fibre was conducted directly to the GC column for 6 min. HS-SPME was performed in triplicate.v/v ratio of 2:1 (1 mL). HD was performed separately on the prepared samples of fresh and air-dried D. vermicularis. The volatile oil dissolved and trapped in the solvent trap was carefully removed with a pipette avoiding taking the water part. It was then slowly concentrated by the gentle flow of nitrogen until the volume of 0.2 mL was reached. An amount of 2 \u00b5L of the sample was used for GC\u2013MS analyses. HD was performed in triplicate.The hydrodistillation (HD) procedure was obtained in a modified Clevenger apparatus for 2 h. Diethyl ether  and pentane  were used as the solvent trap in a n-alkanes (C8\u2013C25), with those reported in the literature  and their mass spectra with the spectra from NIST 17 (D-Gaithersburg) and Wiley 9  mass spectral libraries. The percentage composition of the samples was calculated using the normalisation method without correction factors. The average component percentages in The GC\u2013MS analyses of isolated VOCs were run on an Agilent Technologies  gas chromatograph model 8890 tandem mass spectrometer detector model 5977E MSD . An HP-5MS capillary column with the dimensions of 30 m \u00d7 0.25 mm and 0.25 \u00b5m film thickness  was used for the VOCs separation. The injector temperature was set to 250 \u00b0C and the detector temperature to 300 \u00b0C. The oven temperature was set to 70 \u00b0C isothermal for 2 min. The temperature gradient was obtained by temperature increments from 70 to 200 \u00b0C at 3 \u00b0C/min. After reaching the temperature of 200 \u00b0C, it was held isothermally for 15 min. The flow rate of helium as a carrier gas was 1.0 mL/min and the split ratio was 1:50. The MSD (EI mode) was operated at 70 eV with the scanning mass range from 30 to 300 Amu. The identification of the compounds was achieved by comparing their retention indices (RI), which were defined relative to the retention times of D. vermicularis (FdDV) was extracted in methanol and dichloromethane  with 5 min of sonication  three times. The gained extract (with 6.0377 g/105.3 mg drug:extract ratio) was evaporated under a nitrogen flow  and was then mixed with C18 powder . An SPE cartridge  was conditioned with MeOH and ultrapure water and then covered with dry extract. To obtain the fractions F1 to F4, the sample was eluted by applying the solvents of decreasing polarity [2O), F2 , F3 (MeOH), and F4 ). Less polar compounds were eluted in F3 and F4. The fractions were dried by SpeedVac  and stored at 4 \u00b0C in the dark before further analysis.The freeze-dried The UHPLC-ESI\u2013HRMS analyses were performed using an UHPLC ExionLC AD system , which was equipped with the ExionLC Controller, ExionLC AD Pump, ExionLC AD Degasser, ExionLC solvent delivery system, ExionLC AD Autosampler, and ExionLC AD Column oven tandem quadrupole-time-of-flight (Q-TOF) mass spectrometer TripleTOF 6600+  having a Duospray ion source. The chromatographic separations were achieved on the analytical column Acquity UPLC BEH Phenyl-Hexyl with dimensions of 2.1 mm \u00d7 100 mm and a particle size of 1.7 \u00b5m . The aqueous mobile phase (A) was water containing 0.1% formic acid and the organic mobile phase (B) was acetonitrile with 0.1% formic acid. The mobile phase flow rate was continuously 0.4 mL/min and the oven temperature was 30 \u00b0C. The elution started isocratically for 0.6 min with 2% of B and, later, the gradient program was applied: 0.6\u201318.5 min (B linear gradient to 100%), 18.5\u201325 min (100% B). An amount of 4 \u00b5L of the sample was injected.Positive electrospray ionisation (ESI+) was set. Collision-induced dissociation (CID) in information-dependent acquisition (IDA) mode was applied recording the MS/MS mass spectra. The precursor ions only with the signal intensities above the 200 cps threshold were recorded with the maximum number of 15 precursor ions simultaneously subjected to CID. The parameters set in the ion source were as follows: source temperature of 300 \u00b0C, nebulising gas  pressure of 40 psi, curtain gas (nitrogen) pressure of 30 psi, heater gas  pressure of 15 psi, and ESI capillary voltage of 5.5 kV. Mass spectra were recorded in the m/z range of 100\u20131000 (MS) with the declustering potential of 80 V and accumulation time of 100 ms. MS/MS data were recorded in the m/z range of 20\u20131000, and the collision energy of the collision gas (nitrogen) was 40 eV with a spread of 20 eV. The mass scale calibrations (in the MS and MS/MS modes) were performed prior to each run in an automatic regime using an ESI Positive Calibration Solution 5600 .ACD/Spectrus Processor 2021.1.0.  was used for processing data. The accurate masses of the protonated molecules, their isotopic distributions, and the product ions m/z in MS/MS spectra were used for the compounds\u2019 elemental compositions determination. Detected components were then identified based on their mass spectra and the elemental compositions linked to search in the Chemical Entities of Biological Interest (ChEBI) database. The choice among the suggested hits was based on MS/MS data matching.n = 4). A detailed description of all used methods in this research is described in our recently published manuscript [Antioxidant activity determination was performed using five different methods, namely the Folin\u2013Ciocalteu method, ferric-reducing antioxidant power (FRAP), 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay, oxygen radical absorbance capacity (ORAC), and the reduction in the radical cation (ABTS) assays. All assays were carried out in triplicates in 96-well plates using a UV/Vis microplate reader , while all results were expressed as mean \u00b1 standard deviation . The fluorescence intensity of images was quantified using ImageJ software.To determine the antioxidant potential of DAVE fractions in vivo, the protocol described in Jerkovi\u0107 et al. (2021) was followed . BrieflyAnimal housing and spawning were performed in an aquaria unit approved by the Croatian Ministry of Agriculture and according to the Directive 2010/63/EU. The experiments were conducted on the nonprotected stages of zebrafish development (up to 96 hpf), which do not require permission by animal welfare commissions (Directive 2010/63/EU).t-test was used to examine the significant difference between tested samples for each in vitro antioxidant method. Differences were considered significant at p < 0.05. Prior to LC50 and IC50 determination, the obtained values were subjected to logarithmic transformation.GraphPad Prism software version 8 was used for statistical analysis and graph presentation. A one-way analysis of variance (ANOVA) and Tukey\u2019s multiple comparisons were performed to examine the significant differences between the means. A Welch\u2019s D. vermicularis chemical composition in the literature, except for polyphenols and coumarins analysis, and the present research is a comprehensive investigation of D. vermicularis from the Adriatic Sea (Croatia) regarding VOCs and less polar nonvolatile compounds for the first time. VOCs from both fresh (FrDV) and air-dried (DrDV) samples were presented. Both HS-SPME and HD were used, showing a great variability among HS-FrDV and HD-FrDV, as well as among HS-DrDV and HD-DrDV. Utilising these two methods, VOCs of different polarities and volatility were isolated. There is clearly a noticeable difference in fresh (FrDV) and air-dried (DrDV) samples. Aromatic compounds were dominant in both fresh and air-dried HS samples with benzaldehyde and 2-phenylbut-2-enal as the most abundant in fresh samples. C11-hydrocarbons dictyopterpene C\u2019 and dictyopterpene D\u2019 were detected with great abundance in HS-FrDV. Aliphatic compounds were dominant in both HD-FrDV and HD-DrDV samples. In HD-FrDV, diterpene alcohols -falcarinol) and sesquiterpene alcohol cubenol were dominant. Its amount decreased, but C13-norisoprenoides (\u03b1-ionone and \u03b2-ionone) increased during the air-drying process. The less polar compounds in F3 and F4 were analysed and identified by UHPLC-ESI(+)-HRMS. Identified compounds belonged to a group of pigments (7 compounds), fatty acid derivatives (13 compounds), as well as steroids and terpenes (10 compounds). Porphyrin-based compounds (C55H74N4O5\u20137), xanthophylls, sphingolipid compounds, fatty acid amides, and phytosterols represent the majority of identified compounds.Little data are available on D. vermicularis by implementing five independent assays  showed a high activity of both F3 and F4 fractions. F3 exerted a higher inhibition of reactive oxygen species than F4, which can be related to the presence of pigments, namely xanthophylls. When compared to the results obtained by in vivo analysis, F3 also showed protective effects against the H2O2-induced mortality of zebrafish embryos. The first results on D. vermicularis suggest that this green alga might be a potent source of natural antioxidants such as pigments, terpenes, and porphyrin-based compounds, and further research regarding different biological activities is in progress.In vitro analysis of the antioxidant potential of less polar compounds from"}
+{"text": "Retinal diseases are major causes of irreversible vision loss and blindness. Despite extensive research into their pathophysiology and etiology, pharmacotherapy effectiveness and surgical outcomes remain poor. Based largely on numerous preclinical studies, administration of mesenchymal stem cells (MSCs) as a therapeutic strategy for retinal diseases holds great promise, and various approaches have been applied to the therapies. However, hindered by the retinal barriers, the initial vision for the stem cell replacement strategy fails to achieve the anticipated effect and has now been questioned. Accumulating evidence now suggests that the paracrine effect may play a dominant role in MSC-based treatment, and MSC-derived extracellular vesicles emerge as a novel compelling alternative for cell-free therapy. This review summarizes the therapeutic potential and current strategies of this fascinating class of cells in retinal degeneration and other retinal dysfunctions. The dysfunction or degeneration of retina, such as age-related macular degeneration (AMD), diabetic retinopathy (DR), and other hereditary diseases, are leading causes of irreversible blindness and vision loss which seriously impact the patient\u2019s daily life . RecentlWith the rapid development of stem cell research, stem-cell-based therapy is becoming one of the most promising strategies for untreatable retinal diseases. At present, plenty of stem cell clinical trials have been formally approved or are in progress. The clinical application of stem cell therapy will become an important landmark in the development of human medical history and radically change the whole picture of modern medicine ,5,6,7. EMSCs are adult stem cells derived from embryonic mesoderm with a great capacity for self-renewal while maintaining their multipotency. Today, MSCs can be obtained from a wide variety of tissues including dental tissues, skin and foreskin, bones, muscles, salivary glands, blood, cartilages, and umbilical cords. Generally, MSCs can be isolated by two methods, the explant method and the enzymatic method. The key to the former approach lies in cutting the tissue into small pieces, in order to promote the diffusion of gases and nutrients into the cells. The latter method contains the additional steps of digesting the extracellular matrix with enzymes . Former The retina is a continuation of the central nervous system, which is composed of neurons, glial cells, retinal pigment epithelial cells (RPE), and vasculature. Retinal diseases are involved with inflammation, immunity, trauma, neurodegeneration, angiopathy, and other pathological conditions. The special anatomy of ocular, diverse pathogenesis and remodeling progress of the retinal diseases make retina a more appropriate target for MSC-based therapy than other tissues. The advantages include the following: (i) the transparent refractive media allow us to observe the retina intuitively, while a variety of detection methods can provide accurate and vivid evaluation of retinal function and evaluate the process of therapy in vivo, as well as enable immediate response to the possible adverse effect accordingly; (ii) stem cells are more likely to survive inside the ocular environment, which is relatively immune privileged due to the blood\u2013eye barrier ; (iii) rDue to particular organizational structure of retina and the characteristics of retinal diseases, MSC-based treatment primarily concerns how to sufficiently suppress the inflammatory response, reduce tissue damage, and ameliorate blood supply and nutrition in order to promote tissue remodeling and neurological functions. Thus, appropriate administration routes of MSCs and strategies are required for the diversity of retinal diseases.Generally, there are two administration routes of MSCs: local and systemic intravenous application .For the local administration route, MSCs are usually delivered intravitreally or subretinally by a microscope. Subretinal transplantation of MSCs can anatomically circumvent the blood-retinal barrier to the adjacent area of the outer retina and avoid the challenge of immune rejection. One of the commonly used methods in clinical trials is to inject stem cells directly through the retina after pars plana vitrectomy . In someAn alternative approach to local stem cell therapy is intravitreal injection, an administration route that has been globally applied on account of the prevalence of anti-VEGF therapy. Unlike vitrectomy surgery for subretinal implantation of cells, which requires hospital admission and significant recovery time from surgery, intravitreal injection of cells can be operated in the clinic, with minimal damage and risk to the eye. It seems that intravitreal administration is more suitable for treatment of inner retinal lesions taking account of the anatomic location. Nevertheless, stem cells do not readily incorporate into the retina, partly resulting from the inherent integrity of the internal limiting membrane .Intravenous injection can be repetitively performed at the clinic and barely causes any harm to the recipient. After systemic administration, MSCs can possibly reach the maximum range of optic circulation and benefit the entire retina and choroid ,31. MSCsHowever, with the deepening of MSC-based research, more researchers argued the theory of direct cell replacement, suggesting the revived and regenerative effect of MSCs by introducing its paracrine trophic effect ,34. If MAs we know, there are encouraging outcomes in MSC-based treatments for retinal diseases .Stem cell therapy has opened a new era of tissue regeneration. MSCs may provide an alternative source for retinal regeneration. In addition to the established multipotential of MSCs to differentiate into osteoblasts, adipocytes, and chondrocytes, they were seen to differentiate into retinal cells in early studies. Bone-marrow-derived MSCs (BMSCs) and adipose-tissue-derived MSCs (AMSCs) are shown to express RPE and photoreceptor cell markers by exposure to conditioned medium in vitro ,36. IntrIn contrast, intravitreal administration of MSCs remained in the vitreous cavity and did not differentiate into neural or perivascular-like cells while preventing retinal ganglion cell loss by triggering an effective cytoprotective microenvironment in the models of diabetic and experimental ocular hypertension ,42. HoweIn terms of long-term application, a study reported that 82 participants with retinitis pigmentosa (RP) successfully received 5 million UC-MSCs to the suprachoroidal area. After 6 months, studies showed improvement in best-corrected visual acuity (BCVA), visual field (VF), and multifocal electroretinography (mf-ERG) measurements in most patients, and no ocular or systemic adverse events were observed during the follow-up period . AdditioChronic and recurrent uveitis involving the choroid and retina are usually treated by corticosteroids and immunosuppressants with many serious potential side effects, including myelosuppression, tumor formation, and liver and kidney damage. By targeting different parts of the immune system, MSCs are supposed to inhibit the autoimmune response, allograft rejection, and graft-versus-host disease . TheoretMSCs are currently being tested in preclinical studies for the treatment of experimental autoimmune uveitis (EAU) owing to their immunosuppressive properties . Yang etMSCs are able to regulate the toll-like receptor 4 signaling pathway and pro-inflammatory factors such as tumor necrosis factor-\u03b1, interleukin (IL)-1\u03b2, and reactive oxygen species to reduce retinal cell apoptosis, increase retinal inner layer thickness, and reduce neuroinflammation . FurtherIn the animal model of retinal degeneration, rats suffering from retinitis pigmentosa were given subretinal injection or intravenous injection with MSCs, which successfully inhibits retinal