Combining wastewater surveillance and case data in estimating the time-varying effective reproduction number.

Wastewater surveillance has been increasingly acknowledged as a useful tool for monitoring transmission dynamics of infections of public health concern, including the coronavirus disease (COVID-19). While a range of models have been proposed to estimate the time-varying effective reproduction number (Rt) utilizing clinical data, few have harnessed the viral concentration in wastewater samples to do so, leaving uncertainties about the potential precision gains with its use. In this study, we developed a Bayesian hierarchical model which simultaneously reconstructed the latent infection trajectory and estimated Rt. Focusing on the 2022 and early 2023 COVID-19 transmission trends in Singapore, where mass community wastewater surveillance has become routine, we performed estimations using a spectrum of data sources, including reported case counts, hospital admissions, deaths, and wastewater viral loads. We further explored the performance of our wastewater model across various scenarios with different sampling strategies. The results showed consistent estimates derived from models employing diverse data streams, while models incorporating more wastewater samples exhibited greater uncertainty and variation in the inferred Rts. Additionally, our analysis revealed prominent day-of-the-week effect in reported case counts and substantial temporal variations in ascertainment rates. In response to these findings, we advocate for a hybrid approach leveraging both clinical and wastewater surveillance data to account for changes in case-ascertainment rates. Furthermore, our study demonstrates the possibility of reducing sampling frequency or sample size without compromising estimation accuracy for Rt, highlighting the potential for optimizing resource allocation in surveillance efforts while maintaining robust insights into the transmission dynamics of infectious diseases.

Case report: Zika surveillance complemented with wastewater and mosquito testing.

BACKGROUND: In June 2023, a local cluster of 15 Zika cases was reported in a neighbourhood in Northeastern Singapore. The last significant local transmission of Zika virus (ZIKV) with more than 450 cases was in 2016-2017. To monitor the situation and mitigate further transmission, case, entomological and wastewater-based surveillance were carried out. METHODS: Primary healthcare practitioners and the community were alerted to encourage timely case identification. Surveillance was enhanced through testing of Aedes mosquitoes collected from the National Gravitrap surveillance system, and wastewater samples were collected from a network of autosamplers deployed at manholes across the country. FINDINGS: ZIKV RNA was detected in mosquito pools (3/43; 7%) and individual mosquitoes (3/82; 3.7%) captured, and in wastewater samples (13/503) collected from the vicinity of the cluster of cases. Respective samples collected from other sites across the country were negative. The peak detection of ZIKV RNA in mosquitoes and wastewater coincided temporally with the peak in the number of cases in the area (15-25 May 2023). INTERPRETATION: The restriction of ZIKV signals from wastewater and mosquitoes within the neighbourhood suggested limited ZIKV transmission. The subsequent waning of signals suggested effectiveness of control measures. We demonstrate the utility of wastewater-based surveillance of ZIKV, which complements existing case- and entomological-based surveillance. The non-intrusive approach is particularly useful to monitor diseases such as Zika, which generally causes silent or mild infections, but may cause severe outcomes such as congenital Zika syndrome. FUNDING: This study was funded by Singapore's Ministry of Finance and the National Environment Agency, Singapore.

Degradation effects of dietary solvents on microhardness and inorganic elements of computer-aided design/computer-aided manufacturing dental composites.

