Recovery and Quantification of Norovirus in Air Samples from Experimentally Produced Aerosols.

Norovirus is the leading cause of acute gastroenteritis in humans across all age groups worldwide. Norovirus-infected patients can produce aerosolized droplets which play a role in gastroenteritis transmission. The study aimed to assess bioaerosol sampling in combination with a virus concentrating procedure to facilitate molecular detection of norovirus genogroup (G) II from experimentally contaminated aerosols. Using a nebulizer within an experimental chamber, aerosols of norovirus GII were generated at known concentrations. Air samples were then collected in both 5 mL and 20 mL water using the SKC BioSampler at a flow rate of 12.5 L/min, 15 min. Subsequently, the virus in collected water was concentrated using speedVac centrifugation and quantified by RT-qPCR. The optimal distances between the nebulizer and the SKC BioSampler yielded high recoveries of the virus for both 5 and 20 mL collections. Following nebulization, norovirus GII RNA was detectable up to 120 min in 5 mL and up to 240 min in 20 mL collection. The concentrations of norovirus GII RNA recovered from air samples in the aerosol chamber ranged from 102 to 105 genome copies/mL, with average recoveries of 25 ± 12% for 5 mL and 22 ± 19% for 20 mL collections. These findings provide quantitative data on norovirus GII in aerosols and introduce a novel virus concentrating method for aerosol collection in water, thus enhancing surveillance of this virus.

Prevalence, antimicrobial resistance, and enterotoxin gene profiles of Staphylococcus aureus isolated from mobile phones of the food vendors in Phayao province, Thailand.

BACKGROUND: Mobile phones are widely used and may cause bacterial pathogens to spread among various professionals. Staphylococcus aureus from the mobile phones can contaminate the hands of food vendors and food during the cooking or packaging process. This research aimed to determine the prevalence, enterotoxin genes, and antimicrobial resistance (AMR) profiles of S. aureus contaminating the vendors' mobile phones. METHODS: In this study, 266 mobile phone samples were randomly collected from food vendors selling food on walking streets (n = 139) and in food centers (n = 127) in Phayao province. All samples were identified as S. aureus by the conventional culture method and confirmed species-specific gene by polymerase chain reaction (PCR). Then, all identified S. aureus isolates were tested for antimicrobial susceptibility by broth microdilution method and for the presence of staphylococcal enterotoxin (SE) genes by PCR. RESULTS: The results showed that 12.8% of the mobile phones collected were contaminated with S. aureus. Of 49 S. aureus isolates obtained, 30 (61.2%) were positive for SE genes. The most common SE gene was sea followed by sec, seb, sem, seq, and sel. Moreover, S. aureus was most frequently resistant to penicillin, followed by chloramphenicol and tetracycline, erythromycin, clindamycin, and gentamicin. Methicillin-resistant S. aureus (MRSA), vancomycin-resistant S. aureus (VRSA), and multidrug-resistant (MDR) strains were also detected. CONCLUSIONS: This study showed that mobile phones were an intermediate surface for the transmission of S. aureus, including MDR variants. It indicates that hand hygiene and the decontamination of mobile phones are essential to prevent cross-contamination of S. aureus in food settings.

Molecular characterization and antibiotic resistance of Staphylococcus aureus isolated from clinical specimens in an urban university hospital in Bangkok, Thailand.

Little is known about the properties of the current strains of Staphylococcus aureus associated with human infections in Thailand. This study examined the rate of resistance to various antimicrobial agents, prevalence of virulence genes, and biofilm formation ability of 60 clinical S. aureus isolates from a single Thai hospital. Moreover, the Staphylococcus protein A gene (spa) type was determined among methicillin-resistant S. aureus (MRSA) isolates. Most methicillin-susceptible S. aureus isolates were susceptible to antimicrobials, whereas all MRSA isolates were resistant to erythromycin and clindamycin. The major virulence genes among the isolates were hla (100%), sec (26.7%), and hlb (20%). Meanwhile, 46.7% and 1.7% of the strains exhibited low-grade and high-grade biofilm formation, respectively. Our findings revealed the presence of spa types among MRSA isolates were: t032 (37.5%, 6/16), t088 (25%, 4/16), t001 (12.5%, 2/16), t008 (6.25%, 1/16), t034 (6.25%, 1/16), t439 (6.25%, 1/16), and t1928 (6.25%, 1/16). These findings will be useful for future research on anti-virulence therapies and the epidemiology of the strains circulating in our hospital.

