Assessment of seasonality and normalization techniques for wastewater-based surveillance in Ontario, Canada.

Wastewater-based surveillance is at the forefront of monitoring for community prevalence of COVID-19, however, continued uncertainty exists regarding the use of fecal indicators for normalization of the SARS-CoV-2 virus in wastewater. Using three communities in Ontario, sampled from 2021-2023, the seasonality of a viral fecal indicator (pepper mild mottle virus, PMMoV) and the utility of normalization of data to improve correlations with clinical cases was examined. Wastewater samples from Warden, the Humber Air Management Facility (AMF), and Kitchener were analyzed for SARS-CoV-2, PMMoV, and crAssphage. The seasonality of PMMoV and flow rates were examined and compared by Season-Trend-Loess decomposition analysis. The effects of normalization using PMMoV, crAssphage, and flow rates were analyzed by comparing the correlations to clinical cases by episode date (CBED) during 2021. Seasonal analysis demonstrated that PMMoV had similar trends at Humber AMF and Kitchener with peaks in January and April 2022 and low concentrations (troughs) in the summer months. Warden had similar trends but was more sporadic between the peaks and troughs for PMMoV concentrations. Flow demonstrated similar trends but was not correlated to PMMoV concentrations at Humber AMF and was very weak at Kitchener (r = 0.12). Despite the differences among the sewersheds, unnormalized SARS-CoV-2 (raw N1-N2) concentration in wastewater (n = 99-191) was strongly correlated to the CBED in the communities (r = 0.620-0.854) during 2021. Additionally, normalization with PMMoV did not improve the correlations at Warden and significantly reduced the correlations at Humber AMF and Kitchener. Flow normalization (n = 99-191) at Humber AMF and Kitchener and crAssphage normalization (n = 29-57) correlations at all three sites were not significantly different from raw N1-N2 correlations with CBED. Differences in seasonal trends in viral biomarkers caused by differences in sewershed characteristics (flow, input, etc.) may play a role in determining how effective normalization may be for improving correlations (or not). This study highlights the importance of assessing the influence of viral fecal indicators on normalized SARS-CoV-2 or other viruses of concern. Fecal indicators used to normalize the target of interest may help or hinder establishing trends with clinical outcomes of interest in wastewater-based surveillance and needs to be considered carefully across seasons and sites.

Dhiyebi HA ，Abu Farah J ，Ikert H ，Srikanthan N ，Hayat S ，Bragg LM ，Qasim A ，Payne M ，Kaleis L ，Paget C ，Celmer-Repin D ，Folkema A ，Drew S ，Delatolla R ，Giesy JP ，Servos MR ... -  《Frontiers in Public Health》

Does normalization of SARS-CoV-2 concentrations by Pepper Mild Mottle Virus improve correlations and lead time between wastewater surveillance and clinical data in Alberta (Canada): comparing twelve SARS-CoV-2 normalization approaches.

Wastewater-based surveillance (WBS) data normalization is an analyte measurement correction that addresses variations resulting from dilution of fecal discharge by non-sanitary sewage, stormwater or groundwater infiltration. No consensus exists on what WBS normalization parameters result in the strongest correlations and lead time between SARS-CoV-2 WBS data and COVID-19 cases. This study compared flow, population size and biomarker normalization impacts on the correlations and lead times for ten communities in twelve sewersheds in Alberta (Canada) between September 2020 and October 2021 (n = 1024) to determine if normalization by Pepper Mild Mottle Virus (PMMoV) provides any advantages compared to other normalization parameters (e.g., flow, reported and dynamic population sizes, BOD, TSS, NH3, TP). PMMoV concentrations (GC/mL) corresponded with plant influent flows and were highest in the urban centres. SARS-CoV-2 target genes E, N1 and N2 were all negatively associated with wastewater influent pH, while PMMoV was positively associated with temperature. Pooled data analysis showed that normalization increased ρ-values by almost 0.1 and was highest for ammonia, TKN and TP followed by PMMoV. Normalization by other parameters weakened associations. None of the differences were statistically significant. Site-specific correlations showed that normalization of SARS-CoV-2 data by PMMoV only improved correlations significantly in two of the twelve systems; neither were large sewersheds or combined sewer systems. In five systems, normalization by traditional wastewater strength parameters and dynamic population estimates improved correlations. Lead time ranged between 1 and 4 days in both pooled and site-specific comparisons. We recommend that WBS researchers and health departments: a) Investigate WWTP influent properties (e.g., pH) in the WBS planning phase and use at least two parallel approaches for normalization only if shown to provide value; b) Explore normalization by wastewater strength parameters and dynamic population size estimates further; and c) Evaluate purchasing an influent flow meter in small communities to support long-term WBS efforts and WWTP management.

