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Background: Dengue remains a global health challenge with limited treatment options, highlighting the need for effective vector control strategies. The introduction of Wolbachia pipientis into Aedes aegypti populations has shown success in reducing dengue transmission across global field trials. However, the spillover effectiveness of the technology on untreated areas is not well-known. This study estimates the spillover protective effectiveness (PE) of Wolbachia-mediated introgression on dengue., Methods: We used the synthetic control method (SCM) under assumption of partial interference to evaluate the direct and spillover PEs of Wolbachia-mediated introgression in a long-running operational trial of the intervention in Malaysia. Synthetic controls (SCs), which comprise of a weighted sum of non-spillover controls, were constructed for each directly-treated and spillover site in the pre-intervention period to account for historical imbalances in dengue risk and risk trajectories. SCs were compared to directly/spillover-treated sites to estimate the impact of Wolbachia-introgression on dengue incidence across each site, calendar year and intervention time. Robustness checks, including visual inspections, root-mean-square error (RMSE) calculations, in-space and in-time placebo checks, and permutation tests, were used to inspect the model's ability in attributing dengue incidence reductions to the Wolbachia interventions., Findings: The direct and spillover PEs of Wolbachia on dengue incidence were expressed as a percentage reduction of dengue incidence, or the absolute case reductions, by comparing SCs to actual intervention/spillover sites. Findings indicate a direct reduction in dengue incidence by 64.35% (95% CI: 63.50-66.71, p < 0.05 using permutation tests) in directly treated areas, corresponding to 1802 (95% CI: 1768-1932) cases averted. Meanwhile, spillover effects contributed to a 37.69% (95% CI: 36.45-38.49, p < 0.05) reduction in adjacent non-intervention areas, accounting for 115 (95% CI: 104-132) absolute cases averted. Tracking PEs by intervention time revealed a dose-response relationship, where PEs increased concomitantly with Wolbachia frequency. Model checks confirmed the robustness of these results, and ascertained that these PEs were not an artefact of poor control selection, pre-trends in dengue incidence or poor predictive ability of the fitted SCs., Interpretation: Wolbachia-introgression effectively diminished dengue incidence in directly-treated and surrounding spillover regions. This dual effectiveness highlights the potential of Wolbachia-infected mosquitoes as a sustainable, cost-effective strategy against dengue., Funding: This research is hosted by CNRS@CREATE and supported by the National Research Foundation, Prime Minister's Office, Singapore, under its Campus for Research Excellence and Technological Enterprise (CREATE) program, and is funded by the Lee Kong Chian School of Medicine-Ministry of Education Start-Up Grant. The original Hoffmann et al. (2024) study was funded by the Wellcome Trust Awards 226166, 108508, 202888 and the Ministry of Health Malaysia NMRR-16-297-28898., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Matings between male Aedes aegypti mosquitoes infected with wAlbB strain of Wolbachia and wildtype females yield non-viable eggs. We evaluated the efficacy of releasing wAlbB-infected Ae. aegypti male mosquitoes to suppress dengue., Methods: We specified the protocol of a two-arm cluster-randomized test-negative controlled trial (cRCT) and emulated it using a nationally representative test-negative/positive database of individuals reporting for febrile illness to any public hospital, general practitioner or polyclinic. We retrospectively built a cohort of individuals who reside in Wolbachia locations vs a comparator control group who do not reside in Wolbachia locations, using a nationally representative database of all individuals whom report for febrile illness and were tested for dengue at the Environmental Health Institute/hospital laboratories/commercial diagnostic laboratories, through general practitioner clinic, polyclinic or public/private hospital from epidemiological week (EW) 1 2019 to EW26 2022. We emulated a constrained randomization protocol used in cRCTs to balance dengue risk between intervention and control arms in the pre-intervention period. We used the inverse probability weighting approach to further balance the intervention and control groups using a battery of algorithmically selected sociodemographic, environmental and anthropogenic variables. Intention-to-treat analyses were conducted to estimate the risk reduction of dengue given Wolbachia exposure., Results: Intention-to-treat analyses revealed that, compared with controls, Wolbachia releases for 3, 6 and ≥12 months was associated to 47% (95% confidence interval: 25-69%), 44% (33-77%) and 61% (38-78%) protective efficacy against dengue, respectively. When exposed to ≥12 months of Wolbachia releases, protective efficacies ranged from 49% (13-72%) to 77% (60-94%) across years. The proportion of virologically confirmed dengue cases was lower overall in the intervention arm. Protective efficacies were found across all years, age and sex subgroups, with higher durations of Wolbachia exposure associated to greater risk reductions of dengue., Conclusion: Results demonstrated that Wolbachia-mediated sterility can strengthen dengue control in tropical cities, where dengue burden is the greatest., (© International Society of Travel Medicine 2024. Published by Oxford University Press.)

