Detection of noroviruses in foods: a study on virus extraction procedures in foods implicated in outbreaks of human gastroenteritis.

Disease outbreaks in which foods are epidemiologically implicated as the common source are frequently reported. Noroviruses and enteric hepatitis A viruses are among the most prevalent causative agents of foodborne diseases. However, the detection of these viruses in foods other than shellfish is often time-consuming and unsuccessful. In this study, three virus concentration methods were compared: polyethylene glycol (PEG) plus NaCl, ultracentrifugation, and ultrafiltration. Two RNA extraction methods, TRIzol and RNeasy Mini Kit (Qiagen), were compared for detection of viruses in whipped cream and lettuce (as representatives of the dairy and vegetable-fruit food groups, respectively). A seeding experiment with canine calicivirus was conducted to determine the efficiency of each virus extraction procedure. The PEG-NaCl-TRIzol method was most efficient for the detection of viruses in whipped cream and the ultracentrifugation-RNeasy-Mini Kit procedure was best for detection on lettuce. Based on the seeding experiments, food items implicated in norovirus-associated gastroenteritis outbreaks were subjected to the optimal procedure for a specific composition and matrix. No noroviruses were detected in the implicated food items, possibly because the concentration of virus on the food item was too low or because of the presence of inhibitory factors. For each food group, a specific procedure is optimal. Inhibitory factors should be controlled in these procedures because they influence virus detection in food

Evaluation of 5 '-nuclease and hybridization probe assays for the detection of shiga toxin-producing Escherichia coli in human stools

5'-Nuclease and a hybridization probe assays for the detection of shiga toxin-producing Escherichia coli were validated with regard to selectivity, analytical sensitivity, reproducibility and clinical performance. Both assays were capable of detecting the classical stx(1) and stx(2) genes when challenged with reference strains of E. coli (n=40), although 1 to 4 minority sequence variants, whose clinical relevance is limited (stx(1c), stx(1d), and stx(2f)), were detected less efficiently or not at all by one or both assays. No cross reaction was observed for both assays with 37 strains representing other gastrointestinal pathogens, or normal gastrointestinal flora. Analytical sensitivity ranged from 3.07 to 3.52 log(10) and 3.42 to 4.63 log(10) CFU/g of stool for 5'-nuclease and hybridization probe assay, respectively. Reproducibility was high with coefficients of variation o

Enhanced surveillance of Shiga toxin producing Escherichia coli in 2005

Since January 1999, an enhanced surveillance of Shiga toxin-producing Escherichia coli (STEC) O157 has been implemented in the Netherlands. In 2005, 53 symptomatic patients were diagnosed with STEC O157. This was relatively high compared with the number in previous years (annually 36 to 57), due to a national outbreak with 21 patients involved. Of the patients, 33% were hospitalised, 8% developed the haemolytic-uraemic syndrome (exclusion of outbreak-cases: 13%), including one one-year-old boy who died. Consumption of raw or undercooked beef and contact with farm animals and manure are still most frequently mentioned by the patients as possible cause. In 2005, cluster analyses of the fingerprints of bacterial DNA from the STEC O157 isolates (by pulsed-field gel electrophoresis) nine times suggested a relationship between several patients. For three clusters this was supported by additional epidemiological information. One cluster, consisting of two sub clusters, comprises the national outbreak caused by filet américain, except for two patients who fell ill two and one month before this outbreak. Furthermore, one household cluster was identified for which an indistinguishable PFGE pattern was found in a manure isolate taken from their cattle. In addition, an isolate from one individual case could be matched with an isolate taken from their neighbours cattle. As other serogroups than O157 can cause serious illness, a collaboration between RIVM and eight medical microbiological laboratories to assess the relative importance of non-O157 serogroups was started in the Netherlands in the autumn of 2005

Steak tartare (also known as “filet américain”) cause of first nationwide outbreak of Shiga-toxin producing E. coli O157 infections in the Netherlands

In September 2005, the first nationwide outbreak of Shiga toxin-producing Escherichia coli (STEC) O157 infections was observed. A total of 21 confirmed and 11 probable patients were reported, who fell ill between September 11 and October 10. Preliminary investigation by the local public health services revealed two possible risk factors: consumption of steak tartare and contact with other persons with gastroenteritis. The results of the subsequent case-control study suggested steak tartare as the most likely cause of the outbreak. Samples of steak tartare taken at a supermarket chain where most of the patients bought the product, tested negative for STEC O157. However, sampling took place 3 days after the date of symptom onset of the last outbreak case. Because 88% of the cases became ill within a two-week period and samples taken shortly afterwards tested negative, point source contamination of steak tartare was considered most plausible