We investigated the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater samples in southern Louisiana, USA. Untreated and treated wastewater samples were collected on five occasions over a four-month period from January to April 2020. The wastewater samples were concentrated via ultrafiltration (Method A), and an adsorption–elution method using electronegative membranes (Method B). SARS-CoV-2 RNA was detected in 2 out of 15 wastewater samples using two reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays (CDC N1 and N2). None of the secondary treated and final effluent samples tested positive for SARS-CoV-2 RNA. To our knowledge, this is the first study reporting the detection of SARS-CoV-2 RNA in wastewater in North America, including the USA. However, concentration methods and RT-qPCR assays need to be refined and validated to increase the sensitivity of SARS-CoV-2 RNA detection in wastewater.

Wastewater-based epidemiology (WBE) demonstrates potential for COVID-19 community transmission monitoring; however, data on the stability of SARS-CoV-2 RNA in wastewater are needed to interpret WBE results. The decay rates of RNA from SARS-CoV-2 and a potential surrogate, murine hepatitis virus (MHV), were investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in untreated wastewater, autoclaved wastewater, and dechlorinated tap water stored at 4, 15, 25, and 37 °C. Temperature, followed by matrix type, most greatly influenced SARS-CoV-2 RNA first-order decay rates ( k ). The average T 90 (time required for 1-log 10 reduction) of SARS-CoV-2 RNA ranged from 8.04 to 27.8 days in untreated wastewater, 5.71 to 43.2 days in autoclaved wastewater, and 9.40 to 58.6 days in tap water. The average T 90 for RNA of MHV at 4 to 37 °C ranged from 7.44 to 56.6 days in untreated wastewater, 5.58–43.1 days in autoclaved wastewater, and 10.9 to 43.9 days in tap water. There was no statistically significant difference between RNA decay of SARS-CoV-2 and MHV; thus, MHV is suggested as a suitable persistence surrogate. Decay rate constants for all temperatures were comparable across all matrices for both viral RNAs, except in untreated wastewater for SARS-CoV-2, which showed less sensitivity to elevated temperatures. Therefore, SARS-CoV-2 RNA is likely to persist long enough in untreated wastewater to permit reliable detection for WBE application.

Wastewater-based epidemiology has potential as an early-warning tool for determining the presence of COVID-19 in a community. The University of Arizona (UArizona) utilized WBE paired with clinical testing as a surveillance tool to monitor the UArizona community for SARS-CoV-2 in near real-time, as students re-entered campus in the fall. Positive detection of virus RNA in wastewater lead to selected clinical testing, identification, and isolation of three infected individuals (one symptomatic and two asymptomatic) that averted potential disease transmission. This case study demonstrated the value of WBE as a tool to efficiently utilize resources for COVID-19 prevention and response. Thus, WBE coupled with targeted clinical testing was further conducted on 13 dorms during the course of the Fall semester (Table 3). In total, 91 wastewater samples resulted in positive detection of SARS-CoV-2 RNA that successfully provided an early-warning for at least a single new reported case of infection (positive clinical test) amongst the residents living in the dorm. Overall, WBE proved to be an accurate diagnostic for new cases of COVID-19 with an 82.0% positive predictive value and an 88.9% negative predictive value. Increases in positive wastewater samples and clinical tests were noted following holiday-related activities. However, shelter-in-place policies proved to be effective in reducing the number of daily reported positive wastewater and clinical tests. This case study provides evidence for WBE paired with clinical testing and public health interventions to effectively contain potential outbreaks of COVID-19 in defined communities.

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering additional community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of SARS-CoV-2 in wastewater can 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, culminating in recommended strategies that can be implemented to identify and mitigate these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, amplification 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.

Severe acute respiratory syndrome coronavirus 2 pandemic capacity is derived from the unique structural features on its spike protein: fast viral surfing over the epithelium with flat N‐terminal domain, tight binding to ACE2 entry receptor, and furin protease utilization. In addition, the possible involvement of other components such as lipid rafts, CLRs, and neuropilin is, in combination, mediating the accelerated cell entry and other critical steps in its overwhelming contagious capacity and pandemy.