degeneration progress and protects the function of the retina, but transplanted MSCs did not migrate or integrate into the retina ,65,66. AMany clinical studies have shown that MSC-based surgery holds great potential in retinal diseases owing to its anti-inflammatory and neuroprotective effects. \u00d6zmert et al. observed that the subtenon Wharton\u2019s jelly-derived MSCs transplant can be beneficial to patients with retinitis pigmentosa . InjectiPathological process of inflammatory reaction and tissue damage are involved in the above diseases such as uveitis, laser-induced retinal damage, ischemia injury, and diabetic retinopathy. MSCs play a role in anti-inflammation and promoting restoration of the retinal tissue damage. MSCs have the capacity to home into damaged tissue with inflammation, probably in response to chemokines, adhesion molecules, and matrix metalloproteinases, following intravenous injection ,72. HoweROS levels increase dramatically under environmental stress, which causes serious damage to cellular structures in retinal disease. As hMSCs were able to scavenge free radicals, promote endogenous antioxidant defenses, alter mitochondrial bioenergetics, transfer mitochondria to impaired cells, and effectively regulate oxidative stress, they have been studied as a treatment for oxidative injury. Ohkouchi et al. showed that MSCs elevated the survival rate of A549 cells through Stanniocalcin 1 upregulation . InjectiAbnormal angiogenesis is one of the main reasons for many ocular diseases, including DR, retinopathy of prematurity, and AMD. As MSCs can secrete angiogenesis-related factors and proteins, such as VEGF, fibroblast growth factor, HGF, TGF-\u03b21, and insulin-like growth factor 1, they can promote angiogenesis and repair retinal ischemic injury ,82,83.Studies have shown that adult stem cells , AMSCs , iPSCs , and autIn addition, MSCs also have anti-angiogenic effects in proliferative retinopathy. High levels of TGF-\u03b21 secreted from human placental amniotic membrane derived MSCs demonstrated rescue potential on suppressing retinal neovascularization in a mouse model of oxygen-induced retinopathy .Mitochondria create chemical energy for biochemical activities. A majority of mitochondrial proteins are encoded from nucleus DNA, maintaining mitochondrial functions, while mitochondria also contains its own DNA, known as mitochondrial DNA, which encodes 13 proteins . DysfuncA fascinating and creative way that MSCs rescue impaired neural cells has provoked profound thought. Through different approaches, MSCs can deliver their own mitochondria to injured cells, in order to promote their repair and regeneration. This phenomenon was first described by Spees et al., who cocultured mitochondrial gene-depleted cells with MSCs. The mutant cells with enhanced mitochondria showed expression of mitochondrial proteins, and significantly increased ATP production and decreased lactate levels, a byproduct of anerobic respiration . PreviouThe adult visual pathway can possibly rebuild new synaptic interactions and guide new axons in certain circumstances, which provide an opportunity for cellular therapy. However, being similar to the central nervous system, the retina is not easily influenced by the outside factors, for example, stem cells, due to the retinal barriers. The blood\u2013retinal barrier is part of the blood\u2013ocular barrier that consists of retinal vascular endothelium and RPE . The phyWhether using local or systemic administration, MSCs were not seen to have migrated or integrated into the retina on an ideal scale while the retinal barriers were relatively integrated ,67,68. TMSCs also have limitations for human retinal disease treatment due to the vulnerability of their expression. Some new technologies, such as gene therapy, retinal organoids in vitro, and bio-printing technology, have demonstrated prospective therapeutic capabilities to repair damaged retinal cells.Erythropoietin-engineered human MSCs enhance differentiation into retinal photoreceptors in retinal degenerative diseases . HiPSC-dExosomes are the tiniest extracellular vesicles with bi-lipid membranes shuttling active cargoes  involved in the complex intercellular communication system . Being rRecent discoveries noticed that MSC-derived exosomes, 50\u2013150 nm microvesicles, could inherit the multiple functions from MSCs and might be the key mediators of MSC paracrine activity ,109. TheAlthough preclinical studies of exosomes derived from stem cells have been applied to the treatment for a variety of diseases, very few results came out in ophthalmology. We first observed the therapeutic effect of exosomes derived from MSCs in retinal dysfunction . MSC-derWhile extant obstacles limit the clinical applications of simple stem cell treatment for retinal diseases, such as retinal barriers, the alternative application of MSC-derived exosomes remains more promising, primarily due to their inherited abilities, low immunogenicity, long half-life in circulation, and other cell-free advantages. Additionally, MSCs can produce a higher number of exosomes compared to other cells. However, whether MSC-derived exosomes preserve the same effective properties compared to the cells themselves remains controversial, as MSCs may act by releasing other active soluble factors and the biological features of exosomes may vary with different extracellular environments. Therefore, further studies are required to understand the full dimension of this complex intercellular communication system and how it can be optimized to take full advantage of its therapeutic effects.Despite many clinical advances in mesenchymal stem cell therapy in the past decades , there aCell survival is the key to successful MSC-based therapy. After implantation, ischemia and the inflammatory microenvironment are likely to cause apoptosis of MSCs in vivo. In addition, both enzymatic treatment to separate MSCs from culture plates prior to transplantation and injection procedures are able to destroy cells and reduce their survival rate.Moreover, the application of autologous MSCs is limited by time-consuming preparation and loss of cell viability. To overcome these limitations, allogeneic MSC transplantation has been applied. As it is easy to cause immune rejection, studies have shown that allogeneic BMSCs are easily eliminated . In addiRetinal dysfunctions, especially the ones that lead to retinal degeneration, are the leading causes of vision loss with very few effective treatments. The pleiotropic properties and therapeutic potentials of MSCs, including broad distribution, immune evasion, capacity for self-renewal and multilineage differentiation, and secretion of an array of anti-inflammatory, immunoregulatory, or neuroprotective mediators, bring new hope for the untreatable retinopathies. In general, the use of MSCs has been explored as a cellular therapy for retinal degenerative conditions through replacement of retinal cells and/or the release of protective factors into damaged tissue. However, in direct transplantation without in vitro induction, MSCs showed limited ability and extremely low efficiency for integration or differentiation toward retinal cells, as the main obstacle seems to be the integrity of retinal barriers. Even the ones that exhibited photoreceptor phenotypes lack convincing evidence of synaptic fusion into the damaged neuropil. Meanwhile, increasing lines of evidence point to the paracrine pattern as the key role of MSC-based strategies, displaying beneficial effects on retinal tissue remodeling and functional preservation via secreting cytokines, cell\u2013cell interactions, and releasing extracellular vesicles. As an emerging research field, MSC-derived extracellular vesicles, especially exosomes, hold a great potential for cell-free therapies in retinal dysfunctions that are safer and easier to manipulate than the idea of cell replacement. However, further exploration and elucidation of the biologic features, therapeutic mechanisms, long-term safety and curative effects, and limitations and complications of MSC-based strategies are needed before the full potential can be unearthed."}
+{"text": "Piper nigrum L.) is rich in bioactive compounds that make it an imperative constituent in traditional medicines. Although the unripe fruits have long been used in different Ayurvedic formulations, the mechanism of gene regulation resulting in the production of the bioactive compounds in black pepper is not much investigated. Exploring the regulatory factors favouring the production of bioactive compounds ultimately help to accumulate the medicinally important content of black pepper. The\u00a0factors that enhance the biosynthesis of these compounds could be potential candidates for metabolic engineering strategies to obtain a high level production of significant biomolecules.Black pepper (P. nigrum from which commercially available black pepper is prepared. In this study, the key gene regulations involved in the synthesis of bioactive principles in black pepper was brought out with a focus on the highly expressed phenylpropanoid pathway genes. Quantitative real-time PCR analysis of critical genes and transcription factors in the different developmental stages from bud to the mature green berries provides important information useful for choosing the developmental stage that would be best for the production of a particular bioactive compound. Comparison with a previous study has also been included to understand the relative position of the results obtained from this study.Being a non-model plant, de novo sequencing technology was used to unravel comprehensive information about the genes and transcription factors that are expressed in mature unripe green berries of The current study uncovered significant information regarding the gene expression and regulation responsible for the bioactivity of black pepper. The key transcription factors and enzymes analyzed in this study are promising targets for achieving a high level production of significant biomolecules through metabolic engineering.The online version contains supplementary material available at 10.1186/s12870-022-03878-1. Piper nigrum L.) is called 'Black Gold' or the \u2018King of Spices\u2019 and is a commonly used spice all over the world ATCTCGTATGCCGTCTTCTGCTTG-3\u2019.Here, [INDEX] is the unique sequence to identify sample-specific sequencing data.After ligation with the adapter, cDNA was purified with HighPrep beads. Purified cDNA underwent 12 cycles of Indexing-PCR  for enriching the adapter-ligated fragments. The PCR product is the sequencing library, which was purified with HighPrep beads. Quality control checking was done. The quantification was done using Qubit fluorometer . The fragment size distribution was analyzed on Agilent 2200 Tapestation.P. nigrum transcriptome. Long contig sequences were obtained by continuously joining overlapping reads of particular quality and length. The maximum and minimum length, N50 and average length were determined. CD-HIT open source program [Trinitysoftware package  was used program , 18 was http://www.genome.jp/tools/kaas/), MISA (https://webblast.ipk-gatersleben.de/misa/), GO (http://geneontology.org/) and Uniprot (https://www.uniprot.org/). NCBI-BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) [Arabidopsis thaliana (ath), Theobroma cacao (tcc), Tarenaya hassleriana (thj), Eucalyptus grandis (egr), Glycine max (gmx), Fragaria vesca (fve), Vitis vinifera (vvi), Solanum lycopersicum (sly), Populus trichocarpa (pop) and Oryza sativa japonica (osa). Simple sequence repeats were identified from assembled transcripts using MISA.Functional annotation was done using several databases including KAAS (ast.cgi)  was usedast.cgi) . Cut-offast.cgi)  against P.\u00a0nigrum berries. For this, the total RNA isolated from each developmental stage was used for cDNA synthesis. Forward and reverse primers were designed using IDT (www.idtdna.com) for the selected genes based on the contig sequences obtained from the transcriptomic data .Comparative quantitative real-time Polymerase Chain Reaction (qRT-PCR) experiment was done for selected genes in the different developing stages  and stage 2 (YF2), and unripe berries) . cDNA was synthesized from 1\u00a0\u03bcg of RNA using a high capacity cDNA reverse transcription kit . Each reaction mixture containing 2.5\u00a0\u03bcl SYBR Green Master mix (Applied Biosystems), 1\u00a0\u03bcl cDNA template (50\u00a0ng), and 0.25 ul each of forward and reverse primer (10 \u03c1M) was made up to a total volume of 5\u00a0\u03bcl with nuclease free water. Real-time experiments were performed in Biosystems 7900 HT Fast Real-Time system with Power SYBR Green qPCR Master Mix  in384-well optical reaction plates . The PCR reactions were performed in triplicate under following conditions: 10\u00a0min at 95\u00a0\u00b0C, and 40 cycles of 15\u00a0s at 95\u00a0\u00b0C and 45\u00a0s at 60\u00a0\u00b0C. The cDNA template was replaced with nuclease free water in the case of negative controls. 5.8S ribosomal RNA was used as the reference gene. The analysis was done using the formula 2rror bar . The exprror bar .http://www.ncbi.nlm.nih.gov/Traces/sra/; accession number SRS856941) in which they used a mixture of RNA from different stages of fruit (1\u201310\u00a0months post anthesis).We compared the RPKM of selected genes from our data with that of  retrieveSequencing was performed in Illumina HiSeq sequencer with the total RNA extracted from unripe berry sample  terms were observed Fig.\u00a0.Fig. 3FrUsing Viridiplantae database, 47,085 transcripts with high similarity  were observed while using BLAST i.e., the possible hits of similar score for these transcripts that could be obtained by mere chance ranged from 1e-60 to 1e-5. This low E-value indicates high significance and were considered for further analysis. The E-value of the rest of the transcripts ranged from 1e-5 to 0  in which they used a mixture of RNA from different stages of fruit (1\u201310\u00a0months post anthesis). None of the selected genes showed significant variation in RPKM (more than 2 fold) between the sample of the current study and that of the previous one  is implicated in the accumulation of phenylpropanoids in uberosum . WRKY truberosum . The expuberosum , 70. LIMuberosum . The comuberosum . ContradP. nigrum cv. Reyin 1 whereas we used the mature unripe berries alone from which black pepper is produced commercially. Though there was a general variation in the intensity, a similar trend of expression of genes and transcription factors was observed. Altered source\u2013sink communications are assumed to have resulted in the observed trend of gene expression [There are reports of previous transcriptome attempts in black pepper with variation in sample and other conditions , 33, 34.pression .de novo transcriptome profile of unripe P.\u00a0nigrum berries brought out the prominent secondary metabolic pathways, of which the highest expressed genes belonged to the phenylpropanoid pathway. The differential expression of these genes and significant transcription factors regulating the pathway were analyzed in the various developmental stages from bud to the development of the green berries from which black pepper is prepared for commercial applications. The expression of the selected transcripts was compared with previously published transcriptome data of a pooled sample of P.\u00a0nigrum fruits of different developmental stages. The significant information obtained from the study can be beneficial for the commercial production of natural pharmaceuticals as well as for metabolic engineering studies that aim for black pepper berries enriched in a particular secondary metabolite group.The Additional file 1.Additional file 2.Additional file 3.Additional file 4.Additional file 5.Additional file 6.Additional file 7.Additional file 8.Additional file 9."}
