Development of an Extraction Method to Detect Hepatitis A Virus, Hepatitis E Virus, and Noroviruses in Fish Products

Viruses are a leading cause of foodborne disease worldwide. Hepatitis viruses (hepatitis A (HAV) and hepatitis E (HEV)) and human norovirus are recognized as the main viruses of public health concern in food hygiene. ISO 15216 approved procedures are not validated for detection of HAV and human norovirus in foodstuffs, such as fishes, leading to an inability to ensure the safety of these products. This study aimed to provide a rapid and sensitive method for detecting these targets in fish products. An existing method that includes proteinase K treatment was selected for further validation using artificially contaminated fish products, according to the recent international standard ISO 16140-4. Recovery efficiencies in pure RNA extracts of viruses ranged from 0.2% to 66.2% for HAV, 4.0% to 100.0% for HEV, 2.2% to 100.0% for norovirus GI, and 0.2% to 12.5% for norovirus GII. LOD50 values were between 144 and 8.4 × 104 genome copies/g for HAV and HEV, and 104 and 2.0 × 103 copies/g for norovirus GI and norovirus GII, respectively. LOD95 values were between 3.2 × 103 and 3.6 × 105 genome copies/g for HAV and HEV, and between 8.8 × 103 and 4.4 × 104 genome copies/g for norovirus GI and norovirus GII, respectively. The method developed here was successfully validated in various fish products and can be applied for routine diagnostic needs

Detection of norovirus, hepatitis A and hepatitis E viruses in multicomponent foodstuffs

International audienceAmong the enteric viruses implicated in foodborne outbreaks, the human norovirus and hepatitis viruses A and E (HAV and HEV) represent a serious public health concern. International standard ISO 15216 proposes methods for detecting HAV and norovirus (genogroups I and II) RNA from soft fruit, leaf, stem and bulb vegetables, bottled water or food surfaces. These methods had not previously been validated for detecting the targeted viruses in other foodstuffs such as multicomponent foods, nor for detecting other viruses in foodstuffs. The aim of this study was to characterise a method derived from the vegetable method described in ISO 15216 to detect HAV, HEV and norovirus in artificially-contaminated multicomponent foodstuffs according to the recent international standard ISO 16140-4. Results showed that the mean recovery rates for all settings did not differ according to the operator. The mean extraction yields ranged from 0.35% to 40.44% for HAV, 5.19% to 100% for HEV, 0.10% to 40.61% for norovirus GI and 0.88% to 69.16% for norovirus GII. The LOD(95) was 10(2) genome copies/g for HAV, HEV and norovirus GII and 10(3) genome copies/g for norovirus GI. The LOQ was 2.90 × 10(4), 1.40 × 10(3), 1.60 × 10(4) and 1.30 × 10(4) genome copies/g for HAV, HEV, norovirus GI and norovirus GII respectively. The MNV-1 process control was detected in 120 out of 128 RNA extracts analysed and was recovered with an efficiency of between 3.83% and 50.22%. The mean inhibition rates of quantitative real-time RT-PCR reaction ranged from 3.25% to 28.70% and varied significantly with the type of food matrix. The described method could be used to detect viruses in composite food products for routine diagnosis needs

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

Discrimination of infectious and heat-treated norovirus by combining platinum compounds and real-time RT-PCR

International audienceHuman noroviruses (NoV) are major agents of foodborne outbreaks. Because of the lack of a standardized cell culture method, real-time reverse transcriptase PCR is now commonly used for the detection of NoV in foodstuffs and environmental samples. However, this approach detects the viral nucleic acids of both infectious and non-infectious viruses and needs to be optimized to predict infectivity for public health risk assessment. The aim of this study was to develop a viability PCR method to discriminate between native and heat-treated virus, for both NoV and its surrogate, murine norovirus (MNV).To this end, screening of viability markers (monoazide dyes, platinum and palladium compounds) was performed on viral RNA, native virus or heat-treated virus, and incubation conditions were optimized with PtCl4, the most efficient viability marker. Multiple MNV molecular models were designed: no impact of amplicon length was observed on inactivated MNV genomic titer; but the 5′NTR, ORF1 and 3′UTR regions resulted in higher reductions than central genomic regions. The optimal viability PCR conditions developed (incubation with 2.5 mM PtCl4 in PBS for 10 min at 5 °C) were finally applied to MNV by performing heat inactivation studies and to native and heat-treated NoV clinical strains. The viability PCR discriminated efficiently between native and heat-inactivated MNV at 72 °C and 80 °C, and efficiently reduced the genomic titer of heat-treated NoV strains.This viability PCR method could be useful to study heat inactivation kinetics of NoV and MNV. It could also be evaluated for the identification of infectious enteric viruses in foodstuffs and environmental samples

GUIP1: a R package for dose escalation strategies in phase I cancer clinical trials

International audienceBACKGROUND: The main objective of phase I cancer clinical trials is to identify the maximum tolerated dose, usually defined as the highest dose associated with an acceptable level of severe toxicity during the first cycle of treatment. Several dose-escalation designs based on mathematical modeling of the dose-toxicity relationship have been developed. The main ones are: the continual reassessment method (CRM), the escalation with overdose control (EWOC) method and, for late-onset and cumulative toxicities, the time-to-event continual reassessment method (TITE-CRM) and the time-to-event escalation with overdose control (TITE-EWOC) methods. The objective of this work was to perform a user-friendly R package that combines the latter model-guided adaptive designs. RESULTS: GUIP1 is an R Graphical User Interface for dose escalation strategies in Phase 1 cancer clinical trials. It implements the CRM (based on Bayesian or maximum likelihood estimation), EWOC and TITE-CRM methods using the dfcrm and bcrm R packages, while the TITE-EWOC method has been specifically developed. The program is built using the TCL/TK programming language, which can be compiled via R software libraries (tcltk, tkrplot, tcltk2). GUIP1 offers the possibility of simulating and/or conducting and managing phase I clinical trials in real-time using file management options with automatic backup of study and/or simulation results. CONCLUSIONS: GUIP1 is implemented using the software R, which is widely used by statisticians in oncology. This package simplifies the use of the main model-based dose escalation methods and is designed to be fairly simple for beginners in R. Furthermore, it offers multiple possibilities such as a full traceability of the study. By including multiple innovative adaptive methods in a free and user-friendly program, we hope that GUIP1 will promote and facilitate their use in designing future phase I cancer clinical tria

Comparison of RT-qPCR, RT-dPCR and RT-PCR array assays.

<p>Characteristics of standard curves based on the RT-qPCR assays and limit of detection (LOD) of viral targets by RT-qPCR, by RT-dPCR and RT-PCR array assays. The differences between relative quantification (by RT-qPCR) and absolute quantification (by RT-dPCR) were indicated.</p