The development of viral capture, concentration and molecular detection method for norovirus in foods to establish the risk to public health

Abstract

Norovirus has been identified as a common cause of gastroenteritis worldwide, and food as a transmission vehicle has been well documented. Standardised detection methods exist for the detection of norovirus from fresh produce and molluscan bivalves, whilst detection methods for a wider range of food matrices that may be implicated in transmission of norovirus do not currently exist. The detection of norovirus in foods suspected to be implicated in transmission is paramount for appropriate outbreak investigation. The contamination of foods other than shellfish and fresh produce often occurs via food handlers. The proportion of norovirus that is typically transferred from food handlers to food also remains unknown. Understanding this is necessary in order to estimate the risk of infection and the burden of gastroenteritis caused by norovirus that is attributable to food contaminated by food handlers. These questions were addressed by the development of a combined capture, concentration and quantitative detection protocol with the aim to enhanced norovirus recovery from a range of food types. A food surface wash and norovirus capture method that was sensitive, reduced processing time, and increased throughput capacity was applied to a range of ready to eat foods. An automated nucleic acid extraction method which further reduced processing time and increased throughput was validated. Finally the validated method demonstrated that two real time RT-PCR assays currently used for the detection of norovirus in shellfish and fresh produce or in faecal samples were comparable overall, and hence either could be used in combination with the norovirus capture, concentration and extraction protocol described in this thesis. The protocol was applied to a range of food matrices and resulted in <1% to 55% recovery of norovirus GI and <1% to 25% recovery of norovirus GII. The optimised protocol was then used to quantify virus transfer between food handlers hands and to food, in simulation experiments where food handlers’ gloved hands were artificially contaminated prior to preparation of a sandwich. This enabled norovirus transfer to food items and to other food handlers to be measured at each stage. Quantitative data demonstrated that 5.9 ± (SD ± 0.1) log10 cDNA copies/µl of norovirus GII inoculum, resulted in a percentage recovery of between 3.0% and 0.02% from Food Handlers and 7.8 ± (SD ± 0.1) log10 cDNA copies/µl of norovirus GI inoculum resulted in a percentage recovery between 9.6% and 0.004% from Food Handlers. The average percentage recovered from sandwich pieces over six replicates was 0.2% for norovirus GII and 1.2% for norovirus GI. The method and protocols developed could be rolled out to official control laboratories and aid foodborne outbreak investigation by allowing testing of food categories that currently are not investigated. Furthermore, this work demonstrated the extent of norovirus transfer from hands to food ingredients and the environment and could be used in risk assessment models. Further work applying these protocols to quantify the transfer from contaminated hands using a range of viral loads will be useful in determining risk more accurately, and to monitor and investigate food premises by introducing this as an additional food and hand hygiene marker.