Hand, foot and mouth disease (HFMD) is a public health threat worldwide, particularly in the Asia–Pacific region. Enterovirus A71 (EV-A71), coxsackievirus A16 (CVA16), and CVA6 are the major pathogens causing HFMD outbreaks in several countries, including Thailand. We retrieved 385 VP1 nucleotide sequences, comprising 228 EV-A71, 33 CVA16, and 124 CVA6, deposited in the databases between 2000 and 2022 for molecular evolutionary characterization using Bayesian phylogeny. All EV-A71 identified belonged to genotype B, subgenotypes B4, and B5, and to genotype C, subgenotypes C1, C2, C4a, C4b, and C5. The analyzes demonstrated these viruses’ co-circulation and subgenotypic changes throughout the past two decades. The CVA16 was grouped in genotype B1, predominantly subgenotype B1a, and the CVA6 was grouped in subgenotype D3, clades 1–4. The tMRCA of EV-A71 genotypes B and C, CVA16 B1, and CVA6 D3 dated 1993.79, 1982.62, 1995.86, and 2007.31, respectively, suggesting that the viruses were likely introduced and cryptically circulated in Thailand before the HFMD cases were recognized. We demonstrated these viruses’ fluctuation and cyclical pattern throughout the two decades of observation. This study provided insight into evolutionary dynamics concerning molecular epidemiology and supported the selection of current genotype-matched vaccines, vaccine development, and implementation.

Hand, foot and mouth disease (HFMD) is endemic among population of young children in Thailand. The disease is mostly caused by enterovirus 71 (EV71) and coxsackievirus A16 (CA16). This study conducted serosurveillance for neutralizing (NT) antibodies to EV71 subgenotypes B5 and C4a, and to CA16 subgenotypes B1a and B1b, in 579 subjects of various ages using a microneutralization assay in human rhabdomyosarcoma (RD) cells. These test viruses were the major circulating subgenotypes associated with HFMD in Thailand during the study period. We found that the levels of seropositivity against all 4 study viruses were lowest in the age group of 6–11 months, i.e., 5.5% had antibody to both EV71 subgenotypes, while 14.5% and 16.4% had antibody to CA16 subgenotypes B1a and B1b, respectively. The percentages of subjects with antibodies to these 4 viruses gradually increased with age, but were still less than 50% in children younger than 3 years. These laboratory data were consistent with the epidemiological data collected by the Ministry of Public Health which showed repeatedly that the highest number of HFMD cases was in children aged 1 year. Analyses of amino acid sequences of the test viruses showed 97% identity between the two subgenotypes of EV71, and 99% between the two subgenotypes of CA16. Nevertheless, the levels of seropositivity and antibody titer against the two subgenotypes of EV71 and of CA16 were not significantly different. This study clearly demonstrated NT antibody activity across EV71-B5 and EV71-C4a subgenotypes, and also across CA16-B1a and CA16-B1b subgenotypes. Moreover, there were no significant differences by gender in the seropositive rates and antibody levels to any of the 4 virus subgenotypes.

Wastewater-based epidemiology (WBE) has been employed for monitoring the presence of SARS-CoV-2 infected population. Herein, the study aims to apply the WBE for surveillance and monitoring SARS-CoV-2 in Bangkok, where the highest official covid-19 cases reported in Thailand, during the fully opening for international tourists in early 2023. A total of 200 wastewater samples (100 influent and 100 effluent samples) were collected from 10 wastewater treatment plants (WWTPs) during January–May 2023. SARS-CoV-2 RNA was detected by real time qRT-PCR with accounting for 51% (102/200). Of these, 88% (88/100) and 14% (14/100) were detected in influent and effluent samples, respectively. The SARS-CoV-2 RNA concentration was detected in ranged of 4.76 × 10²–1.48 × 10⁵ copies/L. The amount of SARS-CoV-2 RNA has increased approximately 4 times from the lag phase (January–March) to the log phase (April–May). Spearman’s correlation coefficient revealed that correlation between estimated infected population and weekly reported cases was statistically significant (p-value = 0.017). SARS-CoV-2 RNA in influent had a statistically significant relationship with weekly reported cases (r = 0.481, p-value < 0.001). Lag time analysis revealed early warning 1–3 weeks before rising covid-19 cases observed. GIS was applied for spatial-temporal analysis at the province level, suggesting real time dashboard should be further developed.