+{"text": "HIV self-testing is preferred by many Chinese people for its convenience and confidentiality. However, most studies on HIV self-testing (HIVST) uptake in China overfocused on men who have sex with men and overrelied on obtrusive methods such as surveys and interviews to collect data.We aimed to explore Chinese HIVST kit users\u2019 authentic experience via their feedback comments posted on e-commerce platforms using an unobtrusive approach.In total, 21,018 feedback comments about buying and using HIVST kits posted on Chinese e-commerce platforms  were collected. An inductive thematic analysis based on a random sample of 367 comments yielded several thematic features. These thematic features were developed into coding categories for a quantitative content analysis of another random sample of 1857 comments.Four themes were identified in the first study, including the expression of positive and negative emotions after and before getting the test, respectively, calling for living a clean and moral life in the future, comments on the sellers and HIVST kits, and the reasons for buying HIVST kits. The results from the second study suggested that there were significant associations between different platforms and several thematic features. Nearly 50% of the comments were related to the product itself and the disclosures of HIV-negative test results. More than 25% of the comments showed users\u2019 feelings of gratefulness after receiving negative test results such as \u201cthank heavens for sparing my life.\u201dThe results suggested that Chinese users relied on HIVST kits to reduce and prevent HIV infection, while they also considered HIV infection a punishment related to moral violation such as being sexually promiscuous. The traditional Chinese health belief that health is influenced by one\u2019s morality still persists among some Chinese users. Many users also lacked appropriate knowledge about HIV transmission and self-testing kits. In recent years, there has been an upsurge of HIV infection rates in China, particularly among young people . Early dMany pilot studies showed that HIVST received high acceptability for populations at risk of HIV infection in multiple countries such as Kenya , Canada To lower the increasing HIV infection rate in China, the State Council of China issued Thirteenth Five-Year Plan (2017-22) for HIV and AIDS prevention and control in which the government encourages the expansion of HIVST by exploring  the straThere are robust HIVST studies examining high-risk populations such as men who have sex with men (MSM) in China ,17-19. HOverall, current HIVST studies in China have been overemphasizing MSM while ignoring a more general population who use HIVST. However, the largest group that uses HIVST kits in China might not be MSM. A recent study examined the profiles of those who purchased HIVST kits on the web and found \u226557% of the web-based buyers were heterosexual . In addiThis research included 2 studies examining HIVST kit feedback comments on e-commerce platforms. The first study was an inductive thematic analysis to identify patterns from qualitative data to generate unanticipated insights . Owing tFeedback comments of HIVST kits from Pinduoduo and Tmall were selected for analyzing the HIVST users\u2019 experiences. Tmall and Pinduoduo were chosen because both platforms have a huge number of users in China, but they have a different target market. Tmall is an e-commerce platform for top-quality branded products that guarantees all goods on the platform are authentic  and had Data were collected in early December 2021. We searched the keyword \u201chiv test\u201d in the platforms and identified the top 10\u2013selling HIVST kits at Tmall mobile app and top 15\u2013selling HIVST kits on the Pinduoduo mobile app based on their sales volume within the past month of data collection. The rationale to choose the top-selling products is that, usually, best-selling products generate most feedback comments. There were relatively fewer comments for each HIVST kit posted on Pinduoduo than Tmall, so more HIVST kits sold on Pinduoduo were selected to obtain enough comments. The HIVST kits sold on Tmall or Pinduoduo are either blood based or saliva based. All the HIVST kits discussed in this paper have passed China\u2019s National Medical Device Registration Approval.Feedback comments are defined as information including texts, signs, and emoticons posted publicly on Tmall and Pinduoduo\u2019s comment section by HIVST kit users in this study. There were several comments such as \u201cThis user thinks this product is really good and gives a five-star rating\u201d or \u201cThis user does not leave any comment,\u201d which were automatically generated by the platforms while data were retrieved. These comments were excluded from the data corpus because they yielded little implication for our study and were not made by real commenters. Duplicated comments for a particular kit from the same commenter posted during the same period were also removed from this analysis because they might be fake reviews. The final data corpus pool included 21,018 comments .All the comments retrieved from Pinduoduo and Tmall were posted publicly on the comment sections and were accessible to everyone visiting these 2 platforms. All personally identifiable information (such as commenters\u2019 usernames in these platforms and names of locations mentioned in comments) included in the comments was removed from the analysis to protect commenters\u2019 anonymity and privacy.A sample of 192 comments were randomly selected from the overall data corpus for thematic analysis. We followed the procedure of thematic analysis as suggested by Braun and Clarke  to generFour major themes were identified from 367 comments posted on Pinduoduo (n=81) and Tmall (n=286), including the expression of positive and negative emotions, calling for living a clean and moral life, comments on the sellers and HIVST kits, and the reasons for buying HIVST kits. These themes are not mutually exclusive, so a single comment may include all 4 themes.Under this theme, HIVST kit users expressed their positive emotions specifically focusing on gratefulness and relief after getting their negative test results, their negative emotions before getting the test, and their hope for others to get negative test results and stay healthy. In most cases, commenters tended to express their gratefulness to the heavens such as \u201cThank heavens and I will be definitely more cautious about my sexual activities,\u201d while several commenters express their gratefulness to many targets. For example, 1 user commented as follows:At last, I will thank that scumbag guy for not killing me. Thank heavens for sparing my life. Thank heavens for not killing me. Thank [the brand of HIVST kits].In addition, comments expressing positive emotions such as relief were observed frequently:Thank heavens for not killing me. Now I finally feel relief.Some commenters expressed negative emotions before the test, such as fear and guilt, and positive emotions (gratefulness and relief) after the test, in a single comment, for example:I will never forget the feeling at the day while waiting for shipping [of the HIVST kits] that I would rather be dead. Once recalling this, I was trembling all the time. Luckily, the result turned to be good. Take 20 minutes [to conduct HIVST] to get peace for the eternal life. Thank heavens. If you are afraid of [getting HIV]. Don\u2019t panic. Don\u2019s trust information on the Internet which is exaggerated. Please get [HIV] test.I am looking at the reagent paper and thinking about my life for a while. The result is negative and I feel I get a rebirth inside. Recalling that experience in a few months ago, it might be the most regretful thing I did in my whole life. I would never think that I was associated with \u201cAIDS\u201d a few months ago. AIDS had deeply influenced my life in the past few months, I was bothered by different kinds of fear and worries inside. [AIDS] just surrounded me entirely and almost suffocated me.I have been scared for a few months. It\u2019s like I had been living in a hell in the past few months. After the test today, I finally feel relief. I will live a clean and moral life in the future and not feel worried any longer.In front of [sexual] temptation, you just could not make any reasonable decision. Previously I committed such a sin and I was so frightened. I am too young and have no children. If I really got shot [to get AIDS], I deserved it. Luckily, thanks for heaven\u2019s mercy to spare my life. Since this event, I feel so grateful and I will be a good person.In addition to gratefulness and relief, HIVST kit users also expressed their hope for both themselves and other HIVST users to receive negative results. Two users commented as follows:Thank heavens for not killing me. I hope the test result is accurate. I hope everyone who purchases this [HIVST kits] gets a negative test result!!!If you found yourself getting infected earlier, you can get yourself treated earlier. I hope all of you just have a false alarm! Hope all of you [have] negative [test results]!Jieshenzihao in original Chinese comments) in the future with greater self-control for users themselves and other HIVST kit users. Commenters made promises to resist promiscuous lifestyles and made a determination to live a self-disciplined life. The following are a few examples:This theme features calling for living a clean and moral life  with everyone!AIDS-phobia is so scary. I was so scared while waiting for the test result. I was too young to know how harmful it is when I had these risky acts. I could not fall into sleep these days [because I was] imagining how I should deal with my family members if I really got AIDS. Thank heavens for not killing me. If I was going out for some promiscuous acts in the future, may thunders strike me down.Other HIVST kit users shared their experiences as a warning to raise HIV awareness and to call for others to resist promiscuous lifestyles:I am warning everyone here. Never live promiscuously. You must live a clean and moral life and be careful about your health. I hope everyone gets a bright future, and I will start to live my new life.After this experience, I have learned the importance of living a clean and moral life and will no longer fool around my own life...I hope everyone can live a clean and moral life and don\u2019t fool around with your own life so easily.Under this theme, HIVST kit users provided either positive or negative evaluations for web-based sellers and HIVST kits. As for their web-based shopping experiences with sellers on these e-commerce platforms, most comments were concerned with privacy and convenience about purchasing HIVST kits on the web, which is consistent with the previous findings on the advantages of using HIVST for the sake of privacy and convenience ,22,37. FI feel that a heavy stone was finally removed from my heart. This reagent paper was amazing to protect me from awkwardness for visiting a clinic.The price is really reasonable and much cheaper than visiting a clinic. The [shipping] speed is really fast. I bought it today and it will arrive tomorrow. Shipping service is really good.Cheap and useful. Accuracy is as high as testing at a hospital.An interesting observation from these comments is that few HIVST kit users complained about the cost of buying kits. Unlike previous findings based on MSM samples in which high cost is one of the top barriers for using HIVST ,22, someThere are comments addressing how patient the customer service representatives were. Some buyers posted positive comments about sellers for fast delivery and discreet shipping to protect their privacy. Because of HIV stigma in China , those wVery nice. The customer service representative is very patient. I finally feel relief after this test. One line [appeared on the reagent paper as a negative result] and there is not any [other] problem.Shipping speed is fast. Operation is easy and [test result is] accurate. The package was delivered in discreet shipping.This time of torture is finally over. I will definitely love my life after this. [AIDS] is such terrible. The reagent paper is good. The package [of HIVST kits] was handled in discreet shipping. This seller is considerate and the speed of delivery is fast.As for the user experience of administering HIVST, there are several positive comments on the accuracy of the test and the speed of getting the result. The following are several examples:Reagent paper has good quality. The test result is very accurate. Administering this test at home is so convenient.Very good to check health status. I have been worried for several days and finally I feel relief to work. It\u2019s so frightening. Reagent paper is nice and operation is convenient. I conducted [this test] really quick.The quality is guaranteed. Safe and effective. The package from this seller is complete. The most important is to get the result fast and conveniently. [I] don\u2019t have to worry about this every day!Previously I used HIVST kits of other brands, but I still felt unsure [about test results]. Then I found this [brand name of HIVST kits] and knew many hospitals and disease control centers all used this [brand]. I also knew their HIVST kits got certified from World Health Organization PQ, which makes me trust the accuracy of test results. Thank [this brand] for producing such excellent kits.Accurate, formal, safe, and convenient, but the lancet to collect blood samples is too short to use. It took several times to push blood out. Maybe it\u2019s my own problem.Interestingly, few commenters mentioned visiting a clinical center for conventional voluntary counseling and testing (VCT) to ensure their test results are accurate after obtaining negative results from HIVST. There are also few comments questioning the accuracy of the test result, which is not consistent with the previous findings that MSM in China were worried about the accuracy of HIVST ,22. One There are also a few negative comments on sellers and user experiences. Some comments complained about the shipping speed, customer service, and the quality of tools inside HIVST kits. For example, one user complained about the shipping speed and finally gave up on waiting for the HIVST kits ordered on the web:Shipping is a little bit slow. I ordered [HIVST kits] on Monday night\u2026and received it on Thursday afternoon. I could not wait for that and went to a clinic (to test), which cost me 40 Chinese Yuan and 2 hours to get the result.Another buyer posted a negative comment on a customer service representative who denied the problem caused by the quality of kits:Thumb down (for this product). [Reagent paper] did not absorb the collected blood sample. [The customer service representative] said this was due to my operation error. I bought HIVST kits from many [other] sellers and I bought kits from this seller several times. It is the first time to have this problem\u2026Please do not buy [HIVST kits] from this seller.Several buyers commented on the lancet used to collect blood samples, such as biosafety of using such a lancet and how hurtful it would be. Zhang et al  reportedOne of the lancets looks like broken. Don\u2019t know whether or not it was used by someone else. I was so scared.Commenters also provided their reasons about why they decided to purchase and use HIVST kits. The reasons included risky sexual activities, nonsexual transmission, pure AIDS phobia, and preventive purposes. Most of the reasons are related to commenters\u2019 own or their sexual (ex-) partners\u2019 risky sexual activities, which made them purchase HIVST kits, for example:Thanks! Heavens gave me a chance for a new life. I met a lady\u2026and after that I got neck and spine pain with slight fever latterly. I saw AIDS prevention campaigns in my community and I found I got similar symptoms. I felt so scared. My brothers and sisters, please live a clean and moral life! I would not behave like that after this lesson. At last, thanks for this HIVST kit to bring me a new life.It is necessary to test. Nowadays young people do not have only one partner. This disease has a long incubation period so it is necessary to test.Don\u2019t date young adults anymore in the future. It\u2019s scary. Thank heavens for sparing my life. I will behave myself and I must use a condom. Safety comes first, my sisters.I was once got drunk and had sex with someone. I was so scared but don\u2019t have the courage [to get test]. Thank heavens for sparing my life. I will not drink alone in the future. I will live a clean and moral life.There are also reasons focusing on using HIVST because of fear of nonsexual HIV transmission via blood and oral fluids; for example:I was having a meal with a friend and was kissed suddenly [by this friend]. I felt so scared and decided to get a [HIV] test. I knew he went out for prostitution before. I went to a disease control center for counseling, and the doctor there was stubborn and did not give me any clear suggestions. Then I went to a hospital that specializes in infectious diseases for counseling. The nurse there told me HIV could not be transmitted via kissing, even tongue kissing, and suggested that I seek psychological therapies if I was so fearful.While I was giving an injection to someone who might be a drug addict, [this person\u2019s blood sample or oral fluids (not specified in the comment)] dropped on my fingernail. On the second day, it was found out that this [patient] got AIDS. Although there was no open wound on my skin, I was scared to death. I am a pregnant woman and I am so angry. I hope all AIDS patients should not hide their status while visiting a clinic. This is really dangerous for health practitioners. It\u2019s harmful for both others and patients themselves.Negative. Thank heavens for sparing me. I went to a small beauty salon to remove pimples a few years ago. I was so frightened since the needle used in this beauty salon might not get disinfected. Listen to me, if you want to do some cosmetic surgeries like ear piercing or eyebrow tattoo, you definitely should go to a formal beauty salon. Otherwise, you