Sylvie Perelle

A comparative study of digital RT-PCR and RT-qPCR for quantification of Hepatitis A virus and Norovirus in lettuce and water samples

Sensitive and quantitative detection of foodborne enteric viruses is classically achieved by quantitative RT-PCR (RT-qPCR). Recently, digital PCR (dPCR) was described as a novel approach to genome quantification without need for a standard curve. The performance of microfluidic digital RT-PCR (RT-dPCR) was compared to RT-qPCR for detecting the main viruses responsible for foodborne outbreaks (human Noroviruses (NoV) and Hepatitis A virus (HAV)) in spiked lettuce and bottled water. Two process controls (Mengovirus and Murine Norovirus) were used and external amplification controls (EAC) were added to examine inhibition of RT-qPCR and RT-dPCR. For detecting viral RNA and cDNA, the sensitivity of the RT-dPCR assays was either comparable to that of RT-qPCR (RNA of HAV, NoV GI, Mengovirus) or slightly (around 1 log10) decreased (NoV GII and MNV-1 RNA and of HAV, NoV GI, NoV GII cDNA). The number of genomic copies determined by dPCR was always from 0.4 to 1.7 log10 lower than the expected numbers of copies calculated by using the standard qPCR curve. Viral recoveries calculated by RT-dPCR were found to be significantly higher than by RT-qPCR for NoV GI, HAV and Mengovirus in water, and for NoV GII and HAV in lettuce samples. The RT-dPCR assay proved to be more tolerant to inhibitory substances present in lettuce samples. This absolute quantitation approach may be useful to standardize quantification of enteric viruses in bottled water and lettuce samples and may be extended to quantifying other human pathogens in food samples.

Screening for clostridium botulinum type A, B, and E in cooked chilled foods containing vegetables and raw material using polymerase chain reaction and molecular probes.

A molecular method was used for the detection of Clostridium botulinum spores of type A, B, and E in commercial cooked and pasteurized vegetable purées and in the raw materials (vegetables and other ingredients). The method allowed the detection of less than 8 spores/g of product for C. botulinum type A, less than 1 spore/g for proteolytic type B, less than 21 spores/g for nonproteolytic type B, and less than 0.1 spore/g for type E. Thirty-seven samples of raw vegetables and ingredients were tested for the presence of C. botulinum type A, B, and E; 88 and 90 samples of vegetable purées were tested, respectively, for the presence of C. botulinum type A and B and for the presence of C. botulinum type E. All samples were negative, suggesting that the prevalence of C. botulinum in these vegetable purées and the raw ingredients is probably low.

Method for HEV detection in raw pig liver products and its implementation for naturally contaminated food.

It is now recognized that Hepatitis E virus (HEV) infection is not confined to developing countries. HEV infection is a growing public health concern in industrialized countries where the disease is mainly autochthonous, caused by HEV genotypes 3 and 4 and is today considered to be zoonotic. HEV causes acute hepatitis in humans, predominantly through contamination of food and water. Due to the low concentrations found in food and water samples, an efficient and rapid virus concentration method is required for routine control. Because of the absence of a reliable cell culture method for the main enteric viruses most commonly involved in the outbreaks, reverse transcription quantitative real time PCR (RT-qPCR) is now widely used for the detection of RNA viruses in all types of samples. The aim of this study was to provide a rapid and sensitive method for detecting HEV in pig liver products. A method which includes a virus concentration step by PEG has been chosen from 9 protocols to be further validated. We used a one-step duplex RT-qPCR for detecting HEV and the murine norovirus (MNV-1) used as a process control for monitoring the quality of the whole extraction procedure. The mean recovery rates of the HEV and MNV-1 obtained from pig liver sausages were respectively 3.94% and 2.92%, increasing in figatelli to 18.38% and 13.11% respectively. This method also proved to be effective for HEV detection in naturally contaminated foodstuffs containing raw pig liver.