BACKGROUND: Computer-aided design/computer-aided manufacturing (CAD/CAM) dental composites were introduced with superior mechanical properties than conventional dental composites. However, little is known on effects of dietary solvents on microhardness or inorganic elemental composition of CAD/CAM composites. OBJECTIVES: The objectives of this study were to evaluate the degradation effects of each dietary solvent on the microhardness of the different CAD/CAM dental composites and to observe the degradation effects of dietary solvent on the inorganic elements of the dental composites investigated. METHODS: Fifty specimens with dimensions 12 mm x 14 mm x 1.5 mm were prepared for direct composite (Filtek Z350 XT [FZ]), indirect composite (Shofu Ceramage [CM]), and three CAD/CAM composites (Lava Ultimate [LU], Cerasmart [CS], and Vita Enamic [VE]). The specimens were randomly divided into 5 groups (n = 10) and conditioned for 1-week at 37°C in the following: air (control), distilled water, 0.02 N citric acid, 0.02 N lactic acid and 50% ethanol-water solution. Subsequently, the specimens were subjected to microhardness test (KHN) using Knoop hardness indenter. Air (control) and representative postconditioning specimens with the lowest mean KHN value for each material were analyzed using energy dispersive X-ray spectroscopy (EDX). Statistical analysis was done using one-way ANOVA and post hoc Bonferroni test at a significance level of p = 0.05. RESULTS: Mean KHN values ranged from 39.7 ± 2.7 kg/mm2 for FZ conditioned in 50% ethanol-water solution to 79.2 ± 3.4 kg/mm2 for VE conditioned in air (control). With exception to LU, significant differences were observed between materials and dietary solvents for other dental composites investigated. EDX showed stable peaks of the inorganic elements between air (control) and representative postconditioning specimens. CONCLUSIONS: The microhardness of dental composites was significantly affected by dietary solvents, except for one CAD/CAM composite [LU]. However, no changes were observed in the inorganic elemental composition of dental composites between air (control) and 1-week postconditioning.

Efficacy of Wolbachia-mediated sterility to reduce the incidence of dengue: a synthetic control study in Singapore.

BACKGROUND: Due to the absence of available therapeutics and good vaccines, vector control solutions are needed to mitigate the spread of dengue. Matings between male Aedes aegypti mosquitoes infected with the wAlbB strain of Wolbachia and wildtype females yield non-viable eggs. We evaluated the efficacy of releasing wAlbB-infected A aegypti male mosquitoes to suppress dengue incidence. METHODS: In this synthetic control study, we conducted large-scale field trials in Singapore involving release of wAlbB-infected A aegypti male mosquitoes for dengue control via vector population suppression, from epidemiological week (EW) 27, 2018, to EW 26, 2022. We selected two large towns (Yishun and Tampines) to adopt an expanding release strategy and two smaller towns (Bukit Batok and Choa Chu Kang) to adopt a targeted-release approach. Releases were conducted two times a week in high-rise public housing estates. All intervention and control locations practised the same baseline dengue control protocol. The main outcome was weekly dengue incidence rate caused by any dengue virus serotype. We used incidence data collected by the Singapore Ministry of Health to assess the efficacy of the interventions. To compare interventions, we used the synthetic control method to generate appropriate counterfactuals for the intervention towns using a weighted combination of 30 control towns between EW 1, 2014 and EW 26, 2022. FINDINGS: Our study comprised an at-risk population of 607 872 individuals living in intervention sites and 3 894 544 individuals living in control sites. Interventions demonstrated up to 77·28% (121/156, 95% CI 75·81-78·58) intervention efficacy despite incomplete coverage across all towns until EW 26, 2022. Intervention efficacies increased as release coverage increased across all intervention sites. Releases led to 2242 (95% CI 2092-2391) fewer cases per 100 000 people in intervention sites during the study period. Secondary analysis showed that these intervention effects were replicated across all age groups and both sexes for intervention sites. INTERPRETATION: Our results demonstrated the potential of Wolbachia-mediated incompatible insect technique for strengthening dengue control in tropical cities, where dengue burden is the greatest. FUNDING: Singapore Ministry of Finance, Ministry of Sustainability, and the National Environment Agency, and the Singapore National Robotics Program.

Characterization of size-differentiated airborne particulate matter collected from indoor environments of childcare facilities.

