Wastewater Surveillance Can Function as an Early Warning System for COVID-19 in Low-Incidence Settings.

INTRODUCTION: During the first two years of the COVID-19 pandemic, Australia implemented a series of international and interstate border restrictions. The state of Queensland experienced limited COVID-19 transmission and relied on lockdowns to stem any emerging COVID-19 outbreaks. However, early detection of new outbreaks was difficult. In this paper, we describe the wastewater surveillance program for SARS-CoV-2 in Queensland, Australia, and report two case studies in which we aimed to assess the potential for this program to provide early warning of new community transmission of COVID-19. Both case studies involved clusters of localised transmission, one originating in a Brisbane suburb (Brisbane Inner West) in July-August 2021, and the other originating in Cairns, North Queensland in February-March 2021. MATERIALS AND METHODS: Publicly available COVID-19 case data derived from the notifiable conditions (NoCs) registry from the Queensland Health data portal were cleaned and merged spatially with the wastewater surveillance data using statistical area 2 (SA2) codes. The positive predictive value and negative predictive value of wastewater detection for predicting the presence of COVID-19 reported cases were calculated for the two case study sites. RESULTS: Early warnings for local transmission of SARS-CoV-2 through wastewater surveillance were noted in both the Brisbane Inner West cluster and the Cairns cluster. The positive predictive value of wastewater detection for the presence of notified cases of COVID-19 in Brisbane Inner West and Cairns were 71.4% and 50%, respectively. The negative predictive value for Brisbane Inner West and Cairns were 94.7% and 100%, respectively. CONCLUSIONS: Our findings highlight the utility of wastewater surveillance as an early warning tool in low COVID-19 transmission settings.

Moving forward with COVID-19: Future research prospects of wastewater-based epidemiology methodologies and applications.

Wastewater-based epidemiology (WBE) has been demonstrated for its great potential in tracking of coronavirus disease 2019 (COVID-19) transmission among populations despite some inherent methodological limitations. These include non-optimized sampling approaches and analytical methods; stability of viruses in sewer systems; partitioning/retention in biofilms; and the singular and inaccurate back-calculation step to predict the number of infected individuals in the community. Future research is expected to (1) standardize best practices in wastewater sampling, analysis and data reporting protocols for the sensitive and reproducible detection of viruses in wastewater; (2) understand the in-sewer viral stability and partitioning under the impacts of dynamic wastewater flow, properties, chemicals, biofilms and sediments; and (3) achieve smart wastewater surveillance with artificial intelligence and big data models. Further specific research is essential in the monitoring of other viral pathogens with pandemic potential and subcatchment applications to maximize the benefits of WBE beyond COVID-19.

Comparison of RT-qPCR and RT-dPCR Platforms for the Trace Detection of SARS-CoV-2 RNA in Wastewater.

We compared reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RT digital PCR (RT-dPCR) platforms for the trace detection of SARS-CoV-2 RNA in low-prevalence COVID-19 locations in Queensland, Australia, using CDC N1 and CDC N2 assays. The assay limit of detection (ALOD), PCR inhibition rates, and performance characteristics of each assay, along with the positivity rates with the RT-qPCR and RT-dPCR platforms, were evaluated by seeding known concentrations of exogenous SARS-CoV-2 in wastewater. The ALODs using RT-dPCR were approximately 2-5 times lower than those using RT-qPCR. During sample processing, the endogenous (n = 96) and exogenous (n = 24) SARS-CoV-2 wastewater samples were separated, and RNA was extracted from both wastewater eluates and pellets (solids). The RT-dPCR platform demonstrated a detection rate significantly greater than that of RT-qPCR for the CDC N1 and CDC N2 assays in the eluate (N1, p = 0.0029; N2, p = 0.0003) and pellet (N1, p = 0.0015; N2, p = 0.0067) samples. The positivity results also indicated that for the analysis of SARS-CoV-2 RNA in wastewater, including the eluate and pellet samples may further increase the detection sensitivity using RT-dPCR.

Surveillance of SARS-CoV-2 RNA in wastewater: Methods optimisation and quality control are crucial for generating reliable public health information.