Maal-Bared R ，Qiu Y ，Li Q ，Gao T ，Hrudey SE ，Bhavanam S ，Ruecker NJ ，Ellehoj E ，Lee BE ，Pang X ... -  《-》

The role of catchment population size, data normalization, and chronology of public health interventions on wastewater-based COVID-19 viral trends.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic presented the most challenging global crisis in recent times. A pandemic caused by a novel pathogen such as SARS-CoV-2 necessitated the development of innovative techniques for the monitoring and surveillance of COVID-19 infections within communities. Wastewater surveillance (WWS) is recognized as a non-invasive, cost-effective, and valuable epidemiological tool to monitor the prevalence of COVID-19 infections in communities. Seven municipal wastewater sampling sites representing distinct sewershed communities were selected for the surveillance of the SARS-CoV-2 virus in Durham Region, Ontario, Canada over 8 months from March 2021 to October 2021. Viral RNA fragments of SARS-CoV-2 and the normalization target pepper mild mottle virus (PMMoV) were concentrated from wastewater influent using the PEG/NaCl superspeed centrifugation method and quantified using RT-qPCR. Strong significant correlations (Spearman's rs = 0.749 to 0.862, P < 0.001) were observed between SARS-CoV-2 gene copies/mL of wastewater and clinical cases reported in each delineated sewershed by onset date. Although raw wastewater offered higher correlation coefficients with clinical cases by onset date compared to PMMoV normalized data, only one site had a statistically significantly higher Spearman's correlation coefficient value for raw data than normalized data. Implementation of community stay-at-home orders and vaccinations over the course of the study period in 2021 were found to strongly correspond to decreasing SARS-CoV-2 wastewater trends in the wastewater treatment plants and upstream pumping stations.

Islam G ，Gedge A ，Ibrahim R ，de Melo T ，Lara-Jacobo L ，Dlugosz T ，Kirkwood AE ，Simmons D ，Desaulniers JP ... -  《-》

Longitudinal and quantitative fecal shedding dynamics of SARS-CoV-2, pepper mild mottle virus, and crAssphage.

Arts PJ ，Kelly JD ，Midgley CM ，Anglin K ，Lu S ，Abedi GR ，Andino R ，Bakker KM ，Banman B ，Boehm AB ，Briggs-Hagen M ，Brouwer AF ，Davidson MC ，Eisenberg MC ，Garcia-Knight M ，Knight S ，Peluso MJ ，Pineda-Ramirez J ，Diaz Sanchez R ，Saydah S ，Tassetto M ，Martin JN ，Wigginton KR ... -  《-》

Wastewater SARS-CoV-2 monitoring as a community-level COVID-19 trend tracker and variants in Ohio, United States.

The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 129 million confirm cases. Many health authorities around the world have implemented wastewater-based epidemiology as a rapid and complementary tool for the COVID-19 surveillance system and more recently for variants of concern emergence tracking. In this study, three SARS-CoV-2 target genes (N1 and N2 gene regions, and E gene) were quantified from wastewater influent samples (n = 250) obtained from the capital city and 7 other cities in various size in central Ohio from July 2020 to January 2021. To determine human-specific fecal strength in wastewater samples more accurately, two human fecal viruses (PMMoV and crAssphage) were quantified to normalize the SARS-CoV-2 gene concentrations in wastewater. To estimate the trend of new case numbers from SARS-CoV-2 gene levels, different statistical models were built and evaluated. From the longitudinal data, SARS-CoV-2 gene concentrations in wastewater strongly correlated with daily new confirmed COVID-19 cases (average Spearman's r = 0.70, p < 0.05), with the N2 gene region being the best predictor of the trend of confirmed cases. Moreover, average daily case numbers can help reduce the noise and variation from the clinical data. Among the models tested, the quadratic polynomial model performed best in correlating and predicting COVID-19 cases from the wastewater surveillance data, which can be used to track the effectiveness of vaccination in the later stage of the pandemic. Interestingly, neither of the normalization methods using PMMoV or crAssphage significantly enhanced the correlation with new case numbers, nor improved the estimation models. Viral sequencing showed that shifts in strain-defining variants of SARS-CoV-2 in wastewater samples matched those in clinical isolates from the same time periods. The findings from this study support that wastewater surveillance is effective in COVID-19 trend tracking and provide sentinel warning of variant emergence and transmission within various types of communities.

Ai Y ，Davis A ，Jones D ，Lemeshow S ，Tu H ，He F ，Ru P ，Pan X ，Bohrerova Z ，Lee J ... -  《-》