Inhalation of particulate matter (PM) present in indoor atmospheres has been associated with poor health and wellbeing of occupants. Here we report the characteristics of airborne PM collected from twenty-two air-conditioned childcare centres in Singapore. Airborne PM were collected using cascade impactors and characterized for morphology, elemental composition, endotoxin levels, ability to generate abiotic reactive oxygen species, and oxidative stress-dependent cytotoxicity in BEAS-2B cell lines. The mass concentrations of ultrafine particles (PM0.06-1) were more abundant than that of larger particles (PM1-4, PM4-20, and PM20-35 particles). PM20-35 and PM4-20 were irregularly shaped particles, PM1-4 particles had membranous flaky structures and PM0.06-1 particles were pseudo-spherical with the occasional presence of crystalline structures. Carbonaceous matter dominated PM20-35 particles, and the abundance of inorganic salts, iron and sulfur increased with decreasing PM size. Measured endotoxin levels were especially higher in PM4-20 particles. Compared to other particle size fractions, PM0.06-1 particles generated the highest ROS and were also the most potent in generating intracellular ROS in BEAS-2B cell lines. However, total mass concentrations, elemental compositions, abiotic responses, and PM collected from centres with split air-conditioning systems and no active outdoor air supply (SAC) were not statistically different compared with PM collected from centres with air conditioning with mechanical ventilation (ACMV). In conclusion, our study showed obvious distinctions in mass concentrations, morphology, elemental compositions, and cytotoxic potential of different sized particles collected from childcare centres, where the smallest particles (PM0.06-1) exhibited higher hazard potential.

Positive association of SARS-CoV-2 RNA concentrations in wastewater and reported COVID-19 cases in Singapore - A study across three populations.

Wastewater testing of SARS-CoV-2 has been adopted globally and has shown to be a useful, non-intrusive surveillance method for monitoring COVID-19 trends. In Singapore, wastewater surveillance has been widely implemented across various sites and has facilitated timely COVID-19 management and response. From April 2020 to February 2022, SARS-CoV-2 RNA concentrations in wastewater monitored across three populations, nationally, in the community, and in High Density Living Environments (HDLEs) were aggregated into indices and compared with reported COVID-19 cases and hospitalisations. Temporal trends and associations of these indices were compared descriptively and quantitatively, using Poisson Generalised Linear Models and Generalised Additive Models. National vaccination rates and vaccine breakthrough infection rates were additionally considered as confounders to shedding. Fitted models quantified the temporal associations between the indices and cases and COVID-related hospitalisations. At the national level, the wastewater index was a leading indicator of COVID-19 cases (p-value <0.001) of one week, and a contemporaneous association with hospitalisations (p-value <0.001) was observed. At finer levels of surveillance, the community index was observed to be contemporaneously associated with COVID-19 cases (p-value <0.001) and had a lagging association of 1-week in HDLEs (p-value <0.001). These temporal differences were attributed to differences in testing routines for different sites during the study period and the timeline of COVID-19 progression in infected persons. Overall, this study demonstrates the utility of wastewater surveillance in understanding underlying COVID-19 transmission and shedding levels, particularly for areas with falling or low case ascertainment. In such settings, wastewater surveillance showed to be a lead indicator of COVID-19 cases. The findings also underscore the potential of wastewater surveillance for monitoring other infectious diseases threats.

Screening for extended-spectrum beta-lactamase Escherichia coli in recreational beach waters in Singapore.

Aims: As part of Singapore's One Health antimicrobial resistance (AMR) management, this work was designed to understand the AMR burden in recreational beach waters using extended-spectrum beta-lactamase Escherichia coli (ESBL-EC) as an indicator. Materials & methods: A total of 90 water samples were collected from six different recreational beaches over three different time periods. Only 28/90 (31.3%) water samples yielded E. coli colonies ranging from 1 to 80 colony-forming units/100 ml. Results & conclusion: Screening of all colonies using CHROMID® ESBL agar and Luria-Bertani broth supplemented with ceftriaxone showed that none was ESBL-EC. Further monitoring is required to understand the prevalence of ESBL-EC spatiotemporally, contributing to the national AMR surveillance program and providing timely risk assessment for exposure to ESBL-EC.

