Methods Evaluation for Rapid Concentration and Quantification of SARS-CoV-2 in Raw Wastewater Using Droplet Digital and Quantitative RT-PCR.

Wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging public health tool to understand the spread of Coronavirus Disease 2019 (COVID-19) in communities. The performance of different virus concentration methods and PCR methods needs to be evaluated to ascertain their suitability for use in the detection of SARS-CoV-2 in wastewater. We evaluated ultrafiltration and polyethylene glycol (PEG) precipitation methods to concentrate SARS-CoV-2 from sewage in wastewater treatment plants and upstream in the wastewater network (e.g., manholes, lift stations). Recovery of viruses by different concentration methods was determined using Phi6 bacteriophage as a surrogate for enveloped viruses. Additionally, the presence of SARS-CoV-2 in all wastewater samples was determined using reverse transcription quantitative PCR (RT-qPCR) and reverse transcription droplet digital PCR (RT-ddPCR), targeting three genetic markers (N1, N2 and E). Using spiked samples, the Phi6 recoveries were estimated at 2.6-11.6% using ultrafiltration-based methods and 22.2-51.5% using PEG precipitation. There was no significant difference in recovery efficiencies (p < 0.05) between the PEG procedure with and without a 16 h overnight incubation, demonstrating the feasibility of obtaining same day results. The SARS-CoV-2 genetic markers were more often detected by RT-ddPCR than RT-qPCR with higher sensitivity and precision. While all three SARS-CoV-2 genetic markers were detected using RT-ddPCR, the levels of E gene were almost below the limit of detection using RT-qPCR. Collectively, our study suggested PEG precipitation is an effective low-cost procedure which allows a large number of samples to be processed simultaneously in a routine wastewater monitoring for SARS-CoV-2. RT-ddPCR can be implemented for the absolute quantification of SARS-CoV-2 genetic markers in different wastewater matrices.

Targeted wastewater surveillance of SARS-CoV-2 on a university campus for COVID-19 outbreak detection and mitigation.

Targeted wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been proposed by the United States Centers for Disease Control and Prevention's National Wastewater Surveillance System as a complementary approach to clinical surveillance to detect the presence of Coronavirus Disease 2019 (COVID-19) at high-density facilities and institutions such as university campuses, nursing homes, and correctional facilities. In this study we evaluated the efficacy of targeted wastewater surveillance of SARS-CoV-2 RNA together with individual-level testing for outbreak mitigation on a university campus during Fall 2020 semester. Wastewater samples (n = 117) were collected weekly from manholes or sewer cleanouts that receive wastewater inputs from dormitories, community-use buildings, and a COVID-19 isolation dormitory. Quantitative RT-PCR N1 and N2 assays were used to measure SARS-CoV-2 nucleocapsid genes in wastewater. Due to varying human waste input in different buildings, pepper mild mottle virus (PMMV) RNA was also measured in all samples and used to normalize SARS-CoV-2 N1 and N2 RNA wastewater concentrations. In this study, temporal trends of SARS-CoV-2 in wastewater samples mirrored trends in COVID-19 cases detected on campus. Normalizing SARS-CoV-2 RNA concentrations using human fecal indicator, PMMV enhanced the correlation between N1 and N2 gene abundances in wastewater with COVID-19 cases. N1 and N2 genes were significant predictors of COVID-19 cases in dormitories, and the N2 gene was significantly correlated with the number of detected COVID-19 cases in dormitories. By implementing several public health surveillance programs include targeted wastewater surveillance, individual-level testing, contact tracing, and quarantine/isolation facilities, university health administrators could act decisively during an outbreak on campus, resulting in rapid decline of newly detected COVID-19 cases. Wastewater surveillance of SARS-CoV-2 is a proactive outbreak monitoring tool for university campuses seeking to continue higher education practices in person during the COVID-19 pandemic.