FilmArray GI-panel performance for the rapid and multiple detection of gastrointestinal microorganisms in foodborne illness outbreaks in Shenzhen during 2018-2019.

Foodborne illness outbreaks can be caused by a great many of gastrointestinal microorganisms including bacteria, viruses and parasites. Acute gastroenteritis is most commonly found in such patients infected with at least one pathogen through food intake. The stool culture has been conventionally used to guide a single diagnosis and therapy. However, traditional methods for identification of a pathogen are time-consuming and have limited sensitivity, leading to false negatives and co-infection omission. The aim of this study was to characterize the multiple etiology of each foodborne illness outbreak in Shenzhen during 2018-2019 by the FilmArray GI panel, and to reveal the seasonality of each causative organism incurring outbreaks. All patients included had a FilmArray GI panel performance and the seasonal characteristics were recorded. A total of 173 patients suffered from foodborne illnesses in 32 outbreaks in Nanshan District of Shenzhen. In total, 365 microorganisms were detected of which 83.8% (306/365) corresponded to bacteria and 16.2% (59/365) to viruses. Co-infections with more than one microorganism were detected in 81.3% (26/32) of the outbreaks. In 153 (88.4%) of 173 patients at least two pathogens were identified. The most common diarrheal pathogen related to outbreaks was EPEC (56%), followed by ETEC (38%), Norovirus (34%), EAEC (28%), Vibrio (25%), Salmonella (22%), P. shigelloides (22%), C. difficile (16%), STEC (3%) and Sapovirus (3%). Bacterial outbreaks occurred with a seasonal distribution with the exception of C. difficile whereas Norovirus outbreaks predominated during the autumn-winter months. The use of the FilmArray GI panel has given us worthy information regarding the epidemiology of pathogens detected in patients with acute diarrhea. It also highlights the importance of multi-pathogen infections and the frequency of diarrheogenic E. coli in foodborne disease outbreaks. More significantly, the rapid and multiple findings may help quickly taking an appropriate precaution, control and treatment.

Molecular evolution of GII.P17-GII.17 norovirus associated with sporadic acute gastroenteritis cases during 2013-2018 in Zhoushan Islands, China.

In this study, we investigated the molecular characteristics and spatio-temporal dynamics of GII.P17-GII.17 norovirus in Zhoushan Islands during 2013-2018. We collected 1849 samples from sporadic acute gastroenteritis patients between January 2013 and August 2018 in Zhoushan Islands, China. Among the 1849 samples, 134 (7.24%) samples were positive for human norovirus (HuNoV). The complete sequence of GII.17 VP1 gene was amplified from 31 HuNoV-positive samples and sequenced. A phylogenetic tree was constructed based on the full-length sequence of the VP1 gene. Phylogenetic analysis revealed that the GII.17 genotype detected during 2014-2018 belongs to the new GII.17 Kawasaki variant. Divergence analysis revealed that the time of the most recent common ancestor (TMRCA) of GII.17 in Zhoushan Islands was estimated to be between 1997 and 1998. The evolutionary rate of the VP1 gene of the GII.17 genotype norovirus was 1.14 × 10-3 (95% HPD: 0.62-1.73 × 10-3) nucleotide substitutions/site/year. The spatio-temporal diffusion analysis of the GII.17 genotype identified Hong Kong as the epicenter for GII.17 dissemination. The VP1 gene sequence of Zhoushan Island isolates correlated with that of Hong Kong and Japan isolates.

Rotavirus G9P[4], G9P[6] and G1P[6] strains isolated from children with acute gastroenteritis in Pune, western India, 2013-2015: evidence for recombination in genes encoding VP3, VP4 and NSP1.