Singapore's 5 decades of dengue prevention and control-Implications for global dengue control.

This paper summarises the lessons learnt in dengue epidemiology, risk factors, and prevention in Singapore over the last half a century, during which Singapore evolved from a city of 1.9 million people to a highly urban globalised city-state with a population of 5.6 million. Set in a tropical climate, urbanisation among green foliage has created ideal conditions for the proliferation of Aedes aegypti and Aedes albopictus, the mosquito vectors that transmit dengue. A vector control programme, largely for malaria, was initiated as early as 1921, but it was only in 1966 that the Vector Control Unit (VCU) was established to additionally tackle dengue haemorrhagic fever (DHF) that was first documented in the 1960s. Centred on source reduction and public education, and based on research into the bionomics and ecology of the vectors, the programme successfully reduced the Aedes House Index (HI) from 48% in 1966 to <5% in the 1970s. Further enhancement of the programme, including through legislation, suppressed the Aedes HI to around 1% from the 1990s. The current programme is characterised by 4 key features: (i) proactive inter-epidemic surveillance and control that is stepped up during outbreaks; (ii) risk-based prevention and intervention strategies based on advanced data analytics; (iii) coordinated inter-sectoral cooperation between the public, private, and people sectors; and (iv) evidence-based adoption of new tools and strategies. Dengue seroprevalence and force of infection (FOI) among residents have substantially and continuously declined over the 5 decades. This is consistent with the observation that dengue incidence has been delayed to adulthood, with severity highest among the elderly. Paradoxically, the number of reported dengue cases and outbreaks has increased since the 1990s with record-breaking epidemics. We propose that Singapore's increased vulnerability to outbreaks is due to low levels of immunity in the population, constant introduction of new viral variants, expanding urban centres, and increasing human density. The growing magnitude of reported outbreaks could also be attributed to improved diagnostics and surveillance, which at least partially explains the discord between rising trend in cases and the continuous reduction in dengue seroprevalence. Changing global and local landscapes, including climate change, increasing urbanisation and global physical connectivity are expected to make dengue control even more challenging. The adoption of new vector surveillance and control tools, such as the Gravitrap and Wolbachia technology, is important to impede the growing threat of dengue and other Aedes-borne diseases.

Genetic Diversity and Dispersal of DENGUE Virus among Three Main Island Groups of the Philippines during 2015-2017.

Dengue has been one of the major public health concerns in the Philippines for more than a century. The annual dengue case burden has been increasing in recent years, exceeding 200,000 in 2015 and 2019. However, there is limited information on the molecular epidemiology of dengue in the Philippines. We, therefore, conducted a study to understand the genetic composition and dispersal of DENV in the Philippines from 2015 to 2017 under UNITEDengue. Our analyses included 377 envelope (E) gene sequences of all 4 serotypes obtained from infections in 3 main island groups (Luzon, Visayas, and Mindanao) of the Philippines. The findings showed that the overall diversity of DENV was generally low. DENV-1 was relatively more diverse than the other serotypes. Virus dispersal was evident among the three main island groups, but each island group demonstrated a distinct genotype composition. These observations suggested that the intensity of virus dispersal was not substantive enough to maintain a uniform heterogeneity among island groups so that each island group behaved as an independent epidemiological unit. The analyses suggested Luzon as one of the major sources of DENV emergence and CAR, Calabarzon, and CARAGA as important hubs of virus dispersal in the Philippines. Our findings highlight the importance of virus surveillance and molecular epidemiological analyses to gain deep insights into virus diversity, lineage dominance, and dispersal patterns that could assist in understanding the epidemiology and transmission risk of dengue in endemic regions.

Viable mpox virus in the environment of a patient room.

