The wastewater viral activity level (WVAL) was developed by the United States Centers for Disease Control and Prevention (US CDC) as a standardized metric to aggregate SARS-CoV-2 wastewater data, enabling the assessment of infection levels and trends at state/territorial, regional and national scales. This approach also facilitates comparative analysis of SARS-CoV-2 prevalence across regions. In this study, we developed and evaluated graphical methods to integrate the WVAL metric into interpretable visualizations for public health decision-making. Preliminary analysis demonstrated that WVAL values correlated strongly with clinical case counts, supporting its role as a confirmatory epidemiological measure. The WVAL framework provided a linear quantification method, allowing for the comparison of regional variations in infection patterns. This study leveraged data from the Ontario Wastewater Surveillance Initiative (Ontario WSI), which included over 100 sampling sites across seven geographical regions. Weekly mean WVAL values were computed for each site and aggregated at regional and provincial levels. In total, 59 sites contributed to the provincial WVAL calculation. The computational aggregation method followed the US CDC WVAL approach and was generally comparable to the Public Health Ontario (PHO) aggregation method, with the notable improvement of incorporating a linear level scale. Overall, this study demonstrated that WVAL effectively quantified SARS-CoV-2 differences at a public health regional scale. The WVAL metric proved to be a robust epidemiological tool, complementing other surveillance measures to support public health decision-making. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by the Ontario Wastewater Surveillance Initiative of the Ontario Ministry of the Environment, Conservation and Parks (MECP). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Objectives We evaluated the potential impacts from using a rapid same-day quantitative polymerase chain reaction (qPCR) monitoring method for beach posting outcomes at two Toronto beaches. Methods In total, 228 water samples were collected at Marie Curtis Park East and Sunnyside Beaches over the 2021 summer season. Water samples were processed using the USEPA 1609.1 Enterococcus qPCR-based method. Escherichia coli ( E. coli ) culture data and daily beach posting decisions were obtained from Toronto Public Health. Results No significant correlation was observed between previous-day and same-day (retrospective) E. coli enumeration results at any Sunnyside Beach transect, and only relatively low ( R  = 0.41–0.56) or no significant correlation was observed at sampling transects for Marie Curtis Park East Beach. Comparing our same-day Enterococcus qPCR data to Toronto’s 2-day E. coli geometric mean beach posting decisions, we noted the need for additional postings for 1 (2%) and 3 (8%) missed health-risk days at Sunnyside and Marie Curtis Park East Beaches, respectively. The qPCR data also pointed to incorrect postings for 12 (31%) and 6 (16%) lost beach days at Sunnyside and Marie Curtis Park East Beaches, respectively. Conclusion Application of a rapid Enterococcus qPCR method at two Toronto beaches revealed 5% of beach posting decisions were false negatives that missed health-risk days, while 23% of decisions were false positives resulting in lost beach days. Deployment of the rapid same-day qPCR method offers the potential to reduce both health risks and unnecessary beach postings. Objectifs Nous avons évalué, à deux plages de Toronto, l’effet possible de l’utilisation d’une méthode de surveillance rapide par PCR quantitative (qPCR) le même jour sur les avis de fermeture ou d’ouverture des plages. Méthode En tout, 228 échantillons d’eau ont été prélevés aux plages Marie Curtis Park East et Sunnyside au cours de la saison estivale 2021. La présence d’ Enterococcus dans les échantillons a été détectée par la méthode USEPA 1609.1, utilisant la qPCR. Les données sur les cultures d’ Escherichia coli (E. coli) et les avis quotidiens de fermeture ou d’ouverture des plages ont été obtenus auprès du Bureau de santé de Toronto. Résultats Aucune corrélation significative n’a été observée entre les résultats (rétrospectifs) du dénombrement de E. coli obtenus la veille et le même jour dans les transects de la plage Sunnyside, et une corrélation significative faible (R = 0,41–0,56) ou nulle a été observée dans les transects d’échantillonnage de la plage Marie Curtis Park East. En comparant nos données sur Enterococcus obtenues le même jour par qPCR à la moyenne géométrique des avis de fermeture ou d’ouverture des plages sur deux jours liés à E. coli émis par le Bureau de santé de Toronto, nous avons remarqué qu’il aurait fallu émettre des avis de fermeture pour 1 jour de risques pour la santé manqué (2 %) à la plage Sunnyside et pour 3 jours de risques pour la santé manqués (8 %) à la plage Marie Curtis Park East. Les données de la qPCR ont aussi fait état d’avis de fermeture incorrects ayant entraîné la perte de 12 jours de plage (31 %) à Sunnyside et de 6 jours de plage (16 %) à Marie Curtis Park East. Conclusion L’application d’une méthode de surveillance rapide d’ Enterococcus par qPCR à deux plages de Toronto a montré que 5 % des avis étaient des faux négatifs qui n’ont pas détecté des jours de risques pour la santé, et que 23 % étaient des faux positifs qui ont entraîné des jours de plage perdus. Le déploiement de la méthode rapide par qPCR le même jour offre la possibilité de réduire à la fois les risques pour la santé et les avis de fermeture de plages inutiles.