Species A rotaviruses (RVAs) are genetically diverse pathogens. These are the most evolutionarily adaptable organisms, with a multitude of mechanisms for evolutionary change. To date, full-genome classification has been proved to be an excellent tool for studying the evolution of unusual rotavirus strains. As limited data are available from Pune (Maharashtra), western India, the current study was undertaken with the aim of understanding the genetic diversity in three (G1P[6], G9P[4] and G9P[4]) unusual RVA strains circulating in Pune, India during 2013-2015. Full-genome analysis of these strains classified them as G1-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1, G9-P[4]-I2-R2-C2-[M1-M2_R]-[A1-A2_R]-N2-T2-E6-H2 and G9-[P4-P6_R]-I1-R1-C1-M1-A1-N1-T1-E1-H1. Sequencing and phylogenetic analysis of the structural and non-structural genes of these unusual RVA strains showed nucleotide/amino acid identities of 82.3-98.5 %/77.3-99.8 % and 86.6-97.6 %/89.6-97.8 % between the strains of the study. Evidence of recombination events was found within the genes encoding VP3, VP4 and NSP1, which showed a combination of genetic information for genogroup 1 [M1/P[6]/A1] and genogroup 2 [M2/P[4]/A2] strains. This study will facilitate future investigations into the molecular pathogenesis of such RVAs as the exchange of whole or partial genetic material between rotaviruses through recombination contributes directly to their diversification, adaptation and evolution.

Genetic Diversity of Sapoviruses among Inpatients in Germany, 2008-2018.

Sapovirus enteric disease affects people of all ages across the globe, in both sporadic cases and outbreak settings. Sapovirus is seldom assessed in Germany and its epidemiology in the country is essentially unknown. Thus, sapovirus occurrence and genetic diversity were studied by real-time reverse transcription polymerase chain reaction (RT-PCR) and partial sequencing of major viral structural protein (VP1) gene in two different sets of stool samples: 1) a selection of 342 diarrheal stools collected from inpatient children during 2008-2009, and 2) 5555 stool samples collected during 2010-2018 from inpatients of all age groups with gastrointestinal complaints. Results showed year-round circulation of sapoviruses, with peaks during cooler months. In total, 30 samples (8.8%) of the first and 112 samples of the second set of samples (2.0%) were sapovirus positive. Capsid gene sequencing was successful in 134/142 samples (94.4%) and showed circulation of all known human pathogenic genogroups. Genotype GI.1 predominated (31.8%), followed by GII.1 (16.7%), GII.3 (14.5%), GI.2 (13.8%) and GV.1 (12.3%). Additionally, minor circulation of GI.3, GI.6, GII.2, GII.4, GII.6 and GIV.1 was shown. Consequently, sapovirus diagnostics need broadly reactive RT-PCR protocols and should particularly be considered in infants and young children. Further studies from other sampling sites are essential to extend our knowledge on sapovirus epidemiology in Germany.

Diversity of human parechovirus in infants and children with acute gastroenteritis in Japan during 2014-2016.

A total of 972 stool samples were collected from infants and children with acute gastroenteritis (AGE) in pediatric clinics encompassing six localities (Hokkaido, Tokyo, Shizuoka, Kyoto, Osaka, and Saga) in Japan during the 2-year period from July 2014 to June 2016. Sixty six of the samples (6.8%) were found to be positive for human parechovirus (HPeV) by multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) and subjected to genotyping based on viral protein 1 (VP1) sequences. Four different HPeV genotypes consisting of HPeV1, -3, -4 and -6 were detected, with HPeV1 clade B being predominant and followed by HPeV3 and -6. The first-time presence of HPeV1 clade A in Japan and rare HPeV4 were noted. This study provides up-to-date information on the genetic diversity of HPeV circulating in Japanese infants and children with AGE.

Recombinant GII.Pe-GII.4 Norovirus, Thailand, 2017-2018.

During June 2017-December 2018, norovirus was responsible for 10.9% of acute gastroenteritis cases in Thailand. Genogroup I (GI) was found in 14% of samples, of which 12 were co-infected with genogroup II (GII). In 35.8% of samples, GII.Pe-GII.4 Sydney predominated. Diverse recombinant strains of GI and GII norovirus co-circulated year-round.