We conducted a prospective environmental surveillance study to investigate the air, surface, dust, and water contamination of a room occupied by a patient infected with mpox virus (MPXV) at various stages of the illness. The patient tested positive for MPXV from a throat swab and skin lesions. Environmental sampling was conducted in a negative pressure room with 12 unidirectional high efficiency particulate air filter (HEPA) air changes per hour and daily cleaning of the surfaces. A total of 179 environmental samples were collected on days 7, 8, 13, and 21 of illness. Among the days of sampling, air, surface, and dust contamination showed the highest contamination rates on day 7 and 8 of illness, with a gradual decline to the lowest contamination level by day 21. Viable MPXV was isolated from surfaces and dust samples and no viable virus was isolated from the air and water samples.

Field trial assessing the antimicrobial decontamination efficacy of gaseous ozone in a public bus setting.

The widespread COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) necessitated measures aimed at preventing the spread of SARS-CoV-2. To mitigate the risk of fomite-mediated transmission, environmental cleaning and disinfection regimes have been widely implemented. However, conventional cleaning approaches such as surface wipe downs can be laborious and more efficient and effective disinfecting technologies are needed. Gaseous ozone disinfection is one technology which has been shown to be effective in laboratory studies. Here, we evaluated its efficacy and feasibility in a public bus setting, using murine hepatitis virus (a related betacoronavirus surrogate) and the bacteria Staphylococcus aureus as test organisms. An optimal gaseous ozone regime resulted in a 3.65-log reduction of murine hepatitis virus and a 4.73-log reduction of S. aureus, and decontamination efficacy correlated with exposure duration and relative humidity in the application space. These findings demonstrated gaseous ozone disinfection in field settings which can be suitably translated to public and private fleets that share analogous characteristics.

Co-incidence of BA.1 and BA.2 at the start of Singapore's Omicron wave revealed by Community and University Campus wastewater surveillance.

Wastewater surveillance (WWS) has been globally recognised to be a useful tool in quantifying SARS-CoV-2 RNA at the community and residential levels without biases associated with case-reporting. The emergence of variants of concern (VOCs) have given rise to an unprecedented number of infections even though populations are increasingly vaccinated. This is because VOCs have been reported to possess higher transmissibility and can evade host immune responses. The B.1.1.529 lineage (Omicron) has severely disrupted global plans to return to normalcy. In this study, we developed an allele-specific (AS) RT-qPCR assay which simultaneously targets the stretch of deletions and mutations in the spike protein from position 24-27 for quantitative detection of Omicron BA.2. Together with previous assays that detect mutations associated with Omicron BA.1 (deletion at position 69 and 70) and all Omicron (mutation at position 493 and 498), we report the validation and time series of these assays from September 2021 to May 2022 using influent samples from two wastewater treatment plants and across four University campus sites in Singapore. Viral RNA concentrations at the treatment plants corroborate with locally reported clinical cases, AS RT-qPCR assays revealed co-incidence of Omicron BA.1 and BA.2 on 12 January 2022, almost two months after initial BA.1 detection in South Africa and Botswana. BA.2 became the dominant variant by the end of January 2022 and completely displaced BA.1 by mid-March 2022. University campus sites were similarly positive for BA.1 and/or BA.2 in the same week as first detection at the treatment plants, where BA.2 became rapidly established as the dominant lineage within three weeks. These results corroborate clinical incidence of the Omicron lineages in Singapore and indicate minimal silent circulation prior to January 2022. The subsequent simultaneous spread of both variant lineages followed strategic relaxation of safe management measures upon meeting nationwide vaccination goals.

Features of the urban environment associated with Aedes aegypti abundance in high-rise public apartments in Singapore: An environmental case-control study.

