Molecular detection of noroviruses in ready-to-eat foods and fruit products

In this PhD, three main goals were defined. The first goal consisted of the development and evaluation of a methodology for detection of noroviruses (NoV) in ready-to-eat (RTE) foods and soft red fruits while the second main goal included the evaluation of the murine norovirus 1 (MNV-1) as control reagent for different steps throughout the NoV detection protocols. Finally, a screening study on a selection of fruit produce products towards NoV presence was the third main goal of this PhD. To illustrate these goals, a literature study was performed in chapter 1. In this literature study, a brief overview of the most important food borne viruses was followed by a more detailed description of NoV in terms of classification, virion and genome structure. The NoV genotype most commonly identified in NoV gastroenteritis outbreaks (NoV GII.4) was described as well. The importance of NoV as a food borne pathogen was highlighted by data originating from official bodies such as CDC (Centers for Disease Control and Prevention; USA) and EFSA (European Food Safety Authority; Europe), accompanied with data gathered on own initiatives by research groups. The two main transmission routes of NoV contamination of foods (pre-harvest contamination via contact with contaminated water and (post-) harvest contamination via an infected food handler/picker) were investigated by summarizing and analyzing 59 NoV food borne outbreaks described between 2000 and 2010. Furthermore, the three main steps of NoV detection in food were portrayed in detail to prepare the development and evaluation of the NoV detection methodology in chapters 2, 3, 4 and 5. Finally, the use of adequate positive and negative controls to assure reliable detection of NoV in foods was illustrated. For the first and second goals of this PhD, a molecular assay for detection of the purified NoV genomic material was optimized and subsequently combined with protocols for extraction of (genomic material of) NoV from RTE foods and soft red fruits in chapters 4 and 5. MNV-1 was included in these protocols as control reagent. The molecular detection assay described in chapter 2 was a quantitative two-step multiplex real-time reverse transcriptase (RT-) PCR assay for simultaneous detection of NoV genogroup I (GI) and II (GII) and the murine norovirus 1 (MNV-1), the latter used as internal amplification control (IAC). For this multiplex assay, NoV GI and GII specific primers and hydrolysis probes designed by the European Committee for Standardization/ Technical Committee 275 / Working Group 6 /Task Group 4 on virus detection in foods (CEN/TC275/WG6/TAG4 working group) were combined with primers for murine norovirus 1 designed by Baert and colleagues (2008b). Evaluation of this multiplex assay showed a high concordance between the multiplex assay and the corresponding singleplex PCR assays. Specificity analysis of the multiplex assay by testing a NoV RNA reference panel and clinical GI and GII NoV samples showed that specific amplification of NoV GI and GII was possible. In addition, no cross-amplification was observed when subjecting a collection of bovine NoV and other (non-NoV) enteric viruses to the multiplex assay. Finally, MNV-1 was successfully integrated as IAC, although a sufficiently low concentration was needed to avoid interference with the possibility of the developed multiplex assay to quantitatively and simultaneously detect the presence of GI and GII NoV within one sample. During development of the multiplex real-time RT-PCR assay, contamination issues were encountered and the investigation towards the source of the positive no template controls (NTCs) was described in chapter 3. This investigation was believed to be necessary because of the need for reliable detection of 10 or less NoV genomic copies per PCR reaction, due to the low infectious dose of GI and GII NoV. In this chapter, a suspicion of well-to-well migration of positive control DNA (a short synthetic single stranded DNA (ssDNA) fragment) during real-time PCR runs was uttered as hypothetic cause of the positive NTCs. Results in this chapter showed that evaporation of water occurred during real-time PCR runs regardless of the DNA type, the reaction plate seal type and the use of mineral oil as cover layer. It was also suggested that co-evaporation of DNA took place, with an apparent negative correlation between the size of the DNA type and the extent of this co-evaporation. The use of mineral oil as cover layer and plasmid DNA as quantitative positive PCR control resulted in a complete absence of positive NTCs while only negligible effects were noticed on the performance of the real-time PCR. After development of the multiplex real-time RT-PCR assay and the resolving of the contamination