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.

Synthetic Heparan Sulfate Mimetic Pixatimod (PG545) Potently Inhibits SARS-CoV-2 by Disrupting the Spike–ACE2 Interaction

Heparan sulfate (HS) is a cell surface polysaccharide recently identified as a coreceptor with the ACE2 protein for the S1 spike protein on SARS-CoV-2 virus, providing a tractable new therapeutic target. Clinically used heparins demonstrate an inhibitory activity but have an anticoagulant activity and are supply-limited, necessitating alternative solutions. Here, we show that synthetic HS mimetic pixatimod (PG545), a cancer drug candidate, binds and destabilizes the SARS-CoV-2 spike protein receptor binding domain and directly inhibits its binding to ACE2, consistent with molecular modeling identification of multiple molecular contacts and overlapping pixatimod and ACE2 binding sites. Assays with multiple clinical isolates of SARS-CoV-2 virus show that pixatimod potently inhibits the infection of monkey Vero E6 cells and physiologically relevant human bronchial epithelial cells at safe therapeutic concentrations. Pixatimod also retained broad potency against variants of concern (VOC) including B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Furthermore, in a K18-hACE2 mouse model, pixatimod significantly reduced SARS-CoV-2 viral titers in the upper respiratory tract and virus-induced weight loss. This demonstration of potent anti-SARS-CoV-2 activity tolerant to emerging mutations establishes proof-of-concept for targeting the HS–Spike protein–ACE2 axis with synthetic HS mimetics and provides a strong rationale for clinical investigation of pixatimod as a potential multimodal therapeutic for COVID-19. Heparan sulfate (HS) has emerged as a SARS-CoV-2 coreceptor. Pixatimod (PG545), an HS mimetic, inhibits infectivity of multiple variants offering a novel therapeutic approach against COVID-19.

A versatile reverse genetics platform for SARS-CoV-2 and other positive-strand RNA viruses.

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that despite the large size of the viral RNA genome (~30 kb), infectious full-length cDNA is readily assembled in vitro by a circular polymerase extension reaction (CPER) methodology without the need for technically demanding intermediate steps. Overlapping cDNA fragments are generated from viral RNA and assembled together with a linker fragment containing CMV promoter into a circular full-length viral cDNA in a single reaction. Transfection of the circular cDNA into mammalian cells results in the recovery of infectious SARS-CoV-2 virus that exhibits properties comparable to the parental virus in vitro and in vivo. CPER is also used to generate insect-specific Casuarina virus with ~20 kb genome and the human pathogens Ross River virus (Alphavirus) and Norovirus (Calicivirus), with the latter from a clinical sample. Additionally, reporter and mutant viruses are generated and employed to study virus replication and virus-receptor interactions.

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.

An Optimized High-Throughput Immuno-Plaque Assay for SARS-CoV-2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 and is capable of human-to-human transmission and rapid global spread. The rapid emergence and global spread of SARS-CoV-2 has encouraged the establishment of a rapid, sensitive, and reliable viral detection and quantification methodology. Here, we present an alternative assay, termed immuno-plaque assay (iPA), which utilizes a combination of plaque assay and immunofluorescence techniques. We have extensively optimized the conditions for SARS-CoV-2 infection and demonstrated the great flexibility of iPA detection using several antibodies and dual-probing with two distinct epitope-specific antibodies. In addition, we showed that iPA could be utilized for ultra-high-throughput viral titration and neutralization assay within 24 h and is amenable to a 384-well format. These advantages will significantly accelerate SARS-CoV-2 research outcomes during this pandemic period.

Assessing the potential of unmanned aerial vehicle spraying of aqueous ozone as an outdoor disinfectant for SARS-CoV-2.

The COVID-19 pandemic has revealed gaps in our understanding of safe, effective and efficient means of disinfecting high use public spaces. Whilst this creates an opportunity for development and application of innovative approaches such as unmanned aerial vehicle (UAV) based disinfection, unregulated outdoor disinfection using chlorine has led to environmental and public health risks. This study has quantified the efficiency, safety and efficacy of UAV-based spraying of aqueous ozone. Optimised UAV flight characteristics of 4.7 km/h at 1.7 m elevation spraying 2.4 L/min were able to provide >97% and >92% coverage of a 1 m and 2 m wide swath respectively. During spraying operations using 1 mg/L aqueous ozone, atmospheric concentrations of ozone remained within background levels (<0.04 ppm). Highly efficient inactivation of two different isolates of SARS-CoV-2 virus was achieved at aqueous ozone concentrations of 0.75 mg/L after an incubation period of only 5 min, with 0.375 mg/L achieving 82-91.5% inactivation in this time. Exposure of diamondback moth larvae and parasitic wasps to 1 mg/L aqueous ozone did not significantly affect their survivorship. These results indicate for the first time that aqueous ozone may provide the required balance between human and environmental safety and viral inactivation efficacy for targeted application in high risk outdoor settings.