Aedes aegypti abundance in residential estates is hypothesized to contribute to localised outbreaks of dengue in Singapore. Knowing the factors in the urban environment underlying high Ae. aegypti abundance could guide intervention efforts to reduce Ae. aegypti breeding and the incidence of dengue. In this study, objective data on Ae. aegypti abundance in public apartment blocks estimated by Singapore's nationally representative Gravitrap surveillance system was obtained from the National Environmental Agency. Low and high abundance status public apartment blocks were classified based on the Gravitrap Aegypti Index, corresponding to the lowest and highest quartiles respectively. An environmental case-control study was conducted, wherein a blinded assessment of urban features hypothesised to form breeding habitats was conducted in 50 randomly sampled public apartment blocks with low and high abundance statuses each. Logistic regression was performed to identify features that correlated with abundance status. A multivariable logistic model was created to determine key urban features found in corridors and void decks which were predictive of the Ae. aegypti abundance status of the public apartment block. At a statistical level of significance of 0.20, the presence of gully traps [Odds Ratio (OR): 1.34, 95% Confidence Interval (CI): 1.10, 1.66], age of the public apartment block [OR: 2.23, 95% CI: 1.48, 3.60], housing price [OR: 0.33, 95% CI: 0.16, 0.61] and corridor cleanliness [OR: 0.67, 95% CI: 0.40, 1.07] were identified as important predictors of abundance status. To reduce Ae. aegypti abundance around public apartment blocks and potential onward dengue transmission, gully traps could be remodelled or replaced by other drainage types. Routine inspections of Ae. aegypti breeding should be targeted at older and low-income neighbourhoods. Campaigns for cleaner corridors should be promoted.

Aedes aegypti exhibits a distinctive mode of late ovarian development.

BACKGROUND: Insects live in almost every habitat on earth. To adapt to their diverse environments, insects have developed a myriad of different strategies for reproduction reflected in diverse anatomical and behavioral features that the reproductive systems of females exhibit. Yet, ovarian development remains largely uncharacterized in most species except Drosophila melanogaster (D. melanogaster), a high Diptera model. In this study, we investigated the detailed developmental process of the ovary in Aedes aegypti (Ae. aegypti), a major vector of various disease-causing pathogens that inhabits tropical and subtropical regions. RESULTS: Compared with Drosophila melanogaster, a model of higher Diptera, the processes of pole cell formation and gonad establishment during embryonic stage are highly conserved in Ae. aegypti. However, Ae. aegypti utilizes a distinct strategy to form functional ovaries during larval/pupal development. First, during larval stage, Ae. aegypti primordial germ cells (PGCs) undergo a cyst-like proliferation with synchronized divisions and incomplete cytokinesis, leading to the formation of one tightly packed "PGC mass" containing several interconnected cysts, different from D. melanogaster PGCs that divide individually. This cyst-like proliferation is regulated by the target of rapamycin (TOR) pathway upon nutritional status. Second, ecdysone-triggered ovariole formation during metamorphosis exhibits distinct events, including "PGC mass" breakdown, terminal filament cell degeneration, and pre-ovariole migration. These unique developmental features might explain the structural and behavioral differences between Aedes and Drosophila ovaries. Importantly, both cyst-like proliferation and distinct ovariole formation are also observed in Culex quinquefasciatus and Anopheles sinensis, suggesting a conserved mode of ovarian development among mosquito species. In comparison with Drosophila, the ovarian development in Aedes and other mosquitoes might represent a primitive mode in the lower Diptera. CONCLUSIONS: Our study reveals a new mode of ovarian development in mosquitoes, providing insights into a better understanding of the reproductive system and evolutionary relationship among insects.

Discovery of super-insecticide-resistant dengue mosquitoes in Asia: Threats of concomitant knockdown resistance mutations.

