Pierre Pothier

Increase in viral gastroenteritis outbreaks in Europe and epidemic spread of new norovirus variant

BACKGROUND Highly publicised outbreaks of norovirus gastroenteritis in hospitals in the UK and Ireland and cruise ships in the USA sparked speculation about whether this reported activity was unusual. METHODS We analysed data collected through a collaborative research and surveillance network of viral gastroenteritis in ten European countries (England and Wales were analysed as one region). We compiled data on total number of outbreaks by month, and compared genetic sequences from the isolated viruses. Data were compared with historic data from a systematic retrospective review of surveillance systems and with a central database of viral sequences. FINDINGS Three regions (England and Wales, Germany, and the Netherlands) had sustained epidemiological and viral characterisation data from 1995 to 2002. In all three, we noted a striking increase in norovirus outbreaks in 2002 that coincided with the detection and emergence of a new predominant norovirus variant of genogroup II4, which had a consistent mutation in the polymerase gene. Eight of nine regions had an annual peak in 2002 and the new genogroup II4 variant was detected in nine countries. Also, the detection of the new variant preceded an atypical spring and summer peak of outbreaks in three countries. INTERPRETATION Our data from ten European countries show a striking increase and unusual seasonal pattern of norovirus gastroenteritis in 2002 that occurred concurrently with the emergence of a novel genetic variant. In addition to showing the added value of an international network for viral gastroenteritis outbreaks, these observations raise questions about the biological properties of the variant and the mechanisms for its rapid dissemination.

Detection of Multiple Noroviruses Associated with an International Gastroenteritis Outbreak Linked to Oyster Consumption

ABSTRACT An international outbreak linked to oyster consumption involving a group of over 200 people in Italy and 127 total subjects in 13 smaller clusters in France was analyzed using epidemiological and clinical data and shellfish samples. Environmental information from the oyster-producing area, located in a lagoon in southern France, was collected to investigate the possible events leading to the contamination. Virologic analyses were conducted by reverse transcription-PCR (RT-PCR) using the same primer sets for both clinical and environmental samples. After sequencing, the data were analyzed through the database operated by the scientific network FoodBorne Viruses in Europe. The existence of an international collaboration between laboratories was critical to rapidly connect the data and to fully interpret the results, since it was not obvious that one food could be the link because of the diversity of the several norovirus strains involved in the different cases. It was also demonstrated that heavy rain was responsible for the accidental contamination of seafood, leading to a concentration of up to hundreds of genomic copies per oyster as detected by real-time RT-PCR.

Aichi Virus, Norovirus, Astrovirus, Enterovirus, and Rotavirus Involved in Clinical Cases from a French Oyster-Related Gastroenteritis Outbreak

ABSTRACT Following a flooding event close to a shellfish production lagoon, 205 cases of gastroenteritis were linked to oyster consumption. Twelve stool samples from different individuals were collected. Analysis showed that eight samples were positive for multiple enteric viruses, and one stool sample had seven different enteric viruses. Analysis of shellfish implicated in the outbreak allowed detection of the same diversity of enteric viruses, with some viral genomic sequences being identical to those obtained from stool sample analysis. Shellfish were contaminated by as many as five different enteric viruses. For the first time in Europe, Aichi virus was identified in oyster samples. Shellfish samples collected over 3 weeks following the outbreak showed a progressive decline in the level of virus contamination as measured by the virus diversity detected and by quantitative reverse transcription-PCR.

Use of Murine Norovirus as a Surrogate To Evaluate Resistance of Human Norovirus to Disinfectants

ABSTRACT Murine norovirus (MNV) was used as a surrogate to study resistance of human norovirus to disinfectants used in hospitals. MNV was sensitive to alcohol, alcohol hand rubs, bleach, and povidone iodine-based disinfectant. Real-time reverse transcription-PCR results indicated that the presence of viral RNA did not correlate with the presence of infectious virus.