issues, two protocols for extraction of (genomic material) of NoV from foods were evaluated towards robustness and sensitivity while MNV-1 was evaluated as process control in both protocols. The evaluation of a direct RNA extraction protocol for extraction of NoV genomic material (RNA) from RTE foods was described in chapter 4, while the evaluation of an elution-concentration protocol for extraction of NoV from soft red fruits was illustrated in chapter 5. For the RTE foods, the direct RNA extraction protocol made use of a guanidine isothiocyanate containing reagent 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). The protocol for extraction of NoV from soft red fruits consisted of alkaline elution of NoV particles from the food, followed by polyethylene glycol (PEG) precipitation and organic solvent purification. For both protocols the RNA was subsequently purified. This purified RNA was detected by the multiplex real-time RT-PCR assay as described in chapter 2. To evaluate both NoV extraction methods towards sensitivity and robustness, the influence of (1) the NoV inoculum level and (2) different food types on the recovery of NoV from these foods was investigated. First of all, a significant influence of the NoV inoculum level on the recovery of NoV from foods was demonstrated for both protocols. High level inocula could be recovered from penne salad, selected as typical RTE food, with higher recovery success rates compared to low level inocula. For these high inoculum levels, the TriShort and TriConc protocols resulted in mean recovery efficiencies of >1 % and 0.1 to 10 %, respectively. Recovery of these low and high level NoV inocula from frozen raspberry crumb was possible with high recovery success rates and with mean recovery efficiencies of 10 to 30 % in most cases. Secondly, a significant influence of the food type on the recovery of NoV could be shown for both protocols. For the direct RNA extraction protocol, the TriConc protocol provided better NoV recoveries for soups, while TriShort and TriConc protocols performed likewise for composite meals and deli sandwiches, although NoV recovery from the latter food type was problematic. For the elution-concentration protocol, a significant influence of the soft red fruit product type on the recovery efficiency of NoV GI and MNV-1 was noticeable, while no significant differences could be shown for GII NoV. In general, the recovery of NoV was more efficient and successful from the strawberry puree compared to a frozen forest fruit mix and fresh raspberries. Regarding the evaluation of MNV-1 as control reagent, results from chapter 4 and chapter 5 suggested that a sufficient high concentration of the MNV-1 PC was needed to allow an estimation of possible inhibition of the RT-PCR or of inefficient virus extraction. When used as reverse transcription control or internal amplification control, the concentration should be adjusted to avoid interference with the quantitative properties of the developed multiplex real-time RT-PCR assay. Chapter 6 described the screening of 75 fruit products (raspberries, strawberries, cherry tomatoes and fruit salads) for NoV presence using the virus extraction protocol described in chapter 5 combined with the multiplex real-time RT-PCR assay illustrated in chapter 2. In total, 18 samples tested positive for GI and/or GII NoV genomic material despite a good bacteriological quality. The level of detected NoV genomic copies concentrations ranged between 2.5 and 5.0 logs per 10 grams of fruit sample. NoV GI and/or GII were found in 4/10, 7/30, 6/20 and 1/15 of the tested raspberries, cherry tomatoes, strawberries and fruit salad samples, respectively. However, confirmation of the positive real-time PCR results by sequencing genotyping regions in the NoV genome was not possible. The question whether or not these unexpected high number of NoV positive results obtained should be perceived as a public health threat was raised and discussed. In conclusion, methods for detection of NoV in RTE foods and soft red fruits were developed and evaluated towards sensitivity and robustness. For detection of NoV in soft red fruits and ready-to-eat foods, an elution-precipitation protocol and a direct RNA extraction protocol were combined with an optimized multiplex real-time RT-PCR assay leading to NoV detection protocols with detection limits of ~104 genomic copies / 10g food product. Influence of the NoV inoculum level and food type on NoV recovery was shown. Additionally, MNV-1 was successfully evaluated as control reagent, and suggestions were made towards its use. However, application of the method for NoV detection in fruit products has shown that interpretation of NoV presence by molecular methods is not straightforward and raises several questions, especially towards the public health safety

