Hospital wastewater surveillance for SARS-CoV-2 identifies intra-hospital dynamics of viral transmission and evolution.

Wastewater testing has emerged as an effective, widely used tool for population-level severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surveillance. Such efforts have been implemented primarily at wastewater treatment plants, providing data for large resident populations but hindering the ability to implement targeted interventions or follow-ups. Conversely, building-level wastewater data exhibit increased variability due to rapid daily population dynamics but allow for targeted follow-up or mitigation efforts. Here, we implemented a three-site wastewater sampling strategy on our university-affiliated medical campus from May 2021 to March 2024, comprised of two distinct hospital quadrants and a building primarily consisting of research laboratories and classrooms. We first addressed several limitations in implementing hospital-level wastewater surveillance by optimizing sampling frequency and laboratory techniques. We subsequently improved our ability to model SARS-CoV-2 case counts using wastewater data by performing sensitivity analyses on viral shedding assumptions and testing the utility of internal normalization factors for population size. Our unique infrastructure allowed us to detect intra-hospital dynamics of SARS-CoV-2 prevalence and diversity and confirmed that direct sequencing of wastewater was able to capture corresponding clinical viral diversity. In contrast, research building wastewater sampling showed that for most non-residential settings, despite low overall viral loads, a threshold approach can still be used to identify peaks in cases or transmission among the general population. Our study expands on current wastewater surveillance practices by examining the utility of, and best practices for, upstream and particularly hospital settings, enabling the use of non-municipal, medium-scale wastewater testing to inform efforts for reducing the burden of coronavirus disease 2019 (COVID-19).IMPORTANCESince the onset of the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has been increasingly implemented to track the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Most wastewater testing across the United States occurs at municipal wastewater treatment plants. Yet, this testing method could also be beneficial at non-municipal and non-residential sites, including hospitals, where wastewater data for SARS-CoV-2 signals and viral diversity could directly impact hospital practices to control its spread. We analyzed both hospital and non-residential research building wastewater over a 3-year period to establish optimized methods for collecting and interpreting wastewater data at sites upstream of treatment plants. We found that even within a single hospital building, wastewater testing in different locations showed distinct signatures over time, which corresponded with data from patients hospitalized in those locations. This study provides a framework for the use of wastewater viral surveillance upstream of municipal treatment plants to enable targeted interventions to limit the spread of SARS-CoV-2.