Evaluating quasi-experimental approaches for estimating epidemiological efficacy of non-randomised field trials: applications in Wolbachia interventions for dengue.

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.

Effects of Recent Prior Dengue Infection on Risk and Severity of Subsequent SARS-CoV-2 Infection: A Retrospective Cohort Study.

Background and Aims: Elucidating whether prior dengue potentially confers cross-protection against COVID-19 is of public health importance in tropical countries at risk of overlapping dengue and COVID-19 epidemics. However, studies to date have yielded conflicting results. We aimed to assess effects of recent prior dengue infection on risk and severity of subsequent SARS-CoV-2 infection among adult Singaporeans. Methods: A retrospective cohort study including all adult Singaporeans aged ≥18 years was conducted from 1 July 2021 through 31 October 2022, when a dengue outbreak driven by the DENV3 serotype preceded subsequent waves of SARS-CoV-2 Delta/Omicron transmission in Singapore. SARS-CoV-2 and dengue infection status were classified using national registries. Cox regression models adjusted for demographics, COVID-19 vaccination status, comorbidity, and socioeconomic-status were used to assess risks and severity (hospitalization, severe illness) of SARS-CoV-2 infection occurring after previous recorded dengue infection. Results: A total of 3 366 399 individuals were included, contributing 1 399 696 530 person-days of observation. A total of 13 434 dengue infections and 1 253 520 subsequent SARS-CoV-2 infections were recorded; with an average of 94.7 days (standard deviation = 83.8) between dengue infection and SARS-CoV-2 infection. Preceding dengue infection was associated with a modest increase in risk of subsequent SARS-CoV-2 infection (adjusted hazards ratio [aHR] = 1.13; 95% confidence interval [CI], 1.08-1.17), and significantly elevated risk of subsequent COVID-19 hospitalization (aHR = 3.25; 95% CI, 2.78-3.82) and severe COVID-19 (aHR = 3.39; 95% CI, 2.29-5.03). Conclusions: Increased risk of SARS-CoV-2 infection and adverse COVID-19 outcomes were observed following preceding dengue infection in a national population-based cohort of adult Singaporeans. This observation is of significance in tropical countries with overlapping dengue and COVID-19 outbreaks.

Assessing Wolbachia-mediated Sterility for Dengue Control: Emulation of a Cluster Randomized Target Trial in Singapore.

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 versus 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 EW1 2019-EW 262022. 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 was 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, 12 or more months was associated to 47%(95%CI:25-69%), 44%(33-77%) and 61%(38-78%) protective efficacy against dengue, respectively. When exposed to 12 or more 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.

Update to: Assessing the efficacy of male Wolbachia-infected mosquito deployments to reduce dengue incidence in Singapore.

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.

Dengue versus COVID-19: comparing the incidence of cardiovascular, neuropsychiatric and autoimmune complications.

BACKGROUND: While persistence of chronic symptoms following dengue infection has been documented in small prospective cohorts, population-based studies are limited. The post-acute risk of new-incident multi-systemic complications following dengue infection was contrasted against that following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in a multi-ethnic adult Asian population. METHODS: National testing and healthcare claims that databases in Singapore were utilized to build a retrospective population-based adult cohort with laboratory-confirmed infection during overlapping waves of SARS-CoV-2 and dengue transmission (1 July 2021 to 31 October 2022). Risks of new-incident cardiovascular/neuropsychiatric/autoimmune complications 31-300 days of post-dengue infection, contrasted with SARS-CoV-2 infection, were estimated using Cox regression with overlap weights. Risks were reported in terms of adjusted hazard ratio (aHR) and excess burden per 1000 persons. RESULTS: 11 707 dengue-infected individuals and 1 248 326 contemporaneous coronavirus disease 2019 (COVID-19) cases were included; the majority had mild initial infection not requiring hospitalization. Amongst dengue-infected individuals, there was 21% [aHR = 1.21 (1.06-1.38)] increased risk of any sequelae, with 55% [aHR = 1.55 (1.27-1.89)] increased risk of cardiovascular sequelae. Specifically, increased risk of dysrhythmias [aHR = 1.79(1.35-2.37)], ischemic heart disease [aHR = 1.45(1.12-1.89)], other cardiac disorders [aHR = 2.21(1.54-3.16)] and thrombotic disorders [aHR = 2.55(1.50-4.35)] was noted. Elevated risk of individual neuropsychiatric sequelae, including cerebrovascular disorders [aHR = 1.49(1.09-2.13)], cognition/memory disorders [aHR = 2.13(1.55-2.93)], extrapyramidal/movement disorders [aHR = 1.98(1.33-2.94)] and anxiety disorders [aHR = 1.61(1.01-2.56)], was observed in dengue-infected individuals compared to COVID-19 cases. Elevated risks of post-acute sequelae in dengue survivors were observed when contrasted against COVID-19 survivors infected during Delta/Omicron predominance, as well as across vaccination strata. CONCLUSION: Increased risk of post-acute cardiovascular/neuropsychiatric complications was observed in dengue survivors, when contrasted against COVID-19 survivors infected during Delta/Omicron predominance.