might have to worry about getting AIDS.Clearly, these commenters know of nonsexual HIV transmission on some level, and they may have legitimate concerns to worry about getting HIV infection via medical tools or blood. However, their self-perceived likelihood of getting infected might be overestimated because of their misunderstanding of HIV transmission. They knew the ways of transmission but did not have a clear picture of the likelihood of getting HIV infection for each type of transmission and other HIV information such as how long HIV will be infectious outside the human body, which finally caused their panic and phobia and the buying of HIVST kits. The contagiousness of HIV was exaggerated, which is consistent with the previous study .There are also a few cases in which HIVST kit users tested themselves because of AIDS phobia elicited by the popular science of HIV or they chose to test for preventive purposes, for instance:I was told getting tooth extraction can even cause AIDS from popular science [articles or videos (not specified in the comment)]. I will get a premarital examination really soon. I was scared so I bought HIVST kits to test myself. I was so nervous before testing. My head was buzzing. I wondered what I should do if I really got [infected by HIV]? I put so much effort into dating a good boyfriend right now and I was afraid that [he] would leave me [because of AIDS]. Later, I realized that I just thought too much\u2026 Thank heavens for sparing my life. Although I did not fool around, I am neither homosexual nor a drug addict. However, it is still risky [to get HIV] from wounds if you do not use any bandage. As someone on the Internet said: AIDS-phobia makes me live a discreet life. Same as me.Interestingly, this commenter did not engage in any risky behaviors and did not contact anyone who might carry HIV. She chose to take HIVST only because she was frightened by the popular science of HIV. She wanted to ensure her HIV status to prevent any possible embarrassment and loss before her marriage. This commenter even suggested that AIDS phobia helped her live a discreet life.The aforementioned examples also demonstrated the stereotypes about people who live with HIV or AIDS, such as portraying people who live with HIV or AIDS as homosexual or drug addict. It raises a question about the effectiveness and the side effect of the popular science of HIV and people who live with HIV or AIDS, which may lead to further HIV-related panic, fear, and stigma.In addition to these 4 main themes, there are some minor themes that did not appear frequently from these comments. For instance, there are a few comments concerning the misinformation and rumors about HIV or AIDS that they searched on the internet. One commenter said: \u201cI will suggest to everyone that they should not check [a Chinese-based search engine] because [information searched on this site] frightened me a lot.\u201d Another commenter was so scared by the misinformation on the internet about HIV and was not able to get a test immediately:I found AIDS symptoms on the Internet matched with mine perfectly. I had been panicking for almost a whole year. I was too scared to get a test. I could only get comfort by telling myself that the chance [of getting HIV infection] is so slight via searching information on the Internet. However, it was said that the more you know, the more you fear. If you search [HIV] on [a Chinese-based search engine], you find yourself getting an incurable disease.There is also a comment concerning the effect of using HIVST on mental health: \u201cJust a suggestion, make sure you are psychologically prepared before purchasing [HIVST kits].\u201d This is consistent with one of the drawbacks of taking HIVST that HIVST kit users receive little professional and appropriate psychological support and counseling in a nonclinical setting ,13,22.As a result, 4 main themes were identified from HIVST kit users\u2019 comments. The first theme is concerned with the expression of positive and negative emotions. Many commenters under this theme thanked the heavens for sparing their life and showed their relief after receiving negative test results. Other commenters under this theme expressed hope for everyone else to receive negative test results and stay healthy. In addition, this theme is also characterized by commenters\u2019 panic and fear of HIV infection before receiving negative test results and commenters felt extremely uncertain and unsafe about their future life interrupted by potential HIV infection. The second theme is concerned with living a clean and moral life in the future. Commenters under this theme not only expressed their determination to live a careful life but also offered sincere suggestions for others not to be sexually promiscuous around in the future. The third theme focuses on the evaluative comments on either sellers for their services, shipping speed, discreet shipping, conveniences, and other advantages over visiting clinics or HIVST kits for their quality. The fourth theme focuses on commenters\u2019 reasons about why they chose to purchase HIVST kits. Most of the reasons reflected commenters\u2019 fear of HIV infection by sexual transmission. Other reasons focused on nonsexual HIV transmission via blood or unclean medical tools. The implications for these findings are explained in the Discussion section after the results of the content analysis.The first thematic analysis study explored some features of HIVST kit users\u2019 comments posted on e-commerce platforms. However, owing to the inductive nature of the thematic analysis, this study only provides a basic picture of users\u2019 concerns with a limited data sample. More systematic observations are needed to provide quantitative data about the frequency of each thematic feature based on a larger sample of comments. In addition, the sample yielding these 4 main themes was based on the mixed comments from 2 different e-commerce platforms, so the possible differences in these themes between platforms were not observed in this study. We will address these aforementioned questions in the second study of the quantitative content analysis based on 1857 comments on HIVST kits.The second study provides quantitative data about frequencies of different thematic features of comments based on a larger sample of comments. In addition, we aimed to explore any potential differences regarding HIVST kit users\u2019 comments between different e-commerce platforms. We conducted a quantitative content analysis of 1857 HIVST kits users\u2019 comments posted on Tmall (n=975) and Pinduoduo (n=882). These 1857 comments were randomly selected from the entire data pool including 21,018 comments retrieved in December 2021. Regarding the random selection process for the content analysis, a Microsoft Excel add-in named Fangfanggezi  that enaThe codebook was developed based on the main themes identified in the first study. Different coding categories were elaborated upon based on thematic features under each theme including gratefulness, positive emotions  other than gratefulness, negative emotions, hope for negative test results and good health, calling for living a clean and moral life in the future, comments for sellers, comments for HIVST kits, and the reasons for buying HIVST kits. In addition to these coding categories developed from thematic features mentioned in the first study, there is one newly added coding category, users\u2019 disclosures of their self-testing results, which emerged inductively from coders\u2019 training processes. A single coding unit could be coded for multiple thematic features mentioned in the codebook. Details of the coding categories are presented in Three coders were trained using the codebook to code 50 comments to test the codebook\u2019s clarity and ensure their understanding of it. Then, each of the 3 coders were provided the same set of 50 new comments to code independently. After they completed coding, intercoder reliabilities between them were calculated, and the coders were brought together to discuss and reconcile their discrepancies in terms of coding. Their feedback was also used to modify the coding categories in the codebook. This procedure of independent coding was repeated thrice. One new coding category of disclosing HIVST results mentioned earlier was included in the original codebook during these 3 trials. The training finally yielded accepted intercoder reliability . Then, each of the coders was provided a different set of about 600 new comments to code independently, yielding 1857 comments in total for the quantitative content analysis.The frequencies of different thematic features in total and in each platform are displayed in 21=30.9, P<.001). The association between comments for sellers and platforms was significant . The association between comments for HIVST kits and platforms was also significant . The relationship between each one of the thematic features and e-commerce platforms were examined by using Chi-square tests to explore possible platform differences. Some significant results were observed. The association between the thematic feature of gratefulness and platforms was significant ; negative emotions ; comments for seller ; comments for HIVST kits . In addition, the theme of negative emotions was associated with positive emotions other than gratefulness ; the theme of negative emotions was also associated with comments for HIVST kits .Chi-square tests were also conducted to examine the relationships between different types of thematic features, and some significant relationships were observed. Gratefulness was associated with multiple other themes: positive emotions other than gratefulness , evaluations for sellers and kits, and reasons for purchase. The quantitative content analysis provided the frequencies of different thematic features, explored platform differences of these features, and examined the relationships between thematic features. The implications of both studies are discussed in subsequent sections.On the basis of the results of the first thematic analysis, many commenters expressed their gratefulness and relief for receiving negative results, but few positive-result comments were observed. This theme features HIVST kits users thanking the heavens for sparing their life and giving them rebirth. For these commenters, getting an HIV infection depends on the heavens instead of themselves. This result also implies HIV fatalism, which is the belief that HIV acquisition and mortality is beyond one\u2019s own control . Such a Interestingly, few comments addressed the importance of using condoms before engaging in risky sexual activities, but many comments emphasized the importance of living a clean and moral life in the future (theme 2). This calling for living a better life suggests HIVST kit users, although being influenced by HIV fatalism on some level, still believe that they can control their health in the future by living a moral life. This contradicting phenomenon might result from Chinese moral beliefs based on Confucianism and Buddhism  that misIn addition, few commenters mentioned seeking VCT to ensure their test results are correct, and there were few doubts about the accuracy of testing results. It is plausible that commenters felt no need to confirm since they had received negative results. However, there is also a risk of misoperation or misinterpretation of results, not to mention the poor quality of uncertified kits or kits damaged during shipping that may also result in accurate results. Although previous studies suggested that the consistency between HIVST results and antibody detection results was 90.5% , and mosThe results of content analysis showed that a large number of commenters expressed their gratefulness suggesting these commenters considered that whether they get HIV is not fully controllable but dependent on some spiritual factors outside of their control. Therefore, there is a need to understand the underlying reason why these people like to take their chances rather than refrain from risky activities. The examination of the reasons for buying HIVST kits revealed that the motivations behind HIVST self-testing are mostly pure AIDS phobia or related to risky sexual activities. Many commenters indicated that they suspected they might get infected after watching an AIDS-related video on a short video app. On the one hand, such media-induced AIDS phobia is an effective way to increase HIVST uptake in the general population. On the other hand, media-induced AIDS phobia could cause anxiety and worry, leading to long-term emotional distress. In addition, the percentage of pure phobia reasons is higher than risky sexual activities reasons. One possible explanation is that many Chinese buyers feel uncomfortable sharing their sexual experiences on the web because people do not usually talk about sex publicly in traditional Chinese culture . It is rNearly 50% of the comments were related to the product itself and 32% were related to the seller. It suggested that most HIVST kit users were concerned with the quality of the product and services that the seller provided. Interestingly, it was observed that many users commented that \u201cthe test result is very accurate,\u201d although they might not have the result from the hospital to compare with. Such a comment reflected the users\u2019 sincere hope for a negative result. There were more positive comments (n=562) than negative comments (n=31) for sellers. Similarly, for HIVST kits, more positive comments (n=856) were observed in the data than negative comments (n=50). It is plausible that Chinese users in our data sample felt reluctant to report a negative user experience or that some positive feedback reviews were fabricated. Some firms pay people to write a fake review using fake identities on the web , so the There were some significant differences between Tmall and Pinduoduo data. For example, the percentage of expressing gratefulness in comments is higher for Pinduoduo than Tmall. One explanation is that there are user differences. The user portrait of Pinduoduo is notably different from Tmall as a large portion of Pinduoduo users are older adults looking for affordable products in small cities , and mosTheoretically, this study explored Chinese cultural influences on attitudes toward HIV prevention and acquisition. Although comments such as \u201cthank heavens\u201d implied HIV fatalism that HIV acquisition is out of one\u2019s own control. Many commenters believed they should live a discreet and virtuous life in the future since the heavens had decided to spare their life. This implied a traditional Chinese health belief in which health is influenced by one\u2019s morality . This beMethodologically, relying on authentic user-generated information from a more general population and the multimethod approach, this paper heralded a new direction for HIVST research in China. Previous HIVST studies in China mostly focused on MSM populations and heavily relied on obtrusive methods such as surveys ,53 and iPractically, this study raised a few concerns with the uptake of HIVST in China. Both studies indicated that only a few Chinese users questioned the quality of HIVST kits based on the feedback comments of top-selling HIVST kits sold on popular e-commerce platforms. In addition, very few commenters mentioned visiting hospitals to confirm their test results. There are still possible errors due to misoperation and misinterpretation while taking HIVST. It is crucial to incorporate counseling and confirmation services into HIVST business to avoid potential false reassurances caused by HIVST. In addition, the results of this study suggested that HIVST kit users were overwhelmed by misinformation on the web about HIV transmission, infection, and symptoms, which led to some users\u2019 AIDS phobia. Future public health campaign designers should consider including brochures or flyers with credible HIV-related knowledge inside HIVST kit packages.This study has its limitations. First, the results might be subject to sampling bias as the buyers whose HIVST results were positive felt reluctant to share their results on the web. Only 0.32% (6/1857) of the commenters shared their positive results in the content analysis. In the same vein, the results of the reasons category may not represent the whole picture as only a small percent of commenters shared the reason why they took HIVST. Second, the comments analyzed in this study was based on the most recent comments on December 2021. It is hard to know whether an extended data collection period would yield different results. The comments analyzed in this study were based on the top-selling products from 2 e-commerce websites and may not be representative of the whole HIVST products sold in China. Such limitations may raise some concerns with the external validity of the results. Third, although platform differences were observed, there is a lack of users\u2019 demographic information for further analysis due to methodological limitations of analyzing users\u2019 feedback comments. Fourth, it is difficult to distinguish the comments based on users\u2019 authentic experiences from intentionally produced fake reviews posted by sellers or competitive sellers . Many du"}