AbstractWe present and demonstrate a quantitative statistical linear trend analysis (QTA) approach to analyze and interpret SARS-CoV-2 RNA wastewater surveillance results concurrently with clinical case data. This demonstration is based on the work completed under the Ontario (Canada) Wastewater Surveillance Initiative (WSI) by two laboratories in four large sewersheds within the Toronto Public Health (TPH) jurisdiction. The sewersheds were sampled over a 9-month period and data were uploaded to the Ontario Wastewater Surveillance Data and Visualization Hub (Ontario Dashboard) along with clinical case counts, both on a sewershed-specific basis. The data from the last 5-months, representing a range of high and low cases, was used for this demonstration. The QTA was conducted on a sewershed specific approach using the recommendations for public health interpretation and use of wastewater surveillance data by the United States Centers for Disease Control and Prevention (US CDC). The interpretation of the QTA results was based on the integration of both clinical and wastewater virus signals using an integration matrix in an interim draft guide by the Public Health Agency of Canada (PHAC). The key steps in the QTA consisted of (i) the calculation of Pepper Mild Mottle Virus (PMMoV), flow and flow-PMMoV-normalized virus loads; (ii) computation of the linear trends including interval estimation to identify the key inflection points using a segmented linear regression method and (iii) integrated interpretations based on consideration of both the cases and wastewater signals, as well as end user actionability. This approach is considered a complementary tool to commonly used qualitative analyses of SARS-CoV-2 RNA in wastewater and is intended to directly support public health decisions using a systematic quantitative approach.

We demonstrate a new methodology for quantitative trend analysis (QTA) to analyze and interpret SARS-CoV-2 RNA wastewater surveillance results concurrently with clinical case data. This demonstration is based on the work completed under the Ontario (Canada) Wastewater Surveillance Initiative (WSI) by two laboratories in four wastewater treatment plants (WWTPs) at each of four large sewersheds, which were sampled over a 9-month period, along with sewershed-specific clinical case counts. The data from the last 5-months, representing a range of high and low case counts, was used for this demonstration. The QTA integrated clinical and wastewater virus signals, while combining recommendations from the United States Centers for Disease Control and Prevention (US CDC) and the Public Health Agency of Canada (PHAC). The key steps in the QTA consisted of signal normalization with pepper mild mottle virus (PMMoV), as a fecal biomarker, statistical linear break-point trend analysis and integration of both wastewater virus signal and clinical cases trend results. Using this approach, the wastewater virus and clinical cases trends, direction, and magnitude were clearly identified and provided a unified complementary tool to support public health decisions on a targeted, sewershed-specific basis.

Peracetic acid (PAA) has been used as a municipal wastewater disinfectant for over 2 decades, but information about its virucidal performance is limited. Here, we report on a 2 year study of virus disinfection with PAA and chlorine as hypochlorite (NaClO) in a secondary wastewater treatment plant. During year 1, we conducted a side-stream comparison of PAA dosed at 4 mg/L (minus a demand of about 10%) and NaClO at 7.3 mg/L against indigenous enteroviruses and noroviruses, as well as coliphages and Escherichia coli, while assessing PAA fish toxicity under flow-through conditions. During year 1, PAA and NaClO produced poor median log10 reductions (LRs) against enteroviruses and noroviruses, although NaClO LRs were significantly higher. PAA and NaClO performed better against coliphages, but differences were not significant. Against E. coli, PAA and NaClO performed well. 96 hour toxicity testing was done only during year 1, revealing that PAA residual was lethal to rainbow trout only prior to quenching. The PAA dose was reduced to 3 mg/L (minus a demand of about 10%) during year 2 when we enumerated only coliphages and E. coli. F+ male-specific coliphage LRs significantly dropped from 1.2 to 0.5, while E. coli LR remained unchanged. To ensure protection of aquatic life, an interim 0.27 mg/L PAA residual discharge limit was derived.

