Whole-Genome Sequencing-Based Confirmatory Methods on RT-qPCR Results for the Detection of Foodborne Viruses in Frozen Berries.

Accurate detection, identification, and subsequent confirmation of pathogens causing foodborne illness are essential for the prevention and investigation of foodborne outbreaks. This is particularly true when the causative agent is an enteric virus that has a very low infectious dose and is likely to be present at or near the limit of detection. In this study, whole-genome sequencing (WGS) was combined with either of two non-targeted pre-amplification methods (SPIA and SISPA) to investigate their utility as a confirmatory method for RT-qPCR positive results of foods contaminated with enteric viruses. Frozen berries (raspberries, strawberries, and blackberries) were chosen as the food matrix of interest due to their association with numerous outbreaks of foodborne illness. The hepatitis A virus (HAV) and human norovirus (HuNoV) were used as the contaminating agents. The non-targeted WGS strategy employed in this study could detect and confirm HuNoV and HAV at genomic copy numbers in the single digit range, and in a few cases, identified viruses present in samples that had been found negative by RT-qPCR analyses. However, some RT-qPCR-positive samples could not be confirmed using the WGS method, and in cases with very high Ct values, only a few viral reads and short sequences were recovered from the samples. WGS techniques show great potential for confirmation and identification of virally contaminated food items. The approaches described here should be further optimized for routine application to confirm the viral contamination in berries.

Norovirus GII.17 Caused Five Outbreaks Linked to Frozen Domestic Bilberries in Finland, 2019.

In March 2019, the Finnish Institute for Health and Welfare and Finnish Food Authority started an outbreak investigation after a notification of food business operators' recall of frozen bilberries due to a norovirus finding. A retrospective search was conducted in the food and waterborne outbreak notification system to identify the notifications linked to norovirus and consumption of bilberries in January-March 2019. Five outbreaks were found in which norovirus GII or GII.17 had been detected in patient samples. A pooled retrospective cohort study was performed for those four in which a questionnaire study had been done. A case was defined as a person with diarrhoea or vomiting within 2 days after consuming a meal studied at one of the outbreak locations. Of 79 participants, 45 (57%) cases were identified. Persons that had consumed foods containing unheated bilberries were three times more likely to get ill than those who had not consumed them (RR 3.1, CI 95% 1.2-8.1, p = 0.02). Norovirus GII.17 was found in 16/17 patient samples sent for further typing. Identical norovirus GII.17 was detected in frozen Finnish bilberries and patient samples. At the berry packaging premises, signs of norovirus GII contamination were found in packaging lines. A new procedure for extracting viral nucleic acid from food and environmental samples was used during the outbreak investigation. Consumption of industrially packed frozen berries as heated would be one of the means to prevent norovirus infections.

Occurrence of selected viral and bacterial pathogens and microbiological quality of fresh and frozen strawberries sold in Spain.

Strawberry production and exports have been increasing in Spain in recent decades. However, little information is available about their microbiological quality. Due to the growing concern about the microbial safety of these fruits, the objective of this investigation was to study the microbiological quality and the prevalence of the main foodborne pathogens on strawberries sold in Spain. Fresh (n = 152) and frozen (n = 31) samples were obtained from marketplaces and fields in 2017 and 2018. The samples were assayed for total aerobic mesophilic microorganisms (TAM), moulds and yeasts (M&Y), total coliforms (TC), Escherichia coli, Salmonella spp., Listeria monocytogenes as well as Norovirus (NoV) GI and GII. The microbiological counts ranged from <1.70 (detection limit, dl) - 5.89 log10 CFU/g (mean 3.78 log10 CFU/g) for TAM; 2.10-5.86 log10 CFU/g (mean 3.80 log10 CFU/g) for M&Y; and <0.70 (dl) - 4.91 log10 CFU/g (mean 2.15 log10 CFU/g) for TC in fresh strawberries. In frozen strawberries, the counts were <1.70 (dl) - 3.66 log10 CFU/g (mean 2.30 log10 CFU/g) for TAM; <1.70 (dl) - 2.76 log10 CFU/g (mean 1.82 log10 CFU/g) for M&Y; and <0.70(dl) - 1.74 log10 CFU/g (mean 0.77 log10 CFU/g) for TC. All the samples in this study tested negative for Salmonella spp., L. monocytogenes. E. coli and NoV GI and GII genome. A global overview of all the data was executed using Principal Component Analysis (PCA), and the results showed that the scores and loadings according to principal components 1 (PC1) and 2 (PC2) accounted for 75.9% of the total variance, allowing a distinction between fresh and frozen samples. The presence of moulds was significantly higher in the supermarket samples whereas the presence of total coliforms was significantly higher in the field samples (p < 0.05). Although pathogenic microorganisms were not found, preventative measures and prerequisites in the strawberry production chain must be considered in order to avoid possible foodborne diseases related to the microbiological quality of the fruit.

