Routine national notifications of dengue cases typically do not reflect the true dengue situation due to large proportion of unreported cases. Serosurveys, when conducted periodically, could shed light on the true dengue infections in the population. To determine the magnitude of dengue infections of the adult population in Singapore following the outbreak in 2007, we performed a cross-sectional study on blood donor samples from December 2009 to February 2010. The residual blood of 3,995 donors (aged 16–60 years) was screened for the presence of dengue-specific immunoglobulin G (IgG) and IgM using enzyme-linked immunosorbent assay (ELISA) kits. The age-weighted IgG prevalence of residents was 50.8% (N = 3,627, 95% confidence interval [CI] = 49.4–52.3%). Dengue IgG prevalence increased with age, with the lowest in 16–20 years age group (16.1%) and the highest in 56–60 years age group (86.6%). Plaque reduction neutralization test (PRNT) on samples of young resident adults (aged 16–30 years) revealed lower prevalence of neutralizing antibodies to each serotype, ranging from 5.4% to 20.3% compared with the older age groups. The level of exposure to dengue among the young adults is relatively low despite the endemicity of the disease in Singapore. It partially explains the population’s susceptibility to explosive outbreaks and the high incidence rate among young adults.

Chikungunya virus (CHIKV) is an arthropod-borne virus responsible for recent epidemics in the Asia Pacific regions. A customized gene expression microarray of 18,760 transcripts known to target Aedes mosquito genome was used to identify host genes that are differentially regulated during the infectious entry process of CHIKV infection on C6/36 mosquito cells. Several genes such as epsin I (EPN1), epidermal growth factor receptor pathway substrate 15 (EPS15) and Huntingtin interacting protein I (HIP1) were identified to be differentially expressed during CHIKV infection and known to be involved in clathrin-mediated endocytosis (CME). Transmission electron microscopy analyses further revealed the presence of CHIKV particles within invaginations of the plasma membrane, resembling clathrin-coated pits. Characterization of vesicles involved in the endocytic trafficking processes of CHIKV revealed the translocation of the virus particles to the early endosomes and subsequently to the late endosomes and lysosomes. Treatment with receptor-mediated endocytosis inhibitor, monodansylcadaverine and clathrin-associated drug inhibitors, chlorpromazine and dynasore inhibited CHIKV entry, whereas no inhibition was observed with caveolin-related drug inhibitors. Inhibition of CHIKV entry upon treatment with low-endosomal pH inhibitors indicated that low pH is essential for viral entry processes. CHIKV entry by clathrin-mediated endocytosis was validated via overexpression of a dominant-negative mutant of Eps15, in which infectious entry was reduced, while siRNA-based knockdown of genes associated with CME, low endosomal pH and RAB trafficking proteins exhibited significant levels of CHIKV inhibition. This study revealed, for the first time, that the infectious entry of CHIKV into mosquito cells is mediated by the clathrin-dependent endocytic pathway.

Wastewater-based surveillance for SARS-CoV-2 has been used for the early warning of transmission or objective trending of the population-level disease prevalence. Here, we describe a new use-case of conducting targeted wastewater surveillance to complement clinical testing for case identification in a small community at risk of COVID-19 transmission. On 2 July 2020, a cluster of COVID-19 cases in two unrelated households residing on different floors in the same stack of an apartment building was reported in Singapore. After cases were conveyed to healthcare facilities and six healthy household contacts were quarantined in their respective apartments, wastewater surveillance was implemented for the entire residential block. SARS-CoV-2 was subsequently detected in wastewaters in an increasing frequency and concentration, despite the absence of confirmed COVID-19 cases, suggesting the presence of fresh case/s in the building. Phone interviews of six residents in quarantine revealed that no one was symptomatic (fever/ respiratory illness). However, when nasopharyngeal swabs from six quarantined residents were tested by PCR tests, one was positive for SARS-CoV-2. The positive case reported episodes of diarrhea and the case's stool sample was also positive for SARS-CoV-2, explaining the SARS-CoV-2 spikes observed in wastewaters. After the case was conveyed to a healthcare facility, wastewaters continued to yield positive signals for five days, though with a decreasing intensity. This was attributed to the return of recovered cases, who had continued to shed the virus. Our findings demonstrate the utility of wastewater surveillance as a non-intrusive tool to monitor high-risk COVID-19 premises, which is able to trigger individual tests for case detection, highlighting a new use-case for wastewater testing.

National data on dengue notifications do not capture all dengue infections and do not reflect the true intensity of disease transmission. To assess the true dengue infection rate and disease control efforts in Singapore, we conducted age-stratified serosurveys among residents after a 2013 outbreak that was the largest dengue outbreak on record. The age-weighted prevalence of dengue immunoglobulin G among residents was 49.8% (95% confidence interval: 48.4, 51.1) in 2013 and 48.6% (95% confidence interval: 47.0, 50.0) in 2017; prevalence increased with age. Combining these data with those from previous serosurveys, the year-on-year estimates of the dengue force of infection from 1930 to 2017 revealed a significant decrease from the late 1960s to the mid-1990s, after which the force of infection remained stable at approximately 10 per 1,000 persons per year. The reproduction number (R0) had also declined since the 1960s. The reduction in dengue transmission may be attributed to the sustained national vector program and partly to a change in the age structure of the population. The improved estimated ratio of notified cases to true infections, from 1:14 in 2005–2009 to 1:6 in 2014–2017, signifies that the national notification system, which relies on diagnosed cases, has improved over time. The data also suggest that the magnitudes of dengue epidemics cannot be fairly compared across calendar years and that the current disease control program remains applicable.

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