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This study aims to determine the presence of norovirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and bocavirus in air samples from a tertiary care hospital in Bangkok, Thailand. Air samples were collected in water using the BioSampler and concentrated using speedVac centrifugation. Based on RT-qPCR, norovirus RNA and SARS-CoV-2 RNA were detected in 13/60 (21.7%) and 3/60 (5.0%) of samples, respectively. One air sample had a weak positivity for both norovirus and SARS-CoV-2 RNAs. Detection rate of norovirus genogroup (G) II (13.3%) was higher than norovirus GI (6.7%). One air sample (1.7%) tested positive for GI and GII. The norovirus GI RNA concentration was 6.0 × 10 2 genome copies/m 3 . The norovirus GII RNA concentrations ranged from 3.4 × 10 1 to 5.0 × 10 3 genome copies/m 3 . Based on RT-nested PCR, norovirus GII was detected in two (3.3%) samples. All samples tested negative for GI RNA and bocavirus DNA. By phylogenetic analysis, GII.17, which is closely related to the outbreak Kawasaki308/JPN/2015 strain, was found in the RT-nested PCR-positive samples. This study highlights the potential of aerosols for norovirus and SARS-CoV-2 transmission and probably cause gastrointestinal and respiratory illnesses, respectively., (© 2024. The Author(s).)

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 10 2 to 10 5 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., (© 2024. The Author(s).)

Light penetration depth in the scalp is a key limitation of low-level light therapy for the treatment of androgenetic alopecia (AGA). A novel light emitting diode (LED) microneedle patch was designed to achieve greater efficacy by enhancing the percutaneous light delivery. The study aimed to investigate the efficacy and safety of this device on hair growth in mice. Thirty-five male C57BL/6 mice which their dorsal skin was split into upper and lower parts to receive either LED irradiation alone or LED irradiation with a microneedle patch. Red (629 nm), green (513 nm), and blue light (465 nm) at an energy dose of 0.2 J/cm 2 were applied once daily for 28 days. Outcomes were evaluated weekly using digital photographs. Histopathological findings were assessed using a 6 mm punch biopsy. A significant increase in hair growth was observed in the green light, moderate in the red light, and the lowest in the blue light group. The addition of the microneedle patch to LED irradiation enhanced greater and faster anagen entry in all the groups. Histopathology showed an apparent increase in the number of hair follicles, collagen bundles in the dermis, angiogenesis, and mononuclear cell infiltration after treatment with the green-light LED microneedle patches. No serious adverse effects were observed during the experiment. Our study provides evidence that the newly developed green-light LED microneedle patch caused the optimal telogen-to-anagen transition and could lead to new approaches for AGA. Microneedle stimulation may aid percutaneous light delivery to the target hair follicle stem cells., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The study aimed to assess the presence and molecular characterization of human bocavirus (HBoV) in recycled water and sewage sludge samples in Thailand. One hundred and two recycled water and eighty-six sewage sludge samples collected from a wastewater treatment plant were tested for the presence of HBoV using nested PCR with broad-range primer pairs targeting the capsid proteins VP1 and VP2. HBoV DNA was detected in recycled water of 9/102 (8.8%) samples and sewage sludge of 27/86 (31.4%) samples. Based on DNA sequencing and phylogenetic analysis, the HBoV DNA sequences had 98.8-100.0% nucleotide identity to the sequences from HBoV reported globally. Thirty-five HBoV-positive samples were identified to genotypes as the predominant HBoV2; 26 followed by HBoV3; 8 and the rare HBoV4; 1 sample. Concerning recycled water, HBoV2 was detected in 3 (2.9%) and HBoV3 was detected in 5 (4.9%) of all samples. The sewage sludge samples were characterized as HBoV2 in 23 (26.7%), HBoV3 in 3 (3.5%) and HBoV4 in 1 (1.2%) of all samples. The frequency of HBoV detected in recycled water and sewage sludge samples significantly differed in sample type (p-value = 0.007). The findings of three HBoV genotypes in recycled water and sewage sludge emphasized the circulation of the virus in the environment and the potential source of transmission to the community., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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 × 10 4 genome copies/g of oyster. One sample (1/75, 1.3%) processed by the adsorption-elution method was positive for GII at 5.00 × 10 1 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., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The objective of this study was to describe the epidemiological and molecular surveillance of rotaviruses in tap water, recycled water, and sewage sludge in Thailand from 2007 to 2018. Three hundred and seventy tap water, 202 recycled water, and 72 sewage sludge samples were collected and processed to detect the rotavirus VP7 gene using RT-nested PCR. Rotavirus G genotypes were identified by DNA sequencing and phylogenetic analysis. The frequency of rotavirus detection was 0.54% of the tap water samples, 30.2% of the recycled water samples, and 50.0% of the sewage sludge samples. During the 12-year surveillance, G1 was prevalent most years and constantly predominant in recycled water and sewage sludge. G2 was identified in a tap water sample and in recycled water samples. G3 and G9 were observed in both recycled water and sewage sludge samples. The uncommon G6 rotavirus strain was identified in one recycled water sample. The rotavirus VP4 gene was detected in rotavirus strains with an identified G genotype using RT-multiplex nested PCR. The unusual P[6] genotype was the most frequently detected, followed by mixed P[6]/[4] and P[4] genotypes. Phylogenetic analysis of both G and P genotypes showed a close genetic relationship with sequences of human rotavirus strains. The high nucleotide identity of the rotavirus strains found in this study to human rotavirus strains suggests that the rotaviruses are derived from human source. These results represent useful epidemiological and molecular information for evaluating rotavirus distribution in water for consumption and irrigation, and in biosolids for agricultural application.

