Evaluation of a norovirus detection methodology for ready-to-eat foods

Despite recent norovirus (NoV) foodborne outbreaks related to consumption of ready-to-eat (RTE) foods, a standardized assay to detect NoV in these foods is not available yet. Therefore, the robustness of a methodology for NoV detection in RTE foods was evaluated. The NoV detection methodology consisted of direct RNA extraction with an eventual concentration step, followed by RNA purification and a multiplex RT-qPCR assay for the detection of GI and GII NoV and the murine norovirus-1 (MNV-1), the latter used as process control. The direct RNA extraction method made use of the guanidine-isothiocyanate containing reagent (Tri-reagent®, Ambion) to extract viral RNA from the food sample (basic protocol called TriShort), followed by an eventual concentration step using organic solvents (extended protocol called TriConc).To evaluate the robustness of the NoV detection method, the influence of (1) the NoV inoculum level and (2) different food types on the recovery of NoV from RTE foods was investigated. Simultaneously, the effect of two RNA purification methods (manual RNeasy minikit (Qiagen) and automated NucliSens EasyMAG (BioMérieux)) on the recovery of NoV from these foods was examined. Finally, MNV-1 was evaluated as process control.First of all, high level GI and GII NoV inocula (~ 106 NoV genomic copies/10 g) could be recovered from penne salad samples (10 g) in at least 4 out of 6 PCRs, while low level GI and GII NoV inocula (~ 104 NoV genomic copies/10 g) could be recovered from this food product in maximally 3 out 6 PCRs, showing a significant influence of the NoV inoculum level on its recovery.Secondly, low level GI and GII NoV inocula (104 NoV genomic copies/10 g) were spiked onto 22 ready-to-eat food samples (10 g) classified in three categories (soups, deli sandwiches and composite meals). The GI and GII NoV inocula could be recovered from 20 of the 22 samples.The TriConc protocol provided better recoveries of GI and GII NoV for soups while the TriShort protocol yielded better results for the recovery of GII NoV from composite meals. NoV recovery from deli sandwiches was problematic using either protocol.Thirdly, the simultaneous comparison of two RNA purification protocols demonstrated that automated RNA purification performed equally or better compared to manual RNA extraction.Finally, MNV-1 was successfully evaluated as process control when detecting NoV in RTE foods using this detection methodology.In conclusion, the evaluated NoV detection method was capable of detecting NoV in RTE foods, although recoveries were influenced by the inoculum level and by the food type

Zero risk does not exist: lessons learned from microbial risk assessment related to use of water and safety of fresh produce

Risk assessments related to use of water and safety of fresh produce originate from both water and food microbiology studies. Although the set-up and methodology of risk assessment in these 2 disciplines may differ, analysis of the current literature reveals some common outcomes. Most of these studies from the water perspective focus on enteric virus risks, largely because of their anticipated high concentrations in untreated wastewater and their resistance to common wastewater treatments. Risk assessment studies from the food perspective, instead, focus mainly on bacterial pathogens such as Salmonella and pathogenic Escherichia coli. Few site-specific data points were available for most of these microbial risk assessments, meaning that many assumptions were necessary, which are repeated in many studies. Specific parameters lacking hard data included rates of pathogen transfer from irrigation water to crops, pathogen penetration, and survival in or on food crops. Data on these factors have been investigated over the last decade and this should improve the reliability of future microbial risk estimates. However, the sheer number of different foodstuffs and pathogens, combined with water sources and irrigation practices, means that developing risk models that can span the breadth of fresh produce safety will be a considerable challenge. The new approach using microbial risk assessment is objective and evidence-based and leads to more flexibility and enables more tailored risk management practices and guidelines. Drawbacks are, however, capacity and knowledge to perform the microbial risk assessment and the need for data and preferably data of the specific region