Determination of which virus to use as a process control when testing for the presence of hepatitis A virus and norovirus in food and water

Noroviruses (genogroup I (NoV GI) and genogroup II (NoV GII)) and the hepatitis A virus (HAV) are frequently involved in foodborne infections worldwide. They are mainly transmitted via the fecal-oral route, direct person-to-person contact or consumption of contaminated water and foods. In food virology, detection methods are currently based on identifying viral genomes using real-time reverse transcriptase PCR (RT-qPCR). One of the general requirements for detecting these viruses in food involves the use of a process control virus to monitor the quality of the entire viral extraction procedure as described in the ISO/TS 15216-1 and 15216-2 standards published in 2013. The selected process control virus should have similar morphological and physicochemical properties as the screened pathogenic virus and thus have the potential to provide comparable extraction efficiency. The aim of this study was to determine which virus should be used for process control, murine norovirus (MNV-1) or Mengovirus, when testing for the presence of HAV, NoV GI and NoV GII in bottled water, lettuce and semi-dried tomatoes. Food samples were spiked with HAV, NoV GI or NoV GII alone or in the presence of MNV-1 or Mengovirus. Recovery rates of each pathogenic virus were compared to those of both process control viruses using a multiple comparison procedure. Neither process control virus influenced the recovery of pathogenic virus regardless of the type of food matrix. MNV-1 was the most appropriate virus for validating the detection of HAV and NoV GII in all three food matrices as well as NoV GI in lettuce. Mengovirus proved to be the most appropriate control for NoV GI detection in bottled water and semi-dried tomatoes. The process control virus is essential for validating viral detection in food and the choice of virus depends on food type and the screened pathogenic virus.

Digital RT-PCR method for hepatitis A virus and norovirus quantification in soft berries.

Raw fruits may harbour many pathogens of public health concern including enteric viruses, which are the leading cause of foodborne outbreaks. Recently, consumption of soft berries has been associated with increasing reports of norovirus and hepatitis A virus outbreaks in Europe. Due to their low infectious doses and low concentrations in food samples, an efficient and sensitive analytical method is required for virus detection. In this study we explored two different ways to improve the reference method for the detection of enteric viruses in soft fruits (ISO/TS 15216-1; 15216-2): an additional purification step after RNA extraction; and the detection of enteric viral genome by an absolute quantification method (microfluidic digital RT-PCR). Both of these approaches led to an improvement of enteric virus detection in soft berries by greatly lowering PCR inhibition, raising viral extraction efficiencies and enabling validation of controls using pure RNA extracts. The PCR inhibitor removal step can be easily included in the routine method. Absolute quantification by digital RT-PCR may be a relevant alternative method to standardize quantification of enteric viruses in foodstuffs.

Quantification of Hepatitis E Virus in Naturally-Contaminated Pig Liver Products

Hepatitis E virus (HEV), the cause of self-limiting acute hepatitis in humans, is widespread and endemic in many parts of the world. The foodborne transmission of HEV has become of concern due to the identification of undercooked pork products as a risk factor for infection. Foodborne enteric viruses are conventionally processed by quantitative RT-PCR (RT-qPCR), which gives sensitive and quantitative detection results. Recently, digital PCR (dPCR) has been described as a novel approach to genome quantification with no need for a standard curve. The performance of microfluidic digital RT-PCR (RT-dPCR) was compared to RT-qPCR when detecting HEV in pig liver products. The sensitivity of the RT-dPCR assay was similar to that of RT-qPCR, and quantitative data obtained by both detection methods were not significantly different for almost all samples. This absolute quantification approach may be useful for standardizing quantification of HEV in food samples and may be extended to quantifying other human pathogens in food samples.

Hepatitis A virus subgenotyping based on RT-qPCR assays

BackgroundThe hepatitis A virus (HAV) is the most frequent cause of viral hepatitis worldwide and is recognized as one of the most widespread foodborne pathogens. HAV genotypes and subtypes differ in their geographic distribution and the incidence of HAV infection varies considerably among countries, and is particularly high in areas with poor sanitation and hygiene. Phylogenetic analyses are traditionally used in clinical microbiology for tracing the geographic origin of HAV strains. In food microbiology, this approach is complicated by the low contamination levels of food samples. To date, real-time reverse-transcription PCR has been one of the most promising detection methods due to its sensitivity, specificity and ability to deliver quantitative data in food samples, but it does not provide HAV subtyping information.ResultsSix subtype-specific RT-qPCR assays were developed for human HAV. The limit of detection of HAV was 50 genome copies/assay for subtype IIB, 500 genome copies assay for IA, IB, IIA and IIIB and 5000 genome copies/assay for IIIA. The specificity of the assays was evaluated by testing reference isolates and in vitro HAV RNA transcripts. No significant cross reactivity was observed. Subtyping results concordant with sequencing analysis were obtained from 34/35 clinical samples. Co-infection with a minor strain of a different subtype was suggested in 5 cases and a recombinant event in one case.ConclusionsThese RT-qPCR assays may be particularly useful for accurately tracing HAV in low-level contaminated samples such as food matrices but also to allow co-infection identification in human samples.