Norovirus outbreaks in Beijing, China, from 2014 to 2017.

OBJECTIVES: Noroviruses are a leading cause of acute gastroenteritis (AGE) outbreaks worldwide. This study examined the epidemiology and genetic characteristics of norovirus outbreaks in Beijing, China. METHODS: Epidemiological data and fecal specimens were collected through the AGE outbreak surveillance system in Beijing. GI and GII genogroup noroviruses were detected and genotyped. The data were analyzed using descriptive statistics. RESULTS: Between September 2014 and August 2017, 762 AGE outbreaks were reported in Beijing, of which 661 (86.7%) were laboratory-confirmed as norovirus. Most norovirus outbreaks were reported during the spring (66.9%, 442/661), occurred in kindergartens and elementary schools (92.3%, 610/661), and were caused by GII genogroup noroviruses (95.6%; 632/661). The genotypes of the norovirus strains were determined in 468 outbreaks, and GII.P16-GII.2 and GII.P17-GII.17 strains were the most commonly identified. GII.P17-GII.17 and GII.P16-GII.2 strains predominated in 2014-2015 and 2016-2017 outbreaks, respectively. GII.P16-GII.2 noroviruses were responsible for a steep increase in AGE outbreaks in Beijing: 549 norovirus outbreaks were reported from 2016 to 2017, 9.2 times the number that occurred during the previous year. CONCLUSIONS: Norovirus causes a large disease burden in Beijing, and the prevalence of non-GII.4 noroviruses presents a new challenge for the development of vaccines.

Pediatric norovirus GII.4 infections in Nicaragua, 1999-2015.

OBJECTIVES: Investigate clinical and epidemiological factors of pediatric GII.4 norovirus infections in children with acute gastroenteritis (AGE) in Nicaragua between 1999 and 2015. METHODS: We retrospectively analyzed laboratory and epidemiologic data from 1,790 children≤7years with AGE from 6 hospitals in Nicaragua (n=538), and 3 community clinics (n=919) and households (n=333) in León, between 1999 and 2015. Moreover, asymptomatic children from community clinics (n=162) and households (n=105) were enrolled. Norovirus was detected by real-time PCR and genotyped by sequencing the N-terminal and shell region of the capsid gene. RESULTS: Norovirus was found in 19% (n=338) and 12% (n=32) of children with and without AGE, respectively. In total, 20 genotypes including a tentatively new genotype were detected. Among children with AGE, the most common genotypes were GII.4 (53%), GII.14 (7%), GII.3 (6%) and GI.3 (6%). In contrast, only one (1.4%) GII.4 was found in asymptomatic children. The prevalence of GII.4 infections was significantly higher in children between 7 and 12months of age. The prevalence of GII.4 was lowest in households (38%), followed by community clinics (50%) and hospitals (75%). Several different GII.4 variants were detected and their emergence followed the global temporal trend. CONCLUSIONS: Overall our study found the predominance of pediatric GII.4 norovirus infections in Nicaragua mostly occurring in children between 7 and 12months of age, implicating GII.4 as the main norovirus vaccine target.

Interrelationship of rotavirus infection and Creatine Kinase-MB isoenzyme levels in children hospitalized with acute gastroenteritis in Guangzhou, China, 2012-2015.

Elevated levels of Creatine Kinase-MB (CK-MB) Isoenzyme are a common phenomenon among rotavirus (RV) diarrhea. However, few studies have addressed this issue using large sample size. In current study, 1,118 children (age <5 years) hospitalized with diarrhea in Guangzhou Women and Children's Medical Center from 2012 to 2015 were finally included. Changing pattern of CK-MB and its relationship with RV-infection were analyzed and characterized. Multivariate linear regression models showed that RV-positive cases had a 28% rise in CK-MB compared to RV-negative cases (OR = 1.28, 95% CI: 1.15 to 1.41, P < 0.01) after controlling for age, gender, season of admission, and weight. The pattern of change showed that CK-MB level of RV-positive group started to rise immediately at the 1st day of diarrhea, reached the peak on days 2 to 4, declined during 4-9 days, and then reached a relatively stable level when compared to the RV-negative group. Mediation analyses showed that indirect effect of RV infection on the increase of CK-MB via Vesikari score was significant (ß = 8.01, P < 0.01), but direct effect was not (ß = 9.96, P = 0.12). Thus, elevated CK-MB value is a common finding in RV-infection and completely mediated by the severity of diarrhea. CK-MB monitoring may help to identify children with more severe viral infection.