Virus Diversity in a Winter Epidemic of Acute Diarrhea in France

ABSTRACT In France, an epidemic peak of acute diarrhea is observed each winter. Previous results suggested a viral etiology for these winter epidemics. We investigated the role of enteric viruses in acute diarrhea and their molecular diversity. One hundred sixty-one patients with acute diarrhea and 45 healthy patients (controls) from the general population were given a standardized questionnaire between December 1998 and May 1999. Stool specimens were screened for group A and C rotaviruses, human caliciviruses, astroviruses, and adenovirus types 40 and 41 by reverse transcription-PCR and/or enzyme immunoassay. Virologic analysis was positive for 63 cases (39%). Caliciviruses and group A rotaviruses were the most frequent (19 and 17% of cases, respectively). Two control stool specimens were found positive for group A rotavirus, and one was found positive for astrovirus. Molecular characterization of the strains disclosed a cocirculation of P[8],G1, P[8],G4, and P[4],G2 rotaviruses; type 1, 2, 3, 4, and 8 astroviruses; and Sapporo-like and Norwalk-like human caliciviruses. These four types of viruses accounted for an attributable risk of acute diarrhea of 34.7% for the general population, under the assumption of a causal role of these viruses.

Characterization of New Recombinant Noroviruses

ABSTRACT Noroviruses are important etiologic agents of acute gastroenteritis and show great genetic diversity. To characterize more fully previously detected strains that could not be assigned unequivocally to one particular genotype based on the RNA polymerase, we have sequenced a region in the capsid gene and, in some cases, in the junction between open reading frame 1 (ORF1) and ORF2. The results allowed us to identify several recombinant noroviruses: GGIIb viruses were detected for the first time in France in August 2000 and then spread through France and to Europe during the following winter. Here we present the characterization of three other probable GII recombinants which showed different phylogenetic positions depending on their ORF1 and ORF2 sequences. Analysis of the region located between ORF1 and ORF2 by a nucleotide identity window search showed a sudden shift in similarities. Moreover, recombination breakpoints were identified upstream and downstream of the beginning of ORF2 by using a statistical test, thus confirming the involvement of this region in recombination. Unlike GGIIb, the three recombinants described here do not seem to have diffused widely in the community: one was found in a waterborne outbreak, and the other two were found in sporadic cases. Recombination is important for the evolution of RNA viruses and has already been described for noroviruses. Our results suggest that recombination is not a rare phenomenon among noroviruses, but not all these presumed recombinants that formed during RNA replication are able to spread widely.

European Multicenter Evaluation of Commercial Enzyme Immunoassays for Detecting Norovirus Antigen in Fecal Samples

ABSTRACT A total of 2,254 fecal samples were tested in a European multicenter evaluation of commercially available norovirus antigen detection assays. Two commercial enzyme immunoassays, IDEIA Norovirus (Oxoid; Thermo Fisher Scientific, Ely, United Kingdom) and RIDASCREEN Norovirus (R-Biopharm, Darmstadt, Germany), were included in the evaluation, and their performance was compared with the results of reverse transcription-PCR (RT-PCR). Included in the evaluation were samples collected in sporadic cases of gastroenteritis, samples from outbreaks in which two or more samples were collected, well-characterized samples representing genotypes currently cocirculating within Europe, and samples collected from patients with gastroenteritis caused by a pathogen other than norovirus. The sensitivities and specificities of the IDEIA Norovirus and RIDASCREEN Norovirus assays were 58.93 and 43.81% and 93.91 and 96.37%, respectively, compared with RT-PCR. The sensitivities of both assays for outbreak investigations improved when six or more samples from an outbreak were examined. The IDEIA Norovirus assay exhibited reactivity to a broader range of norovirus genotypes than the RIDASCREEN Norovirus assay, which showed genotype-dependent sensitivities. The results indicate that, if used, these assays should serve as screening assays and the results should be confirmed by RT-PCR.