Prevalence of Norovirus in produce sold at retail in the United Kingdom.

To acquire data on contamination with Norovirus in berry fruit and salad vegetables in the United Kingdom, one thousand one hundred and fifty two samples of fresh produce sold at retail in the UK were analysed for Norovirus. Of 568 samples of lettuce, 30 (5.3%) were Norovirus-positive. Most (24/30) lettuce samples which tested positive for Norovirus were grown in the UK and 19 of those 24 samples contained NoV GI. Seven/310 (2.3%) samples of fresh raspberries were Norovirus-positive. Most (6/7) of the positively-testing fresh raspberry samples were imported, but no predominance of a genogroup, or any seasonality, was observed. Ten/274 (3.6%) samples of frozen raspberries were Norovirus-positive. The country of origin of the positively-testing frozen raspberry samples was not identified in most (7/10) instances. The collected data add to the currently limited body of prevalence information on Norovirus in fresh produce, and indicate the need for implementation of effective food safety management of foodborne viruses.

Minimal risk of tilapia lake virus transmission via frozen tilapia fillets.

Recent outbreaks of a novel tilapia lake virus (TiLV) have raised concerns regarding the international spread of TiLV in frozen tilapia products. This study investigated the potential risks of frozen tilapia fillet as a source of TiLV transmission. It revealed that TiLV genomic RNA could be detected in tilapia fillet and the virus isolated from non-frozen and frozen fillets with clinical TiLV infection stored up to 28 days caused a cytopathic effect (CPE) formation in the susceptible cell line in vitro. However, frozen fillets from clinical TiLV infection stored for 90 and 120 days did not cause CPE in the susceptible cell line. Similarly, CPE was not observed in TiLV isolated from subclinically TiLV-infected fish fillets. In addition, in vivo bioassay revealed that despite the presence of TiLV isolated from subclinically TiLV-infected fillet stored at -20°C for 14 days, there was no evidence of TiLV disease in naïve red hybrid tilapia based on the absence of clinical signs and mortality and without the detection of TiLV genomic RNA using reverse transcription-quantitative polymerase chain reaction assay. Collectively, these findings suggested minimal risk of transmission of TiLV via frozen tilapia fillets.

Hepatitis A outbreak linked to imported frozen strawberries by sequencing, Sweden and Austria, June to September 2018.

Between June-September 2018, 20 hepatitis A cases were notified in six counties in Sweden. Combined epidemiological and microbiological investigations identified imported frozen strawberries produced in Poland as the source of the outbreak. Sequence analysis confirmed the outbreak strain IB in the strawberries with 100 % identity and the respective batch was withdrawn. Sharing the sequence information internationally led to the identification of 14 additional cases in Austria, linked to strawberries from the same producer.

Successful Use of Advertisement Pictures to Assist Recall in a Food-Borne Hepatitis A Outbreak in The Netherlands, 2017.

This study describes an outbreak investigation of 14 hepatitis A cases in the Netherlands. The hepatitis A virus (HAV) genotype IB sequences in cases were highly similar (459/460 nt). The origin of strains could be narrowed to Bulgaria based on information from EPIS-FWD. As an association with consumption of soft fruit was suspected, a case-control study was initiated using a questionnaire and a list of pictures of soft fruit available at the supermarket chain involved. Twelve out of 13 cases consumed a specific frozen raspberry/blueberry product shown on the list (OR 46.0, 95% CI 5.0-27). In multivariable regression analysis this product was the only risk factor (aOR 26.6, 95% CI 2.0-263). Laboratory analyses could not demonstrate HAV-RNA in batches that had been on the market in the incubation period of patients. Trace back of frozen fruit showed that raspberries had been traded by a producer in Bulgaria. After withdrawal of the product from the supermarket no new cases were reported. Use of advertisement pictures of consumed food was helpful in this investigation. Suspicion of the source was strengthened by data from molecular typing and food trace back activities, underlining the importance of good (inter)national cooperation between public health and food safety organisations.

Comparison and optimization of detection methods for noroviruses in frozen strawberries containing different amounts of RT-PCR inhibitors.