Global Spread of Norovirus GII.17 Kawasaki 308, 2014-2016.

Analysis of complete capsid sequences of the emerging norovirus GII.17 Kawasaki 308 from 13 countries demonstrated that they originated from a single haplotype since the initial emergence in China in late 2014. Global spread of a sublineage SL2 was identified. A new sublineage SL3 emerged in China in 2016.

Norovirus GII.17 as Major Epidemic Strain in Italy, 2015-16.

In winter 2015-16, norovirus GII.17 Kawasaki 2014 emerged as a cause of sporadic gastroenteritis in children in Italy. Median patient age was higher for those with GII.17 than GII.4 infection (55 vs. 24 months), suggesting limited cross-protection for older children.

Genotype diversity and molecular evolution of noroviruses: A 30-year (1982-2011) comprehensive study with children from Northern Brazil.

A chronologically comprehensive 30-year study was conducted that involved children living in Belém, in the Amazon region of Northern Brazil, who participated in eight different studies from October 1982 to April 2011. The children were followed either in the community or in health units and hospitals in order to identify the norovirus genotypes involved in infections during this time. A total of 2,520 fecal specimens were obtained and subjected to RT-PCR and nucleotide sequencing for regions A, B, C, D and P2 of the viral genome. An overall positivity of 16.9% (n = 426) was observed, and 49% of the positive samples were genotyped (208/426), evidencing the presence of several genotypes as follows: Polymerase gene (GI.P4, GII.Pa, GII.Pc, GII.Pe, GII.Pg, GII.Pj, GII.P3, GII.P4, GII.P6, GII.P7, GII.P8, GII.P12, GII.P13, GII.P14, GII.P21, GII.P22), and VP1 gene (GI.3, GI.7, GII.1, GII.2, GII.3, GII.4, GII.6, GII.7, GII.8, GII.10, GII.12, GII.14, GII.17, GII.23). The GII.P4/GII.4 genotype determined by both open reading frames (ORFs) (partial polymerase and VP1 genes) was found for 83 samples, and analyses of the subdomain P2 region showed 10 different variants: CHDC (1970s), Tokyo (1980s), Bristol_1993, US_95/96, Kaiso_2003, Asia_2003, Hunter_2004, Yerseke_2006a, Den Haag_2006b (subcluster "O") and New Orleans_2009. Recombination events were confirmed in 47.6% (n = 20) of the 42 samples with divergent genotyping by ORF1 and ORF2 and with probable different breakpoints within the viral genome. The evolutionary analyses estimated a rate of evolution of 1.02 x 10-2 and 9.05 x 10-3 subs./site/year using regions C and D from the VP1 gene, respectively. The present research shows the broad genetic diversity of the norovirus that infected children for 30 years in Belém. These findings contribute to our understanding of noroviruses molecular epidemiology and viral evolution and provide a baseline for vaccine design.

Recombinant GII.P16-GII.2 Norovirus, Taiwan, 2016.

In Taiwan, acute gastroenteritis outbreaks caused by a new norovirus genotype GII.2 increased sharply toward the end of 2016. Unlike previous outbreaks, which often involved restaurants, GII.2 outbreaks mainly occurred in schools. Phylogenetic analysis indicates that these noroviruses are recombinant GII.P16-GII.2 strains.

Norovirus GII.P16/GII.2-Associated Gastroenteritis, China, 2016.

