Understanding the efficacy of wastewater surveillance for SARS-CoV-2 in two diverse communities.

During the COVID-19 pandemic, wastewater-based surveillance has been shown to be a useful tool for monitoring the spread of disease in communities and the emergence of new viral variants of concern. As the pandemic enters its fourth year and clinical testing has declined, wastewater offers a consistent non-intrusive way to monitor community health in the long term. This study sought to understand how accurately wastewater monitoring represented the actual burden of disease between communities. Two communities varying in size and demographics in Michigan were monitored for SARS-CoV-2 in wastewater between March of 2020 and February of 2022. Additionally, each community was monitored for SARS-CoV-2 variants of concern from December 2020 to February 2022. Wastewater results were compared with zipcode and county level COVID-19 case data to determine which scope of clinical surveillance was most correlated with wastewater loading. Pearson r correlations were highest in the smaller of the two communities (population of 25,000) for N1 GC/person/day with zipcode level case data, and date of the onset of symptoms (r = 0.81). A clear difference was seen with more cases and virus signals in the wastewater of the larger community (population 110,000) when examined based on vaccine status, which reached only 50%. While wastewater levels of SARS-CoV-2 had a lower correlation to cases in the larger community, the information was still seen as valuable in supporting public health actions and further data including vaccination status should be examined in the future.

Connecting microbial, nutrient, physiochemical, and land use variables for the evaluation of water quality within mixed use watersheds.

As non-point sources of pollution begin to overtake point sources in watersheds, source identification and complicating variables such as rainfall are growing in importance. Microbial source tracking (MST) allows for identification of fecal contamination sources in watersheds; when combined with data on land use and co-occuring variables (e.g., nutrients, sediment runoff) MST can provide a basis for understanding how to effectively remediate water quality. To determine spatial and temporal trends in microbial contamination and correlations between MST and nutrients, water samples (n = 136) were collected between April 2017 and May of 2018 during eight sampling events from 17 sites in 5 mixed-use watersheds. These samples were analyzed for three MST markers (human - B. theta; bovine - CowM2; porcine - Pig2Bac) along with E. coli, nutrients (nitrogen and phosphorus species), and physiochemical paramaters. These water quality variables were then paired with data on land use, streamflow, precipitation and management practices (e.g., tile drainage, septic tank density, tillage practices) to determine if any significant relationships existed between the observed microbial contamination and these variables. The porcine marker was the only marker that was highly correlated (p value <0.05) with nitrogen and phosphorus species in multiple clustering schemes. Significant relationships were also identified between MST markers and variables that demonstrated temporal trends driven by precipitation and spatial trends driven by septic tanks and management practices (tillage and drainage) when spatial clustering was employed.

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.