+{"text": "Objective: To monitor severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA contamination in samples linked to imported cold-chain food and assess the situation from the implementation of a centralized supervision warehouse system in Guangzhou, Guangdong Province, China. Methods: Swabs of workers and frozen-food-related samples were collected between July 2020 and December 2023 in Guangzhou, Guangdong Province. SARS-CoV-2 RNA was extracted and analyzed by a real-time quantitative polymerase chain reaction using the commercially available SARS-CoV-2 nucleic acid test kit. The risk level and food source were monitored simultaneously. Results: A total of 283 positive cold-chain events were found in Guangzhou since the first cold-chain-related event of the coronavirus disease 2019 pandemic was identified in July 2020. Most positive samples were a low-to-medium risk, and the cycle threshold value was >30. No live virus was detected, and no staff came into direct contact with a live virus. In total, 87.63% of positive events were identified through sampling and testing at the centralized food warehouse. Conclusion: Cold-chain food has a relatively low risk of transmitting SARS-CoV-2. Centralized food storage can be used as an effective method to control this risk, and this measure can also be used for other food-related, contact-transmitted diseases. Some laboratory studies have shown that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains highly stable under refrigerated i.e., 4 \u00b0C) and freezing  conditions in fish, meat, poultry, and swine skin for 14\u201321 days; therefore, contaminated cold-stored food may present a systematic risk for the transmission of SARS-CoV-2 between countries and regions . Researc \u00b0C and fBefore the COVID-19 outbreak, China had implemented a food safety supervision and management system and formulated the National Food Safety Law in 2009 to manage food safety at the highest level in the form of national legislation. Under the framework of the National Food Safety Law, a series of national food safety standards and more detailed regulations include very specific management requirements for food production, processing, circulation, and consumption. Guangzhou port is one of the ports that receives the largest amount of imported food in China. In Guangzhou, the customs department is responsible for the supervision and management of the entry of imported cold-chain food. Before food is imported, food manufacturers must register with Guangzhou customs and provide the required certification materials. For example, imported meat products must be accompanied by official certificates, and imported pre-packaged food should carry a Chinese label that meets the requirements. Upon importation, customs officials conduct a sampling inspection and risk monitoring of imported food that includes the monitoring of 180 items of chemical contaminants and microorganisms.Customs issues an inspection and quarantine certificate for the imported goods after the conformity assessment, and the imported food can then be sold and used. After import, Guangzhou Province requires that manufacturers keep a record of food import and sales; food name, specifications, quantity, production date, production batch number or import batch number, and shelf life; names of overseas exporters and buyers; addresses and contact information; delivery date; and other information. Manufacturers are also required to save relevant vouchers.To prevent the transmission of COVID-19, Guangzhou customs began implementing SARS-CoV-2 nucleic acid testing for imported cold-chain food in March 2020 and comprehensive nucleic acid testing after June 2020. Since July 2020, Guangzhou Municipality has established a prevention and control system for imported cold-chain food. A centralized supervision warehouse system was established in January 2021. The following associated policies were subsequently implemented: (1) each batch of imported cold-chain goods must be individually sampled, with samples taken of the inner and outer packaging of each product; and (2) the centralized closed-loop management of employees and positive goods should be managed promptly.In this study, we analyzed the data from the monitoring program for imported cold-chain food that aimed to screen for SARS-CoV-2 RNA contamination in samples linked to imported cold-chain food and assessed the situation since the implementation of the centralized supervision warehouse system in Guangzhou, Guangdong Province, China. The study results will guide the improvement of cold-chain food management and decrease food-related infectious diseases that are transmitted through food contamination.The establishment of a centralized supervision warehouse system for cold-chain food in Guangzhou required that each batch of imported cold-chain food be sent to a centralized supervision warehouse for disinfection and sampling before the batch entered market circulation. The sampling quantity was in proportion to the total number of the same type and batch of goods.The sampling method was in accordance with the Notice on Issuing Technical Guidelines on the Classification and Disposal of Cold-Chain Food for COVID-19 Prevention and Control, which was published by The Joint Prevention and Control Mechanism of The State Council for COVID-19 . The samAccordingly, the outer packaging of each sample of imported cold-chain food was checked before storage, the inner packaging and product surface of imported cold-chain food in each batch was inspected in accordance with the method of proportional sampling, and the surface of samples were confirmed to be in a non-frozen state before sampling and disinfection. Each sampling tube contained five or 10 swabs, and each swab was applied to the surfaces of 5\u201310 boxes of goods. Each box of goods was tested on at least two sides. In addition, environmental sampling covered the chain from cold storage, transportation, and processing to the sale of imported cold-chain food. Environmental sampling also encompassed cold-chain logistics enterprises, farmers\u2019 markets, food production enterprises, shopping malls, supermarkets, and other locations. The surface of a table that had the most frequent contact with personnel or goods was selected for sampling. Samples were transported to the testing agency at a temperature of 4 \u00b0C within 12 h of sampling. Sample transportation and testing complied with relevant regulations on biosafety. If imported cold-chain food, the environment, or employees were found to be positive for COVID-19 nucleic acid, the food was discarded, in accordance with relevant regulations.Each batch of newly arrived cold-chain food was disinfected before being stored in the warehouse. Peracetic acid (0.1~0.2%), hydrogen peroxide (3%), 1000 mg/L chlorine-containing disinfectant, or 2000 mg/L quaternary ammonium salt disinfectant were used to fully wipe, spray, or soak goods for 30 min. All of the six sides of the items were disinfected, and surface ice was removed before disinfection. Disinfection supplies, such as disinfectants and disinfection instruments, were selected in accordance with national health standards. Selected disinfection products were registered on the national online information platform.After handling a batch of goods in the working area with the appropriate tools, 500\u20131000 mg/L of chlorine-containing disinfectant, peracetic acid (0.1\u20130.2%), or hydrogen peroxide (3%) was applied to the surface inside the transportation container, the floor of the working area, and equipment such as carts, shelves, forklifts, and related tools, utensils, and containers. The treatment time was 30 min; the disinfected surface was then washed with clean water to remove residual disinfectant. Cold-storage facilities, refrigerators, and freezers were sterilized weekly. Any visible contaminants on tables and relevant tools and appliances and in the work area environment or cold-storage refrigerators were cleaned before disinfection.When cold-chain goods entered market circulation, comprehensive preventive disinfection was performed in accordance with the principle of \u201cwhoever unpacks must disinfect\u201d and on the premise of \u201cnot affecting food safety and quality.\u201d Outer packaging was disinfected to ensure food safety.Disinfectants that were used in antifreeze conditions were verified using simulated field tests. The disinfection unit established ledgers to ensure the detailed recording\u2014including disinfection date, personnel, location, disinfection object, disinfectant name, concentration, action time, and the amount of disinfectant used\u2014of the disinfection of goods and the environment. Relevant data and records were retained for a specified period.The staff in the centralized supervision warehouse were managed by closed-loop management and underwent daily nucleic acid testing and health monitoring. If test results from the daily monitoring of staff were abnormal, the staff stopped working immediately and were isolated. Secondary self-protection was adopted in the work that contact with cold-chain-food directly, and supervisors who were responsible for staff protection provided timely reminders to staff. The staff performed relevant work in a specified area in accordance with a fixed process. Staff in the centralized supervision warehouse were responsible for six activities  and three special measures  related to imported cold-chain food. The storage and sale of cold-chain food with other food was prohibited.The testing agency tested samples from the centralized supervision warehouse, and the test results were reported to the Guangzhou Municipal Administration of Market Supervision and the Guangzhou Center for Disease Control and Prevention (GZCDC). The GZCDC then issued a weekly assessment report to the Guangzhou Health Commission and Guangzhou Municipal Administration of Market Supervision and proposed targeted recommendations. All positive specimens were sent to the Guangzhou GZCDC for reexamination within 2 h, and the reexamined samples were then sent to the Guangdong CDC within 24 h for virus isolation and gene sequencing. Concurrently, several associated departments were prepared to respond to emergencies by collecting information, screening and controlling personnel, conducting a patriotic health campaign, identifying prevention and control units, expanding sampling, tracing the flow of contaminated products, conducting terminal disinfection, disposing of nucleic acid positive products, and releasing information . If requTo meet the needs for rapid disposal of cold chain events the National Health Commission issued new technical guidelines for the classification and disposal of cold-chain food on 2 June 2022. The basis for risk assessment and the requirements of hierarchical disposal were moderately changed in the new guidelines. The main changes were the classification of risk levels; no changes were made to the treatment of each type of risk.Laboratory detection methods included nucleic acid detection using polymerase chain reaction (PCR), virus whole-genome sequencing, and isolation. The extracted sample RNAs were divided into separate packages and analyzed using real-time quantitative PCR with a commercially available SARS-CoV-2 nucleic acid test kit. The TaqMan probe-based kit , Guangzhou Da\u2019an Gene Co., Ltd. , and Jiangsu Shuoshi Biotechnology Co., Ltd. ) was designed to detect the ORF1ab and N genes of SARS-CoV-2 in one reaction using the procedure described by the Joint Prevention and Control Mechanism of the State Council of the People\u2019s Republic of China. All test kits for SARS-CoV-2 nucleic acid extraction sequencing, isolation, and real-time quantitative PCR analysis were approved by the State Food and Drug Administration of China.The data were periodically collected from a centralized supervision warehouse and were analyzed by the Guangzhou Center for Disease Control and Prevention. The main analysis recorded sample size, risk classification, and the COVID-19 positive rate following the establishment of the centralized supervision warehouse. The risk classification of positive samples, risk grading, cycle threshold (Ct) value, and other aspects were analyzed. Virus isolation and whole sequencing were conducted for high-risk samples. The results of outbreak management were also analyzed.A total of 283 positive cold-chain events were found in Guangzhou since the first cold-chain-related event of the COVID-19 pandemic was identified in July 2020. The number of positive cold-chain events increased annually, with 220 (77.74%) of the events observed in 2022. Positive cold-chain events occurred most frequently in autumn and winter, whereas fewer events occurred from March to April. The highest number of positive events  occurred in October 2022. Positive samples were mainly associated with meat products . The numbers of events that involved aquatic products, fruit products, and other food products were 65 (22.97%), 17 (6.01%), and 26 (9.19%), respectively. The positive results in the cold chain across food were consistent with the distribution of overall positive events .The products associated with positive events were from Asia , South America , North America , Europe , and Australia . Australian imports had the highest number of positive samples (4.75) per positive event, followed by imports from Asian countries , which reduced downstream communication risks in the sales process of cold-chain food. All of the imported cold-chain food was classified into four categories . The most positive events were observed in meat products (61.84% of positive events and 56.53% of positive samples). The number of positive samples (3.50) per positive event was highest in the centralized supervision warehouse .A total of 272 positive events and 886 positive samples were observed. Positive events and samples were found in product packaging, internal packaging, product surfaces, and environmental surfaces. In some events, two types of positive results were found. Four (1.4%) events were positive for outer packaging and inner packaging, one (0.35%) event was positive for outer packaging and the environment, and three (1.06%) events were positive for inner packaging and environmental surfaces. The positive events and samples were mainly associated with product packaging. Meat products accounted for 62.13% and 63.64% of the positive results associated with outer and inner packaging, respectively .According to the cold-chain event risk classification strategy, 226 events were considered low-risk events (79.86%), whereas medium- and high-risk events accounted for 19.79% and 0.35% of events, respectively. The first medium-risk event was reported in January 2021; medium-risk events were reported monthly after August 2021. Only one high-risk event was reported in the regulatory cycle; the product associated with the event was chicken feet imported from Brazil in November 2021. The Ct value of the positive specimen was between 27.23 and 38.01. A total of five positive specimens were found in 225 samples that were collected using preliminary testing and expanded sampling .Five centralized supervision cold-storage facilities in Guangzhou were distributed across four districts . A total of 13,094 high-risk individuals and 75,962 low-risk individuals were monitored, and none were found to be positive. A total of 972,738 cold-chain food samples were monitored, and 904 were found to be positive (positive rate = 0.09%). A total of 14,896 environmental samples were monitored, and six were found to be positive (positive rate = 0.04%; Given that the SARS-CoV-2 virus can survive in low temperatures for a long period, imported cold-chain food poses a risk of infection across all cold-chain links, including processing, transportation, storage, downstream market flow, and consumption. The risk of transmission is highest in trans-regional transportation and downstream market consumption ,8. DurinDisinfection plays an important role in sealing off the cold chain as a transmission route for SARS-CoV-2 but faces many challenges. For example, disinfection of the outer packaging of cold-chain food with disinfectants is influenced by low temperatures\u2014especially those of the ice that forms on the outer packaging. Therefore, achieving optimal disinfection is difficult. Furthermore, the erosion of food and external packaging affects the quality of food and packaging ,19. As cSince July 2020, the city of Guangzhou has comprehensively operationalized the centralized supervision warehouse system for imported frozen food and has implemented centralized testing, disinfection, and supervision of imported frozen food that enters the city and each element of relevant warehousing. Guangzhou gradually introduced electronic traceability, implemented classified management, and centralized the residences, with high-frequency monitoring and whole-process supervision of the personnel who worked in the centralized supervision warehouse. The city\u2019s production and import of cold-chain food units were also regularly supervised ,12. We wThe results of this study showed that the considerable increase in positive cold-chain time in 2022 compared with those in 2020 and 2021 was related to the growing global COVID-19 epidemic and improved supervision ,21. No sFocusing attention on the health risks caused by cold-chain food remains necessary even in the absence of COVID-19. To reduce the risk of infection caused by contact with all pathogens including COVID-19, we suggest that additional focus be applied to the health risks of cold-chain food after the COVID-19 pandemic . Future Our study has some limitations. First, samples that taken in the market or during traffic interception were detected by a testing company, of which we were informed only when positive samples were detected; thus, the whole sample size was not obtained, resulting in the actual positive rate being lower than in our monitoring data. Second, during the COVID-19 epidemic, the management of cold-chain food and personnel by the centralized supervision warehouse was based on several prevention and control measures, and its effect on preventing the infection risk from cold-chain imports was difficult to quantitatively evaluate. Finally, throughout the monitoring period, the SARS-CoV-2 virus had not been successfully sequenced and isolated from positive samples; therefore, direct evidence of transmission from contaminated cold-chain food to humans was lacking.Cold-chain food has a relatively low risk of transmitting SARS-CoV-2. Centralized food storage can be used as an effective method to control this risk, and this measure can also be used for other food-related contact-transmitted diseases."}
