Building-level wastewater surveillance using tampon swabs and RT-LAMP for rapid SARS-CoV-2 RNA detection

Wastewater surveillance for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA has demonstrated useful correlation with both coronavirus disease 2019 (COVID-19) cases and clinical testing positivity at the community level. Wastewater surveillance on college campuses has also demonstrated promising predictive capacity for the presence and absence of COVID-19 cases. However, to date, such monitoring has most frequently relied upon composite samplers and reverse transcription quantitative PCR (RT-qPCR) techniques, which limits the accessibility and scalability of wastewater surveillance, particularly in low-resource settings. In this study, we trialed the use of tampons as passive swabs for sample collection and reverse transcription loop-mediated isothermal amplification (RT-LAMP), which does not require sophisticated thermal cycling equipment, to detect SARS-CoV-2 RNA in wastewater. Results for the workflow were available within three hours of sample collection. The RT-LAMP assay is approximately 20 times less analytically sensitive than RT-droplet digital PCR. Nonetheless, during a building-level wastewater surveillance campaign concurrent with independent weekly clinical testing of all students, the method demonstrated a three-day positive predictive value (PPV) of 75% (excluding convalescent cases) and same-day negative predictive value (NPV) of 80% for incident COVID-19 cases. These predictive values are comparable to that reported by wastewater monitoring using RT-qPCR. These observations suggest that even with lower analytical sensitivity the tampon swab and RT-LAMP workflow offers a cost-effective and rapid approach that could be leveraged for scalable building-level wastewater surveillance for COVID-19 potentially even in low-resource settings.

Within- and between-Day Variability of SARS-CoV-2 RNA in Municipal Wastewater during Periods of Varying COVID-19 Prevalence and Positivity

Wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is being used to monitor Coronavirus Disease 2019 (COVID-19) trends in communities;however, within- and between-day variation of SARS-CoV-2 RNA concentration in primary influent remains largely uncharacterized. In the current study, grab sampling of primary influent was performed every 2 h over two 24-h periods at two wastewater treatment plants (WWTPs) in northern Indiana, USA. The recovery efficiency of endogenous SARS-CoV-2 RNA in wastewater was confirmed to be similar to the recovery efficiency of the process control, bovine respiratory syncytial virus (BRSV). Recovery-corrected SARS-CoV-2 RNA concentrations in primary influent indicate diurnal loading patterns and confirm monitoring dependent on grab samples should target daytime periods with high fecal loading. Importantly, manual compositing performed at the WWTP resulted in concentrations that were consistently lower than grab sample averages indicating potential bias. Uncorrected, recovery-corrected, and pepper mild mottle virus (PMMoV)-normalized SARS-CoV-2 RNA concentrations demonstrated an ordinal agreement with increasing clinical COVID-19 positivity but not COVID-19 cases. In areas where geolocated COVID-19 case data are not available, the COVID-19 positivity rate could provide a useful county-level metric for comparison with wastewater. Nonetheless, large variation both within- and between-days may preclude robust quantitative analyses beyond correlation.

Contribution of SARS-CoV-2 RNA shedding routes to RNA loads in wastewater.

A portion of those infected with SARS-CoV-2 shed the virus and its genetic material in respiratory fluids, saliva, urine, and stool, thus giving the potential to monitor for infections via wastewater. Wastewater surveillance efforts to date have largely assumed that stool shedding has been the primary source of SARS-CoV-2 RNA signal; however, there are increasing questions about the possible contribution of other shedding routes, with implications for wastewater surveillance design and feasibility. In this study we used clinical SARS-CoV-2 RNA shedding data and a Monte Carlo framework to assess the relative contribution of various shedding routes on SARS-CoV-2 RNA loads in wastewater. Stool shedding dominated total SARS-CoV-2 RNA load for community-level surveillance, with mean contributions more than two orders of magnitude greater than other shedding routes. However, RNA loads were more nuanced when considering building-level monitoring efforts designed to identify a single infected individual, where any shedding route could plausibly contribute a detectable signal. The greatest source of model variability was viral load in excreta, suggesting that future modeling efforts may be improved by incorporating specific modeling scenarios with precise SARS-CoV-2 shedding data, and beyond that wastewater surveillance must continue to account for large variability during data analysis and reporting. Importantly, the findings imply that wastewater surveillance at finer spatial scales is not entirely dependent on shedding via feces for sensitive detection of infections thus enlarging the potential use cases of wastewater as a non-intrusive surveillance methodology.

Wastewater Surveillance during Mass COVID-19 Vaccination on a College Campus

The suitability of wastewater monitoring following widespread vaccination against COVID-19 remains uncertain. SARS-CoV-2 RNA levels were monitored in wastewater solids during a university mass vaccination campaign in which >90% of the 12280 students were fully vaccinated (Pfizer-BioNTech, BNT162b2). SARS-CoV-2 RNA levels in wastewater solids correlated with the 7-day average of COVID-19 cases when lagged by 1-3 days (ρ = 0.51-0.55;p = 0.023-0.039). During and after the second vaccine dose, SARS-CoV-2 RNA was not detected in wastewater solids on 19 of 21 days (12 consecutive days of nondetection at the end of the semester), a significant decrease (p = 0.027) in positivity rate. A large influx of outside visitors (move out and commencement) led to an immediate increase in wastewater SARS-CoV-2 RNA positivity (seven detections over seven days). Wastewater solids offer a sensitive matrix for environmental surveillance of COVID-19 at the subsewershed level (50% probability of RNA detection with two cases) both during and after mass vaccination. Mass vaccination was coincident with decreased shedding of SARS-CoV-2 RNA into wastewater. This suggests the absence of a large population of shedding infections, symptomatic or not, following mass vaccination among a university campus population. © 2021 American Chemical Society.