Background: Ongoing outbreaks of H5N1 highly pathogenic avian influenza (HPAI) viruses and the emergence of the genetic-related hemagglutinin (HA) gene of reassortant H5Nx viruses currently circulating in wild birds and poultries pose a great global public health concern. In this study, we comprehensively analyzed the genetic evolution of Thai H5N1 HA and neuraminidase (NA) genes between 2003 and 2010. The H5N1 Thailand virus clade 2.3.4 was also genetically compared to the currently circulating clade 2.3.4.4 of H5Nx viruses. Methods: Full-length nucleotide sequences of 178 HA and 143 NA genes of H5N1 viruses circulating between 2003 and 2010 were phylogenetically analyzed using maximum likelihood (ML) phylogenetic construction. Bayesian phylogenetic trees were reconstructed using BEAST analysis with a Bayesian Markov chain Monte Carlo (MCMC) approach. The maximum clade credibility (MCC) tree was determined, and the time of the most recent common ancestor (tMRCA) was estimated. The H5N1 HA nucleotide sequences of clade 2.3.4 Thailand viruses were phylogenetically analyzed using ML phylogenetic tree construction and analyzed for nucleotide similarities with various subtypes of reassortant H5Nx HA clade 2.3.4.4. Results: ML phylogenetic analysis revealed two distinct HA clades, clade 1 and clade 2.3.4, and two distinct NA groups within the corresponding H5 clade 1 viruses. Bayesian phylogenetic reconstruction for molecular clock suggested that the Thai H5N1 HA and NA emerged in 2001.87 (95% HPD: 2001.34-2002.49) and 2002.38 (95% HPD: 2001.99-2002.82), respectively, suggesting that the virus existed before it was first reported in 2004. The Thai H5N1 HA clade 2.3.4 was grouped into corresponding clades 2.3.4, 2.3.4.1, 2.3.4.2, and 2.3.4.3, and shared nucleotide similarities to reassortant H5Nx clade 2.3.4.4 ranged from 92.4-96.8%. Phylogenetic analysis revealed monophyletic H5Nx clade 2.3.4.4 evolved from H5N1 clade 2.3.4. Conclusion: H5N1 viruses existed, and were presumably introduced and circulated in avian species in Thailand, before they were officially reported in 2004. HA and NA genes continuously evolved during circulation between 2004 and 2010. This study provides a better understanding of genetic evolution with respect to molecular epidemiology. Monitoring and surveillance of emerging variants/reassortants should be continued.

Fomite-mediated viral transmission through using cash might be a potential risk to human health. Persistence of SARS-CoV-2, and other medically important viruses was investigated. A total of 300 samples (i.e., 150 banknotes and 150 coins) were randomly collected from nineteen fresh markets distributed across seventeen districts of Bangkok, Thailand. Every banknote or coin was entirely swabbed and generated a total of 100 pool samples. Total viral nucleic acid was extracted and subjected for multiplex real-time qRT-PCR using Allplex™ SARS-CoV-2/FluA/FluB/RSV assay and Allplex™ GI-virus assay. The results revealed detection rate of 4% (4/100), and they were only detected in banknote pooled samples. Two samples collected from fish shops tested positive for SARS-CoV-2 (2%, 2/100); meanwhile, two samples (2%, 2/100) from pork and chicken shops tested positive for rotavirus A. None of pool samples were detected for influenza A and B viruses, respiratory syncytial virus, norovirus genogroup I and II, adenovirus, astrovirus, and sapovirus. Phylogenetic analysis demonstrated that rotavirus A belonged to genotype G8; meanwhile, SARS-CoV-2 resembled omicron GRA JN.1 sub variant. Our finding is the first report for demonstrating the presence of SARS-CoV-2 and rotavirus A in Thai banknotes on real-world situation, implying the potential risk to human health and safety.

Background: The existence and transmission of pathogenic and antibiotic-resistant bacteria through currency banknotes and coins poses a global public health risk. Banknotes and coins are handled by people in everyday life and have been identified as a universal medium for potentially microbial contamination. Methods: To ascertain existence of medically important bacteria, a total of 300 samples including 150 banknotes and 150 coins were randomly collected at onsite retail fresh meat stores, i.e., pork and chicken, fish, and seafood stores, from nineteen fresh markets distributed across Bangkok, Thailand. An individual banknote or coin was entirely swabbed, and bacterial culture was carried out using tryptic soy agar (TSA), sheep blood agar (SBA) and MacConkey agar (Mac). A colony count was performed and bacterial species identification was conducted using matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry. Phenotypic antimicrobial susceptibility testing was carried out using the Kirby–Bauer disc diffusion methods. Results: The results demonstrated that the bacterial contamination rate was higher on banknotes than on coins (93.33% vs. 30.00%) in all three store types. A substantial number of colonies of >3,000 colony forming units (CFU) was predominantly found in banknotes (70.00%), especially from fish store (83.3%); meanwhile, <1,000 CFU was observed in coin sample (76.67%). MALDI-TOF mass spectrometry could identify 107 bacterial species, most of them were Staphylococcus kloosii (14.02%, 15/107), Staphylococcus saprophyticus (12.15%, 13/107), and Macrococcus caseolyticus (8.41%, 9/107). The prevalence based on genera were Staphylococcus (36.45%, 39/107), Acinetobacter (20.56%, 22/107), and Macrococcus (10.28%, 11/107). Almost all Staphylococcus isolates had low susceptibility to penicillin (21%). Notably, Staphylococcus arlettae, Staphylococcus haemolyticus and M. caseolyticus were multidrug-resistant (MDR). It is notable that none of the staphylococci and macrococci isolates exhibited inducible clindamycin resistance (D-test negative). Escherichia coli and Pseudomonas putida isolates were carbapenem-resistant, and Acinetobacter baumannii isolates were MDR with showing carbapenem resistance. Conclusion: Our data demonstrated a high prevalence of medically important bacteria presented on Thai currency, which may pose a potential risk to human health and food safety. Food vendors and consumers should be educated about the possible cross-contamination of bacteria between the environment, food item, and currency.