Detection of Norovirus Recombinant GII.2[P16] Strains in Oysters in Thailand.

Human norovirus causes sporadic and epidemic acute gastroenteritis worldwide, and the predominant strains are genotype GII.4 variants. Recently, a novel GII.17[P17] and a recombinant GII.2[P16] strain have been reported as the causes of gastroenteritis outbreaks. Outbreaks of norovirus are frequently associated with foodborne illness. In this study, each of 75 oyster samples processed by a proteinase K extraction method and an adsorption-elution method were examined for noroviruses using RT-nested PCR with capsid primers. Thirteen (17.3%) samples processed by either method tested positive for norovirus genogroup II (GII). PCR amplicons were characterized by DNA sequencing and phylogenetic analysis as GII.2 (n = 6), GII.4 (n = 1), GII.17 (n = 3), and GII.unclassified (n = 3). Norovirus-positive samples were further amplified by semi-nested RT-PCR targeting the polymerase-capsid genes. One nucleotide sequence revealed GII.17[P17] Kawasaki strain. Five nucleotide sequences were identified as belonging to the recombinant GII.2[P16] strains by recombination analysis. The collected oyster samples were quantified for norovirus GII genome copy number by RT-quantitative PCR. Using the proteinase K method, GII was found in 13/75 (17.3%) of samples with a range of 8.83-1.85 × 104 genome copies/g of oyster. One sample (1/75, 1.3%) processed by the adsorption-elution method was positive for GII at 5.00 × 101 genome copies/g. These findings indicate the circulation of a new variant GII.17 Kawasaki strain and the recombinant GII.2[P16] in oyster samples corresponding to the circulating strains reported at a global scale during the same period of time. The detection of the recombinant strains in oysters emphasizes the need for continuing systematic surveillance for control and prevention of norovirus gastroenteritis.

Norovirus Monitoring in Oysters Using Two Different Extraction Methods.

Detection of noroviruses in bivalve shellfish is difficult because of the low concentration of norovirus and the presence of reverse transcription (RT)-PCR inhibitors. This study aimed to assess the presence of noroviruses in oysters extracted using a proteinase K extraction (ISO 15216 method) and an adsorption-elution method. Seventy oyster samples were extracted using the two extraction methods and evaluated using RT-nested PCR. The results showed norovirus detection rates at an equal frequency of 28.6%, of which a total of 48 (68.6%) samples had corresponding positive or negative results, while there were 22 (31.4%) samples with discrepant results. Norovirus genogroup (G)I, GII, and mixed GI and GII were detected in 20%, 4.3%, and 4.3% of samples, respectively, by the proteinase K extraction method, which comprised of GI.2, GI.5b, GI.6b, GII.4, and GII.17 genotypes. With the adsorption-elution method noroviruses were detected in 17.1%, 8.6%, and 2.9% of samples, respectively, which comprised of GI.2, GII.2, GII.4, and GII.17 genotypes. All norovirus-positive oyster samples were further estimated for genome copy number using RT-quantitative PCR. The oyster samples processed using the adsorption-elution method contained norovirus GI of 3.36 × 101-1.06 × 105 RNA copies/g of digestive tissues and GII of 1.29 × 103-1.62 × 104 RNA copies/g. Only GII (2.20 × 101 and 7.83 × 101 RNA copies/g) could be quantified in samples prepared using the proteinase K extraction method. The results demonstrate the different performance of the two sample-processing methods, and suggest the use of either extraction method in combination with RT-nested PCR for molecular surveillance of norovirus genotypes in oysters.

Prevalence of Human Noroviruses in Commercial Food Establishment Bathrooms.

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.

Evaluation of real-time RT-PCR assays for detection and quantification of norovirus genogroups I and II.

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 (103 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.

Genetic diversity of rotavirus strains circulating in environmental water and bivalve shellfish in Thailand.

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.