Frozen berries have been repeatedly identified as vehicles for norovirus (NoV) transmission causing large gastroenteritis outbreaks. However, virus detection in berries is often hampered by the presence of RT-PCR-inhibiting substances. Here, several virus extraction methods for subsequent real-time RT-PCR-based NoV-RNA detection in strawberries were compared and optimized. NoV recovery rates (RRs) between 0.21 ± 0.13% and 10.29 ± 6.03% were found when five different artificially contaminated strawberry batches were analyzed by the ISO/TS15216-2 method indicating the presence of different amounts of RT-PCR inhibitors. A comparison of five different virus extraction methods using artificially contaminated strawberries containing high amounts of RT-PCR inhibitors revealed the best NoV RRs for the ISO/TS15216 method. Further improvement of NoV RRs from 2.83 ± 2.92% to 15.28 ± 9.73% was achieved by the additional use of Sephacryl(®)-based columns for RNA purification. Testing of 22 frozen strawberry samples from a batch involved in a gastroenteritis outbreak resulted in 5 vs. 13 NoV GI-positive and in 9 vs. 20 NoV GII-positive samples using the original ISO/TS15216 method vs. the extended protocol, respectively. It can be concluded that the inclusion of an additional RNA purification step can increase NoV detection by the ISO/TS15216-2 method in frozen berries containing high amounts of RT-PCR inhibitors.

Non-heat-treated frozen raspberries the most likely vehicle of a norovirus outbreak in Oslo, Norway, November 2013.

In November 2013, the Norwegian Institute of Public Health was notified of a gastroenteritis outbreak following two meetings held at a conference centre. Identical food and beverages were served during the meetings. We investigated in order to identify the vehicle of infection and implement control measures. Meeting participants completed an online questionnaire on consumption of foods and beverages. We asked symptomatic participants to provide a stool sample. We defined a case as diarrhoea and/or vomiting in a participant who became ill within 3 days after the meeting. We calculated attack rates (AR) and adjusted risk ratios (aRR) with 95% confidence intervals (CI) using binomial regression. We conducted environmental investigations. Overall, 147/168 (88%) participants responded, of which 74 (50%) met the case definition. All five stool samples provided were norovirus positive. No kitchen staff reported being sick. Risk of illness was higher in those who consumed raspberry mousse (aRR 3·4, 95% CI 1·4-8·2) and sliced fresh fruit (aRR 1·9, 95% CI 1·3-2·8). Seventy cases (95%) ate raspberry mousse. Frozen raspberries used for the mousse were imported and not heat-treated before consumption. Non-heat-treated frozen raspberries were the most likely outbreak vehicle. Contamination by a food handler could not be excluded. We recommend heat-treatment of imported frozen berries before consumption.

Monitoring of Extraction Efficiency by a Sample Process Control Virus Added Immediately Upon Sample Receipt.

When analysing food samples for enteric viruses, a sample process control virus (SPCV) must be added at the commencement of the analytical procedure, to verify that the analysis has been performed correctly. Samples can on occasion arrive at the laboratory late in the working day or week. The analyst may consequently have insufficient time to commence and complete the complex procedure, and the samples must consequently be stored. To maintain the validity of the analytical result, it will be necessary to consider storage as part of the process, and the analytical procedure as commencing on sample receipt. The aim of this study was to verify that an SPCV can be recovered after sample storage, and thus indicate the effective recovery of enteric viruses. Two types of samples (fresh and frozen raspberries) and two types of storage (refrigerated and frozen) were studied using Mengovirus vMC0 as SPCV. SPCV recovery was not significantly different (P > 0.5) regardless of sample type or duration of storage (up to 14 days at -20 °C). Accordingly, samples can be stored without a significant effect on the performance of the analysis. The results of this study should assist the analyst by demonstrating that they can verify that viruses can be extracted from food samples even if samples have been stored.

Large and prolonged food-borne multistate hepatitis A outbreak in Europe associated with consumption of frozen berries, 2013 to 2014.

In May 2013, Italy declared a national outbreak of hepatitis A, which also affected several foreign tourists who had recently visited the country. Molecular investigations identified some cases as infected with an identical strain of hepatitis A virus subgenotype IA. After additional European Union/European Economic Area (EU/EEA) countries reported locally acquired and travel-related cases associated with the same outbreak, an international outbreak investigation team was convened, a European outbreak case definition was issued and harmonisation of the national epidemiological and microbiological investigations was encouraged. From January 2013 to August 2014, 1,589 hepatitis A cases were reported associated with the multistate outbreak; 1,102 (70%) of the cases were hospitalised for a median time of six days; two related deaths were reported. Epidemiological and microbiological investigations implicated mixed frozen berries as the vehicle of infection of the outbreak. In order to control the spread of the outbreak, suspected or contaminated food batches were recalled, the public was recommended to heat-treat berries, and post-exposure prophylaxis of contacts was performed. The outbreak highlighted how large food-borne hepatitis A outbreaks may affect the increasingly susceptible EU/EEA general population and how, with the growing international food trade, frozen berries are a potential high-risk food.

Batch testing for noroviruses in frozen raspberries.