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 × 10 1 -1.06 × 10 5 RNA copies/g of digestive tissues and GII of 1.29 × 10 3 -1.62 × 10 4 RNA copies/g. Only GII (2.20 × 10 1 and 7.83 × 10 1 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.

This study evaluated different tissues of naturally contaminated oysters (Crassostrea belcheri) for the presence of noroviruses. RNA from digestive tissues, gills, and mantle of the oysters was extracted and tested for norovirus genogroup (G) I, GII, and GIV using RT-nested PCR. In spiking experiments with a known norovirus, GII.4, the detection limits were 2.97 × 10 2 RNA copies/g of digestive tissues, 2.62 × 10 2 RNA copies/g of gills, and 1.61 × 10 3 RNA copies/g of mantle. A total of 85 oyster samples were collected from a fresh market in Bangkok, Thailand. Noroviruses were found in the oyster samples (40/85, 47%): GI (29/85, 34.1%), GII (9/85, 10.5%), mixed GI and GII (1/85, 1.2%), and GIV (1/85, 1.2%). All three genogroups were found in the digestive tissues of oysters. Norovirus GI was present in all three tissues with the highest frequency in the mantle, and was additionally detected in multiple tissues in some oysters. GII was also detected in all three tissues, but was not detected in multiple tissues in the same oyster. For genogroup I, only GI.2 could be identified and it was found in all tissues. For genogroup II, three different genotypes were identified, namely GII.4 which was detected in the gills and the mantle, GII.17 which was detected in the digestive tissues, and GII.21 which was detected in the mantle. GIV.1 was identified in the digestive tissues of one oyster. This is the first report on the presence of human GIV.1 in oyster in Thailand, and the results indicate oyster as a possible vehicle for transmission of all norovirus genogroups in Thailand.

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 3 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.

Noroviruses are the most common cause of acute gastroenteritis associated with bivalve shellfish consumption. This study aimed to detect and characterize noroviruses in three bivalve shellfish species: oysters (Saccostrea forskali), cockles (Anadara nodifera), and mussels (Perna viridis). The virus concentration procedure (adsorption-twice elution-extraction) and a molecular method were employed to identify noroviruses in shellfish. RT-nested PCR was able to detect known norovirus GII.4 of 8.8 × 10(-2) genome copies/g of digestive tissues from oyster and cockle concentrates, whereas in mussel concentrates, the positive result was seen at 8.8 × 10(2) copies/g of digestive tissues. From August 2011 to July 2012, a total of 300 shellfish samples, including each of 100 samples from oysters, cockles, and mussels were collected and tested for noroviruses. Norovirus RNA was detected in 12.3 % of shellfish samples. Of the noroviruses, 7.7 % were of the genogroup (G) I, 2.6 % GII, and 2.0 % were mixed GI and GII. The detection rate of norovirus GI was 2.1 times higher than GII. With regards to the different shellfish species, 17 % of the oyster samples were positive, while 14.0 and 6.0 % were positive for noroviruses found in mussels and cockles, respectively. Norovirus contamination in the shellfish occurred throughout the year with the highest peak in September. Seventeen norovirus-positive PCR products were characterized upon a partial sequence analysis of the capsid gene. Based on phylogenetic analysis, five different genotypes of norovirus GI (GI.2, GI.3, GI.4, GI.5, and GI.9) and four different genotypes of GII (GII.1, GII.2, GII.3, and GII.4) were identified. These findings indicate the prevalence and distribution of noroviruses in three shellfish species. The high prevalence of noroviruses in oysters contributes to the optimization of monitoring plans to improve the preventive strategies of acute gastroenteritis.