+{"text": "Subsequently, CST was established as a pleiotropic hormone. In 2005, it was reported that N-terminal 15 amino acids of bovine CST (bCST1\u201315 aka cateslytin) exert antibacterial, antifungal, and antiyeast effects without showing any hemolytic effects. In 2017, D-bCST1\u201315 (where L-amino acids were changed to D-amino acids) was shown to exert very effective antimicrobial effects against various bacterial strains. Beyond antimicrobial effects, D-bCST1\u201315 potentiated (additive/synergistic) antibacterial effects of cefotaxime, amoxicillin, and methicillin. Furthermore, D-bCST1\u201315 neither triggered bacterial resistance nor elicited cytokine release. The present review will highlight the antimicrobial effects of CST, bCST1\u201315 (aka cateslytin), D-bCST1\u201315, and human variants of CST (Gly364Ser-CST and Pro370Leu-CST); evolutionary conservation of CST in mammals; and their potential as a therapy for antibiotic-resistant \u201csuperbugs\u201d.The rapid increase in drug-resistant and multidrug-resistant infections poses a serious challenge to antimicrobial therapies, and has created a global health crisis. Since antimicrobial peptides (AMPs) have escaped bacterial resistance throughout evolution, AMPs are a category of potential alternatives for antibiotic-resistant \u201csuperbugs\u201d. The Chromogranin A (CgA)-derived peptide Catestatin (CST: hCgA Likewisbacteria . The higde (LPS) . Beyond de (LPS)  and cholde (LPS)  that wor1\u201315 was reported to exert more effective antimicrobial effects against various bacterial strains than L-bCST1\u201315 [1\u201315 was reported to potentiate (additive/synergistic) the antibacterial effects of cefotaxime, amoxicillin, and methicillin [1\u201315 neither triggered bacterial resistance nor elicited cytokine release [1\u201315 was reported to be more resistant to degradation by secreted bacterial protease than L-bCST1\u201315 [1\u201315 can be used as a monotherapy or as a combination therapy with currently prescribed antibiotics to counteract various diseases associated with bacterial infection [D-bCSTbCST1\u201315 . In addihicillin . Further release . In addibCST1\u201315 . Thus, infection .While antibiotics target specific cellular activities , AMPs target the LPS layer of the cell membrane. Extensive studies have been conducted to learn the composition of the bacterial membrane. The bacterial cytoplasmic membrane consists of zwitterionic phospholipids  and anionic phospholipids , providing them with a negative charge ,30,31,32342\u2013370) performed in a water shell led to a \u03b2-strand-loop-\u03b2-strand structure. Molecular dynamics and computer simulations of human CST1\u201321 revealed the following: R10, A11, and Y12 contribute to a 310 helix [7, R8, A9, F14, R15, G16, P17, and G18 contribute to the antiparallel \u03b2-sheet [1\u201321 could start by interacting with negatively charged moieties such as LPS in the outer membranes of Gram-negative bacteria and lipoteichoic acid in the wall of Gram-positive bacteria. The primary structure of CST reveals that CST contains cationic and hydrophobic residues and adopt a \u03b2-sheet secondary structure via intermolecular forces [Based on their secondary structure, AMPs are generally categorized into four groups: (i) \u03b1-helical AMPs, (ii) \u03b2-sheet AMPs, (iii) extended AMPs, and cationic loop AMPs . Homologr forces . This for forces . Since tr forces . In addir forces ,41  [100 values of CST or its human variants against the above fungal species ranged from 0.8 \u00b5M to 100 \u00b5M [100 of CST and its variants against the above yeasts ranged from 6 \u00b5M to 75 \u00b5M [Fungal infections are common on the surface of skin, nails, or mucous membranes , underneath the skin (subcutaneous), or in the lungs, brain, or heart (deep infection). Deep fungal infections include Histoplasmosis , Coccidineumonia , Mucormyneumonia ,67, and neumonia ,69. It ineumonia ,71, whicneumonia . To this rubrum) ,24  with fungal (A. fumigatus) and yeast  membranes [The composition of fungal cell membranes is similar to that of bacteria, comprising zwitterionic and anionic phospholipids. In contrast, the fungal cell wall is composed of chitin, glucan, ergosterol, and mannoprotein, which reside on the surface of the cytoplasmic membrane. Because of the negative charge on the cytoplasmic membrane, CST could exert its anti-fungal activities in a similar way to its antibacterial activity. Metz-Boutigue\u2019s group used confocal laser microscopy to analyze the interaction of the synthetic rhodamine-labeled cateslytin , such as acetate, butyrate, and propionate [Bacteroides thetaiotaomicron produces significant amounts of glycosylhydrolases, which prevent obesity [Bacteroides species are also reported to prevent obesity and increase insulin sensitivity [Bacteroides fragilis produces zwitterionic polysaccharide, which activates CD4+ T cells to produce interleukin 10 (IL-10). IL-10 plays crucial roles in preventing abscess formation and other unchecked inflammatory responses [Recent studies have identified a larger role of gut microbiota in gut-immune homeostasis and in intestinal pathology. The human intestinal microbiota is dominated by five phyla: high-abundant >80%) (1) Bacillota (aka Firmicutes) and (2) Bacteroidota; less-abundant (3) Actinomycetota (aka Actinobacteria), (4) Pseudomonadota (aka Proteobacteri), and (5) Verrucomicrobiota  as compaopionate ,80,81, w% (1) Bacopionate ,83. In aopionate ,85. SCFAopionate ,87,88 anopionate ,90,91. B obesity . Other Bsitivity ,94. Furtesponses ,96. The Akkermansia species. Since A. muciniphila modulates obesity by regulating metabolism and energy homeostasis to improve insulin sensitivity and glucose homeostasis [CST knockout (CST-KO) mice were generated in 2018 and are: insulin-resistant on a normal chow diet , hyperadeostasis , low Vereostasis .H and inverted Simpson\u2019s index [Alistipes, Akkermansia, and Roseburia genera only in CST-KO mice [Decreased amplicon sequence variants and abundance-based coverage estimator indices in CST-KO mice were restored after supplementation with CST for 15 days . Akin to\u2019s index . At the \u2019s index . In cont-KO mice .Clostridium difficile colitis with a success rate of up to 95% [FMT-CST-KO) encompassed a reduction of Clostridia and Akkermansia [FMT-WT) showed an increase in richness, a notable reduction of Staphylococcus, and an increase in the butyrate-producing Intestinimonas [FMT is now established as an effective therapeutic modality in the treatment of the following diseases: (i) antibiotic-refractory recurrent p to 95% ,104,105,p to 95% ,107,108.ermansia , which aermansia ,111 and ermansia ,113. Of ermansia . In continimonas   dereceptor ; (iii) dreceptor ; (iv) blreceptor ; (v) decreceptor ; (vi) inreceptor ; (vii) dreceptor ; and  in association with van der Walls interactions of apolar  amino acids provided a global stability for CST [Sequence alignment of CST in 53 mammalian species belonging to eight orders revealed >80% homology in 52 species, except in Platypus  where the homology with the primates  was >58% , indicatne rings ,136,137.ne rings ,140,141.ne rings . Therefo for CST ,144. In 364Ser  [367Val (only in Indian populations) [370Leu (US and Indian populations) [374Gln (US populations only) [370Leu-CST has the highest potency of inhibiting catecholamine secretion and desensitizing catecholamine secretion, followed by WT-CST and Gly364-Ser-CST [Four non-synonymous SNPs have been identified in CST domain of CgA: Glylations) ,145,146,lations) , and Argns only) . Pro370L-Ser-CST . As a sh-Ser-CST , Gly364S-Ser-CST .3, L5, F7, F14, and G18) are 100% conserved; nine (~43%) amino acids  are 90\u201396% conserved; and three (~14%) amino acids  are >80% conserved. The least conserved sequences are G13 (>66%) and P19 (>58%), where human variants of CST were reported for G13 (G13S) and P19 (P19L), indicating that natural selection pressures still exist on those two amino acids [(i) High conservation of CST in mammals: Alignment of CST sequences from 53 mammalian species belonging to eight orders revealed that CST sequence is highly conserved (>90% in 90% species) in mammals: Five (~24%) amino acids  CST as an immunomodulatory peptide: Existing literature  CST as an antimicrobial peptide: Prominent effects of CST in the low micromolar range on inhibition of growth of Gram-positive and Gram-negative bacteria, fungi, and yeast establish CST as an antimicrobial peptide .1\u201315 as a potential therapy for microbial infection: D-bCST1\u201315 could be used as a monotherapy or as a combination therapy with cefotaxime, amoxicillin, and methicillin against the \u201csuperbugs\u201d because it has more effective antibacterial activity compared to L-bCST1\u201315, penetration through the bacterial cell wall, resistance to bacterial proteases, undetectable susceptibility to resistance, and potentiation/synergic action of commonly prescribed antibiotics [(iv) D-bCSTibiotics .(v) CST as a cell permeable peptide: Penetration of CST (pI 12.03\u201312.48) in bacteria, fungus, yeast, and neutrophils ,152, couAkkermansia spp.) in CST-KO mice [Bacilotta to Bacteroidota coupled with increased abundance of Verrucomicrobiota after supplementation of CST-KO mice with CST [Akkermansia and increased population of Proteobacteria in WTFMT-CST-KO coupled with increased population of butyrate producing Intestimonas in CST-KOFMT-WT [(vi) Gut microbiome-mediated improvement in insulin sensitivity by CST: The increased ratio of Bacilotta to Bacteroidota, together with low levels of Verrucomicrobiota  Improvement in antimicrobial effect of CST by cyclization: Based on the existing literature ,41, we p"}
+{"text": "Pelates sexlineatus) communities across six temperate Australian estuaries spanning \u223c500\u00a0km. We find that spatial and environmental factors have different influences on these communities, with seawater demonstrating strong distance-decay relationships (R = \u22120.69) and significant associations with a range of environmental variables. Distance-decay relationships were weak for sediment communities but became stronger over smaller spatial scales , potentially reflecting environmental filtering across biogeochemical gradients or stochastic processes within estuary sediments. Finally, P. sexlineatus hindgut microbiome communities displayed weak distance-decay relationships (R = \u22120.36), and limited variation explained by environmental variables, indicating the significance of host-related factors in driving community variation. Our findings provide important ecological insights into the spatial distributions and driving forces of both free-living and host-associated bacterial patterns across temperate estuarine systems.A major goal of microbial ecology is to establish the importance of spatial and environmental factors in driving community variation. Their relative importance likely varies across spatial scales, but focus has primarily been on free-living communities within well-connected aquatic environments rather than less connected island-like habitats such as estuaries, and key host-associated communities within these systems. Here we sampled both free-living (seawater and sediment) and host-associated (estuarine fish hindgut microbiome,  Environmental and spatial factors have greater influence on estuarine free-living bacterial communities in comparison to host-associated communities across six temperate estuaries spanning 500km. Microorganisms play crucial roles in global biogeochemical cycles, particularly critical to the functioning of dynamic environments such as estuaries  : Hastings River, Wallis Lake, Lake Macquarie, Brisbane Water, Lake Illawarra, and Burrill Lake , salinity (0.1 ppt), pH (0.01), turbidity (0.1 NTU), and dissolved oxygen (0.01\u00a0mg/L). Seawater was collected in sterile bottles (rinsed with 10% bleach solution) for amplicon sequencing (n = 5), with \u223c800\u00a0ml from each sample filtered onto 0.2 \u03bcm pore-sized Sterivex filters, depending on the amount of particulate organic matter present. Sediment samples (50\u201360\u00a0ml) were collected using a 50\u00a0ml Luer Lock syringe plunged vertically into the sediment to \u223c100\u00a0mm depth, for sediment granulometry and the determination of organic matter. Surface sediments were also collected for amplicon sequencing (n = 5) by scraping the upper 1\u00a0cm layer into sterile 15\u00a0ml tubes. Pelates sexlineatus were collected using a 10-m seine net pulled through seagrass beds for amplicon sequencing of the hindgut microbiome (n = 5). All samples were immediately stored on ice and processed within 6 h of collection.This research was conducted under the University of Newcastle Animal Ethics Protocol A-2020-026. Six estuaries along the NSW coastline were selected for sampling, based on available seagrass habitat and distribution of the estuarine fish ake Fig.\u00a0. Pelates Pollard . SeawatePelates sexlineatus total length (mm) and weight (g) were recorded, and fish hindgut contents removed (\u223c0.25\u00a0g). A 0.25\u00a0g sample from collected sediments were stored at \u221280\u00b0C until DNA extraction. Qiagen DNeasy PowerSoil Kits were used to extract DNA from fish and sediment samples, and Qiagen DNeasy PowerWater Kits were used for seawater samples. Extraction blanks containing no sample were processed with each batch to serve as controls. Sample quantity and quality were checked using a NanoPhotometer NP80. Bacterial communities were characterized using 16S rDNA gene amplicon sequencing. The V1\u2013V3 region of the 16S rDNA gene from prokaryotes was amplified using universal primers 27F (AGAGTTTGATCMTGGCTCAG) and 519R (GWATTACCGCGGCKGCTG) attached with Illumina adaptors in PCR with the following cycling conditions: 95\u00b0C for 10\u00a0min, then 35 cycles of: 94\u00b0C for 30\u00a0s, 55\u00b0C for 10\u00a0s, and 72\u00b0C for 45\u00a0s, with a final extension of 72\u00b0C for 10\u00a0min. PCR products were sequenced using the Illumina Miseq v3 (2 \u00d7 300\u00a0bp) platform at the Ramaciotti Centre for Genomics at the University of New South Wales. Resultant amplicons were processed using the R pipeline DADA2 with default parameters  and environmental variables (Euclidean distances), and distance-decay slopes were visualized using scatterplots. PERMANOVA with pairwise comparisons was used to investigate significant differences in community composition (based on Bray\u2013Curtis dissimilarity) between estuaries, and PERMDISP was further employed to investigate differences in community variance across estuaries using the \u201cbetadisper\u201d function , catchment cleared (%), urbanization (%), estuary surface area (km2), and estuary volume (ML). NSW estuarine macrophyte data was obtained from the Fisheries NSW Spatial Data Portal ([dataset]* NSW Department of Primary Industries), and seagrass, mangrove, and saltmarsh area (km2) for each estuary was calculated in QGIS 3  were removed from further statistical analyses. Mantel and partial mantel tests were used to test the correlation between environmental variability and geographic distance on variation of bacterial alpha diversity between sites (Euclidean distances). Redundancy analysis (RDA) was used to model the effect of environmental variables on entire microbial communities, with forward selection used to select statistically significant variables. Significance of RDA models and explanatory variables were determined using ANOVAs. Finally, Spearman rank correlations were employed to test associations between environmental parameters on dominant phyla (>1% relative abundance) and ASVs (50 most abundant), with significant correlations visualized using correlation heatmaps.The following environmental variables were extracted from the NSW Estuary Health Monitoring, Evaluating, and Reporting (MER) program on the SEED NSW database  sequences from 48 samples spanning five estuaries were obtained for seawater, 1\u00a0712\u00a0831 (22\u00a0537.25 \u00b1 1004.20) sequences from 75 samples spanning six estuaries were obtained for sediments, and 880\u00a0706 (13\u00a0761.03 \u00b1 1259.76) sequences from 65 samples spanning six estuaries were obtained for fish hindgut samples.R = \u22120.69, P < 0.01; Fig.