Impact of Norovirus/Sapovirus-Related Diarrhea in Renal Transplant Recipients Hospitalized for Diarrhea

Background. Diarrhea of unspecified cause frequently occurs after renal transplantation and is usually ascribed to mycophenolic acid toxicity. Norovirus (NoV) and sapovirus (SaV) have been sporadically reported to cause chronic diarrhea in immunocompromised patients. Methods. We undertook a retrospective study (2008–2009) to examine the clinical and epidemiologic significance of NoV and SaV infections in adult renal transplant recipients hospitalized for acute or chronic diarrhea. Results. Ninety-six renal transplant recipients were hospitalized for diarrhea at our institution during a 16-month period, 87 of whom were included in the study, including 46 patients with chronic diarrhea. Among 41 patients with unexplained diarrhea, 20 patients were screened for NoV/SaV, 16 of whom were positive. Fifteen of them (94%) had chronic diarrhea. When compared with bacterial and parasitic infections, NoV/SaV infections were associated with a greater weight loss at the time of admission, a 8.7-fold longer duration of symptoms and a more frequent need for mycophenolic acid dosage reduction. Eighty-one percent of patients hospitalized for NoV/SaV-associated diarrhea experienced acute renal failure. Five and one patients subsequently had biopsy-diagnosed active graft rejection and oxalate nephropathy, respectively. Ten of the 14 patients who underwent a longitudinal study of NoV/SaV stools clearance exhibited a prolonged viral shedding period with a median time of 289 days (107–581 days). Conclusions. Our study indicates that NoV/SaV infection causes posttransplant chronic diarrhea potentially complicated by severe kidney graft impairment.

Diversity and zoonotic potential of rotaviruses in swine and cattle across Europe

Group A rotaviruses can infect both humans and animals. Individual rotavirus strains can occasionally cross species barriers and might hereby contribute to the emergence of new genotypes in heterologous hosts. The incidence and impact of zoonotic rotavirus are not well defined, and one reason for this is a lack of data about strains circulating in suspected reservoir animal hosts. In this study we report the incidence, genetic diversity, and molecular epidemiology of rotaviruses detected in domestic cattle and swine in 6 European countries. From 2003 to 2007, 1101 and more than 2000 faecal specimens were collected from swine and cattle, both healthy and diarrhoeic, and tested for rotaviruses. Viruses from positive stools were genotyped and a subset of strains was characterized by nucleotide sequencing and phylogenetic analysis of the VP7 (G) and VP4 (P) genes. Rotaviruses were detected in 43% of bovine samples and in 14% of porcine samples. In cattle, 10 different combinations of G and P types were identified and the most common strains were G6P[11] and G6P[5]. In swine, the number of identified G-P combinations was higher (n=21), however, no single combination was predominant across Europe. Newly described genotype specificities, P[27] and P[32], were identified in swine. When compared at the nucleotide sequence level, the identified porcine rotavirus strains and contemporary human strains grouped together phylogenetically, whereas bovine rotavirus strains formed separate clades. These data demonstrate large genetic diversity of porcine and bovine rotavirus strains across Europe, and suggest that livestock herds may serve as potential reservoirs for human infections.

Comprehensive Analysis of a Norovirus-Associated Gastroenteritis Outbreak, from the Environment to the Consumer

ABSTRACT Noroviruses have been recognized to be the predominant agents of nonbacterial gastroenteritis outbreaks in humans, and their transmission via contaminated shellfish consumption has been demonstrated. Norovirus laboratory experiments, volunteer challenge studies, and community gastroenteritis outbreak investigations have identified human genetic susceptibility factors related to histo-blood group antigen expression. Following a banquet in Brittany, France, in February 2008, gastroenteritis cases were linked to oyster consumption. This study identified an association of the norovirus illnesses with histo-blood group expression, and oyster contamination with norovirus was confirmed by qualitative and quantitative analyses. The secretor phenotype was associated with illness, especially for the non-A subgroup. The study showed that, in addition to accidental climatic events that may lead to oyster contamination, illegal shellfish collection and trading are also risk factors associated with outbreaks.