Norovirus transfer between foods and food contact materials

Human infective noroviruses (NoVs) are a worldwide leading cause of foodborne illness and are frequently spread via infected food handlers preparing and manipulating food products such as deli sandwiches. The objective of the current study was to determine the efficiencies whereby NoV could be transferred between surfaces associated with the preparation of manually prepared foods such as deli sandwiches. Nonfood surfaces included gloves and stainless steel discs, and boiled ham, lettuce, and a sandwich bun were the ingredients of the deli sandwich. Both NoV GII.4 and the murine NoV 1 (MNV-1, a cultivable human NoV surrogate) were included in the presented study. Transfer of NoV GII.4 and MNV-1 between surfaces was performed by pressing an inoculated donor surface against an acceptor surface. To evaluate the effect of subsequent contact, donor surfaces were pressed a second time to an identical acceptor surface. Subsequently, NoV GII.4 and MNV-1 were detected using real-time reverse transcription PCR assays and plaque assays, respectively. Transfer of both viruses from gloves to stainless steel was inefficient, and virus transfer from food products to stainless steel occurred with inure variability for NoV GII.4 than for MNV-1. Virus transfer from the stainless steel discs to the gloves was substantially more efficient than from the gloves to the stainless steel. NoV GII.4 and MNV-1 transfer from food products to the doves occurred with varying efficiencies, although this variation was more evident for NoV GII.4. The MNV-1 inoculum was significantly less efficiently transferred to the acceptor surface at the second contact, which was not the case for NoV GII.4. The obtained transfer efficiency data may provide insights into the transfer of NoV during preparation of foods and can be included in risk assessment models describing the transmission of NoVs in this context

The need for harmonization in detection of human noroviruses in food

Noroviruses (NoV) have been recognized worldwide as a leading cause of foodborne gastroenteritis over the last decade. A broad range of foods-shellfish, fresh produce, and ready-to-eat/catered foods has been implicated in NoV foodborne outbreaks. The recognition of NoV as an important food pathogen has been aided by the development of sensitive molecular methods for detection of the NoV genome. However, despite advances, NoV detection is still hampered by several limitations. First, NoV detection can often only be implemented by expert laboratories due to the complexity of the virus extraction step, which in most protocols is cumbersome and labor-intensive. Moreover, a very wide selection of automated methods for virus extraction from foods is available, so selection of an adequate method is not straightforward. On the other hand, automated systems have been made available or the RNA purification and real-time RT-PCR (RT-qPCR) is considered the gold standard for detection of NoV. Second, correct interpretation of real-time PCR results is often difficult. From a technical point of view, the interpretation of the often nonsigmoidal amplification curves remains difficult, even for experts. From a food safety perspective, interpretation of very high Cq (or Ct) values and thus, of low viral genomic copy numbers is not straightforward, as RT-(q)PCR merely detects the presence of viral genomic material that is not necessarily linked to the presence of infectious viral particles. Despite efforts, both limitations have not completely resolved thus far. Harmonization may be a first step to comprehend and deal with these limitations. The current review provides an overview of a number of validated methods that have been published by food safety and other authorities

Viral genes everywhere: public health implications of PCR-based testing of foods

Food borne viruses such as norovirus and hepatitis A virus are increasingly recognized worldwide as the most important cause of food borne gastro-intestinal illness. Food borne outbreaks, often involving multiples cases, have been reported and associated with food products of both animal and non-animal origin. Most foods are contaminated with food borne viruses during preparation and service. However, bivalve molluscs and occasionally produce (in particular leafy vegetables and soft red fruits) may be contaminated during production and processing. Owing to the low infectious dose of these viruses, the presence of few viral particles on the food is often sufficient for an infection. Over the past decade, molecular methods - such as RT-(q)PCR - have therefore been developed for rapid detection of viral contamination on foods. The availability of these detection methods has led to an increased detection of viral contamination in foods. However, RT-(q)PCR and other molecular methods detect the mere presence of an RNA (or DNA) fragment and are unable to differentiate between infectious and non-infectious viral particles in the monitoring of food products for viral contamination which makes interpretation of these results not straightforward. The current review aims to summarize recent efforts made for a more correct interpretation of these positive RT-(q)PCR results. First of all, RT-(q)PCR test results should be analyzed taking into account the results of various appropriate controls in place to assure well-functioning of good laboratory practices. Subsequently, approaches that may aid to facilitate acceptation and that may aid to put RT-(q)PCR positive food products into context from a public health perspective are discussed. These approaches include (1) the use of a critical acceptance limit, (2) the confirmation of positive RT-(q)PCR results and (3) the potential correlation with faecal indicators. Finally, the current review provides insights in a selection of methods currently under development that may be able to facilitate the specific detection of infectious food borne viruses

