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. Graphical abstract Unlabelled Image

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.

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.

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.

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.

Development of an efficient wastewater testing protocol for high-throughput country-wide SARS-CoV-2 monitoring.

Wastewater-based surveillance has been widely used as a non-intrusive tool to monitor population-level transmission of COVID-19. Although various approaches are available to concentrate viruses from wastewater samples, scalable methods remain limited. Here, we sought to identify and evaluate SARS-CoV-2 virus concentration protocols for high-throughput wastewater testing. A total of twelve protocols for polyethylene glycol (PEG) precipitation and four protocols for ultrafiltration-based approaches were evaluated across two phases. The first phase entailed an initial evaluation using a small sample set, while the second phase further evaluated five protocols using wastewater samples of varying SARS-CoV-2 concentrations. Permutations in the pre-concentration, virus concentration and RNA extraction steps were evaluated. Among PEG-based methods, SARS-CoV-2 virus recovery was optimal with 1) the removal of debris prior to processing, 2) 2 h to 24 h incubation with 8% PEG at 4 °C, 3) 4000 xg or 14,000 xg centrifugation, and 4) a column-based RNA extraction method, yielding virus recovery of 42.4-52.5%. Similarly, the optimal protocol for ultrafiltration included 1) the removal of debris prior to processing, 2) ultrafiltration, and 3) a column-based RNA extraction method, yielding a recovery of 38.2%. This study also revealed that SARS-CoV-2 RNA recovery for samples with higher virus concentration were less sensitive to changes in the PEG method, but permutations in the PEG protocol could significantly impact virus yields when wastewater samples with lower SARS-CoV-2 RNA were used. Although both PEG precipitation and ultrafiltration methods resulted in similar SARS-CoV-2 RNA recoveries, the former method is more cost-effective while the latter method provided operational efficiency as it required a shorter turn-around-time (PEG precipitation, 9-23 h; Ultrafiltration, 5 h). The decision on which method to adopt will thus depend on the use-case for wastewater testing, and the need for cost-effectiveness, sensitivity, operational feasibility and scalability.

Standardizing data reporting in the research community to enhance the utility of open data for SARS-CoV-2 wastewater surveillance.

SARS-CoV-2 RNA detection in wastewater is being rapidly developed and adopted as a public health monitoring tool worldwide. With wastewater surveillance programs being implemented across many different scales and by many different stakeholders, it is critical that data collected and shared are accompanied by an appropriate minimal amount of metainformation to enable meaningful interpretation and use of this new information source and intercomparison across datasets. While some databases are being developed for specific surveillance programs locally, regionally, nationally, and internationally, common globally-adopted data standards have not yet been established within the research community. Establishing such standards will require national and international consensus on what metainformation should accompany SARS-CoV-2 wastewater measurements. To establish a recommendation on minimum information to accompany reporting of SARS-CoV-2 occurrence in wastewater for the research community, the United States National Science Foundation (NSF) Research Coordination Network on Wastewater Surveillance for SARS-CoV-2 hosted a workshop in February 2021 with participants from academia, government agencies, private companies, wastewater utilities, public health laboratories, and research institutes. This report presents the primary two outcomes of the workshop: (i) a recommendation on the set of minimum meta-information that is needed to confidently interpret wastewater SARS-CoV-2 data, and (ii) insights from workshop discussions on how to improve standardization of data reporting.

Non-intrusive wastewater surveillance for monitoring of a residential building for COVID-19 cases.

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

Environmental Contamination of SARS-CoV-2 in a Non-Healthcare Setting.

Fomite-mediated transmission has been identified as a possible route for the spread of COVID-19 disease caused by SARS-CoV-2. In healthcare settings, environmental contamination by SARS-CoV-2 has been found in patients' rooms and toilets. Here, we investigated environmental presence of SARS-CoV-2 in non-healthcare settings and assessed the efficacy of cleaning and disinfection in removing virus contamination. A total of 428 environmental swabs and six air samples was taken from accommodation rooms, toilets and elevators that have been used by COVID-19 cases. By using a reverse transcription polymerase chain reaction assay, we detected two SARS-CoV-2 RNA positive samples in a room where a COVID-19 patient stayed prior to diagnosis. The present study highlights the risk of fomite-mediated transmission in non-healthcare settings and the importance of surface disinfection in spaces occupied by cases. Of note, neither air-borne transmission nor surface contamination of elevators, which were transiently exposed to infected individuals, was evident among samples analyzed.

Environmental Contamination of SARS-CoV-2 in a Non-Healthcare Setting

Fomite-mediated transmission has been identified as a possible route for the spread of COVID-19 disease caused by SARS-CoV-2. In healthcare settings, environmental contamination by SARS-CoV-2 has been found in patients’rooms and toilets. Here, we investigated environmental presence of SARS-CoV-2 in non-healthcare settings and assessed the efficacy of cleaning and disinfection in removing virus contamination. A total of 428 environmental swabs and six air samples was taken from accommodation rooms, toilets and elevators that have been used by COVID-19 cases. By using a reverse transcription polymerase chain reaction assay, we detected two SARS-CoV-2 RNA positive samples in a room where a COVID-19 patient stayed prior to diagnosis. The present study highlights the risk of fomite-mediated transmission in non-healthcare settings and the importance of surface disinfection in spaces occupied by cases. Of note, neither air-borne transmission nor surface contamination of elevators, which were transiently exposed to infected individuals, was evident among samples analyzed.