The objectives of this study were to develop a method for concentrating rotavirus, to assess the detection rate, and to characterize the genotype of naturally occurring rotavirus in bivalve shellfish species; including oysters (Saccostrea forskali), cockles (Anadara nodifera), and mussels (Perna viridis). The results demonstrated that an adsorption-twice elution-extraction method was less-time consuming method of concentrating the spiked rotavirus, yielding high sensitivity of 1.14 genome copies/g of digestive tissues from all three shellfish species, as detected using an RT-nested PCR. In seeding experiments, rotavirus as low as 1.39 genome copies was able to be detected in 4 g of digestive tissues or per sample. In the period of August 2011 to July 2012, of the 300 bivalve shellfish samples collected and tested, 24 (8.0%) were found to be contaminated with rotavirus, the figures being: oysters, 13/100 samples; mussels, 10/100 samples; and cockles, 1/100 samples. By DNA sequencing of the RT-nested PCR products and phylogenetic analysis, the rotaviruses detected were classified into G1, lineage II (4 samples); G3 (10 samples): lineage I (3 samples), lineage IIIc (3 samples), lineage IIId (3 samples), lineage IV (1 sample); G9 (6 samples); and G12, lineage III (1 sample). These findings suggest that this virus concentration method provides high sensitivity for the detection of rotavirus from the three bivalve shellfish species. The prevalence of rotavirus and the identified genotypes contribute to the molecular epidemiology of rotavirus in different shellfish species., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

and young children, but rotavirus gastroenteritis in adults is uncommon. In this study, 260 stool samples collected in Thailand from January 2006 to February 2007 from patients, of all ages with acute gastroenteritis, were tested for group A rotavirus and compared with rotavirus infections in children and adults. Rota- virus was detected in 42% of the patients' samples, but children (< 18 years old) have a significantly higher prevalence (57%) of rotavirus infection than adults (≥ 18 years old) (27%) (OR 3.55; 95% CI: 2.11-5.96; p < 0.001). The highest attack rate was found in the age group of < 2 years old (14%), followed by 2-4 years of age (9%), 18-59 years of age (8%), 5-17 years of age (6%) and ≥ 60 years of age (5%). The dominant genotype was G1P[8] (27%), followed by G2P[4] (7%), G3P[8] (1%), and G9P[8] (1%). The rare genotypes identified were G1P[4], G1P[6], G2P[6], G2P[8], and G3P[6]. Mixed infections mostly occurred in children, comprising G1P[4]/P[8], G1P[4]/P[6], G1P[6]/P[8], G1/G2P[4], G1/G3P[4], and G1/G3P[4]/P[8]. Rotaviruses G3, G9, and P[4] were found only in children and genotype P[6] was found in adults (75%) at a higher frequency than in children (25%) (p < 0.001). The number of rotavirus in children was 1.99x10(8)/ml and in adult patients was 7.32x10(6)/ ml. The present study highlights the higher prevalence of rotavirus infection in children compared to adults and rotavirus genetic heterogeneity. Rotaviruses are the most important cause of severe diarrhea in infants

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.

The aim of this study was to detect and characterize noroviruses (NoVs) in environmental water samples. One hundred and fourteen water samples were collected from a river and irrigation canals in central Thailand during 2006-2007. NoVs were detected by RT-nested PCR in 13% of the samples. The river samples (22%) contained NoVs at a higher frequency than the irrigation canal samples (4%). Among the 15 NoV-positive samples, 9 harbored genogroup (G) I, 2 samples with GII, and 4 samples with mixed GI and GII. DNA sequencing of PCR amplicons and phylogenetic analysis of partial capsid gene revealed that 5 samples were of genotype GI-2, 1 sample was GI-6, and 1 sample was a mix of GI-2 and GII-unclassified genotypes. NoVs in water samples quantified using quantitative RT-PCR were in the range of 4.91 x 10(2) -1.26 x 10(3) copies/ml for NoV GI and 3.51 x 10(3) copies/ml for NoV GII. This is the first study demonstrating the presence of NoV variants in water samples collected from a river and the adjacent canals of Thailand.