Background: Incompatible insect technique (IIT) coupled with sterile insect technique (SIT) via the release of sterile male Wolbachia-infected mosquitoes is a promising tool for Aedes-borne disease control. Yet, real-world evidence on the suppressive effectiveness of IIT-SIT on mosquito abundance remains mostly limited to small semi-rural village and suburban localities over short trial durations. However, a large proportion of Aedes-borne diseases occur in dense, urban, and high-rise locations, limiting the applicability of previous studies for these settings with high disease burden. The sustainability and use of this technology over multiple years is also unknown., Methods: In this synthetic control study, we conducted a large-scale, field trial of IIT-SIT targeting Aedes aegypti among high-rise public housing estates in Singapore, an equatorial city state. Routinely collected data from a large, nationwide surveillance system of 57 990 unique mosquito traps, combined with a high-dimensional set of anthropogenic and environmental confounders were collected to ascertain mosquito abundance and its key drivers. Four townships were selected as the intervention groups (approximate population size of 607 872 residents as of 2022), wherein interventions that combined ITT with SIT over the course of the study period were conducted. Townships were subject to releases of wAlbB-SG male A aegypti mosquitoes twice a week. Data were assessed over the course of epidemiological weeks (EWs), which provide the finest temporal resolution of recorded Wolbachia release schedule and mosquito abundance data. A novel synthetic control framework was then developed to account for the non-randomised and staggered adoption setting of the intervention across trial sectors to identify the direct suppressive effectiveness of IIT-SIT on female A aegypti populations, the spillover effects in non-release areas, and the effect of the intervention on other mosquito populations such as Aedes albopictus. Furthermore, we recalculated effectiveness in terms of calendar time, time since intervention, and over multiple sites to examine heterogeneities in IIT-SIT effectiveness., Findings: Between EW27 2018 and EW26 2022, Wolbachia releases were conducted across 117 sectors, of which 97 had sufficient trap data, which were collected between EW8 2019 and EW26 2022. We found that Wolbachia-based IIT-SIT reduced wild-type female A aegypti populations by a mean of 62·01% (95% CI 60·68 to 63·26) by 3 months, 78·40% (77·56 to 79·18) by 6 months, and 91·32% (90·95 to 91·66) by at least 18 months of releases. We also found a smaller but non-negligible spillover suppression effect that gradually increased over time (mean spillover intervention effectiveness 61·02% [95% CI 57·89 to 63·72] in adjacent, non-intervention sectors). Although no consistent change in A albopictus populations was seen across the four intervention townships after Wolbachia releases, the average intervention effectiveness on the A albopictus population across all release sectors was -25·80% (95% CI -30·93 to -21·05), which was driven by increases in two towns., Interpretation: Our results demonstrate the potential of IIT-SIT for strengthening long-term, large-scale vector control in tropical cities, where dengue burden is the greatest. The effect of these interventions in different geographical settings should be assessed in future work., Funding: Singapore's Ministry of Finance, Ministry of Sustainability and the Environment, National Environment Agency, and National Robotics Program., Competing Interests: Declaration of interests DDY is a paid employee of Orinno Technology. EGK and YY are paid employees of Verily Life Sciences. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: Wolbachia symbiosis in Aedes aegypti is an emerging biocontrol measure against dengue. However, assessing its real-world efficacy is challenging due to the non-randomised, field-based nature of most intervention studies. This research re-evaluates the spatial-temporal impact of Wolbachia interventions on dengue incidence using a large battery of quasi-experimental methods and assesses each method's validity., Methods: A systematic search for Wolbachia intervention data was conducted via PUBMED. Efficacy was reassessed using commonly-used quasi-experimental approaches with extensive robustness checks, including geospatial placebo tests and a simulation study. Intervention efficacies across multiple study sites were computed using high-resolution aggregations to examine heterogeneities across sites and study periods. We further designed a stochastic simulation framework to assess the methods' ability to estimate intervention efficacies (IE)., Results: Wolbachia interventions in Singapore, Malaysia, and Brazil significantly decreased dengue incidence, with reductions ranging from 48.17% to 69.19%. IEs varied with location and duration. Malaysia showed increasing efficacy over time, while Brazil exhibited initial success with subsequent decline, hinting at operational challenges. Singapore's strategy was highly effective despite partial saturation. Simulations identified Synthetic Control Methods (SCM) and its variant, count Synthetic Control Method (cSCM), as superior in precision, with the smallest percentage errors in efficacy estimation. These methods also demonstrated robustness in placebo tests., Conclusions: Wolbachia interventions exhibit consistent protective effects against dengue. SCM and cSCM provided the most precise and robust estimates of IEs, validated across simulated and real-world settings., (© 2024. The Author(s).)

