Evaluation of three alternative methods to the plaque reduction neutralizing assay for measuring neutralizing antibodies to dengue virus serotype 2.

BACKGROUND: Dengue is a global public health challenge which requires accurate diagnostic methods for surveillance and control. The gold standard for detecting dengue neutralizing antibodies (nAbs) is the plaque reduction neutralization test (PRNT), which is both labor-intensive and time-consuming. This study aims to evaluate three alternative approaches, namely, the MTT-based (or (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) microneutralization assay, the xCELLigence real-time cell analysis (RTCA), and the immuno-plaque assay-focus reduction neutralization test (iPA-FRNT). METHODS: Twenty-two residual serum samples were tested for DENV-2 nAbs using all four assays at three neutralization endpoints of 50%, 70% and 90% inhibition in virus growth. For each neutralization endpoint, results were compared using linear regression and correlation analyses. Test performance characteristics were further obtained for iPA-FRNT using 38 additional serum samples. RESULTS: Positive correlation of DENV-2 neutralization titers for the MTT-based microneutralization assay and the PRNT assay was only observed at the neutralization endpoint of 50% (r = 0.690). In contrast, at all three neutralization end points, a linear trend and positive correlation of DENV-2 neutralization titers for the xCELLigence RTCA and the PRNT assays were observed, yielding strong or very strong correlation (r = 0.829 to 0.967). This was similarly observed for the iPA-FRNT assay (r = 0.821 to 0.916), which also offered the added advantage of measuring neutralizing titers to non-plaque forming viruses. CONCLUSION: The xCELLigence RTCA and iPA-FRNT assays could serve as suitable alternatives to PRNT for dengue serological testing. The decision to adopt these methods may depend on the laboratory setting, and the utility of additional applications offered by these technologies.

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.

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.

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.

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.

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.

Wastewater surveillance of SARS-CoV-2 and chemical markers in campus dormitories in an evolving COVID - 19 pandemic.

In this study, we report the implementation of a comprehensive wastewater surveillance testing program at a university campus in Singapore to identify Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infected individuals and the usage of pharmaceuticals and personal care products (PPCPs) as well as other emerging contaminants (ECs). This unique co-monitoring program simultaneously measured SARS-CoV-2 with chemical markers/contaminants as the COVID-19 situation evolved from pandemic to endemic stages, following a nationwide mass vaccination drive. SARS-CoV-2 RNA concentrations in wastewater from campus dormitories were measured using real-time reverse transcription-polymerase chain reaction (RT-qPCR) and corroborated with the number of symptomatic COVID-19 cases confirmed with the antigen rapid test (ART). Consistent results were observed where the concentrations of SARS-CoV-2 RNA detected in wastewater increased proportionately with the number of COVID-19 infected individuals residing on campus. Similarly, a wide range of ECs, including disinfectants and antibiotics, were detected through sensitive liquid chromatography with tandem mass spectrometry (LC-MS/MS) techniques to establish PPCPs consumption patterns during various stages of the COVID-19 pandemic in Singapore. Statistical correlation of SARS-CoV-2 RNA was observed with few ECs belonging to disinfectants, PCPs and antibiotics. A high concentration of disinfectants and subsequent positive correlation with the number of reported cases on the university campus indicates that disinfectants could serve as a chemical marker during such unprecedented times.

Rapid Diagnostic Tests for the Detection of the Four Dengue Virus Serotypes in Clinically Relevant Matrices.

The efficient and accurate diagnosis of dengue, a major mosquito-borne disease, is of primary importance for clinical care, surveillance, and outbreak control. The identification of specific dengue virus serotype 1 (DENV-1) to DENV-4 can help in understanding the transmission dynamics and spread of dengue disease. The four rapid low-resource serotype-specific dengue tests use a simple sample preparation reagent followed by reverse transcription-isothermal recombinase polymerase amplification (RT-RPA) combined with lateral flow detection (LFD) technology. Results are obtained directly from clinical sample matrices in 35 min, requiring only a heating block and pipettes for liquid handling. In addition, we demonstrate that the rapid sample preparation step inactivates DENV, improving laboratory safety. Human plasma and serum were spiked with DENV, and DENV was detected with analytical sensitivities of 333 to 22,500 median tissue culture infectious doses (TCID50)/mL. The analytical sensitivities in blood were 94,000 to 333,000 TCID50/mL. Analytical specificity testing confirmed that each test could detect multiple serotype-specific strains but did not respond to strains of other serotypes, closely related flaviviruses, or chikungunya virus. Clinical testing on 80 human serum samples demonstrated test specificities of between 94 and 100%, with a DENV-2 test sensitivity of 100%, detecting down to 0.004 PFU/µL, similar to the sensitivity of the PCR test; the other DENV tests detected down to 0.03 to 10.9 PFU/µL. Collectively, our data suggest that some of our rapid dengue serotyping tests provide a potential alternative to conventional labor-intensive RT-quantitative PCR (RT-qPCR) detection, which requires expensive thermal cycling instrumentation, technical expertise, and prolonged testing times. Our tests provide performance and speed without compromising specificity in human plasma and serum and could become promising tools for the detection of high DENV loads in resource-limited settings. IMPORTANCE The efficient and accurate diagnosis of dengue, a major mosquito-borne disease, is of primary importance for clinical care, surveillance, and outbreak control. This study describes the evaluation of four rapid low-resource serotype-specific dengue tests for the detection of specific DENV serotypes in clinical sample matrices. The tests use a simple sample preparation reagent followed by reverse transcription-isothermal recombinase polymerase amplification (RT-RPA) combined with lateral flow detection (LFD) technology. These tests have several advantages compared to RT-qPCR detection, such as a simple workflow, rapid sample processing and turnaround times (35 min from sample preparation to detection), minimal equipment needs, and improved laboratory safety through the inactivation of the virus during the sample preparation step. The low-resource formats of these rapid dengue serotyping tests have the potential to support effective dengue disease surveillance and enhance the diagnostic testing capacity in resource-limited countries with both endemic dengue and intense coronavirus disease 2019 (COVID-19) transmission.