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.

Detection and quantification of hepatitis E virus RNA in ready to eat raw pork sausages in the Netherlands.

The aim of the present study was to assess raw pork sausages collected on the Dutch market for the presence of hepatitis E virus (HEV) RNA. 46 of 316 (14.6%) products sampled from Dutch retail stores in 2017-2019 were positive for HEV RNA. HEV RNA was detected in 10.8% of "cervelaat" (n = 74), 18.5% of salami (n = 92), 26.1% of "metworst" (n = 46), 16.3% of "snijworst" (n = 43) samples. This was significantly more often than in other raw pork sausages like dried sausages, fuet or chorizo (3.3%, n = 61). The percentage of HEV RNA positive products was not significantly different for products sold as either sliced or unsliced deli meat. The average viral load in positive tested products was 2.76 log10 genome copies per 5 g, incidentally reaching up to 4.5 log10 genome copies per 5 g. The average HEV RNA level was significantly higher in samples collected in 2017 than those in samples collected in 2018, and most of the samples in 2019. Typing by sequence analysis was successful for 33 samples, all revealing genotype 3c. The results support recent epidemiological studies that identified specific raw pork sausages as risk factor for hepatitis E virus infection in the Netherlands. Persons at risk, including Dutch transplant recipients, have been advised to avoid the consumption of raw pork sausages. The study warrants a continuation of monitoring to follow the HEV RNA levels in pork products for use in risk assessments and risk management.