Objective: To investigate the efficacy of a vaccine formulation of the 19 kDa conserved carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (PyMSP1(19)) formulated with CpG ODN 1826 and Montanide ISA51 or ISA720 when used to immunize mice by a single injection., Methods: Groups of BALB/c mice were immunized parenterally with one, two or four injections with PBS or PyMSP1(19) formulated with CpG ODN in ISA51 or ISA720. Sera were collected weekly and assessed for total IgG and IgG subclass titers. Protection was tested by challenge infection with P. yoelii YM., Results: Interestingly, single injection immunization showed the same kinetics of antibody responses as two- or four-injection immunization. However, the peak antibody response induced by PyMSP1(19) in CpG ODN and ISA51 appeared earlier than that induced by PyMSP1(19) in CpG ODN and ISA720 (28 days vs 41 days). At day 63 after the first injection, the PyMSP1(19)-specific IgG antibody levels by single injection and four-injection immunizations were not different. However, the levels of the IgG2a antibody subclass were significantly lower by single injection immunization with PyMSP1(19) in CpG ODN and ISA720. The antibodies were sustained at high levels for at least 20 weeks. After challenge infection, all mice immunized by a single injection of PyMSP1(19) in CpG ODN and ISA51 survived with low-grade parasitemia, while 50% of mice immunized with PyMSP1(19) in CpG ODN and ISA720 died with high levels of parasitemia., Conclusion: These findings suggest that MSP1(19) immunization by a single injection can induce protective immunity, particularly when formulated with an appropriate strong adjuvant.

One hundred and eighteen oyster samples collected from local markets and oyster farms in southern Thailand were examined for noroviruses (NoVs) and bacterial indicators of fecal contamination (fecal coliforms and Escherichia coli). Using a virus concentration procedure followed by RT-nested PCR, NoVs were detected in 38% of the samples. Oysters collected from oyster farms were found with NoVs at a higher detection rate (25/53 samples) than oysters from local markets (20/65 samples). Of the 45 NoV-positive oyster samples, 67% belonged to NoV genogroup I (GI), 15% to GII, and 18% to both GI and GII. DNA sequencing showed that 2 NoVs belonged to NoV GI-2 genotype. Fecal coliforms in NoV-positive oyster samples were in the range of < 3.0 to 1.5 x 10(4) most probable number (MPN)/g and 33% of NoV-positive oyster samples contained fecal coliforms within the standard acceptable level of raw shellfish (< 20 MPN/g). E. coli was found in the range of < 3.0 to 1.5 x 10(4) MPN/g and 9% of NoV-positive oyster samples were within acceptable levels of E. coli contamination (< 3 MPN/g). These findings indicate that NoV contamination in oysters obtained from both markets and oyster farms might pose a potential risk of acute gastroenteritis associated with raw oyster consumption. Examination for both fecal bacterial indicators and enteric viruses should be conducted for microbiological food safety of shellfish.

Noroviruses (NoVs) are recognized as a significant cause of acute gastroenteritis in children and adults. A 14-month study, from January 2006 to February 2007, was undertaken in a hospital in Thailand to determine the prevalence and genetic characterization of NoVs in patients of all ages with acute gastroenteritis. Based on reverse transcription-nested polymerase chain reaction (RT-nested PCR), NoVs were detected in 122 of 273 (44.7%) collected stool samples. Of the 122 NoV-positive samples, 28 (23%) belonged to GI, 79 (64.8%) belonged to GII, and 15 (12.2%) were mixed infections of GI and GII strains. Three NoV GI-positive and 42 NoV GII-positive samples were characterized successfully by DNA sequencing of the RT-nested PCR products and phylogenetic analysis. For NoV GI, two genotypes were identified: GI-2 (one sample) and GI-6 (two samples). NoV GII could be classified further into five distinct genotypes: GII-2 (1 sample), GII-3 (3 samples), GII-4 (14 samples), GII-6 (3 samples), and GII-17 (2 samples), and one unclassified genotype (19 samples). All NoV GII-4 strains showed 88-98% nucleotide identity with NoV GII-4 2006b variants reported worldwide. Among genotypes of NoV characterized, one co-infected stool sample exhibited NoVs GI-6 and GII-4 2006b. This study suggests that there is an important role of NoVs as etiologic agents in patients with acute gastroenteritis. The predominant circulating genotype of NoV infections is GII-4 2006b variant., ((c) 2010 Wiley-Liss, Inc.)

