Hand, foot, and mouth disease (HFMD) is a contagious childhood illness and annually affects millions of children aged less than 5 years across the Asia–Pacific region. HFMD transmission mainly occurs through direct contact (person-to-person) and indirect contact with contaminated surfaces and objects. Therefore, public health measures to reduce the spread of HFMD in kindergartens and daycare centers are essential. Based on the guidelines by the Department of Disease Control, a school closure policy for HFMD outbreaks wherein every school in Thailand must close when several HFMD classrooms (more than two cases in each classroom) are encountered within a week, was implemented, although without strong supporting evidence. We therefore conducted a prospective cohort study of children attending five kindergartens during 2019 and 2020. We used molecular genetic techniques to investigate the characteristics of the spreading patterns of HFMD in a school-based setting in Bangkok, Thailand. These analyses identified 22 index cases of HFMD (symptomatic infections) and 25 cases of enterovirus-positive asymptomatic contacts (24 students and one teacher). Enterovirus (EV) A71 was the most common enterovirus detected, and most of the infected persons (8/12) developed symptoms. Other enteroviruses included coxsackieviruses (CVs) A4, CV-A6, CV-A9, and CV-A10 as well as echovirus. The pattern of the spread of HFMD showed that 45% of the subsequent enteroviruses detected in each outbreak possessed the same serotype as the first index case. Moreover, we found a phylogenetic relationship among enteroviruses detected among contact and index cases in the same kindergarten. These findings confirm the benefit of molecular genetic assays to acquire accurate data to support school closure policies designed to control HFMD infections.

• Scientific question: Hand, foot, and mouth disease (HFMD) is a contagious disease that mainly affects young children in the Asia–Pacific region. The viral transmission can occur through direct contact with nose and throat secretions, surfaces, fomites and the environment. Moreover, children’s school toys may serve an important role in HFMD surface-to-hand transmission. Heat inactivation can be an alternative to control HFMD transmission at schools. • Evidence before this study: Stability and heat inactivation of enteroviruses on different surfaces have been investigated in previous studies. However, studies regarding entervirus-A71 (EV-A71) and coxsackievirus A16 (CVA16) on children’s school toy surfaces have been rarely reported. • New findings: In this study, we showed that the infectivity of EV-A71 and CVA16 declined within a few hours on all three surfaces, whereas the viral RNA could be detected for up to 28 days. In both assays, viral stability was higher on wood than on the other surfaces. Meanwhile, heat inactivation at 60°C for 15 min, which can be repeatable without damaging toys, was sufficient to completely inactivate both enterovirus serotypes. • Significance of the study: Results of the study clearly demonstrated the prolonged persistence of enteroviruses on commonly encountered surfaces, highlighting the potential of indirect spread of these viruses among children. The heat inactivation is an applicable way for controlling the spread of pathogens in school environments, including complimented disinfection programs which normally required detergents and water.

Zika virus (ZIKV) is the mosquito-transmitted virus that the WHO declared a Public Health Emergency of International Concern in 2016 due to the consequence of microcephaly from infected pregnancies. The incidence of Zika infection has been unclear in many countries because most infected people have nonspecific febrile illnesses. This study’s aim is to investigate the incidence of symptomatic Zika virus infections from the archived samples of a dengue cohort study of children in central Thailand from 2006 to 2009. We performed Zika NS1 immunoglobulin (Ig)G enzyme-linked immunosorbent assay (ELISA) screening to identify symptomatic Zika infections in paired acute/convalescent serum samples. Symptomatic Zika infections were confirmed by reverse transcription polymerase chain reactions (RT-PCR) of acute serum samples. The comparison of the Zika NS1 IgG ELISA results between acute and convalescent samples showed 290/955 (30.4%) seropositive cases. Zika RT-PCR results were positive in 28 febrile cases (15 females, 13 males). Zika RT-PCR showed that symptomatic Zika infection occurred in children aged 4–11 years in Ratchaburi province, Thailand (2007–2009, first case in April 2007), and the symptomatic Zika:dengue infection ratio was 28 Zika:394 dengue (1:14). Phylogenetic analysis showed that all Zika viruses were of Asian lineage. Zika NS1 IgG ELISA identified Zika-infected patients and showed a low Zika:dengue ratio.