During October-December 2016, the number of norovirus outbreaks in China increased sharply from the same period during the previous 4 years. We identified a recombinant norovirus strain, GII.P16-GII.2, as the cause of 44 (79%) of the 56 outbreaks, signaling that this strain could replace the predominant GII.4 viruses.

The molecular epidemiology of norovirus outbreaks in Victoria, 2014 to 2015
.

Noroviruses are a leading cause of outbreaks of gastroenteritis. This study examined the incidence and molecular characteristics of norovirus outbreaks in healthcare and non-healthcare settings in Victoria, Australia, over 2 years (2014-2015). Norovirus was detected in 65.7% and 60.4% of gastroenteritis outbreaks investigated for the years 2014 and 2015 respectively. There was a significant decline in the number of norovirus outbreaks in the period 2014 to 2015 although in both years norovirus outbreaks peaked in the latter part of the year. Norovirus Open Reading Frame (ORF) 2 (capsid) genotypes identified included GI.2, GI.3, GI.4, GI.5, GI.6, GI.9, GII.2, GII.3, GII.4, GII.6, GII.7, GII.8, GII.13 and GII.17. GII.4 was the most common genotype detected. In addition, the following ORF 1/ORF 2 recombinant forms were confirmed: GII.P4_NewOrleans_2009/GII.4_Sydney_2012, GII.P12/GII.3, GII.Pb (GII.21)/GII.3, GII.Pe/GII.2 and GII.Pe/GII.4_Sydney_2012. A significant decline was noted in the chief norovirus strain GII.Pe/GII.4_Sydney_2012 between 2014 and 2015 but there was a re-emergence of a GII.P4_ NewOrleans _2009 norovirus strain. Outbreaks involving the GII.P17/GII.17 genotype were also detected for the first time in Victoria. GI genotypes circulating in Victoria for the 2 years 2014 and 2015 underwent a dramatic change between the 2 years of the survey. Many genotypes could occur in both healthcare and non-healthcare settings although GI.3, GII.6, and GII.4 were significantly more common in healthcare settings. The study emphasises the complex way in which norovirus circulates throughout the community.

OzFoodNet quarterly report, 1 April to 30 June 2015.

The Australian Government Department of Health established the OzFoodNet network in 2000 to collaborate nationally to investigate foodborne disease. In each Australian state and territory, OzFoodNet epidemiologists investigate outbreaks of enteric infection. In addition, OzFoodNet conducts studies on the burden of illness and coordinates national investigations into outbreaks of foodborne disease. This quarterly report documents investigations of outbreaks of gastrointestinal illness and clusters of disease potentially related to food, which commenced in Australia between 1 April and 30 June 2015. Data were received from OzFoodNet epidemiologists in all Australian states and territories. The data in this report are provisional and subject to change. During the 2nd quarter of 2015 (1 April to 30 June), OzFoodNet sites reported 352 outbreaks of enteric illness, including those transmitted by contaminated food or water. Outbreaks of gastroenteritis are often not reported to health authorities, which results in current figures under-representing the true burden of enteric disease outbreaks within Australia. There were 5,214 people affected in these outbreaks and 192 hospitalisations. There were 11 deaths reported during these outbreaks. This represents a decrease in the number of people affected compared with the 5-year average from 2010 to 2014 for the 2nd quarter (8,191). The majority of reported outbreaks of gastrointestinal illness in Australia are due to person-to-person transmission. In this quarter, 72% (255/352) of outbreaks were transmitted via this route (see Table 1). This percentage was similar to the same quarter in 2014 (73%, 305/419) but the total number is lower than the 5-year average (2nd quarter, 2010-2014) of 360 outbreaks transmitted person-to-person. Of the person-to-person outbreaks in the 2nd quarter of 2015, 47% (119/255) occurred in child care facilities and 40% (102/255) occurred in aged care facilities.

OzFoodNet quarterly report, 1 July to 30 September 2015.