This study was conducted to determine the presence of hepatitis A virus (HAV) in raw oysters (Crassostrea belcheri) using a virus concentration method and reverse transcription-nested polymerase chain reaction (RT-nested PCR). A total of 220 oyster samples were collected from oyster farms and local markets in Thailand. HAV was found in three oyster samples. Nested PCR products of HAV detected in oysters were characterized further by DNA sequencing of the VP1/2A region and subjected to phylogenetic analysis. All HAV sequences (168 basepairs) were associated with human HAV subgenotype IB (GIB). Fecal coliforms and Escherichia coli were determined using the multiple tube fermentation method, to assess the microbiological quality of collected oysters. Among oyster samples tested, 65% had fecal coliforms higher than the standard level for raw shellfish [< 20 Most Probable Numbers (MPN)/g]; MPN values in the range of 21.0-4.6 x 10(4)/g. Most oyster samples (85%) were contaminated with E. coli in the range of 3.0-4.6 x 10(4) MPN/g. One oyster sample with an acceptable level of fecal coliforms contained HAV GIB. E. coli was found in all HAV-positive oyster samples. The results suggest a significant presence of HAV and bacterial indicators of fecal contamination in raw oysters, which are a health risk for consumers and a source of gastrointestinal illness. Enteric viruses should also be tested to assess the microbiological quality of oysters.

Outbreaks of viral gastroenteritis occur worldwide including Thailand. Unfortunately, there is limited information since etiologic agents have not been identified in several outbreaks of nonbacterial gastroenteritis. The genotype of enteric viruses causing acute gastroenteritis in Thailand was determined using reverse transcription-multiplex polymerase chain reaction and DNA sequencing. From January 2006 to February 2007, stool samples were collected from patients with acute gastroenteritis of all age groups attending a hospital in Thailand, and patients with nonbacterial acute gastroenteritis (262 patients) were tested for enteric viruses. The overall positive detection rate of enteric viruses was 14.9%; group A rotaviruses (6.1%), noroviruses (6.5%): GI (0.8%) and GII (5.7%), adenoviruses (1.5%), and sapoviruses (0.8%) were found. Group B and C rotaviruses, and astroviruses were not detected in the enrolled patients. Viral acute gastroenteritis occurred in children less than 15 years of age (25.2%, 33/131) with higher frequency than in adults (4.6%, 6/131), P-value <0.001. Rotavirus G1 was the most predominant genotype, followed by G3, and G9. Among noroviruses, GI-2 was identified; whereas, GII was predominant with a high frequency of GII-4 observed, followed by GII-16, GII-2, GII-3, and GII-12. Sapovirus GII-3 and human adenoviruses were identified. This study suggests that enteric viruses play an essential role in patients with acute gastroenteritis attending hospital and mainly in children who have a higher prevalence of group A rotaviruses and noroviruses. The genetic analyses provide molecular epidemiological data for viruses important to public health., ((c) 2008 Wiley-Liss, Inc.)

Background: Dengue haemorrhagic fever is an important public health problem and mainly occurs in children less than 15 years of age. Recently, the incidence of the disease have increased in adults but data on clinical and laboratory presentations of those affected are limited., Objectives: To assess and compare clinical manifestations and laboratory findings of dengue virus infected children and adults in Thailand., Study Design: A 1-year study was conducted from September 2003 to August 2004 for dengue virus infected patients admitted to Phetchabun Provincial Hospital, Thailand. Physical signs, symptoms, and laboratory features were recorded. All dengue patients were confirmed using immunochromatographic test on convalescent sera., Results: Based on serology-confirmed dengue virus infection, there was 286 dengue patients including 15 (5.3%) dengue fever and 271 (94.7%) dengue haemorrhagic fever (DHF). Among DHF cases, clinical classifications were DHF I, 40.9%; DHF II, 43%; and DHF III or dengue shock syndrome (DSS), 10.8%. Of all dengue patients, 231 cases (80.8%) were children aged less than 15 years and 55 cases (19.2%) were adults. The highest proportion of child cases was DHF I (42.9%), whereas that of adults was DHF II (51%). Some clinical manifestations were more common in adult patients, such as petechiae, melena, headache, retro-orbital pain, joint pain, myalgia, nausea and vomiting (p-value<0.05). Signs found commonly in children were epistaxis, oliguria, and liver enlargement (p-value<0.05). Haemoconcentration, thrombocytopenia, increased alanine aminotransferase, and longer prothrombin time were found to be significantly higher in adults than in children (p-value<0.05)., Conclusions: Some clinical presentations of dengue disease and laboratory findings in adults are different from those in children. Therefore, adults as well as pediatric cases of DHF need appropriate and prompt case management to reduce the mortality rate of DHF.

