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Fecal indicator bacteria currently used for water quality monitoring inadequately represent viral fate in water systems, motivating the development of viral fecal pollution indicators. Molecular viral fecal pollution indicators such as crAssphage and pepper mild mottle virus (PMMoV) have emerged as leading viral fecal pollution indicator candidates due to ease and speed of measurement and target specificity. Elucidating the fate of molecular viral fecal indicators in water systems is necessary to facilitate their development, broader adoption, and ultimately their association with infectious risk. A significant mechanism controlling the behavior of viral indicators in environmental waters is association with particles, as this would dictate removal via settling and transport characteristics. In this study, we investigated the particle associations of six molecular fecal pollution targets (crAssphage, PMMoV, adenovirus, human polyomavirus, norovirus, HF183/BacR287) in wastewater using a cascade filtration approach. Four different filters were employed representing large settleable particles (180 μm), larger (20 μm) and smaller suspended particles (0.45 μm), and non-settleable particles (0.03 μm). All molecular targets were detected on all particle size fractions; however, all targets had their highest concentrations on the 0.45 μm (percent contribution ranging from 40% to 80.5%) and 20 μm (percent contribution ranging from 3.9% to 39.4%) filters. The association of viral fecal pollution targets with suspended particles suggests that particle association will dictate transport in environmental waters and that sample concentration approaches based upon particle collection will be effective for these targets. 

ABSTRACT Wastewater-based epidemiology (WBE) expanded rapidly in response to the COVID-19 pandemic. As the public health emergency has ended, researchers and practitioners are looking to shift the focus of existing wastewater surveillance programs to other targets, including bacteria. Bacterial targets may pose some unique challenges for WBE applications. To explore the current state of the field, the National Science Foundation-funded Research Coordination Network (RCN) on Wastewater Based Epidemiology for SARS-CoV-2 and Emerging Public Health Threats held a workshop in April 2023 to discuss the challenges and needs for wastewater bacterial surveillance. The targets and methods used in existing programs were diverse, with twelve different targets and nine different methods listed. Discussions during the workshop highlighted the challenges in adapting existing programs and identified research gaps in four key areas: choosing new targets, relating bacterial wastewater data to human disease incidence and prevalence, developing methods, and normalizing results. To help with these challenges and research gaps, the authors identified steps the larger community can take to improve bacteria wastewater surveillance. This includes developing data reporting standards and method optimization and validation for bacterial programs. Additionally, more work is needed to understand shedding patterns for potential bacterial targets to better relate wastewater data to human infections. Wastewater surveillance for bacteria can help provide insight into the underlying prevalence in communities, but much work is needed to establish these methods. IMPORTANCEWastewater surveillance was a useful tool to elucidate the burden and spread of SARS-CoV-2 during the pandemic. Public health officials and researchers are interested in expanding these surveillance programs to include bacterial targets, but many questions remain. The NSF-funded Research Coordination Network for Wastewater Surveillance of SARS-CoV-2 and Emerging Public Health Threats held a workshop to identify barriers and research gaps to implementing bacterial wastewater surveillance programs. 

Abstract Since the start of the coronavirus disease-2019 (COVID-19) pandemic, there has been interest in using wastewater monitoring as an approach for disease surveillance. A significant uncertainty that would improve the interpretation of wastewater monitoring data is the intensity and timing with which individuals shed RNA from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into wastewater. By combining wastewater and case surveillance data sets from a university campus during a period of heightened surveillance, we inferred that individual shedding of RNA into wastewater peaks on average 6 days (50% uncertainty interval (UI): 6–7; 95% UI: 4–8) following infection, and that wastewater measurements are highly overdispersed [negative binomial dispersion parameter, k = 0.39 (95% credible interval: 0.32–0.48)]. This limits the utility of wastewater surveillance as a leading indicator of secular trends in SARS-CoV-2 transmission during an epidemic, and implies that it could be most useful as an early warning of rising transmission in areas where transmission is low or clinical testing is delayed or of limited capacity. 

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.