Method-dependent variability in determination of prevalence of Campylobacter jejuni and Campylobacter coli in Canadian retail poultry.

Campylobacter is the most frequent cause of bacterial gastroenteritis in Canada, and the illness is commonly associated with poultry consumption. Whereas Canadian retail poultry is often contaminated with campylobacters, studies on the prevalence of this organism are inconsistent due to variability in sampling and microbiological methodology. To determine the current microbiological status of Canadian poultry, and to evaluate two commonly used microbiological methods, 348 raw poultry samples were collected at retail across Canada over a period of 3 years (2007 to 2010) and were analyzed for the presence of thermophilic Campylobacter species. The overall prevalence of Campylobacter spp. was found to be 42.8% by a combination of the two testing methods, with 33.9% of the samples positive for C. jejuni, 3.7% of the samples positive for C. coli, and 5.2% of the samples positive for both. Variability in Campylobacter spp. prevalence was observed in samples obtained from different regions across Canada and from poultry with or without skin, but this was not statistically significant. In co-contaminated samples, C. jejuni was preferentially recovered from Preston agar compared with mCCDA and Campy-Cefex agar, with an increase in recovery of C. coli on all selective media after 48 h of enrichment. A subset of 214 of the poultry rinses were analyzed by both Health Canada's standard method, MFLP-46 (enrichment in Park and Sanders broth), and a second method requiring enrichment in Bolton broth. Significantly more positive samples were obtained with the MFLP-46 method (40.6%) than with the alternate method (35.0%). This improved recovery with MFLP-46 may be due to the omission of cycloheximide from this method. These results demonstrate that determination of prevalence of Campylobacter spp. on poultry products may be significantly impacted by the choice of microbiological methods used. Canadian poultry continues to be a source of exposure to Campylobacter spp.

Microbiological contamination in counterfeit and unapproved drugs.

BACKGROUND: Counterfeit and unapproved medicines are inherently dangerous and can cause patient injury due to ineffectiveness, chemical or biological contamination, or wrong dosage. Growth of the counterfeit medical market in developed countries is mainly attributable to life-style drugs, which are used in the treatment of non-life-threatening and non-painful conditions, such as slimming pills, cosmetic-related pharmaceuticals, and drugs for sexual enhancement. One of the main tasks of health authorities is to identify the exact active pharmaceutical ingredients (APIs) in confiscated drugs, because wrong API compounds, wrong concentrations, and/or the presence of chemical contaminants are the main risks associated with counterfeit medicines. Serious danger may also arise from microbiological contamination. We therefore performed a market surveillance study focused on the microbial burden in counterfeit and unapproved medicines. METHODS: Counterfeit and unapproved medicines confiscated in Canada and Austria and controls from the legal market were examined for microbial contaminations according to the US and European pharmacopoeia guidelines. The microbiological load of illegal and legitimate samples was statistically compared with the Wilcoxon rank-sum test. RESULTS: Microbial cultivable contaminations in counterfeit and unapproved phosphodiesterase type 5 inhibitors were significantly higher than in products from the legal medicines market (p < 0.0001). Contamination levels exceeding the USP and EP limits were seen in 23% of the tested illegal samples in Canada. Additionally, microbiological contaminations above the pharmacopoeial limits were detected in an anabolic steroid and an herbal medicinal product in Austria (6% of illegal products tested). CONCLUSIONS: Our results show that counterfeit and unapproved pharmaceuticals are not manufactured under the same hygienic conditions as legitimate products. The microbiological contamination of illegal medicinal products often exceeds USP and EP limits, representing a potential threat to consumer health.

Comparison of commercial viral genomic extraction kits for the molecular detection of foodborne viruses.

When genetic material is extracted from viruses responsible for food illnesses, two broad types of possibilities are offered: conventional methods, which are well established but usually long and exacting to perform, or commercial kits, which are faster and easy to use but much more expensive. Thus, it is important to evaluate some performance parameters such as the analytical sensitivity to be able to select the optimal technique for each situation. The principal objective of this study was to establish and compare the analytical sensitivities of three commercial genetic material extraction methods (TRIzol reagent, FTA cards, and QIAGEN kits) along with three selected viruses, adenovirus, hepatitis A virus, and rotavirus. Viral detection was carried out using a standard PCR technique for adenovirus and reverse transcription PCR for rotavirus and hepatitis A virus. The results obtained showed that with the QIAGEN kit, the sensitivity was 2 logs lower than with the two other methods for all three viruses studied. Nevertheless, despite their lower analytical sensitivities, the other two extraction methods should not be overlooked and ought to be considered when evaluating the most efficient approach suitable for a specific commodity, since food-related outbreaks may be traced to a wide variety of food types.

The feline calicivirus as a sample process control for the detection of food and waterborne RNA viruses.