Circulating hsa-miRNA-126 (CmiR-126) has been reported to involve in the pathogenesis of many infectious diseases including dengue virus infection. However, no prior study has been conducted to describe more details in dengue-infected pediatric patients. This study aimed to describe CmiR-126-3p in dengue-infected pediatric patients during the febrile and convalescent phases. Additionally, the correlations between CmiR-126-3p and other relevant clinical laboratory factors were investigated. Sixty paired-serum specimens collected during febrile and convalescent phases were retrieved from patients with dengue fever (DF) (n = 30) and dengue hemorrhagic fever (DHF) (n = 30). Thirty paired-serum specimens collected from non-dengue acute febrile illness patients (AFI) were included as the control group. CmiR-126-3p was determined using reverse transcription quantitative real-time polymerase-chain reaction (RT-qPCR). Relative miRNA expression was calculated as 2^−ΔCt using CmiR-16-5p for data normalization. CmiR-126-3p expression during febrile and convalescent phases in dengue-infected patients was significantly lower than AFI (p < 0.05). However, miRNA levels were not different (p > 0.05) compared between DF and DHF and between primary and secondary infection. CmiR-126-3p levels in DF in the convalescent were significantly higher than in the febrile phase (p = 0.025). No association between CmiR-126-3p and hematocrit, WBC level, platelet count, WBC differential count or dengue viral load was observed (p > 0.05). The data suggest that hsa-miR-126-3p involved in pathogenesis of dengue infection and may be a promising early and late biomarker for DENV infection. However, hsa-miR-126-3p alone cannot be used as a predictor for dengue severity.

Zika virus (ZIKV) is a mosquito-borne flavivirus that has recently emerged as a global health threat. The rise in ZIKV infections has driven an increased incidence of neonates born with microcephaly or other neurological malformations. Therefore, screening for ZIKV infection can considerably impact pregnant women, especially during the first trimester. The majority of ZIKV infections are mild or asymptomatic, and clinical diagnosis is inaccurate. Moreover, given the high level of cross-reactivity among flaviviruses, serological approaches to distinguish ZIKV from dengue virus (DENV) infections are complicated. We used the combination of DENV and ZIKV nonstructural protein 1 (NS1) IgG enzyme-linked immunosorbent assay (ELISA) and ZIKV NS1 blockade-of-binding (BOB) ELISA to test the convalescent sera of non-flavivirus, primary DENV, secondary DENV, and ZIKV infections. Our findings indicate that primary testing using a ZIKV NS1 IgG ELISA, the test of choice for large-scale ZIKV serosurvey studies, provided relatively high sensitivity. Moreover, the confirmation of positive ELISA results using the ZIKV NS1 BOB ELISA increased average specificity to 94.59% across serum samples. The combined use of two simple ELISAs for ZIKV serosurveys and the monitoring of ZIKV infection during pregnancy can elucidate the epidemiology, pathogenesis, and complications of ZIKV in DENV-endemic areas.

The frequency of Zika virus (ZIKV)-specific IgA and IgM and the cytokine expression profile of ZIKV-infected patients in hyperendemic areas remain unclear. This study investigated the rates of ZIKV non-structural protein 1 (NS1)-specific IgA and IgM and evaluated serum cytokine levels of ZIKV and Dengue virus (DENV) cases in Thailand to identify potential diagnostic biomarkers, elucidate the immunity against ZIKV and DENV, and investigate the association between cytokine levels and ZIKV symptoms. Low rates of positivity for ZIKV NS1-specific IgA and IgM were detected in our study. ZIKV NS1 IgA/M (11%, 11/101) in combination was more frequently detected than ZIKV NS1 IgM (2%, 2/101) or ZIKV NS1 IgA (4%, 4/96) alone, especially in acute ZIKV cases with previous DENV exposure (14%, 10/72). Cytokine analysis showed that both ZIKV and DENV infections induced polyfunctional immunity, and the latter triggered more prolonged responses. The existence of significant differences in IL-4 and IL-10 levels between acute ZIKV and acute DENV cases suggested that IL-4 (p = 0.0176) and IL-10 (p = 0.0003) may represent biomarkers for acute ZIKV and acute DENV infections, respectively. Analysis of the association between increased cytokine levels and ZIKV symptoms indicated that CXCL10 (p = 0.0029) was associated with exanthema, while IL-5 (p = 0.0496) was linked to headache. The detection of ZIKV NS1 IgA and IgM in combination may enhance the diagnosis of early ZIKV infection, particularly when levels of IgM or IgA alone are low or undetectable. IL-4 and IL-10 may serve as targets for the development of diagnostic tools to detect ZIKV and DENV infections early, respectively, in flavivirus-endemic regions.