Aedes aegypti (Linnaeus, 1762) is the main mosquito vector for dengue and other arboviral infectious diseases. Control of this important vector highly relies on the use of insecticides, especially pyrethroids. The high frequency (>78%) of the L982W substitution was detected at the target site of the pyrethroid insecticide, the voltage-gated sodium channel (Vgsc) of A. aegypti collected from Vietnam and Cambodia. Alleles having concomitant mutations L982W + F1534C and V1016G + F1534C were also confirmed in both countries, and their frequency was high (>90%) in Phnom Penh, Cambodia. Strains having these alleles exhibited substantially higher levels of pyrethroid resistance than any other field population ever reported. The L982W substitution has never been detected in any country of the Indochina Peninsula except Vietnam and Cambodia, but it may be spreading to other areas of Asia, which can cause an unprecedentedly serious threat to the control of dengue fever as well as other Aedes-borne infectious diseases.

Isolation and Genetic Characterization of Japanese Encephalitis Virus Two Decades after Its Elimination in Singapore.

Japanese encephalitis virus (JEV) is an important arbovirus in Asia that can cause serious neurological disease. JEV is transmitted by mosquitoes in an enzootic cycle involving porcine and avian reservoirs, in which humans are accidental, dead-end hosts. JEV is currently not endemic in Singapore, after pig farming was abolished in 1992; the last known human case was reported in 2005. However, due to its location along the East-Asian Australasian Flyway (EAAF), Singapore is vulnerable to JEV re-introduction from the endemic regions. Serological and genetic evidence in the last decade suggests JEV's presence in the local fauna. In the present study, we report the genetic characterization and the first isolation of JEV from 3214 mosquito pools consisting of 41,843 Culex mosquitoes, which were trapped from April 2014 to May 2021. The findings demonstrated the presence of genotype I of JEV (n = 10), in contrast to the previous reports of the presence of genotype II of JEV in Singapore. The genetic analyses also suggested that JEV has entered Singapore on several occasions and has potentially established an enzootic cycle in the local fauna. These observations have important implications in the risk assessment and the control of Japanese encephalitis in non-endemic countries, such as Singapore, that are at risk for JEV transmission.

Assessing the efficacy of male Wolbachia-infected mosquito deployments to reduce dengue incidence in Singapore: study protocol for a cluster-randomized controlled trial.

BACKGROUND: Dengue is a severe environmental public health challenge in tropical and subtropical regions. In Singapore, decreasing seroprevalence and herd immunity due to successful vector control has paradoxically led to increased transmission potential of the dengue virus. We have previously demonstrated that incompatible insect technique coupled with sterile insect technique (IIT-SIT), which involves the release of X-ray-irradiated male Wolbachia-infected mosquitoes, reduced the Aedes aegypti population by 98% and dengue incidence by 88%. This novel vector control tool is expected to be able to complement current vector control to mitigate the increasing threat of dengue on a larger scale. We propose a multi-site protocol to study the efficacy of IIT-SIT at reducing dengue incidence. METHODS/DESIGN: The study is designed as a parallel, two-arm, non-blinded cluster-randomized (CR) controlled trial to be conducted in high-rise public housing estates in Singapore, an equatorial city-state. The aim is to determine whether large-scale deployment of male Wolbachia-infected Ae. aegypti mosquitoes can significantly reduce dengue incidence in intervention clusters. We will use the CR design, with the study area comprising 15 clusters with a total area of 10.9 km2, covering approximately 722,204 residents in 1713 apartment blocks. Eight clusters will be randomly selected to receive the intervention, while the other seven will serve as non-intervention clusters. Intervention efficacy will be estimated through two primary endpoints: (1) odds ratio of Wolbachia exposure distribution (i.e., probability of living in an intervention cluster) among laboratory-confirmed reported dengue cases compared to test-negative controls and (2) laboratory-confirmed reported dengue counts normalized by population size in intervention versus non-intervention clusters. DISCUSSION: This study will provide evidence from a multi-site, randomized controlled trial for the efficacy of IIT-SIT in reducing dengue incidence. The trial will provide valuable information to estimate intervention efficacy for this novel vector control approach and guide plans for integration into national vector control programs in dengue-endemic settings. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT05505682 . Registered on 16 August 2022. Retrospectively registered.

