Noroviruses are the leading cause of acute gastroenteritis in humans. Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) is a promising molecular method for the detection of noroviruses. In this study, the performance of three TaqMan real-time RT-PCR assays was assessed, which were one commercially available real-time RT-PCR kit (assay A: Norovirus Real Time RT-PCR kit) and two in-house real-time RT-PCR assays (assay B: LightCycler RNA Master Hybprobe and assay C: RealTime ready RNA Virus Master). Assays A and B showed higher sensitivity than assay C for norovirus GI, while they all had the same sensitivity (10 DNA copies/mL) for GII DNA standard controls. Assay B had the highest efficiency for both genogroups. No cross-reactivity was observed among GI and GII noroviruses, rotavirus, hepatitis A virus, and poliovirus. The detection rates of these assays in GI and GII norovirus-positive fecal samples were not significantly different. However, the mean quantification cycle (Cq) value of assay B for GII was lower than assays A and C with statistical significance (P-value, 0.000). All three real-time RT-PCR assays could detect a variety of noroviruses including GI.2, GII.2, GII.3, GII.4, GII.6, GII.12, GII.17, and GII.21. This study suggests assay B as a suitable assay for the detection and quantification of noroviruses GI and GII due to good analytical sensitivity and higher performance to amplify norovirus on DNA standard controls and clinical samples.
Rotavirus is a common cause of acute diarrhea in young children worldwide. This study investigated the prevalence and molecular characterization of rotavirus in environmental water and oyster samples in Thailand. A total of 114 water samples and 110 oyster samples were collected and tested for group A rotavirus using RT-nested PCR. Rotavirus genotype was identified by phylogenetic analysis of the VP7 genetic sequences. Group A rotavirus was detected in 21 water samples (18.4%) and six oyster samples (5.4%). Twenty five rotavirus strains were successfully sequenced and classified into four genotypes; G1, G2, G3, and G9. Rotavirus G1 (three strains), G2 (three strains), and G9 (two strains) demonstrated the genetic sequences similar to human strains (90%–99% nucleotide identity), whereas G3 (17 strains) was closely related to animal strains (84%–98% nucleotide identity). G1 strains belonged to lineages I (sub-lineage c) and II. G2 strains belonged to lineage II. G9 strains belonged to lineages III (sub-lineage b) and IV. G3 strains belonged to lineages I, III (sub-lineage c), and IV with a predominance of lineage I. The present study provides important information on the rotavirus strains circulating in the environment.
Aims: To investigate the presence of noroviruses in recycled water and sewage sludge obtained from a wastewater treatment plant in Bangkok Metropolitan Region, Thailand. Methods and Results: Twenty-seven recycled water and twenty-three sewage sludge samples were tested for the presence of norovirus genogroup (G)I and GII using RT-nested PCR. Molecular characterization of noroviruses was undertaken by DNA sequencing and phylogenetic analysis. The level of the RNA genome of the noroviruses was determined using quantitative real-time RT-PCR. Noroviruses were detected in 44Á4% of recycled water samples and 73Á9% of sewage sludge samples. Norovirus GI.2 and GII.4 were identified in recycled water samples at levels of 2Á19 9 10 1 and 3Á26 9 10 4 RNA copies per litre. Six different genotypes of GI (GI.1, GI.2, GI.5a, GI.5b, GI.6b and GI.7) and GII.17 were identified in sewage sludge samples at levels ranging from 1Á99 9 10 1 -1Á43 9 10 5 RNA copies per gram wet weight. Four recombinant norovirus strains were detected in sewage sludge samples, namely GII.P16-GII.2, GII.P16-GII.4, GII.P16-GII.13 and GII.P21-GII.13. Conclusions: These findings provide evidence that noroviruses may be spread to the community and environment via the use of recycled water for plant areas, and sewage sludge for land application. Significance and Impact of the Study: This is the first study demonstrating recombinant norovirus strains in sewage sludge samples. The presence of noroviruses in recycled water and sewage sludge contributes to a health risk of environmental exposure.
Although transmission of human norovirus in food establishments is commonly attributed to consumption of contaminated food, transmission via contaminated environmental surfaces, such as those in bathrooms, may also play a role. Our aim was to determine the prevalence of human norovirus on bathroom surfaces in commercial food establishments in New Jersey, Ohio, and South Carolina under nonoutbreak conditions and to determine characteristics associated with the presence of human norovirus. Food establishments (751) were randomly selected from nine counties in each state. Four surfaces (underside of toilet seat, flush handle of toilet, inner door handle of stall or outer door, and sink faucet handle) were swabbed in male and female bathrooms using premoistened macrofoam swabs. A checklist was used to collect information about the characteristics, materials, and mechanisms of objects in bathrooms. In total, 61 (1.5%) of 4,163 swabs tested were presumptively positive for human norovirus, 9 of which were confirmed by sequencing. Some factors associated with the presence of human norovirus included being from South Carolina (odd ratio [OR], 2.4; 95% confidence interval [CI], 1.2 to 4.9; P < 0.05) or New Jersey (OR, 1.7; 95% CI, 0.9 to 3.3; 0.05 < P < 0.10), being a chain establishment (OR, 1.9; 95% CI, 1.1 to 3.3; P < 0.05), being a unisex bathroom (versus male: OR, 2.0; 95% CI, 0.9 to 4.1; 0.05 < P < 0.10; versus female: OR, 2.6; 95% CI, 1.2 to 5.7; P < 0.05), having a touchless outer door handle (OR, 3.3; 95% CI, 0.79 to 13.63; 0.05 < P < 0.10), and having an automatic flush toilet (OR, 2.5, 95% CI, 1.1 to 5.3; 0.05 < P < 0.10). Our findings confirm that the presence of human norovirus on bathroom surfaces in commercial food establishments under nonoutbreak conditions is a rare event. Therefore, routine environmental monitoring for human norovirus contamination during nonoutbreak periods is not an efficient method of monitoring norovirus infection risk.
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