Many food and waterborne outbreaks of infectious disease are caused by viruses. While numerous methods exist and are being developed to test food and water for the presence of enteric viruses, there is no standard control for the comparison of different methods. Potential control viruses should be well characterized, share the physical characteristics of the enterically infecting viruses and not normally be associated with foods. Here, the feline calicivirus (FCV) is proposed as a sample process control for methods aimed at the extraction and detection of RNA viruses in food and water. FCV is shown to be useful as a control for the extraction of hepatitis A virus (HAV) from water using filtration technology and from strawberries using the Pathatrix system. The FCV standard provides a valuable quality control tool when testing potentially contaminated food samples.

Molecular characterization of hepatitis E virus detected in swine farms in the province of Quebec.

Swine Hepatitis E virus (HEV) could be a zoonotic agent for HEV infection in humans. In Canada, approximately 60% of 6-mo-old commercial pigs are seropositive for HEV; the prevalence is higher in the provinces of Quebec and Ontario. A study was set up to evaluate the presence of swine HEV in Quebec farms and to compare the strains detected in fecal samples with human and swine HEV strains reported worldwide. Fecal samples were collected randomly from May 2003 to January 2004 from 70 swine farms in Quebec. In 24 specimens, representing 34% of the visited farms, HEV RNA was extracted and detected by nested reverse-transcription polymerase chain reaction (RT-PCR). All amplified nested RT-PCR products were purified, cloned, and sequenced. The nucleotide sequences of a 304 base pair fragment at the 5' end of the open reading frame 2 gene were determined. Phylogenetic analysis revealed that the 24 amplified fragments clustered in genotype 3 and had 85% to 99% nucleotide-sequence similarity with HEV strains identified in Japan, the United States, and Canada. Three strains identified in the study (swSTHY1, swSTHY31, and swSTHY47) showed 95% homology with 2 Japanese (swJ1-1 and HE-JA10) and 1 American (US1) HEV human strains.

An outbreak of norovirus caused by consumption of oysters from geographically dispersed harvest sites, British Columbia, Canada, 2004.

OBJECTIVES: In January 2004, an increase in gastrointestinal illness following oyster consumption was reported in British Columbia. An investigation was initiated to explore the association between norovirus infection and consumption of British Columbia oysters and to identify the source of oyster contamination. METHODS: The outbreak investigation included active surveillance for human cases, two cohort studies, trace-back of oysters, and laboratory testing of oysters and human stools. RESULTS: Enhanced surveillance identified 26 confirmed and 53 clinical cases over 3 months. Oyster consumption was associated with illness in one cohort and suggestive in the other. Oysters were traced to 14 geographically dispersed harvest sites, 18 suppliers, and 45 points of purchase. Norovirus BCCDC03-028 (genotype I.2) was detected in 50% of human specimens. Experimental methods detected norovirus in 12 oyster samples. Sequencing identified mixed clonal patterns in the oysters with one direct sequence match between an oyster sample and the associated human specimen. CONCLUSIONS: The consumption of raw oysters led to norovirus infection. The source of oyster contamination remained unidentified. The geographical dispersion of implicated harvest sites was unusual. APPLICATIONS: This outbreak is unlike most shellfish outbreaks that can be traced back to a common source and challenges conventional thinking that all oyster-related norovirus outbreaks of are a result of point source contamination.

Presence of hepatitis E virus in a naturally infected swine herd from nursery to slaughter.

Hepatitis E virus has recently been recognized as having zoonotic potential and could be transmitted from pig to human. Pigs are identified as a potential animal reservoir and HEV is highly prevalent in the swine population around the world. In this study, the presence of HEV was investigated in 51 subjects reared on a simulated commercial farm setting from the age of 2 weeks up to slaughter. Samples were collected on four occasions: at 2, 8, and 18 weeks and between 22-29 weeks of age. Anti-HEV IgG in plasma samples, presence of HEV RNA in plasma samples and feces were monitored. At 2 weeks of age, HEV RNA was detected in feces of 6 subjects (11.8%) but not in their plasma. At 8 weeks, HEV was detected in feces of 27 subjects (52.9%) and in plasma of one subject. At 18 weeks, HEV was detected in feces of 44 subjects (86.2%) and in plasma of 24 subjects (47.1%). At slaughter time (22-29 weeks), HEV was present in plasma of 6 subjects (11.8%) and in stools of 21 subjects (41.2%). Spread of the virus inside the population was evaluated by comparison of means (paired t-test, P<0.05) of anti-HEV IgG ELISA results from the 4 bleedings. Significant differences were noted between the results of populations at 8 and 18 weeks and also between 18 and 22 to 29 weeks indicating an immune response to the virus. Based on the comparison of a 304 nucleotides sequence of the 5' ORF 2 gene, all amplified fragments clustered in genotype 3a.

Comparative evaluation of new TaqMan real-time assays for the detection of hepatitis A virus.