Background: This trial is a parallel, two-arm, non-blinded cluster randomised controlled trial that is under way in Singapore, with the aim of measuring the efficacy of male Wolbachia-infected Aedes aegypti deployments in reducing dengue incidence in an endemic setting with all four dengue serotypes in circulation. The trial commenced in July 2022 and is expected to conclude in September 2024. The original study protocol was published in December 2022. Here, we describe amendments that have been made to the study protocol since commencement of the trial., Methods: The key protocol amendments are (1) addition of an explicit definition of Wolbachia exposure for residents residing in intervention sites based on the duration of Wolbachia exposure at point of testing, (2) incorporation of a high-dimensional set of anthropogenic and environmental characteristics in the analysis plan to adjust for baseline risk factors of dengue transmission, and (3) addition of alternative statistical analyses for endpoints to control for post hoc imbalance in cluster-based environmental and anthropogenic characteristics., Discussion: The findings from this study will provide the first experimental evidence for the efficacy of releasing male-Wolbachia infected mosquitoes to reduce dengue incidence in a cluster-randomised controlled trial. The trial will conclude in 2024 and results will be reported shortly thereafter., Trial Registration: ClinicalTrials.gov, identifier: NCT05505682. Registered on 16 August 2022. Retrospectively registered. Last updated 11 November 2023., (© 2024. The Author(s).)

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., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Ho et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Fernandez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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 km 2 , 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., (© 2022. The Author(s).)

The Incompatible Insect Technique (IIT) strategy involves the release of male mosquitoes infected with the bacterium Wolbachia . Regular releases of male Wolbachia -infected mosquitoes can lead to the suppression of mosquito populations, thereby reducing the risk of transmission of vector-borne diseases such as dengue. However, due to imperfect sex-sorting under IIT, fertile Wolbachia -infected female mosquitoes may potentially be unintentionally released into the environment, which may result in replacement and failure to suppress the mosquito populations. As such, mitigating Wolbachia establishment requires a combination of IIT with other strategies. We introduced a simple compartmental model to simulate ex-ante mosquito population dynamics subjected to a Wolbachia -IIT programme. In silico, we explored the risk of replacement, and strategies that could mitigate the establishment of the released Wolbachia strain in the mosquito population. Our results suggest that mitigation may be achieved through the application of a sterile insect technique. Our simulations indicate that these interventions do not override the intended wild type suppression of the IIT approach. These findings will inform policy makers of possible ways to mitigate the potential establishment of Wolbachia using the IIT population control strategy.

Background: Despite the licensure of the world's first dengue vaccine and the current development of additional vaccine candidates, successful Aedes control remains critical to the reduction of dengue virus transmission. To date, there is still limited literature that attempts to explain the spatio-temporal population dynamics of Aedes mosquitoes within a single city, which hinders the development of more effective citywide vector control strategies. Narrowing this knowledge gap requires consistent and longitudinal measurement of Aedes abundance across the city as well as examination of relationships between variables on a much finer scale., Methods: We utilized a high-resolution longitudinal dataset generated from Singapore's islandwide Gravitrap surveillance system over a 2-year period and built a Bayesian hierarchical model to explain the spatio-temporal dynamics of Aedes aegypti and Aedes albopictus in relation to a wide range of environmental and anthropogenic variables. We also created a baseline during our model assessment to serve as a benchmark to be compared with the model's out-of-sample prediction/forecast accuracy as measured by the mean absolute error., Results: For both Aedes species, building age and nearby managed vegetation cover were found to have a significant positive association with the mean mosquito abundance, with the former being the strongest predictor. We also observed substantial evidence of a nonlinear effect of weekly maximum temperature on the Aedes abundance. Our models generally yielded modest but statistically significant reductions in the out-of-sample prediction/forecast error relative to the baseline., Conclusions: Our findings suggest that public residential estates with older buildings and more nearby managed vegetation should be prioritized for vector control inspections and community advocacy to reduce the abundance of Aedes mosquitoes and the risk of dengue transmission.