Influence of public hesitancy and receptivity on reactive behaviours towards releases of male Wolbachia-Aedes mosquitoes for dengue control.

Singapore, a highly urbanized Asian tropical country that experiences periodic dengue outbreaks, is piloting field releases of male Wolbachia-carrying Aedes aegypti mosquitoes with the aim of suppressing urban populations of the primary dengue vector Aedes aegypti. This study proposes and assesses a model to explain the roles of hesitancy and receptivity towards Project Wolbachia-Singapore in influencing reactive mosquito prevention behaviors (reactive behaviors) towards the release of Wolbachia-Aedes mosquitoes for residents living in the release sites. Interestingly, both hesitancy and receptivity predicted greater instances of reactive behaviors. The model also examines the roles of general knowledge about Wolbachia technology, perceived severity of mosquito bites, perceived density of mosquitoes, and social responsibility as predictors of hesitancy, receptivity, and reactive behaviors towards the release of Wolbachia-Aedes mosquitoes. Hesitancy towards the project mediated the effects of general knowledge, perceived severity of mosquito bites, and perceived density of mosquitoes on reactive behaviors towards the releases, although receptivity towards the project did not. Having less knowledge about Project Wolbachia-Singapore was associated with higher hesitancy towards the project and higher likelihood of performing reactive behaviors towards the releases. Individuals who perceive mosquito bites to be more severe and think that there are more mosquitoes in their living environments were also more likely to be hesitant about the project and practice reactive behaviors. However, both hesitancy and receptivity towards the project mediated the effect of social responsibility on reactive behaviors. Receptivity towards the project was driven by social responsibility, which was also associated with reduced hesitancy towards the project. Our findings suggest that, to address the hesitancy reported by a minority of participants, future outreach efforts should focus on strengthening the public's sense of social responsibility and on tailored education campaigns targeting groups with low levels of knowledge of the project.

Development of Differentiating Infected from Vaccinated Animals (DIVA) Real-Time PCR for African Horse Sickness Virus Serotype 1.

African horse sickness (AHS) is a highly infectious and often fatal disease caused by 9 serotypes of the orbivirus African horse sickness virus (AHSV). In March 2020, an AHS outbreak was reported in Thailand in which AHSV serotype 1 was identified as the causative agent. Trivalent live attenuated vaccines serotype 1, 3, and 4 were used in a targeted vaccination campaign within a 50-km radius surrounding the infected cases, which promptly controlled the spread of the disease. However, AHS-like symptoms in vaccinated horses required laboratory diagnostic methods to differentiate infected horses from vaccinated horses, especially for postvaccination surveillance. We describe a real-time reverse transcription PCR-based assay for rapid characterization of the affecting field strain. The development and validation of this assay should imbue confidence in differentiating AHS-vaccinated horses from nonvaccinated horses. This method should be applied to determining the epidemiology of AHSV in future outbreaks.

Geographical clusters of dengue outbreak in Singapore during the Covid-19 nationwide lockdown of 2020.

Dengue, a mosquito-transmitted viral disease, has posed a public health challenge to Singaporean residents over the years. In 2020, Singapore experienced an unprecedented dengue outbreak. We collected a dataset of geographical dengue clusters reported by the National Environment Agency (NEA) from 15 February to 9 July in 2020, covering the nationwide lockdown associated with Covid-19 during the period from 7 April to 1 June. NEA regularly updates the dengue clusters during which an infected person may be tagged to one cluster based on the most probable infection location (residential apartment or workplace address), which is further matched to fine-grained spatial units with an average coverage of about 1.35 km2. Such dengue cluster dataset helps not only reveal the dengue transmission patterns, but also reflect the effects of lockdown on dengue spreading dynamics. The resulting data records are released in simple formats for easy access to facilitate studies on dengue epidemics.