Berries, in particular raspberries, have been associated with multiple recalls due to norovirus contamination and were linked to a number of norovirus (NoV) outbreaks. In the present study a total of 130 samples of frozen raspberries were collected from 26 batches in four different raspberry processing companies. In two companies the samples consisted of bulk frozen raspberries serving as raw material for the production of raspberry puree (an intermediate food product in a business to business setting). In two other companies, the samples consisted of bulk individually quick frozen (IQF) raspberries serving as raw material for the production of frozen fruit mixes (as a final food product for consumer). Enumeration of Escherichia coli and coliforms was performed as well as real-time reverse transcription PCR (RT-qPCR) detection of GI and GII NoV (in 2 × 10 g). In addition, in cases where positive NoV GI or GII RT-qPCR signals were obtained, an attempt to sequence the amplicons was undertaken. Six out of 70 samples taken from the 14 batches of frozen raspberries serving raspberry puree production provided a NoV RT-qPCR signal confirmed by sequencing. Four of these six positive samples clustered in one batch whereas the other two positive samples clustered in another batch from the same company. All six positive samples showed NoV RT-qPCR signals above the limit of quantification of the RT-qPCR assay. These two positive batches of frozen raspberries can be classified as being of insufficient sanitary quality. The mean NoV level in 20 g of these raspberry samples was 4.3 log genomic copies NoV GI/20 g. The concern for public health is uncertain as NoV RT-qPCR detection is unable to discriminate between infectious and non-infectious virus particles. For the IQF raspberries, one batch out of 12 tested NoV positive, but only 1 out of the 5 samples analyzed in this batch showed a positive RT-qPCR GI NoV signal confirmed by sequencing. The RT-qPCR signal was below the limit of quantification of the assay used (<3.7 log genomic copies/20g). It was shown that the applied protocol for sequencing of the amplicon to confirm the specificity of the RT-qPCR signal was successful for GI NoV amplicons but often failed and provided an inconclusive result for GII NoV amplicons.

Outbreak of hepatitis A infection associated with the consumption of frozen berries, Ireland, 2013--linked to an international outbreak.

In May 2013, a European alert was issued regarding a hepatitis A virus (HAV) outbreak in Italy. In June 2013, HAV subgenotype IA with an identical sequence was identified in Ireland in three cases who had not travelled to Italy. The investigation consisted of descriptive epidemiology, a case-control study, microbiological testing of human and food specimens, molecular typing of positive specimens and food traceback. We identified 21 outbreak cases (14 confirmed primary cases) with symptom onset between 31 January and 11 October 2013. For the case-control study, we recruited 11 confirmed primary cases and 42 matched controls. Cases were more likely than controls to have eaten berry cheesecake (matched odds ratio (mOR): 12; 95% confidence interval (CI): 1.3-114), whole frozen berries (mOR: 9.5; 95% CI: 1.0-89), yoghurt containing frozen berries (mOR: 6.6, 95% CI: 1.2-37) or raw celery (mOR: 4; 95% CI: 1.2-16). Among cases, 91% had consumed at least one of four products containing frozen berries (mOR: 12; 95% CI: 1.5-94). Sixteen food samples tested were all negative for HAV. As products containing frozen berries were implicated in the outbreak, the public were advised to heat-treat frozen berries before consumption.

Lessons from Norovirus Outbreak in Warsaw, Poland, December 2012.

Efficient foodborne outbreak investigations are important for identification of gaps in food safety and public health practice. This article reports on an investigation of a gastroenteritis outbreak linked to catering food following a Christmas reception at the National Institute of Public Health-National Institute of Hygiene (NIPH-NIH) in Warsaw in December 2012. Of 192 employees eating food at the catering event, 97 (50.5%) developed symptoms. Persons eating dishes with recipes containing frozen carrots were five times more likely to develop gastrointestinal symptoms compared to those who did not eat carrots. Laboratory analysis identified norovirus in stool samples taken from symptomatic persons. Leftover food was not available for testing. The investigators did not collect stool specimens from food handlers and did not conduct trace backs for the suspected food ingredients. This investigation underlines the need for a revision of an existing procedures and importance of their complementation with detailed instructions for the local public health authorities for effective completion of foodborne outbreaks investigations in Poland.

Hepatitis A Outbreak in Europe: Imported Frozen Berry Mix Suspected to be the Source of At least One Infection in Austria in 2013.

We tested 19 sera from Austrian patients with acute hepatitis A. A serum from a 48-year-old female patient yielded HAV-nucleic acid that showed 99.7% homology to the HAV-sequence obtained from samples taken during the current outbreak in several European countries, which is associated with consumption of frozen berries. So far, Austria was considered not to be affected by this hepatitis A outbreak.