A Novel High-Throughput Method for Molecular Detection of Human Pathogenic Viruses Using a Nanofluidic Real-Time PCR System

Human enteric viruses are recognized as the main causes of food- and waterborne diseases worldwide. Sensitive and quantitative detection of human enteric viruses is typically achieved through quantitative RT-PCR (RT-qPCR). A nanofluidic real-time PCR system was used to develop novel high-throughput methods for qualitative molecular detection (RT-qPCR array) and quantification of human pathogenic viruses by digital RT-PCR (RT-dPCR). The performance of high-throughput PCR methods was investigated for detecting 19 human pathogenic viruses and two main process controls used in food virology. The conventional real-time PCR system was compared to the RT-dPCR and RT-qPCR array. Based on the number of genome copies calculated by spectrophotometry, sensitivity was found to be slightly better with RT-qPCR than with RT-dPCR for 14 viruses by a factor range of from 0.3 to 1.6 log10. Conversely, sensitivity was better with RT-dPCR than with RT-qPCR for seven viruses by a factor range of from 0.10 to 1.40 log10. Interestingly, the number of genome copies determined by RT-dPCR was always from 1 to 2 log10 lower than the expected copy number calculated by RT-qPCR standard curve. The sensitivity of the RT-qPCR and RT-qPCR array assays was found to be similar for two viruses, and better with RT-qPCR than with RT-qPCR array for eighteen viruses by a factor range of from 0.7 to 3.0 log10. Conversely, sensitivity was only 0.30 log10 better with the RT-qPCR array than with conventional RT-qPCR assays for norovirus GIV detection. Finally, the RT-qPCR array and RT-dPCR assays were successfully used together to screen clinical samples and quantify pathogenic viruses. Additionally, this method made it possible to identify co-infection in clinical samples. In conclusion, given the rapidity and potential for large numbers of viral targets, this nanofluidic RT-qPCR assay should have a major impact on human pathogenic virus surveillance and outbreak investigations and is likely to be of benefit to public health.

Prevalence of Human Noroviruses in Frozen Marketed Shellfish, Red Fruits and Fresh Vegetables

Noroviruses (NoVs), currently recognised as the most common human food-borne pathogens, are ubiquitous in the environment and can be transmitted to humans through multiple foodstuffs. In this study, we evaluated the prevalence of human NoV genogroups I (GI) and II (GII) in 493 food samples including soft red fruits (nxa0=xa0200), salad vegetables (nxa0=xa0210) and bivalve mollusc shellfish (nxa0=xa083), using the Bovine Enterovirus type 1 as process extraction control for the first time. Viral extractions were performed by elution concentration and genome detection by TaqMan Real-Time RT-PCR (RT-qPCR). Experimental contamination using hepatitis A virus (HAV) was used to determine the limit of detection (LOD) of the extraction methods. Positive detections were obtained from 2xa0g of digestive tissues of oysters or mussels kept for 16xa0h in seawater containing 2.0–2.7 log10 plaque-forming units (PFU)/L of HAV. For lettuces and raspberries, the LOD was, respectively, estimated at 2.2 and 2.9 log10xa0PFU per 25xa0g. Of the molluscs tested, 8.4 and 14.4xa0% were, respectively, positive for the presence of GI NoV and GII NoV RNA. Prevalence in GI NoVs varied from 11.9xa0% for the salad vegetables samples to 15.5xa0% for the red soft fruits. Only 0.5xa0% of the salad and red soft fruits samples were positive for GII NoVs. These results highlight the high occurrence of human NoVs in foodstuffs that can be eaten raw or after a moderate technological processing or treatment. The determination of the risk of infection associated with an RT-qPCR positive sample remains an important challenge for the future.

Norovirus GII.17 Outbreak Linked to an Infected Post-Symptomatic Food Worker in a French Military Unit Located in France

In February 2016, an outbreak of gastroenteritis occurred in a French military unit located in Poitiers, France. Attack rate was of 34% (103/300). A case–control study identified association between illness and cake consumption. Stool samples were tested positive for Norovirus GII.17 for one patient and one post-symptomatic food worker (FW). The FW presented vomiting one day before cake preparation. The NoV strain was probably spread through food worker hand contact. Prevention of Norovirus foodborne outbreaks implies new guidelines for FWs management in France and Europe.