The Australian Government Department of Health established the OzFoodNet network in 2000 to collaborate nationally to investigate foodborne disease. In each Australian state and territory, OzFoodNet epidemiologists investigate outbreaks of enteric infection. In addition, OzFoodNet conducts studies on the burden of illness and coordinates national investigations into outbreaks of foodborne disease. This quarterly report documents investigations of outbreaks of gastrointestinal illness and clusters of disease potentially related to food, which commenced in Australia between 1 July and 30 September 2015.

Caliciviruses in hospitalized children, São Luís, Maranhão, 1997-1999: detection of norovirus GII. 12

ABSTRACT Gastroenteritis is one of the most common diseases during childhood, with norovirus (NoV) and sapovirus (SaV) being two of its main causes. This study reports for the first time the incidence of these viruses in hospitalized children with and without gastroenteritis in São Luís, Maranhão. A total of 136 fecal samples were tested by enzyme immunoassays (EIA) for the detection of NoV and by reverse transcription-polymerase chain reaction (RT-PCR) for detection of both NoV and SaV. Positive samples for both agents were subjected to sequencing. The overall frequency of NoV as detected by EIA and RT-PCR was 17.6% (24/136) and 32.6% (15/46), respectively in diarrheic patients and 10.0% (9/90) in non-diarrheic patients (p < 0.01). Of the diarrheic patients, 17% had fever, vomiting and anorexia, and 13% developed fever, vomiting and abdominal pain. Of the 24 NoV-positive samples, 50% (12/24) were sequenced and classified as genotypes GII.3 (n = 1), GII.4 (6), GII.5 (1), GII.7 (2), GII.12 (1) and GII.16 (1). SaV frequency was 9.8% (11/112), with 22.6% (7/31) in diarrheic patients and 4.9% (4/81) in nondiarrheic (p = 0.04) ones. In diarrheic cases, 27.3% had fever, vomiting and anorexia, whereas 18.2% had fever, anorexia and abdominal pain. One SaV-positive sample was sequenced and classified as GII.1. These results show a high genetic diversity of NoV and higher prevalence of NoV compared to SaV. Our data highlight the importance of NoV and SaV as enteropathogens in São Luís, Maranhão.

Clinico-epidemiology and genetic diversity of Salivirus in acute gastroenteritis cases from Pune, Western India: 2007-2011.

Acute gastroenteritis is a leading cause of mortality in children from developing countries. Recently, Salivirus has been frequently detected in acute gastroenteritis patients, suggesting its possible aetiological role. Conflicting reports available on disease association of Salivirus have made it difficult to ascertain their causative role. The overall epidemiology and clinical features of Salivirus infections are poorly understood. The present five year study was undertaken to investigate the presence and genetic diversity of Salivirus in acute gastroenteritis cases from Pune, Western India and to determine the clinico-epidemiological features of Salivirus infections. A total of 985 faecal samples (778 acute gastroenteritis and 207 asymptomatic controls), collected from three local hospitals (Jan2007-Dec2011) were examined for the presence of Salivirus by RT-PCR. Molecular characterization was performed by PCR amplification of the 3D and VP regions. Frequency of Salivirus detection in cases (2.6%) and controls (1.93%) was not significantly different (p = 0.57). Co-infection with other enteric viruses was seen in 50% of the cases. Comparison of clinical features between Salivirus mono and mixed infections revealed that Salivirus alone did not exacerbate gastroenteritis. The frequency of diarrhoea and overall clinical severity of mixed infections was significantly greater than mono infections (p = 0.02). Based on clinical findings, our study suggests that Salivirus does not cause severe gastroenteritis. Phylogenetic analysis indicated that study strains belonged to Salivirus A1 and formed 2 distinct clusters which shared nucleotide identities of 94.1-96.2% and 88.9-93.8% between themselves in 3D and VP regions, respectively. Interestingly, the more divergent Cluster2 strains shared a low nucleotide identity with the closest reference strain in both regions (~95% in 3D and ~92% in VP) suggesting that they could represent a variant type of Salivirus A1. The genetic diversity in strains detected from study region, emphasizes the need for Salivirus surveillance from other regions of India.