Development and application of a tool to assess laboratory hygiene in contained-use facilities.

To gain information on laboratory hygiene in contained-use laboratories, a method was developed to study the presence of microorganisms on laboratory equipment. Focusing detection on genetically modified organisms (GMOs) containing the universal M13 primer binding sites enabled the detection of a broad range of GMOs using a single PCR. Swabbing surfaces in three different contained-use laboratories led to detection of M13-containing PCR products in 26 out of 34 swabs. Most sequences (up to five per sample) were detected in swabs from the centrifuge and sink, followed by swabs taken from the bin and incubator (up to four sequences per sample). The obtained sequences varied in length from 171 nucleotides (nt) to 878 nt. In most cases, sequences were only partially similar to sequences published in GenBank. The lengths of the regions with high similarity varied from 94 nt to 795 nt, and these similarities ranged from 81% to 100%. Similarities with more than one sequence were commonly found, complicating the identification of detected sequences. Nonetheless, 84% of the detected sequences were actually handled in the laboratory at the time of sampling. This demonstrates that the method may be used as a quality control tool to assess the efficacy of decontamination and cleaning of commonly used surfaces, such as laboratory benches, freezer doors, and centrifuge rotors, without prior knowledge of the identity or characteristics of the GMOs.

Seroprevalence and molecular detection of hepatitis E virus in wild boar and red deer in The Netherlands.

To date, sources of hepatitis E virus (HEV) in the Netherlands, including swine and wild boar, have been identified, but no direct attribution to Dutch hepatitis E cases have been demonstrated. Other animal sources may exist. To identify these species, HEV RNA detection by RT-PCR is required, but complicated. A preselection based on serology may be useful. Therefore, wildlife species were studied by serology and molecular methods. Using a species-independent double-antigen sandwich ELISA, HEV-specific antibodies were detected in sera from 12% of 1029 wild boar (Sus scrofa scrofa), in 5% of 38 red deer (Cervus elaphus) and in none of 8 studied roe deer (Capreolus capreolus). Differences in background signals were observed between species and accounted for by fitting finite mixture distributions. HEV RNA was detected in 8% of 106 wild boars, in 15% of 39 red deer and in none of 8 roe deer. In conclusion, HEV was shown to be present in European red deer for the first time. This preselection based on species-independent serological assays may be beneficial to identify new potential animal reservoirs of HEV. The consumption of Dutch undercooked wild boar and red deer meat may lead to human exposure to HEV.

Year-round screening of noncommercial and commercial oysters for the presence of human pathogenic viruses.

Consumption of virus-contaminated shellfish has caused numerous outbreaks of gastroenteritis and hepatitis worldwide. In The Netherlands, oysters are cultured and imported both for consumption and export; therefore, the presence of noroviruses, rotaviruses, astroviruses, hepatitis A viruses, and enteroviruses was determined in 64 commercial and noncommercial oyster samples. Oysters were collected monthly for 13 months from four different harvesting areas in the Oosterschelde Delta. Oyster samples were classified by determining Escherichia coli levels according to the standards set by the Councils Directive (91/ 492/EEC). Two of 36 commercial and 2 of 28 noncommercial oyster samples were B-classified and therefore not ready for consumption. All other oyster samples were A-classified. For the detection of viral RNA, 150 mg of hepatopancreatic tissue was subjected to the Qiagen RNeasy Mini Kit, followed by reverse transcriptase (RT)-PCR and Southern blot hybridization. Enterovirus RNA was detected in 14 of 64 oyster samples, of which 4 were from noncommercial oyster harvesting areas and 10 were from commercial harvesting areas. None of the other human pathogenic viruses were detected. The levels of somatic coliphages and F-specific phages were also determined in all 64 oyster samples, with some samples containing high phage levels (>50 PFU/g of hepatopancreatic tissue), but with most samples containing low phage levels (<50 PFU/g of hepatopancreatic tissue). However, independent of these high or low phage levels, enterovirus RNA could be detected. Thus, commercial oysters can be contaminated with pathogenic viruses, and monitoring only fecal indicators might not sufficiently protect human health.