\u00a0R = \u22120.32, P < 0.01; Fig.\u00a0R = \u22120.39, P < 0.01; Fig.\u00a0R = \u22120.71, P < 0.01; Fig.\u00a0R = \u22120.5, P < 0.01; Fig.\u00a0R = \u22120.37, P < 0.01; Fig.\u00a0P < 0.01) and within estuaries , but not environmental variables (P > 0.05); however, there were significant interactions between geographic distance and environmental variables , seawater , or sediments . Despite this, the results from the PERMANOVA tests and the nMDS plots indicate that for all sample types, bacterial community composition demonstrated significant separation by estuary , but not environmental factors . Spearman rank correlations were employed to investigate associations between individual environmental variables and seawater bacterial alpha diversity measures, with pH, sediment silt, latitude, average estuary depth, flushing time, catchment area, area of saltmarsh and mangrove in the estuary, and estuary surface area most strongly associated with alpha diversity measures  or environmental variables , Spearman rank correlations identified significant associations between sediment bacterial alpha diversity measures and particular environmental variables, specifically salinity, pH, sediment organic matter, estuary volume, catchment size, urbanization percentage, and area of seagrass and mangroves , but not environmental variables . Significant associations between individual environmental variables and fish hindgut bacterial alpha diversity were identified, particularly sediment silt percentage, latitude, estuary flushing time, catchment area, urbanization percentage, and area of mangroves  , Bacteroidota (21.97% \u00b1 1.35), Cyanobacteria (16.76% \u00b1 2.20), Actinobacteria (11.80% \u00b1 0.84), Verrucomicrobiota (0.56% \u00b1 0.08), Planctomycetota (0.37% \u00b1 0.05), Chloroflexota (0.15% \u00b1 0.04), and the SAR324 clade (0.15% \u00b1 0.04) Fig.\u00a0. Dominan2) Fig.\u00a0. Among t2) Fig.\u00a0. Specifilatitude .Woeseia and Methyloceanibacter) and Actinobacteria phyla  had the strongest associations with environmental variables, particularly negative relationships with water column salinity, latitude, and estuary catchment area (Sediment samples were dominated by Proteobacteria (43.95% \u00b1 0.58), Actinobacteria (18.70% \u00b1 0.87), Bacteroidota (11.36% \u00b1 0.87), Desulfobacterota (6.53% \u00b1 0.32), Chloroflexota (4.80% \u00b1 0.27), Planctomycetota (3.66% \u00b1 0.24), Cyanobacteria (2.76% \u00b1 0.48), Acidobacteriota (2.48% \u00b1 0.15), and Myxococcota (2.49% \u00b1 0.14) Fig.\u00a0. Some ofent area .Methyloceanibacter, Rhodobaculum, and Rickettsiales had significant associations with pH and salinity, while some ASVs identified as Synechococcus CC902 and Cyanobium PCC-6307 (within the Cyanobacteria phylum) had negative associations with latitude (Fish hindgut samples were dominated by Proteobacteria (59.28% \u00b1 2.74), Actinobacteria (20.12% \u00b1 1.82), Cyanobacteria (9.24% \u00b1 1.72), Chloroflexota (5.95% \u00b1 0.52), Firmicutes (2.37% \u00b1 0.57), Planctomycetota (1.43% \u00b1 0.15), Desulfobacterota (0.97% \u00b1 0.25), Campylobacterota (0.27% \u00b1 0.19), Bacteroidota (0.14% \u00b1 0.06), and Acidobacteriota (0.12% \u00b1 0.03) Fig.\u00a0. Dominanlatitude .Unveiling the relative importance of spatial and environmental factors in the biogeographic distribution and assembly of microbial communities is a central issue in microbial ecology  and steep biogeochemical gradients within coastal sediments, potentially resulting in strong habitat heterogeneity  and Actinobacteria  were significantly correlated with salinity and are abundant in estuarine sediments, likely carrying out diverse ecological functions including denitrification, sulfur oxidation, and aerobic ammonium oxidation , ASVs belonging to the bacterial families Propionibacteriaceae, Rhodobacteraceae, and Vibrionaceae were also abundant, consistent with previous findings (Larios\u2010Soriano et al. P. sexlineatus is an opportunistic carnivore that feeds on dominant food sources within seagrass meadows, environmental filtering may play an important role in structuring and maintaining the gut microbiome across estuaries through diet; however, this was not measured here (Sanchez\u2010Jerez et al. While many of the dominant ASVs reported here are common marine and estuarine environmental taxa (e.g. P. sexlineatus, across six eastern Australian estuaries spanning \u223c500\u00a0km. Seawater communities exhibited strong distance-decay relationships as well as significant associations with a range of environmental variables. Conversely, sediment and P. sexlineatus hindgut bacterial communities displayed weak distance-decay relationships and limited variation explained by measured environmental variables, potentially reflecting environmental filtering across biogeochemical gradients or stochastic processes within estuary sediments and that host-associated communities are governed most strongly by host-related factors. These results provide important ecological insights into the spatial distribution and some of the driving factors of bacterial community composition across temperate estuarine systems for multiple sample types.A central goal in microbial ecology is understanding the contribution of spatial and environmental factors in driving patterns of microbial composition and diversity. Biogeography has been shown to apply to free-living microbial communities across interconnected aquatic environments, with the relative importance of specific environmental variables identified at a range of spatial scales. Patterns in free-living bacterial communities are likely to differ from those in host-associated communities, which may be buffered from environmental variation and under stronger selective pressure from the niche habitat provided by the host and are comparatively less well understood. We found that spatial and environmental factors have different influences on the bacterial communities associated with estuary seawater, sediments, and a common estuarine fish, fiad061_Supplemental_FilesClick here for additional data file."}
+{"text": "India suffers ~58,000 annual deaths due to snakebites. The \u2018Big Four\u2019 snakes  that are responsible for most bites cause diverse clinical effects. Delayed treatment increases the risk of serious complications and treatment costs. Although government hospitals offer free treatment for snakebites in India, most patients opt for private healthcare, which is an out-of-pocket expense as they often lack health insurance coverage. This study aims to analyse snakebite treatment costs in private tertiary care hospitals in Tamil Nadu, India and identifies the key factors contributing to treatment costs.The treatment cost details for 913 snakebite victims were collected from 10 private tertiary care hospitals across Tamil Nadu. The data were classified into hospital, pharmacy, investigation, and laboratory costs, and analysed to determine various factors that contribute to the costs. The results demonstrate that the average treatment costs vary widely for different snakes. The hospital and pharmacy costs are higher than investigation and laboratory costs for all snakebites. Notably, Russell\u2019s viper bites cost significantly more than the bites from other snakes. Overall, the type of snake, nature of complications, specialist treatments required, and arrival time to hospitals were identified as some of the key factors for higher treatment costs.These data demonstrate that ~80% of snakebite patients can be treated with INR 100,000 (~GBP 1000 or USD 1200) or less. This study emphasises the urgent need to improve rural medical care by providing appropriate training for healthcare professionals and essential resources to facilitate early assessment of patients, administer the initial dose of antivenom and refer the patients to tertiary care only when needed. Moreover, the outcome of this study forms a basis for developing appropriate policies to regulate snakebite treatment costs and provide affordable medical insurance for vulnerable communities. Snakebite envenoming (SBE) predominantly affects poor communities living in rural areas of developing countries. As SBE induces a wide range of pathological effects in patients, they need a broad spectrum of treatment approaches to tackle those issues. Therefore, antivenom alone is not sufficient to treat SBE patients. In India, most people (including SBE patients) seek treatments from private healthcare settings, although government hospitals provide free treatments. Hence the treatment costs pose a significant burden on the SBE victims and their families leading to severe socioeconomic impacts. To develop better policies to support the clinical management of SBE, it is critical to estimate the costs of SBE treatment in private healthcare settings. In this study, we analysed the treatment costs of 913 snakebite patients who were treated in 10 different private tertiary care hospitals in Tamil Nadu, India, and identified various factors that contribute to treatment costs. For example, snake type, specialist treatments and the time of arrival to the hospital following bites are some of the key factors leading to increased treatment costs. These results form the basis for developing policies to regulate SBE treatment costs, provide health insurance coverage and ensure timely treatment for SBE victims in rural healthcare settings. Daboia russelii), Indian cobra  (Naja naja), common krait (Bungarus caeruleus), and saw-scaled viper (Echis carinatus)] are responsible for most of the incidents, resulting deaths, and disabilities  conducted on the patients. A total treatment cost was calculated by adding up each of these individual parts. All the prices charged to participants in this study were mentioned in the form of Indian rupees (INR).A generalised linear model with a gamma distribution and a log-link function for the mean component was used to model treatment costs with the snake, age, sex, number of vials of antivenom and interactions as covariates. This modelling approach is due to the fact that the treatment costs are positive, and it allows us to address the observed heterogeneity in the data detected during the model validation process. Before fitting any model, data exploration was applied using the protocols that were previously described , 30. TheAll statistical analyses were performed using the R  and SPSS  statistical packages and GraphPad Prism .Fig 1A). Among the study population, 600 (65.7%) males and 313 (34.3%) females were included indicating that males are more vulnerable to snakebites than females, probably due to their increased involvement in agricultural activities. Further classification of these data based on the type of snake involved in the incident revealed that 355 [38.9% of total incidents: 242 (68.2%) males and 113 (31.8%) females] patients were bitten by Russell\u2019s vipers , 133  patients were bitten by common kraits, 108  patients were bitten by saw-scaled vipers and 69  patients were bitten by cobras. Moreover, 87  patients were bitten by non-venomous snakes such as Indian rat snakes (Ptyas mucosa), wolf snake (Lycodon aulicus), and common bronzeback tree snake (Dendrelaphis tristis). A total of 161 patients  were bitten by unidentified snake species as their identities could not be ascertained based on the available details. This category may include a few cases of Malabar , hump nosed  and bamboo  pit vipers as these cases are also being reported in distinct parts of Tamil Nadu .In total, 913 snakebite patients who were admitted to 10 different private tertiary care hospitals in Tamil Nadu were included in this study . The youngest patient included in this study was 2 years old and the oldest patient was 91 years old with a median of 40 years  and a mean of 39.9. For Russell\u2019s viper bites, the minimum age was 2.5 years old, and the maximum age was 91 with a median age of 40 and a mean of 40.6  . For common krait bites, the minimum age was 11 and the maximum age was 81 with a median age of 47 and a mean of 46.9 . For cobra bites, the minimum age was 4 years old, and the maximum age was 80 with a median age of 35 and a mean of 36.2 . For saw-scaled viper bites, the minimum age was 2 years old, and the maximum was 70 with a median age of 41.5 and mean of 41.8 . For non-venomous snakebites, the minimum age was 3 years old, and the maximum age was 75 with a median age of 33 and a mean of 32.2 . For unknown snakes, the minimum age was 2 years old, and the maximum age was 75 with a median age of 39 and a mean of 37.2 . This data indicates that males of working age (from 21 to 60) are most likely to suffer snakebites.To determine the specific age groups that are most affected by snakebites, the data were categorised based on the age of the patients. In total, 38  people were in the age range of 0\u201310, 92 (10%) in 11\u201320, 143 (15.7%) in 21\u201330, 200 (21.9%) in 31\u201340, 196 (21.5%) in 41\u201350, 146 (16%) in 51\u201360, and 98 (10.7%) were above 60 years old ], swelling [286 (80.6%)], vomiting [173 (48.7%)], blurred vision [119 (33.5%)] and bleeding [117 (33%)] as the most prevalent signs/symptoms. Common krait bites induced ptosis [62 ] and blurred vision [50 (37.6%)]. Cobra bites induced pain [67 ], swelling [42 (60.9%)], vomiting [28 (40.6%)], blurred vision [22 (31.9%)] and ptosis [21 (30.4%)]. Saw-scaled viper bites induced pain [107 ], swelling [66 (61.1%)], and blurred vision [30 (27.8%)]. Non-venomous snakes mainly induced pain [72 ]. Bites from snakes within the unknown category induced pain [131 (81.4% of unknown bite cases)], swelling [84 (52.2%)], vomiting [54 (33.5%)], and bleeding [30 (18.6%)].Fig 2A). The costs for common krait bites ranged from INR 10,504 to INR 856,688 with an average cost of INR 47,276 . For cobra bites, the costs ranged from INR 11,029 to INR 309,683 with an average of INR 86,833 . Treating saw-scaled viper bites costs between INR 5,814 to INR 105,223 with an average of INR 28,863 . The costs of non-venomous bites ranged from INR 2,937 to INR 108,057 with an average of INR 17,554 . The unknown snake category costs ranged from INR 2,101 to INR 674,884 with an average of INR 77,342 . These data suggest that the average treatment costs for Russell\u2019s viper bites are higher than the costs for treating bites from other snake species.The total treatment costs were classified into hospital, investigation, laboratory, and pharmacy costs. The total treatment costs for Russell\u2019s viper bites ranged from INR 2,502 to INR 694,620 with an average of INR 111,496   and pharmacy  costs are considerably higher than the laboratory  and investigation  costs. For example, the hospital costs ranged from INR 500 to INR 357,300  and pharmacy costs ranged from INR 1,111 to INR 253,713  for Russell\u2019s viper patients. However, the average costs of investigation  and laboratory tests  were only INR 8,742 and INR 10,377, respectively, for Russell\u2019s viper patients. These data suggest that hospital and pharmacy costs are the most significant factors contributing to the overall treatment costs for snakebite patients. The average percentages of different treatment costs compared to the total costs for bites from diverse snake species are shown in Table 1.Further evaluation of treatment costs revealed that hospital . However, when the total treatment costs of different age groups were compared, age was determined to be a significant predictor for the total treatment cost  . Similarly, when the treatment costs of patients from different snake species were compared, the offending snake was confirmed to be a significant predictor of total treatment cost . The results indicate that all snake species\u2019 total treatment costs vary significantly from each other. However, the total treatment costs are not significantly different between Russell\u2019s viper and the cobra, or between the unknown category and cobra. The data also indicate that the overall treatment cost decreases with age in both males and females .When the total treatment costs for males and females were compared, there was no significant difference between the cohorts, indicating that gender is not a significant predictor for total treatment cost [XTable 2). For example, 13 patients who were bitten by Russell\u2019s viper received dialysis and therefore, their average total treatment costs were INR 172,018 which is higher than the average  total costs for Russell\u2019s viper patients. A single patient who developed parotitis along with excessive levels of bilirubin paid a total treatment cost of INR 292,502. A total of 26 patients bitten by Russell\u2019s viper stayed in intensive care units and they paid a total of INR 188,950. 