Impact of coagulation on SARS-CoV-2 and PMMoV viral signal in wastewater solids.

Wastewater surveillance (WWS) of SARS-CoV-2 has become a crucial tool for monitoring COVID-19 cases and outbreaks. Previous studies have indicated that SARS-CoV-2 RNA measurement from testing solid-rich primary sludge yields better sensitivity compared to testing wastewater influent. Furthermore, measurement of pepper mild mottle virus (PMMoV) signal in wastewater allows for precise normalization of SARS-CoV-2 viral signal based on solid content, enhancing disease prevalence tracking. However, despite the widespread adoption of WWS, a knowledge gap remains regarding the impact of ferric sulfate coagulation, commonly used in enhanced primary clarification, the initial stage of wastewater treatment where solids are sedimented and removed, on SARS-CoV-2 and PMMoV quantification in wastewater-based epidemiology. This study examines the effects of ferric sulfate addition, along with the associated pH reduction, on the measurement of SARS-CoV-2 and PMMoV viral measurements in wastewater primary clarified sludge through jar testing. Results show that the addition of Fe3+ concentrations in the conventional 0 to 60 mg/L range caused no effect on SARS-CoV-2 N1 and N2 gene region measurements in wastewater solids. However, elevated Fe3+ concentrations were shown to be associated with a statistically significant increase in PMMoV viral measurements in wastewater solids, which consequently resulted in the underestimation of PMMoV-normalized SARS-CoV-2 viral signal measurements (N1 and N2 copies/copies of PMMoV). The observed pH reduction from coagulant addition did not contribute to the increased PMMoV measurements, suggesting that this phenomenon arises from the partitioning of PMMoV viral particles into wastewater solids.

Rapidly developed, optimized, and applied wastewater surveillance system for real-time monitoring of low-incidence, high-impact MPOX outbreak.

Recent MPOX viral resurgences have mobilized public health agencies around the world. Recognizing the significant risk of MPOX outbreaks, large-scale human testing, and immunization campaigns have been initiated by local, national, and global public health authorities. Recently, traditional clinical surveillance campaigns for MPOX have been complemented with wastewater surveillance (WWS), building on the effectiveness of existing wastewater programs that were built to monitor SARS-CoV-2 and recently expanded to include influenza and respiratory syncytial virus surveillance in wastewaters. In the present study, we demonstrate and further support the finding that MPOX viral fragments agglomerate in the wastewater solids fraction. Furthermore, this study demonstrates that the current, most commonly used MPOX assays are equally effective at detecting low titers of MPOX viral signal in wastewaters. Finally, MPOX WWS is shown to be more effective at passively tracking outbreaks and/or resurgences of the disease than clinical testing alone in smaller communities with low human clinical case counts of MPOX.

Understanding the dynamic relation between wastewater SARS-CoV-2 signal and clinical metrics throughout the pandemic.

Wastewater surveillance (WWS) of SARS-CoV-2 was proven to be a reliable and complementary tool for population-wide monitoring of COVID-19 disease incidence but was not as rigorously explored as an indicator for disease burden throughout the pandemic. Prior to global mass immunization campaigns and during the spread of the wildtype COVID-19 and the Alpha variant of concern (VOC), viral measurement of SARS-CoV-2 in wastewater was a leading indicator for both COVID-19 incidence and disease burden in communities. As the two-dose vaccination rates escalated during the spread of the Delta VOC in Jul. 2021 through Dec. 2021, relations weakened between wastewater signal and community COVID-19 disease incidence and maintained a strong relationship with clinical metrics indicative of disease burden (new hospital admissions, ICU admissions, and deaths). Further, with the onset of the vaccine-resistant Omicron BA.1 VOC in Dec. 2021 through Mar. 2022, wastewater again became a strong indicator of both disease incidence and burden during a period of limited natural immunization (no recent infection), vaccine escape, and waned vaccine effectiveness. Lastly, with the populations regaining enhanced natural and vaccination immunization shortly prior to the onset of the Omicron BA.2 VOC in mid-Mar 2022, wastewater is shown to be a strong indicator for both disease incidence and burden. Hospitalization-to-wastewater ratio is further shown to be a good indicator of VOC virulence when widespread clinical testing is limited. In the future, WWS is expected to show moderate indication of incidence and strong indication of disease burden in the community during future potential seasonal vaccination campaigns.

Wastewater to clinical case (WC) ratio of COVID-19 identifies insufficient clinical testing, onset of new variants of concern and population immunity in urban communities.

Clinical testing has been the cornerstone of public health monitoring and infection control efforts in communities throughout the COVID-19 pandemic. With the anticipated reduction of clinical testing as the disease moves into an endemic state, SARS-CoV-2 wastewater surveillance (WWS) will have greater value as an important diagnostic tool. An in-depth analysis and understanding of the metrics derived from WWS is required to interpret and utilize WWS-acquired data effectively (McClary-Gutierrez et al., 2021; O'Keeffe, 2021). In this study, the SARS-CoV-2 wastewater signal to clinical cases (WC) ratio was investigated across seven cities in Canada over periods ranging from 8 to 21 months. This work demonstrates that significant increases in the WC ratio occurred when clinical testing eligibility was modified to appointment-only testing, identifying a period of insufficient clinical testing (resulting in a reduction to testing access and a reduction in the number of daily tests) in these communities, despite increases in the wastewater signal. Furthermore, the WC ratio decreased significantly in 6 of the 7 studied locations, serving as a potential signal of the emergence of the Alpha variant of concern (VOC) in a relatively non-immunized community (40-60 % allelic proportion), while a more muted decrease in the WC ratio signaled the emergence of the Delta VOC in a relatively well-immunized community (40-60 % allelic proportion). Finally, a significant decrease in the WC ratio signaled the emergence of the Omicron VOC, likely because of the variant's greater effectiveness at evading immunity, leading to a significant number of new reported clinical cases, even when community immunity was high. The WC ratio, used as an additional monitoring metric, could complement clinical case counts and wastewater signals as individual metrics in its potential ability to identify important epidemiological occurrences, adding value to WWS as a diagnostic technology during the COVID-19 pandemic and likely for future pandemics.

Municipal and neighbourhood level wastewater surveillance and subtyping of an influenza virus outbreak.

Recurrent influenza epidemics and pandemic potential are significant risks to global health. Public health authorities use clinical surveillance to locate and monitor influenza and influenza-like cases and outbreaks to mitigate hospitalizations and deaths. Currently, global integration of clinical surveillance is the only reliable method for reporting influenza types and subtypes to warn of emergent pandemic strains. The utility of wastewater surveillance (WWS) during the COVID-19 pandemic as a less resource intensive replacement or complement for clinical surveillance has been predicated on analyzing viral fragments in wastewater. We show here that influenza virus targets are stable in wastewater and partitions favorably to the solids fraction. By quantifying, typing, and subtyping the virus in municipal wastewater and primary sludge during a community outbreak, we forecasted a citywide flu outbreak with a 17-day lead time and provided population-level viral subtyping in near real-time to show the feasibility of influenza virus WWS at the municipal and neighbourhood levels in near real time using minimal resources and infrastructure.