Sylvain Parnaudeau

Evaluation of Removal of Noroviruses during Wastewater Treatment, Using Real-Time Reverse Transcription-PCR: Different Behaviors of Genogroups I and II

ABSTRACT Noroviruses, an important cause of gastroenteritis, are excreted by infected individuals and are therefore present in wastewater. We quantified norovirus genogroup I (GI) and GII in wastewater at different locations in France and evaluated removal by a range of treatment types, including basic (waste stabilization pond), current industry standard (activated sludge), and state-of-the-art (submerged membrane bioreactor) treatments. Noroviruses were quantified using real-time reverse transcription-PCR (rRT-PCR). Mengovirus was used as a virus extraction control, and internal controls were used to verify the level of GI and GII rRT-PCR inhibition. A total of 161 (81 influent and 79 effluent) samples were examined; GI and GII were detected in 43 and 88% of the influent samples, respectively, and in 24 and 14% of the effluent samples, respectively. Physicians in France report far more cases of GII than GI during outbreaks; thus, the frequent presence of GI was unexpected. The GI influent concentrations were more variable, the peak GI influent concentrations were higher than the peak GII influent concentrations at all four sites (up to 1 × 109 and 6 × 107 genome copies/liter, respectively), and the average positive influent concentrations of GI were higher than the average positive influent concentrations of GII. The maximum effluent breakthrough concentrations were 6 × 106 and 3 × 106 genome copies/liter for GI and GII, respectively, indicating that the four treatment systems studied decreased the norovirus contamination load in receiving waters.

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.

Detection and quantification of noroviruses in shellfish.

ABSTRACT Noroviruses (NoVs) are the most common viral agents of acute gastroenteritis in humans, and high concentrations of NoVs are discharged into the environment. As these viruses are very resistant to inactivation, the sanitary consequences are contamination of food, including molluscan shellfish. There are four major problems with NoV detection in shellfish samples: low levels of virus contamination, the difficulty of efficient virus extraction, the presence of interfering substances that inhibit molecular detection, and NoV genetic variability. The aims of this study were to adapt a kit for use with a method previously shown to be efficient for detection of NoV in shellfish and to use a one step real-time reverse transcription-PCR method with addition of an external viral control. Comparisons of the two methods using bioaccumulated oysters showed that the methods reproducibly detected similar levels of virus in oyster samples. Validation studies using naturally contaminated samples also showed that there was a good correlation between the results of the two methods, and the variability was more attributable to the level of sample contamination. Magnetic silica very efficiently eliminated inhibitors, and use of extraction and amplification controls increased quality assurance. These controls increased the confidence in estimates of NoV concentrations in shellfish samples and strongly supported the conclusion that the results of the method described here reflected the levels of virus contamination in oysters. This approach is important for food safety and is under evaluationfor European regulation.

Strain-Dependent Norovirus Bioaccumulation in Oysters

ABSTRACT Noroviruses (NoVs) are the main agents of gastroenteritis in humans and the primary pathogens of shellfish-related outbreaks. Some NoV strains bind to shellfish tissues by using carbohydrate structures similar to their human ligands, leading to the hypothesis that such ligands may influence bioaccumulation. This study compares the bioaccumulation efficiencies and tissue distributions in oysters (Crassostrea gigas) of three strains from the two principal human norovirus genogroups. Clear differences between strains were observed. The GI.1 strain was the most efficiently concentrated strain. Bioaccumulation specifically occurred in digestive tissues in a dose-dependent manner, and its efficiency paralleled ligand expression, which was highest during the cold months. In comparison, the GII.4 strain was very poorly bioaccumulated and was recovered in almost all tissues without seasonal influence. The GII.3 strain presented an intermediate behavior, without seasonal effect and with less bioaccumulation efficiency than that of the GI.1 strain during the cold months. In addition, the GII.3 strain was transiently concentrated in gills and mantle before being almost specifically accumulated in digestive tissues. Carbohydrate ligand specificities of the strains at least partly explain the strain-dependent bioaccumulation characteristics. In particular, binding to the digestive-tube-specific ligand should contribute to bioaccumulation, whereas we hypothesize that binding to the sialic acid-containing ligand present in all tissues would contribute to retain virus particles in the gills or mantle and lead to rapid destruction.

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.

Calicivirus Removal in a Membrane Bioreactor Wastewater Treatment Plant

ABSTRACT To evaluate membrane bioreactor wastewater treatment virus removal, a study was conducted in southwest France. Samples collected from plant influent, an aeration basin, membrane effluent, solid sludge, and effluent biweekly from October 2009 to June 2010 were analyzed for calicivirus (norovirus and sapovirus) by real-time reverse transcription-PCR (RT-PCR) using extraction controls to perform quantification. Adenovirus and Escherichia coli also were analyzed to compare removal efficiencies. In the influent, sapovirus was always present, while the norovirus concentration varied temporally, with the highest concentration being detected from February to May. All three human norovirus genogroups (GI, GII, and GIV) were detected in effluent, but GIV was never detected in effluent; GI and GII were detected in 50% of the samples but at low concentrations. In the effluent, sapovirus was identified only once. An adenovirus titer showing temporal variation in influent samples was identified only twice in effluent. E. coli was always below the limit of detection in the effluent. Overall, the removal of calicivirus varied from 3.3 to greater than 6.8 log units, with no difference between the two main genogroups. Our results also demonstrated that the viruses are blocked by the membrane in the treatment plant and are removed from the plant as solid sludge.

Detection of human enteric viruses in shellfish collected in Tunisia

Aims:  The aim of this study was to detect the main pathogenic human RNA enteric viruses able to persist in the environment such as astrovirus, enterovirus, norovirus and hepatitis A virus (HAV) in shellfish collected from two locations in northern Tunisia.

Environmental Detection of Genogroup I, II, and IV Noroviruses by Using a Generic Real-Time Reverse Transcription-PCR Assay

ABSTRACT Norovirus is the most common agent implicated in food-borne outbreaks and is frequently detected in environmental samples. These viruses are highly diverse, and three genogroups (genogroup I [GI], GII, and GIV) infect humans. Being noncultivable viruses, real-time reverse transcription-PCR (RT-PCR) is the only sensitive method available for their detection in food or environmental samples. Selection of consensus sequences for the design of sensitive assays has been challenging due to sequence diversity and has led to the development of specific real-time RT-PCR assays for each genogroup. Thus, sample screening can require several replicates for amplification of each genogroup (without considering positive and negative controls or standard curves). This study reports the development of a generic assay that detects all three human norovirus genogroups on a qualitative basis using a one-step real-time RT-PCR assay. The generic assay achieved good specificity and sensitivity for all three genogroups, detected separately or in combination. At variance with multiplex assays, the choice of the same fluorescent dye for all three probes specific to each genogroup allows the levels of fluorescence to be added and may increase assay sensitivity when multiple strains from different genogroups are present. When it was applied to sewage sample extracts, this generic assay successfully detected norovirus in all samples found to be positive by the genogroup-specific RT-PCRs. The generic assay also identified all norovirus-positive samples among 157 archived nucleic acid shellfish extracts, including samples contaminated by all three genogroups.

Digital PCR for Quantifying Norovirus in Oysters Implicated in Outbreaks, France

Using samples from oysters clearly implicated in human disease, we quantified norovirus levels by using digital PCR. Concentrations varied from 43 to 1,170 RNA copies/oyster. The analysis of frozen samples from the production area showed the presence of norovirus 2 weeks before consumption.