Monitoring for SARS-CoV-2 RNA in wastewater through the process of wastewater-based epidemiology (WBE) provides an additional surveillance tool, contributing to community-based screening and prevention efforts as these measurements have preceded disease cases in some instances. Numerous detections of SARS-CoV-2 RNA have been reported globally using various methods, demonstrating the technical feasibility of routine monitoring. However, in order to reliably interpret data produced from these efforts for informing public health interventions, additional quality control information and standardization in sampling design, sample processing, and data interpretation and reporting is needed. This review summarizes published studies of SARS-CoV-2 RNA detection in wastewater as well as available information regarding concentration, extraction, and detection methods. The review highlights areas for potential standardization including considerations related to sampling timing and frequency relative to peak fecal loading times; inclusion of appropriate information on sample volume collected; sample collection points; transport and storage conditions; sample concentration and processing; RNA extraction process and performance; effective volumes; PCR inhibition; process controls throughout sample collection and processing; PCR standard curve performance; and recovery efficiency testing. Researchers are recommended to follow the Minimum Information for Publication of Quantitative Real-Time PCR (MIQE) guidelines. Adhering to these recommendations will enable robust interpretation of wastewater monitoring results and improved inferences regarding the relationship between monitoring results and disease cases.

Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance.

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases.

Recommendations for the clinical and laboratory diagnosis of VITT against COVID-19: Communication from the ISTH SSC Subcommittee on Platelet Immunology.

Vaccine administration is under way worldwide to combat the current COVID-19 pandemic. The newly developed vaccines are highly effective with minimal adverse effects. Recently, the AstraZeneca ChadOx1 nCov-19 vaccine has raised public alarm with concerns regarding the rare, but serious, development of thrombotic events, now known as vaccine-induced immune thrombotic thrombocytopenia (VITT). These thrombotic events appear similar to heparin-induced thrombocytopenia, both clinically and pathologically. In this manuscript, the ISTH SSC Subcommittee on Platelet Immunology outlines guidelines on how to recognize, diagnose and manage patients with VITT.

SARS-CoV-2 RNA monitoring in wastewater as a potential early warning system for COVID-19 transmission in the community: A temporal case study.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus which causes coronavirus disease (COVID-19), has spread rapidly across the globe infecting millions of people and causing significant health and economic impacts. Authorities are exploring complimentary approaches to monitor this infectious disease at the community level. Wastewater-based epidemiology (WBE) approaches to detect SARS-CoV-2 RNA in municipal wastewater are being implemented worldwide as an environmental surveillance approach to inform health authority decision-making. Owing to the extended excretion of SARS-CoV-2 RNA in stool, WBE can surveil large populated areas with a longer detection window providing unique information on the presence of pre-symptomatic and asymptomatic cases that are unlikely to be screened by clinical testing. Herein, we analysed SARS-CoV-2 RNA in 24-h composite wastewater samples (n = 63) from three wastewater treatment plants (WWTPs) in Brisbane, Queensland, Australia from 24th of February to 1st of May 2020. A total of 21 samples were positive for SARS-CoV-2, ranging from 135 to 11,992 gene copies (GC)/100 mL of wastewater. Detections were made in a Southern Brisbane WWTP in late February 2020, up to three weeks before the first clininal case was reported there. Wastewater samples were generally positive during the period with highest caseload data. The positive SARS-CoV-2 RNA detection in wastewater while there were limited clinical reported cases demonstrates the potential of WBE as an early warning system to identify hotspots and target localised public health responses, such as increased individual testing and the provision of health warnings.

First confirmed detection of SARS-CoV-2 in untreated wastewater in Australia: A proof of concept for the wastewater surveillance of COVID-19 in the community.

Infection with SARS-CoV-2, the etiologic agent of the ongoing COVID-19 pandemic, is accompanied by the shedding of the virus in stool. Therefore, the quantification of SARS-CoV-2 in wastewater affords the ability to monitor the prevalence of infections among the population via wastewater-based epidemiology (WBE). In the current work, SARS-CoV-2 RNA was concentrated from wastewater in a catchment in Australia and viral RNA copies were enumerated using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) resulting in two positive detections within a six day period from the same wastewater treatment plant (WWTP). The estimated viral RNA copy numbers observed in the wastewater were then used to estimate the number of infected individuals in the catchment via Monte Carlo simulation. Given the uncertainty and variation in the input parameters, the model estimated a median range of 171 to 1,090 infected persons in the catchment, which is in reasonable agreement with clinical observations. This work highlights the viability of WBE for monitoring infectious diseases, such as COVID-19, in communities. The work also draws attention to the need for further methodological and molecular assay validation for enveloped viruses in wastewater.