An international inter-laboratory study to compare digital PCR with ISO standardized qPCR assays for the detection of norovirus GI and GII in oyster tissue.

An optimized digital RT-PCR (RT-dPCR) assay for the detection of human norovirus GI and GII RNA was compared with ISO 15216-conform quantitative real-time RT-PCR (RT-qPCR) assays in an interlaboratory study (ILS) among eight laboratories. A duplex GI/GII RT-dPCR assay, based on the ISO 15216-oligonucleotides, was used on a Bio-Rad QX200 platform by six laboratories. Adapted assays for Qiagen Qiacuity or ThermoFisher QuantStudio 3D were used by one laboratory each. The ILS comprised quantification of norovirus RNA in the absence of matrix and in oyster tissue samples. On average, results of the RT-dPCR assays were very similar to those obtained by RT-qPCR assays. The coefficient of variation (CV%) of norovirus GI results was, however, much lower for RT-dPCR than for RT-qPCR in intra-laboratory replicates (eight runs) and between the eight laboratories. The CV% of norovirus GII results was in the same range for both detection formats. Had in-house prepared dsDNA standards been used, the CV% of norovirus GII could have been in favor of the RT-dPCR assay. The ratio between RT-dPCR and RT-qPCR results varied per laboratory, despite using the distributed RT-qPCR dsDNA standards. The study indicates that the RT-dPCR assay is likely to increase uniformity of quantitative results between laboratories.

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.

Rapid Detection of Human Norovirus in Frozen Raspberries.

Raspberries have lately caused several human norovirus (HuNoV) outbreaks in Europe. In this study, we developed and evaluated for HuNoV reverse transcription (RT)-PCR detection in frozen raspberries extraction methods that have equal sensitivity but are less time-consuming than widely used methods based on polyethylene glycol (PEG) precipitation and chloroform-butanol purification. One method was applied to stored frozen raspberries linked to previous HuNoV outbreaks and berries on sale. In the virus elution-based Method 1, sparkling water eluted viruses most efficiently from the berries. Method 2, based on direct nucleic acid extraction with minor PEG supplement, yielded the highest number of positive findings (4 out of 9) at low virus concentration level of 100 genome copies HuNoV genogroup II per 25 g raspberries. Both methods showed approximately equal sensitivity to a method including PEG precipitation and chloroform-butanol purification. Two naturally contaminated berry samples linked to HuNoV outbreaks in 2006 and 2009 were still positive for HuNoV genogroup I, but all berry products purchased from a local store remained negative for HuNoV. In conclusion, this study presents two efficient and rapid methods which can be used in urgent HuNoV outbreak investigations, since the results of the virus analysis are available in a few hours.

Evaluation of four virus recovery methods for detecting noroviruses on fresh lettuce, sliced ham, and frozen raspberries.

Although noroviruses play a significant role in causing foodborne illness in developed countries, no standardised method for detecting noroviruses in foodstuffs is currently available. This study compared four virus recovery methods based on ultrafiltration, immunomagnetic separation, ultracentrifugation and PEG precipitation techniques using identical real-time RT-PCR protocols for detection of RNA in eluates from lettuce, sliced ham and raspberries inoculated artificially with genogroup II norovirus. Noroviruses in all the food source matrices were successfully detected by all four methods. Ultracentrifugation yielded the highest recovery efficiencies in lettuce and ham, whereas PEG precipitation recovered the highest yield of noroviruses from raspberries. The repeatability of the results and the applicability of the methods to all food matrices were best with PEG precipitation, which had average virus recoveries of 19%, 47% and 28% for lettuce, ham and raspberries (viral RNA in dilution 1:10), respectively. In each case, a tenfold dilution of the extracted RNA clearly reduced the level of PCR inhibitors, which were released from raspberries in particular. The results of this study show that the detection of noroviruses in food is challenging, and more efforts to develop sensitive methods are still needed to detect noroviruses in food containing viruses in low numbers.

Pet dogs--a transmission route for human noroviruses?

BACKGROUND: Human noroviruses (HuNoVs) are one of the leading causes of diarrhoeal diseases worldwide in all age groups. Virus transmission can occur via the faecal-oral route from person to person or via contaminated food, water, or surfaces. The most common NoV strains circulating among humans belong to genogroup GII. Thus far, to our knowledge, no HuNoVs have been detected in pets. OBJECTIVES: We investigated whether pet dogs could serve as carriers for HuNoVs and thereby transmit the infection to humans. STUDY DESIGN: Ninety-two faecal samples of indoor pet dogs were obtained. The main criteria for sample collection were that the dog or humans in the household had suffered from diarrhoea or vomiting. All samples were screened for HuNoV genogroups GI, GII, and GIV by real-time one-step RT-PCR. RESULTS: We detected HuNoV in four faecal samples from pet dogs that had been in direct contact with symptomatic persons. Three of the positive samples contained genotype GII.4 variant 2006b or 2008 and one GII.12. All NoV-positive dogs lived in households with small children and two dogs showed mild symptoms. CONCLUSIONS: Our results suggest that HuNoVs can survive in the canine gastrointestinal tract. Whether these viruses can replicate in dogs remains unresolved, but an association of pet dogs playing a role in transmission of NoVs that infect humans is obvious.