During September/October 2012, a norovirus gastroenteritis outbreak affecting about 11,000 people occurred in Germany. Epidemiological studies suggested that frozen strawberries represented the vehicle of infection. We describe here the analysis of frozen strawberries for the presence of norovirus. Samples were taken by applying a stratified subsampling scheme. Two different methods for virus extraction from strawberries were compared. First, viruses were eluted from strawberries under alkaline conditions and concentrated using a polyethylene glycol precipitation. Second, ultrafiltration was applied for concentration of viruses rinsed off of the berries. In both cases, RNA was extracted and analyzed by real-time RT-PCR. Application of the ultrafiltration method generally resulted in a lower detection rate. Noroviruses were detected in 7/11 samples derived from the lot of strawberries implicated in the outbreak using the precipitation method. Typing of norovirus revealed three different genotypes including a combination of norovirus genotype II.16 (viral polymerase) and II.13 (viral capsid). This genotype combination was also found in some of the patients that were involved in the outbreak, but that had not been reported in Germany so far. In conclusion, heterogeneously distributed noroviruses in frozen strawberries can be detected by applying an optimized combination of sampling procedures, virus extraction methods, and real-time RT-PCR protocols. The detection of several different genotypes in the strawberries may suggest contamination from sewage rather than from a single infected food handler.

Hepatitis A virus (HAV) and norovirus are important agents of food-borne human viral illness, with common vehicles including bivalve molluscan shellfish, soft fruit and various vegetables. Outbreaks of viral illness due to contamination of the surfaces of foods, or food preparation surfaces by for example infected food handlers are also common. Virus analysis of food matrices can contribute towards risk management for these hazards and a two-part technical specification for determination of Hepatitis A virus and norovirus in food matrices (ISO/TS 15216:2013) was published jointly by the European Committee for Standardisation and the International Organization for Standardization in 2013. As part of the European Mandate No. M381 to validate 15 standards in the field of food microbiology, an international validation study involving 18 laboratories from 11 countries in Europe was conducted between 2012 and 2014. This study aimed to generate method characteristics including limit of detection, limit of quantification, repeatability and reproducibility for ISO 15216 - Part 1, the method for quantification, in seven food matrices. The organization and results of this study, including observations that led to improvements in the standard method are presented here. After its conclusion, the method characteristics generated were added to the revised international standard, ISO 15216-1:2017, published in March 2017.

Noroviruses and rotaviruses are the leading causes of non-bacterial gastroenteritis in humans worldwide. Virus-contaminated food and surfaces represent an important risk to public health. However, established detection methods for the viruses in food products are laborious and time-consuming. Here, we describe a detailed swabbing protocol combined with real-time RT-PCR for norovirus and rotavirus detection on artificially contaminated food and environmental surfaces. Recovery rates between 2 and 78% for norovirus and between 8 and 42% for rotavirus were determined for contaminated food surfaces of apple, pepper, cooked ham and salami. From contaminated environmental surfaces (stainless steel, ceramic plate, polyethylene, wood), recovery rates between 26 and 52% (norovirus) and between 10 and 58% (rotavirus) were determined. The results demonstrate the suitability of the swab sample method for virus detection on food and environmental surfaces. Compared to other methods, it is easy to perform and significantly time-saving, predestining it for routine testing.

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Aims:  The chromosomal ail gene (attachment and invasion locus) is commonly used as target gene for the detection of pathogenic Y. enterocolitica strains in food testing. The ail PCR does not detect strains of biotype 1A (BT1A), which are regarded as non‐pathogenic because BT1A strains lack the virulence plasmid and chromosomally encoded virulence genes. In some recent reports, however, BT1A strains were discovered that harboured the ail gene. We isolated an ail‐positive strain and characterized this strain with phenotypic and genotypic methods to study its possible relation to pathogenic Y. enterocolitica strains. Methods and Results:  The ail region of the BT1A strain was sequenced and compared with the corresponding region of nonpathogenic BT1A strains and pathogenic strains. Pulsed field gel electrophoresis (PFGE) analysis was applied revealing no similarity of the PFGE pattern of this strain to the patterns of pathogenic strains. Virulence‐gene‐based PCR analyses showed the strain to be positive for ystB, but negative for virulence genes ystA, virF and yadA. Whole‐cell MALDI‐TOF MS combined with a shrinkage discriminant analysis approach was applied and clearly classified the ail‐positive biotype 1A strain within the cluster of BT1A strains. Conclusions:  PCR detection of ail sequences in food matrices should be followed by the isolation of the responsible strain and its characterization using phenotypic or genotypic methods. Significance and Impact of the Study:  The ail gene may be present in Y. enterocolitica BT1A strains, which are commonly considered as nonpathogenic. Efficient methods such as PCR typing of other virulence genes or rapid MALDI‐TOF MS‐based bacterial profiling allow a more comprehensive assessment of the pathogenicity potential of Yersinia strains.