About 25 golf courses in Ontario, Canada have environmental compliance approvals to use reclaimed water for irrigation, where disinfection is confirmed through E. coli limits. A previous study at five Ontario municipal wastewater treatment plants (WWTPs) confirmed that enteric viruses are less susceptible to disinfection than E. coli, when plants provided conventional (secondary or tertiary) treatment and routine (chlorine or UV) disinfection. Here we query whether these four treatment-disinfection scenarios plus 60-day lagoon storage of disinfected effluent would be sufficient to reduce norovirus genogroups I and II (NoV GI and GII) risk of infection to tolerable levels for a golfer who incidentally ingests NoV after handling wet golf balls at a golf course irrigated with reclaimed water. We used our RT-qPCR NoV enumeration datasets from the four treatment-disinfection scenarios above and modeled detected and non-detected NoV by Bayesian inference in 'R'. Monte Carlo simulation included pre-disinfection NoV GI and GII gene copy densities; Ontario WWTP-derived chlorine and UV log10 reductions; literature-derived effluent storage decay parameters and golfer ingested volumes, followed by six different NoV dose-response (DR) models. Quantitative Microbial Risk Assessment (QMRA) results suggest that there is an unacceptable NoV infection risk when using the conservative assumption that all detected NoV particles (RT-qPCR gene copies) are infectious, in both aggregated or disaggregated form. However, after adjusting for PCR target sequences and for infectiousness using data from recently published studies on cultivation of human NoV in human intestinal enteroids, we noted a significant reduction of infection risk. However, this less conservative (i.e., less protective) assumption for water reuse applications such as golf course irrigation may not be corroborated until human NoV are efficiently and routinely grown in cell cultures. In addition, further studies on drivers of NoV risk estimation by DR models are needed, e.g., the extent of NoV particle aggregation resulting from wastewater treatment, as well as the role of immunity. Meantime, regulatory agencies could consider more stringent treatment-disinfection requirements that target enteric viruses rather than E. coli and testing of actual reclaimed irrigation waters.

In Ontario, Canada, information is lacking on chlorine and ultraviolet (UV) light disinfection performance against enteric viruses in wastewater. We enumerated enteroviruses and noroviruses, coliphages, and Escherichia coli per USEPA methods 1615, 1602, and membrane filtration, respectively, in pre- and post-disinfection effluent at five wastewater treatment plants (WWTPs), with full-year monthly sampling, and calculated log10 reductions (LRs) while WWTPs complied with their monthly geometric mean limit of 200 E. coli/100 mL. Modeling of densities by left-censored estimation and Bayesian inference gave very similar results. Polymerase chain reaction (PCR)-detected enteroviruses and noroviruses were abundant in post-disinfection effluent (mean concentrations of 2.1 × 10+4-7.2 × 10+5 and 2.7 × 10+4-3.6 × 10+5 gene copies (GC)/L, respectively). Chlorine or UV disinfection produced modest LRs for culture- (0.3-0.9) and PCR-detected enteroviruses (0.3-1.3), as well as noroviruses GI + GII (0.5-0.8). Coliphages and E. coli were more susceptible, with LRs of 0.8-3.0 and 2.5, respectively. Sand-filtered effluent produced significantly higher enteric virus LRs (except cultured enteroviruses). Coliphage and human enteric virus densities gave significantly positive correlations using Kendall's Tau test. Enteric viruses are abundant in wastewater effluent following routine chlorine or UV disinfection processes that target E. coli. Coliphages appear to be good indicators for evaluating wastewater disinfection of enteric viruses.

ABSTRACT: Culturable bacterial pathogens ( Campylobacter , Salmonella , Listeria , Yersinia ) and indicators ( E. coli , enterococci, Clostridium perfringens ) were quantified at six water resource recovery facilities that land apply anaerobically digested biosolids in Ontario, Canada. Cryptosporidium parvum and Giardia lamblia were also quantified by polymerase chain reaction (PCR). Salmonella and Listeria were frequently detected in sludge and liquid biosolids (70–100% of samples) but less often in fresh dewatered cake biosolids (50–60%); with low levels in fresh cake (<100 cells/g dw). Yersinia were in 20 to 30% of samples, typically at very low levels (<10 cell/g dw). Giardia and Cryptosporidium were detected in 80 and 20% of cake biosolids at geometric means of 270 cysts/g dw and 70 oocysts/g dw, respectively. E. coli reduction was typically >2‐log 10 while pathogen reduction was variable. “Sudden increase” of pathogens was not observed, however, Salmonella and E. coli showed regrowth (at 1 to 3 orders of magnitude) after 2‐ to 3‐day storage at 30 °C.

Pathogen Characterization of Fresh and Stored Biosolids and Implications of a Screening Level Microbial Risk AssessmentA limited study of enteric pathogens from different stages of mesophilic anaerobic digestion (MAD), i.e., primary sludge, and liquid and dewatered (cake) biosolids, was conducted in six wastewater treatment plants (WWTP) in Ontario, Canada. In addition, bacterial pathogens were re-analysed for regrowth after 2 to 3 days storage in a laboratory at 30°C. Pathogen datasets were used in a...Author(s)Cecily FlemmingJeffrey SollerAlbert SimhonEdmund SetoSourceProceedings of the Water Environment FederationSubjectSession 3 - Pathogen ReductionDocument typeConference PaperPublisherWater Environment FederationPrint publication date Jan, 2009ISSN1938-6478SICI1938-6478(20090101)2009:3L.131;1-DOI10.2175/193864709793846556Volume / Issue2009 / 3Content sourceResiduals and Biosolids ConferenceFirst / last page(s)131 - 160Copyright2009Word count297Subject keywordsbiosolidspathogensregrowthquantitative microbial risk assessmentQMRAListeriaSalmonellaGiardiaCryptosporidiumwastewater treatment