Merozoite surface protein 1 (MSP1) is the major protein on the surface of the plasmodial merozoite, and its carboxy terminus, the 19-kDa fragment (MSP1(19)), is highly conserved and effective in induction of a protective immune response against malaria parasite infection in mice and monkeys. However, the duration of the immune response has not been elucidated. As such, we immunized BALB/c mice with a standard four-dose injection of recombinant Plasmodium yoelii MSP1(19) formulated with Montanide ISA51 and CpG oligodeoxynucleotide (ODN) and monitored the MSP1(19)-specific antibody levels for up to 12 months. The antibody titers persisted constantly over the period of time without significant waning, in contrast to the antibody levels induced by immunization with Freund's adjuvant, where the antibody levels gradually declined to significantly lower levels 12 months after immunization. Investigation of immunoglobulin G (IgG) subclass longevity revealed that only the IgG1 antibody level (Th2 type-driven response) decreased significantly by 6 months, while the IgG2a antibody level (Th1 type-driven response) did not change over the 12 months after immunization, but the boosting effect was seen in the IgG1 antibody responses but not in the IgG2a antibody responses. After challenge infection, all immunized mice survived with negligibly patent parasitemia. These findings suggest that protective immune responses to MSP1(19) following immunization using oil-based Montanide ISA51 and CpG ODN as an adjuvant are very long-lasting and encourage clinical trials for malaria vaccine development.

Water samples were concentrated by the modified adsorption-elution technique followed by speedVac reconcentration of the filter eluates. Reverse transcriptase-nested polymerase chain reaction (RT-nested PCR) was used to detect rotavirus RNA in concentrates of the water. The detection limit of the rotavirus determined by RT-nested PCR alone was about 1.67 plaque forming units (PFU) per RT-PCR assay and that by RT-nested PCR combined with concentration from 1l seeded tap water sample was 1.46 plaque forming units per assay. Water samples were collected from various sources, concentrated, and determined rotavirus RNA. Of 120 water samples, rotavirus RNA was detected in 20 samples (16.7%); 2/10 (20%) of the river samples, 8/30 (26.7%) of the canal samples, and 10/40 (25%) of the sewage samples but was not found in any tap water samples (0/40). Only three water samples were positive for rotavirus antigen determined using an enzyme-linked immunosorbent assay (ELISA). Alignment analysis of the sequenced PCR product (346-bp fragment) was performed in eight rotavirus-positive samples using the rotavirus sequence deposited in the GenBank. All samples gave the correct VP7 sequence. Results of analysis showed two samples similar to human rotavirus (97-98%), five similar to rotavirus G9 sequence (94-99%), and one sample similar to animal rotavirus (97%). PCR inhibitors were not observed in any concentrated water samples. In all 20 (of 120) samples where rotaviruses were found, fecal coliforms including Escherichia coli were also found, but of the samples testing negative for rotaviruses, 76 were fecal coliforms positive and 69 were E. coli positive. The combination of the virus concentration method and RT-nested PCR described below made it possible to effectively detect rotaviruses in environmental water samples.

A cross-sectional study was carried out between July 1998 and June 1999 to identify dengue virus-infected patients under age 15 admitted to seven government hospitals in Ang Thong Province, a central region of Thailand, and to assess the knowledge, attitude, and practice (KAP) of their care takers. To differentiate dengue cases, clinical evaluation and laboratory diagnosis were used. Serum samples were collected from 90 admitted children and also from 80 healthy students. The dengue cases were classified as dengue fever (9 cases, 12.2%) and dengue hemorrhagic fever (DHF: 65 cases, 87.8%). Nine patients had dengue shock syndrome, but no death occurred. With serological confirmation, primary antibody response was observed in 8 (11.3%) and definite secondary infection in 49 (69%). Out of 41 serum samples, 14 (34.1%) were positive for dengue virus isolation: dengue serotypes 1, 2 or 3. A total of 131 care takers of enrolled children were interviewed in the context of KAP in DHF. The majority of them were mothers with primary school education level. Half of the care takers were workers. DHF knowledge of the care takers of the dengue cases, non-cases, and healthy students was almost the same. However, the care takers of dengue cases recognized petechiae as a danger sign, p-value of 0.006. They had a higher response in prevention, control and treatment of DHF than the other two groups after their children were admitted to hospital, p-value of 0.000. The results indicated that DHF remains a public health problem in this area and the people need more understanding of the disease. Continuous campaigns are required for community participation so as to prevent and control DHF successfully.