Molecular epidemiology of dengue in Malaysia: 2015-2021.

Dengue has been one of the major public health problems in Malaysia for decades. Over 600,000 dengue cases and 1,200 associated fatalities have been reported in Malaysia from 2015 to 2021, which was 100% increase from the cumulative total of dengue cases reported during the preceding 07-year period from 2008 to 2014. However, studies that describe the molecular epidemiology of dengue in Malaysia in recent years are limited. In the present study, we describe the genetic composition and dispersal patterns of Dengue virus (DENV) by using 4,004 complete envelope gene sequences of all four serotypes (DENV-1 = 1,567, DENV-2 = 1,417, DENV-3 = 762 and DENV-4 = 258) collected across Malaysia from 2015 to 2021. The findings revealed that DENV populations in Malaysia were highly diverse, and the overall heterogeneity was maintained through repetitive turnover of genotypes. Phylogeography analyses suggested that DENV dispersal occurred through an extensive network, mainly among countries in South and East Asia and Malaysian states, as well as among different states, especially within Peninsular Malaysia. The results further suggested Selangor and Johor as major hubs of DENV emergence and spread in Malaysia.

Usefulness of aircraft and airport wastewater for monitoring multiple pathogens including SARS-CoV-2 variants.

BACKGROUND: As global travel resumed in coronavirus disease 2019 (COVID-19) endemicity, the potential of aircraft wastewater monitoring to provide early warning of disease trends for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and other infectious diseases, particularly at international air travel hubs, was recognized. We therefore assessed and compared the feasibility of testing wastewater from inbound aircraft and airport terminals for 18 pathogens including SARS-CoV-2 in Singapore, a popular travel hub in Asia. METHODS: Wastewater samples collected from inbound medium- and long-haul flights and airport terminals were tested for SARS-CoV-2. Next Generation Sequencing was carried out on positive samples to identify SARS-CoV-2 variants. Airport and aircraft samples were further tested for 17 other pathogens through quantitative reverse transcription polymerase chain reaction. RESULTS: The proportion of SARS-CoV-2-positive samples and the average virus load was higher for wastewater samples from aircraft as compared with airport terminals. Cross-correlation analyses indicated that viral load trends from airport wastewater led local COVID-19 case trends by 2-5 days. A total of 10 variants (44 sub-lineages) were successfully identified from aircraft wastewater and airport terminals, and four variants of interest and one variant under monitoring were detected in aircraft and airport wastewater 18-31 days prior to detection in local clinical cases. The detection of five respiratory and four enteric viruses in aircraft wastewater samples further underscores the potential to expand aircraft wastewater to monitoring pathogens beyond SARS-CoV-2. CONCLUSION: Our findings demonstrate the feasibility of aircraft wastewater testing for monitoring infectious diseases threats, potentially detecting signals before clinical cases are reported. The triangulation of similar datapoints from aircraft wastewater of international travel nodes could therefore serve as a useful early warning system for global health threats.

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.

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.

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.