Semi-direct lysis of swabs and evaluation of their efficiencies to recover human noroviruses GI and GII from surfaces

Enteric viruses such as noroviruses (NoVs) continue to be the cause of widespread viral outbreaks due to person-to-person transmission, contaminated food, and contaminated surfaces. In order to optimize swabbing methodology for the detection of viruses on (food) contact surfaces, three swab elution/extraction strategies were compared in part one of this study, out of which, one strategy was based on the recently launched ISO protocol (ISO/TS 15216-1) for the determination of hepatitis A virus and NoV in food using real-time RT-PCR (RT-qPCR). These three swab elution/extraction strategies were tested for the detection of GI.4 and GII.4 NoV on high-density polyethylene (HD-PE) surfaces with the use of cotton swabs. For detection of GI.4 and GII.4, the sample recovery efficiency (SRE) obtained with the direct lysis strategy (based on ISO/TS 15216-1) was significantly lower than the SRE obtained with both other strategies. The semi-direct lysis strategy was chosen to assess the SRE of two common swabs (cotton swab and polyester swab) versus the biowipe (Biom,rieux, Lyon, France) on three surfaces (HD-PE, neoprene rubber (NR), and nitrile gloves (GL)). For both surfaces, HD-PE and GL, no significant differences in SREs of GI.4 and GII.4 NoVs were detected between the three different swabs. For the coarser NR, biowipes turned out to be the best option for detecting both GI.4 and GII.4 NoV

Performance of two real-time RT-PCR assays for the quantification of GI and GII noroviruses and hepatitis A virus in environmental water samples

In this study, the performance of two real-time reverse transcription polymerase chain reaction (RT-qPCR) assays for the detection of hepatitis A viruses (HAV) and GI and GII noroviruses (NoV) was tested in the presence of an environmental matrix by analyzing 15 inoculated environmental water samples. For the detection of HAV, an in-house two-step RT-qPCR from literature was compared with a commercial one-step real-time RT-PCR of Ceeram (La Chapelle-sur-Erdre, France). For the detection of GI and GII NoV, an in-house duplex two-step RT-qPCR assay was used and compared with the results obtained using two commercial singleplex one-step RT-qPCR assays of Ceeram (France). The performance of the two RT-qPCR assays was determined by comparing (1) standard curves, (2) the number of detected genomic copies, and (3) the influence of inhibition by RNA dilution. Both assays for the detection of GI and GII NoV performed likewise. For the detection of HAV, the differences in genomic copies detected were to some extent more apparent and in favor of the commercial one-step assay. When the HAV RT-qPCR assays were compared in terms of inhibition, the performance of the commercial one-step RT-qPCR kit was less affected for the detection of HAV in undiluted RNA in comparison to the in-house two-step RT-qPCR assay. On the other hand, inhibition had only a marginal influence on the performance of both assays for detection of HAV in the 1/10 diluted RNA. In conclusion, only minor differences were observed between the in-house RT-qPCR assays and the commercial one-step assays for the detection of HAV and NoV in environmental water samples

Detection of Noroviruses in shellfish and semiprocessed fishery products from a Belgian seafood company

Shellfish have been implicated in norovirus (NoV) infection outbreaks worldwide. This study presents data obtained from various batches of shellfish and fishery products from a Belgian seafood company over a 6-month period. For the intact shellfish (oysters, mussels, and clams), 21 of 65 samples from 12 of 34 batches were positive for NoVs; 9 samples contained quantitative NoV levels at 3,300 to 14,300 genomic copies per g. For the semiprocessed fishery products (scallops and common sole rolls with scallop fragments), 29 of 36 samples from all eight batches were positive for NoVs; 17 samples contained quantitative NoV levels at 200 to 1,800 copies per g. This convenience study demonstrated the performance and robustness of the reverse transcription quantitative PCR detection and interpretation method and the added value of NoV testing in the framework of periodic control of seafood products bought internationally and distributed by a Belgian seafood processing company to Belgian food markets