A modified adsorption-elution method for the concentration of seeded rotavirus from water samples was used to determine various factors which affected the virus recovery. An enzyme-linked immunosorbent assay was used to detect the rotavirus antigen after concentration. Of the various eluents compared, 0.05M glycine, pH 11.5 gave the highest rotavirus antigen recovery using negatively charged membrane filtration whereas 2.9% tryptose phosphate broth containing 6% glycine; pH 9.0 was found to give the greatest elution efficiency when a positively charged membrane was used. Reconcentration of water samples by a speedVac concentrator showed significantly higher rotavirus recovery than polyethylene glycol precipitation through both negatively and positively charged filters (p-value <0.001). In addition, speedVac concentration using negatively charged filtration resulted in greater rotavirus recovery than that using positively charged filtration (p-value = 0.004). Thirty eight environmental water samples were collected from river, domestic sewage, canals receiving raw sewage drains, and tap water collected in containers for domestic use, all from congested areas of Bangkok. In addition, several samples of commercial drinking water were analyzed. All samples were concentrated and examined for rotavirus antigen. Coliforms and fecal coliforms (0->1,800 MPN/100 ml) were observed but rotavirus was not detected in any sample. This study suggests that the speedVac reconcentration method gives the most efficient rotavirus recovery from water samples.

A modified adsorption-elution technique for concentration of enteric viruses from sewage and water samples was developed. The viruses in water were concentrated by negatively charged membrane filtration, eluted with 2.9% tryptose phosphate broth containing 6% glycine pH 9.0, and reconcentrated using centrifugation by a speedVac concentrator. The presence of poliovirus, hepatitis A virus (HAV) RNA, and rotavirus antigen was determined by cell culture isolation, nested polymerase chain reaction (nested PCR), and enzyme-linked immunosorbent assay (ELISA), respectively. A total of 100 sewage and water samples were collected from various sources in congested communities in Bangkok, concentrated and examined for those enteric viruses. Of 20 surface water samples from canals which located near sewage drains, 15% were positive for HAV RNA by nested PCR. Of 48 domestic sewage samples from man-holes of underground sewers, 8% were positive for rotavirus antigen by ELISA. Even though the samples were concentrated 256-2,000 fold, poliovirus was not found by isolation in cell culture.

This study describes the rapid detection of polioviruses in environmental waters by a simple reverse transcriptase-polymerase chain reaction (RT-PCR) using two primer pairs for differentiation of poliovirus from non-polio enteroviruses in a single reaction by a one-step method, combining RT and PCR in a single tube. The detection by agarose gel electrophoresis yielded 2 bands of 153-bp and 293-bp for poliovirus tested without the need for further hybridization. The detection sensitivity of this one-step duplex RT-PCR, as measured with RNA extracted by heat treatment from supernatant of infected cell extracts, was 10(-1) 50% tissue culture effective doses (TCID50). This assay was used to evaluate the ability of sample concentration by membrane filter-based adsorption and elution, and purification by a simple RNA isolation based on guanidine isothiocyanate-phenol-chloroform extraction; the system yielded a detection limit of 5 x 10(-1) TCID50 seeded in 5 liters of tap water. This protocol was applied to the poliovirus detection in environmental water collected from 2 communities in Bangkok, Thailand during February and May 1998. Of 100 samples tested, 2 water samples collected from the same open sewage pipeline at one location were positive for polioviruses and one sample collected from another sewage pipeline was positive for non-polio enterovirus while a further 97 water samples were negative for both polioviruses and non-polio enteroviruses. With poliovirus detection by cell culture technique, none of the 100 samples tested was positive for poliovirus type 1, 2 or 3. RT-PCR was more sensitive, rapid, simple and cost-effective than the cell culture technique since the two water samples which were positive for polioviruses by RT-PCR failed to be detected by cell culture. Sequence data of 293-bp amplicons from positive samples were compared with those of reference poliovirus strains in the Genbank and the EMBL databases and identity to the sequence of type 1 strain Sabin was found to be 99%.