Three novel real-time TaqMan RT-PCR assays targeting the 5'-UTR, the viral protease and the viral polymerase regions of the hepatitis A virus (HAV) were developed, evaluated and compared against a new published 5'-UTR TaqMan assay (JN) and a widely used conventional RT-PCR assay (HAVc). All conventional RT-PCR (HAV, SH-Prot and SH-Poly systems) and TaqMan (SH-Prot, SH-Poly, JN and SH-5U systems) assays evaluated were consistent for the detection of the three different HAV strains (HM-175, HAS-15 and LSH/S) used and reproducible for both RNA duplicates with the exception of two reproducibility discrepancies observed with both 5'-UTR real-time systems (JN and SH-5U). Limits of detection for conventional HAV, SH-Prot and SH-Poly RT-PCR systems were found to be equivalent when tested with serially diluted suspensions of the HM-175 strain. Although the four real-time RT-PCR TaqMan assays evaluated herein produced similar and consistent quantification data down to the level of one genomic equivalent copy with their respectively cloned amplicons, significant and important differences were observed for the detection of HAV genomic RNA. Results showed that the new real-time TaqMan SH-Poly and SH-Prot primer and probe systems were more consistent and sensitive by 5 logs as compared to both 5'-UTR designs (JN and SH-5U) used for the detection of HAV genomic RNA as well as for the detection in cell culture by cytopathic effect. Considering their higher analytical sensitivity, the proposed SH-Poly and SH-Prot amplification systems could therefore represent valuable tools for the detection of HAV in clinical, environmental and food samples.

Comparative evaluation of RT-PCR, nucleic acid sequence-based amplification (NASBA) and real-time RT-PCR for detection of noroviruses in faecal material.

Using different primer and probe sets, RT-PCR, NASBA and TaqMan RT-PCR molecular methods were compared to detect NoV GII in 13 clinical stool samples. The RT-PCR results observed by gel electrophoresis (Ando, Kageyama and Anderson amplification and probe systems), dot blot hybridization (Ando and Kageyama) and real-time TaqMan assay (Ando and Kageyama) were shown to be consistent and reproducible for the detection of NoV GII. Whereas, the NASBA assay using Ando primers showed some reproducibility discrepancies. Detection limits of the NoV GII/Kageyama system were found to be equal or significantly higher than the Ando system. Real-time TaqMan RT-PCR assay showed similar detection limits to that of NASBA with the Kageyama amplification and detection system, while it was 1log less sensitive than the Ando system. In a clinical context, RT-PCR, NASBA and real-time TaqMan RT-PCR methods using undiluted samples were all suitable for the detection of NoV GII, however the NASBA assay provided less consistent signals. The NoV GII Kageyama real-time TaqMan RT-PCR assay was reliable with a high analytical sensitivity and has shown the capability of detecting one genomic equivalent copy.

Development of an extraction and concentration procedure and comparison of RT-PCR primer systems for the detection of hepatitis A virus and norovirus GII in green onions.

Vegetables can be considered as a vector of transmission for human hepatic and enteric viruses such as hepatitis A virus (HAV) and noroviruses when contaminated by spoiled irrigation water or when prepared by infected food handlers. Recently, outbreaks of HAV have been reported in the USA involving fresh green onions. A viral elution-concentration method was developed for the detection of HAV and norovirus contaminated green onions by RT-PCR. Repeated pipetting/washings of the surface with a pH 9.5 glycine-buffered solution allowed the elution of viruses from the vegetables. Concentration of the viral load was performed by a polyethylene glycol (PEG) precipitation procedure. Viral RNAs were extracted and purified using a combination of Trizol-chloroform and poly(dT) magnetic beads methods. Different sets of primers, including two newly designed primers sets for HAV RT-PCR, were tested in order to achieve the best analytical sensitivity. Using the new primer design, it was possible to detect 10(0) TCID(50%)/25 g of HAV in fresh green onions, while 1 RT-PCRU/25 g was detected for noroviruses GII using previously described primers. This method, based on molecular tools, would be useful for diagnostic laboratories in order to perform viral analyses of such commodities as fresh vegetables in cases of foodborne infections.

Concentration and detection of hepatitis A virus and rotavirus in spring water samples by reverse transcription-PCR.

Every year, enteric viruses such as hepatitis A virus (HAV), rotaviruses, and noroviruses are responsible for viral gastro-enteritis and hepatitis reported worldwide. These viruses are mostly transmitted via the faecal-oral route, from direct contact between people, or by ingestion of contaminated food and water. Since only a few viral particles may cause disease, detection of low concentration of these viruses in food matrices is usually complex. The development of methods to concentrate viruses from food matrices is crucial in collecting data for the development of control strategies or for diagnostic purposes. In the present study, samples of bottled spring water were inoculated with known amounts of HAV (strain HM-175), and rotaviruses (strain Wa) viral particles and filtered through positively charged membranes. Elution of viruses attached to the membranes was achieved with a tryptose phosphate broth-glycine buffer. Eluates were further concentrated using Microsep 100. Finally, RNA was extracted using the Qiagen RNeasy kit followed by an evaporation step with a SpeedVac instrument. The detection limit by reverse-transcription (RT-PCR) was at least 10(-1) TCID50%/ml and at least 10(-3) TCID50%/ml for HAV and rotavirus, respectively.