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 ... -  《-》

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》

Biomarkers selection for population normalization in SARS-CoV-2 wastewater-based epidemiology.

Wastewater-based epidemiology (WBE) has been one of the most cost-effective approaches to track the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) levels in the communities since the coronavirus disease 2019 (COVID-19) outbreak in 2020. Normalizing SARS-CoV-2 concentrations by the population biomarkers in wastewater is critical for interpreting the viral loads, comparing the epidemiological trends among the sewersheds, and identifying the vulnerable communities. In this study, five population biomarkers, pepper mild mottle virus (PMMoV), creatinine (CRE), 5-hydroxyindoleacetic acid (5-HIAA), caffeine (CAF) and its metabolite paraxanthine (PARA) were investigated and validated for their utility in normalizing the SARS-CoV-2 loads through two normalizing approaches using the data from 64 wastewater treatment plants (WWTPs) in Missouri. Their utility in assessing the real-time population contributing to the wastewater was also evaluated. The best performing candidate was further tested for its capacity for improving correlation between normalized SARS-CoV-2 loads and the clinical cases reported in the City of Columbia, Missouri, a university town with a constantly fluctuating population. Our results showed that, except CRE, the direct and indirect normalization approaches using biomarkers allow accounting for the changes in wastewater dilution and differences in relative human waste input over time regardless flow volume and population of the given WWTP. Among selected biomarkers, PARA is the most reliable population biomarker in determining the SARS-CoV-2 load per capita due to its high accuracy, low variability, and high temporal consistency to reflect the change in population dynamics and dilution in wastewater. It also demonstrated its excellent utility for real-time assessment of the population contributing to the wastewater. In addition, the viral loads normalized by the PARA-estimated population significantly improved the correlation (rho=0.5878, p < 0.05) between SARS-CoV-2 load per capita and case numbers per capita. This chemical biomarker complements the current normalization scheme recommended by CDC and helps us understand the size, distribution, and dynamics of local populations for forecasting the prevalence of SARS-CoV2 within each sewershed.

Hsu SY ，Bayati M ，Li C ，Hsieh HY ，Belenchia A ，Klutts J ，Zemmer SA ，Reynolds M ，Semkiw E ，Johnson HY ，Foley T ，Wieberg CG ，Wenzel J ，Johnson MC ，Lin CH ... -  《-》

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

Hegazy N ，Tian X ，D'Aoust PM ，Pisharody L ，Towhid ST ，Mercier É ，Zhang Z ，Wan S ，Thakali O ，Kabir MP ，Fang W ，Nguyen TB ，Ramsay NT ，MacKenzie AE ，Graber TE ，Guilherme S ，Delatolla R ... -  《-》

Beyond COVID-19: Wastewater-based epidemiology for multipathogen surveillance and normalization strategies.

Wastewater-based epidemiology (WBE) is a critical tool for monitoring community health. Although much attention has focused on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a causative agent of coronavirus disease 2019 (COVID-19), other pathogens also pose significant health risks. This study quantified the presence of SARS-CoV-2, influenza A virus (Inf-A), and noroviruses of genogroups I (NoV-GI) and II (NoV-GII) in wastewater samples collected weekly (n = 170) from July 2023 to February 2024 from five wastewater treatment plants (WWTPs) in Yamanashi Prefecture, Japan, by quantitative PCR. Inf-A RNA exhibited localized prevalence with positive ratios of 59 %-82 % in different WWTPs, suggesting regional outbreaks within specific areas. NoV-GI (94 %, 160/170) and NoV-GII (100 %, 170/170) RNA were highly prevalent, with NoV-GII (6.1 ± 0.8 log10 copies/L) consistently exceeding NoV-GI (5.4 ± 0.7 log10 copies/L) RNA concentrations. SARS-CoV-2 RNA was detected in 100 % of the samples, with mean concentrations of 5.3 ± 0.5 log10 copies/L in WWTP E and 5.8 ± 0.4 log10 copies/L each in other WWTPs. Seasonal variability was evident, with higher concentrations of all pathogenic viruses during winter. Non-normalized and normalized virus concentrations by fecal indicator bacteria (Escherichia coli and total coliforms), an indicator virus (pepper mild mottle virus (PMMoV)), and turbidity revealed significant positive associations with the reported disease cases. Inf-A and NoV-GI + GII RNA concentrations showed strong correlations with influenza and acute gastroenteritis cases, particularly when normalized to E. coli (Spearman's ρ = 0.70-0.81) and total coliforms (ρ = 0.70-0.81), respectively. For SARS-CoV-2, non-normalized concentrations showed a correlation of 0.61, decreasing to 0.31 when normalized to PMMoV, suggesting that PMMoV is unsuitable. Turbidity normalization also yielded suboptimal results. This study underscored the importance of selecting suitable normalization parameters tailored to specific pathogens for accurate disease trend monitoring using WBE, demonstrating its utility beyond COVID-19 surveillance.

Malla B ，Shrestha S ，Sthapit N ，Hirai S ，Raya S ，Rahmani AF ，Angga MS ，Siri Y ，Ruti AA ，Haramoto E ... -  《-》