The presence of dengue virus antigens in acute sera and peripheral blood mononuclear cells (PBMC) from dengue infected patients were determined by a biotin-streptavidin enzyme-linked immunosorbent assay (BS-ELISA). The frequency of the antigens detected in PBMC was higher than that in sera (53.8% vs 18.9%). In comparison with sera, the detection rate in PBMC was greater than six times: 7 cases were positive only in sera whereas 44 cases were positive only in PBMC, p < 0.001. The presence of the antigens in the sera did not depend on the severity of the disease, i.e. dengue fever, dengue hemorrhagic fever (grades I and II) or dengue shock syndrome (grades III and IV). In contrast, the presence of the antigens in PBMC increased from 36.8% to 100% when the infection was more severe. The dengue virus antigens could be detected in the samples collected between day 2 and day 7 after onset of the disease with the highest rate of detection (68.8%) in PBMC collected on day 4. The data suggest the use of PBMC with access to the appropriate acute-phase specimen for detection of dengue virus antigens.

A cross-sectional study of 165 school-age children who had no history of HBV vaccination was carried out in a low socioeconomic community of Din-Daeng, Bangkok. Blood specimens were collected for determination of HBV seromarkers (HBsAg, Anti-HBs and Anti-HBc) by EIA commercial kits. The results showed that the prevalence of HBV seromarkers was 24.85%, the HBsAg carrier rate was 3.64%, the anti-HBs positive rate was 15.15%, and the prevalence of only anti-HBc was 6.06%. To investigate factors associated with the positivity of HBV seromarkers, children were divided into two groups--the first group consisted of 41 children with HBV seromarkers and the second consisted of 124 children without HBV seromarkers. The study variables between the two groups were compared and analysed. The results revealed that factors associated with HBV positivity were (a) child factors: child's age, child's sex, ear piercing in female, sharing blade during haircutting, contact wound from other persons, using wares with other persons, searching things in garbage, and (b) family factors: older parent, low education in parent, low family income per month, low parent's knowledge and attitude about HBV infection and vaccination, (P < 0.05). After using stepwise regression analysis, the factor of ear piercing in female was only one significant variable.

Reverse passive hemagglutination (RPHA) tests for the detection of six major poisonous snake venoms of Thailand were studied. Three different species of red blood cells i.e., sheep (SRBC), human (HRBC) and chicken (CRBC) were sensitized with protein A-affinity purified rabbit antivenom IgG using chromic chloride as a coupling reagent. The properties of these sensitized erythrocytes with regard to sensitivity, specificity, stability to venom enzymes and storage etc., were studied and compared. The sensitivities of the RPHA tests in venom detection were 2 to 635 ng/ml. Cross-reactions were observed with heterologous venoms at concentrations at least 62 times higher than those observed with homologous venoms. After treatment with glutaraldehyde, the coupled red blood cells showed reduced sensitivity but were stable at 4 degrees C from 1 to 12 months depending upon the antibody and the species of erythrocytes. The entire test required 60 to 120 min. The RPHA using fresh SRBC correctly identified various venoms in 48 of 59 (81.3%) serum samples and 16 of 26 (61.5%) wound swabs. Venom mis-identifications were made in 2 sera (3.4%). In a comparison of 24 paired serum and wound swab samples, more positive identifications were made with serum than with swab samples but the difference was not statistically significant (p > 0.05).

The investigators conducted a clinical study on antithrombotic effectiveness in ischemic stroke at Siriraj Hospital Medical School, Mahidol University from May 1987 to May 1989. Twenty-nine patients, 16 males and 13 females were enrolled in the study. The ages of the patients ranged from 30-87 years with a mean age of 63 +/- 11 years. Ticlopidine (250 mg) could significantly inhibit platelet aggregation induced by ADP and collagen within 24 hours of drug administration. After 1 week to 6 months, only aggregation by ADP was still inhibited significantly without significant effects on fibrinolytic activity and prostacyclin. Hematocrit was significantly decreased at the 1st and 2nd month of treatment. Serious side effects were skin rash and severe headache while the other common ones were dizziness, and diarrhea but these effects disappeared without discontinuing the drug. Most patients who suffered from nausea, diarrhea and headache, had temporary elevated SGPT. It may be concluded that only half of the recommended dose of ticlopidine has inhibitory effects on both phases of ADP-induced aggregation without interfering with fibrinolytic activity and can maintain prostacyclin. However, it also possesses either serious or common side-effects. This drug, therefore, should be used with the awareness of the clinician.