55 Russell\u2019s viper patients received blood transfusions and paid a total average cost of INR 139,541. 38 Russell\u2019s viper patients had some form of operating intervention such as wound debridement and paid an average cost of INR 133,148. Similarly, seven cobra patients had wound debridement and they paid INR 91,002 which is higher than the total average  costs for treating cobra patients (Table 2). Moreover, two patients bitten by saw-scaled vipers developed anaphylaxis and they paid higher  than the average  total treatment costs. Nine saw-scaled viper patients who stayed in intensive care units paid a total of INR 40,736 which is higher than the total average costs (Table 2). 12 patients of saw-scaled vipers had blood transfusions, and they paid INR 39,959, which is also higher than the average total treatment costs. 17 patients bitten by unknown snakes stayed in the intensive care units and paid an average of INR 83,766 which is more than the average  total treatment costs. Similarly, two patients of unknown snake categories had a tracheostomy and therefore their total treatment costs were increased to INR 87,120, higher than the total average costs.All associated treatment costs for Russell\u2019s viper bites are higher than the bites from other snakes, which can be attributed to the specialist treatments that these patients received . The number of antivenom vials received by patients varies significantly depending on the offending snake . Neither the age  nor the gender  of the patients had any significant bearing on the number of vials received. However, males received 11% (or 1.1 times) more vials than females although this was not found to be statistically significant due to large variations within the data set. The total number of antivenom vials used was calculated as a proportion of the total treatment cost, and it was found to be a lesser contributor to the total treatment cost. In most hospitals, antivenom was provided for around INR 700/vial. Therefore, its contribution to the total treatment cost ranged from INR 1,400 to INR 35,000. Compared to the total treatment costs, this is only a small factor in determining the final treatment costs in tertiary healthcare settings. The cost of an average number of antivenom vials received by Russell\u2019s viper bite patients represents 15.7%  of the total treatment cost. Similarly, it accounts for 13.1%,  of cobra, 24.3%  of common krait, 12.1%  of saw-scaled viper, 17.9%  of non-venomous and 15.7%  of unknown category treatment costs. Notably, if patients received up to 10 vials of antivenom in the primary or local healthcare settings prior to their admission in tertiary care settings, then a significant decrease in the number of vials received at the tertiary private hospitals was observed .We then analysed the data to determine if the volume of antivenom administered is a key predictor of total treatment costs for SBE. The number of vials of antivenom used for Russell\u2019s viper patients varied from 3 to 50 vials . Cobra bites required 5 to 35 vials , common krait bites used 5 to 20 vials , saw-scaled viper bites used 2 to 10 vials , non-venomous snakebites used 3 to 5 vials  and the unknown category required 3 to 35 vials  . Similarly, patients bitten by cobras required a stay of 0.5 to 18 days . Common krait bite patients required between 1 to 26 days , non-venomous bites ranged from 2 hours to 4 days , and unknown bite patients ranged from one hour to 27 days . Although the length of stay in hospitals can contribute to increased treatment costs, this alone is not the sole predictor of total treatment costs for any specific snake. For example, the patients bitten by common kraits stayed an average of 9 days which is higher than any other snakebite patients but paid comparatively lower treatment costs. On the other hand, Russell\u2019s viper bite patients who had shorter stays in the hospital paid more treatment costs than patients bitten by common kraits. Therefore, there are other contributory factors that determine the overall treatment costs for snakebite patients.For Russell\u2019s viper bites, the minimum stay in the hospital was 30 minutes and the maximum stay was 30 days  (Table 3). Therefore, a total of 546 (60%) patients arrived at hospitals within the recommended 4-hour period following bites [Fig 4C). These data suggest that a majority of SBE patients are seeking treatment at hospitals within the recommended time either in local primary/secondary or tertiary healthcare settings.Out of the 913 patients, 252 went directly to tertiary care hospitals from where the data were collected in this study. 661 patients visited at least one local primary or secondary healthcare setting prior to reaching these tertiary care hospitals. Moreover, 185 patients arrived at the hospitals within one hour following the bite, 147 arrived between 1 and 2 hours, and 214 arrived between 2 to 4 hours following the bites (ng bites . In addiTable 3). However, the data indicate that Russell\u2019s viper patients who arrived after 12 hours following bites paid more  than the total average costs  for Russell\u2019s viper patients although it is not significant. Similarly, common krait patients who arrived eight hours following bites paid more  than the total average  treatment costs. Cobra patients who arrived after 4 hours following bites paid more  than the average total costs . Saw-scaled viper patients who arrived four hours following bites paid more  than the average total treatment costs . Notably, there was a significant difference  when comparing the total treatment costs for common krait patients who arrived after 48 hours following bites  with patients who arrived within one hour  following bites although there were only two patients admitted to the hospitals after 48 hours. Similarly, there was a significant  difference in total treatment costs of cobra patients who arrived after 24 hours  compared to patients who arrived within one hour  although only one cobra patient arrived after 24 hours. These data reemphasise that the delay in seeking hospital treatment is a key factor in contributing to increased treatment costs.We found that the time of arrival is not a significant predictor of total treatment costs for most SBE patients (Table 4). However, common krait patients who were bitten after 10 pm paid significantly more  than the total average treatment costs . The increase in treatment costs in late nights could be due to high transport costs, and investigation charges as the specialists may need to come from home or other hospitals for expert investigation and emergency intubation. As the common krait is a nocturnal snake, its bites are likely to be high at night times leading to late-night hospital admissions.Moreover, when analysing the total treatment costs of patients who were bitten during day times (between 6 am and 6 pm), early evenings (6 pm to 10 pm) and late night (10 pm to 6 am), there was no significant difference in the total treatment costs for Russell\u2019s viper, saw-scaled viper, and cobra patients although the values are high for cobra and saw-scaled viper patients who arrived after 10 pm .Finally, the total amount of treatment costs was analysed for all snakebite patients. This reveals that 711 (78%) patients paid INR 100,000 (~GBP 1000 or USD 1200) or less for their hospital treatment. This includes 223 (63%) Russell\u2019s viper, 49 (71%) cobra, 123 (93%) common krait, 107 (99%) saw-scaled viper, 86 (99%) non-venomous and 123 (76%) unknown bite patients. 120 (13%) patients paid between INR 100,001 to INR 200,000 for their treatment. 47 (5%) patients paid between INR 200,001 to INR 300,000, and 19 (2%) paid INR 300,001 to INR 400,000 for their treatments. The higher cost of more than INR 400,000 was paid by a minority of patients. These data suggest that despite gender, age, the offending snake species, time of the bite, time taken to reach tertiary care hospitals, administration of antivenom and specialist treatments used, around 80% of patients can be treated with INR 100,000 or less in private tertiary healthcare settings  to underpin the key contributors to the overall treatment costs. This has simplified the discussion about the nature of treatments involved and the rationale for expensive treatments. The hospital costs that cover intensive/critical care units, wards, operating theatres, and surgical procedures appeared to be the major contributor to total treatment costs. Pharmacy cost as a category covered all medications used during the treatment of SBE patients including antivenom and was the second significant contributor to SBE treatment costs. Each vial of antivenom only costs around INR 700 to INR 1,000 but additional costs such as antibiotics, blood products, consumables and other relevant materials are key contributors to treatment costs. Since the government sets the price for antivenom in India, this helps to minimise the costs of antivenom to the patients. The laboratory investigation and the fees for clinicians including specialists were found to be the least significant contributors to the total treatment costs compared to hospital and pharmacy costs. In this study, nearly 95% of people did not have medical insurance and therefore, they had to pay the full treatment costs. However, a small number of people had medical insurance although it did not cover the full treatment costs, so they still had to contribute towards the total treatment costs. In some hospitals, small discretionary discounts were offered based on the financial situation of patients, however, this is not the most common option offered to all patients.Among the different snakebites analysed in this study, Russell\u2019s viper bites appear to incur greater treatment costs compared to the other snakes analysed. The amount of venom injected and therefore, resulting complications following Russell\u2019s viper bites vary widely. In some patients, a small amount of tissue damage and coagulation abnormalities occur, however, in others, it results in extensive tissue damage which demands specialist interventions. Surgical procedures such as debridement, fasciotomy and amputation require operating theatres and consumables, which results in increased costs. For example, a simple debridement and fasciotomy may cost around INR 5000 to cover staff and operating theatre costs. However, amputation of a limb may increase this cost up to ten times . Due to the impacts of Russell\u2019s viper venom on the coagulation system, the patients often need plasma or whole blood transfusions, and other related products . NotablySnakebite patients and their families and communities have different perspectives in seeking prompt hospital treatment. In some places, snakebite has been considered a fate, and therefore, they should not seek any treatment. For several individuals, the traditional treatment is the only solution for SBE although this significantly delays the hospital treatment ,39,40. SDue to the complex nature of this study, it has several limitations. Since the treatments in government hospitals are provided free of cost to patients, we did not collect the actual costs incurred by the governments to treat SBE in these settings as this was outside of the scope of this work. However, it would have been helpful to analyse these data to determine the contributions of governments to tackle SBE. This study was conducted by collecting only direct treatment costs from 913 snakebite patients who were treated in 10 different private tertiary care hospitals within Tamil Nadu. Therefore, future studies with more patients and hospitals including the ones managed by the governments and charitable organisations are required to get a better understanding of the treatment costs for SBE across Tamil Nadu. Similar studies are also warranted in other states of India as the healthcare system may vary widely across the country. Moreover, we mainly analysed the data under four major categories due to the small sample size specifically for snakes other than Russell\u2019s viper. A larger sample size would allow further characterisation of the treatment costs, allowing for a more in-depth analysis to be performed on the specific factors associated with increased treatment costs. We also did not thoroughly analyse the number of antivenom vials administered in local healthcare centres prior to arriving at these tertiary care hospitals due to the poor memory of patients or the unavailability of these data in several cases. These data will add more to estimate the overall treatment costs for SBE. Therefore, data and costs presented in this study should only be considered as guidance for further studies and policy development as this may vary widely in different hospitals in India and other countries. Moreover, inflation, increasing costs of medications and equipment and availability of medications may ultimately play a role in the increased treatment costs for SBE. The indirect costs associated with SBE treatments were not analysed in this study, and this is another key factor that should be considered in future studies. This study did not discriminate between brands of antivenom used as national standards dictate the potency requirements across brands. Additionally, we did not attempt to analyse the social patterns associated with gender, age, or occupational differences in the presentation of different kinds of snakebites although these are areas of interest for epidemiological and educational purposes. The use of clinical symptoms by treating clinicians was mainly used to ascertain the snake species, and this could have been inaccurate in some cases. Notably, women may not have visited hospitals in a similar manner to men although there is no evidence to ascertain this notion. However, this is another key factor that should be addressed in future.Overall, this study suggests that there are numerous factors associated with the treatment costs for SBE in private tertiary care hospitals. However, it appears that a vast majority (~80%) of SBE patients can be treated with around INR 100,000 or less. Further increases in costs are attributable to complications resulting in operative interventions, dialysis, prolonged ventilation support, extended hospitalisation, and medications. Major healthcare insurance providers could consider SBE as a disease of poverty and offer cheap insurance schemes to cover these people from unexpected SBE treatment costs. Members of the public should also consider taking out these insurance schemes by paying a small fee, protecting themselves from SBE treatment costs. Necessary awareness should be created for members of the public to emphasise the need for taking such medical insurance to access prompt care. Moreover, there are currently no strict regulations or guidelines for SBE treatment costs in private healthcare settings. This results in hospitals deciding on the treatment cost themselves and can often appear unjust to the patients. While patients struggle to survive SBE complications, the treatment costs often come as another major shock, leading to serious downstream socioeconomic impacts. The societal impacts arising from SBE-associated financial burden can be a contributor to causing a shift in the social status of patients and their families within their communities for generations to come. Therefore, the health authorities could investigate this system and try to balance it. The prime minister\u2019s and chief minister\u2019s comprehensive insurance schemes in India could also be extended to more people and cover the entire treatment costs associated with SBE as it mostly affects poor rural farmers. India is a nation that largely relies on its agricultural and related workforce, and therefore, protecting these key workers from unexpected medical costs such as due to SBE is a priority. A key finding of the study is consistent with other studies in that antivenom is a significantly minor driver of costs while still being the only pharmacological preparation known to be effective in the treatment of snakebites ,53. The"
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