Metagenomics of Wastewater Influent from Wastewater Treatment Facilities across Ontario in the Era of Emerging SARS-CoV-2 Variants of Concern

We report metagenomic sequencing analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in composite wastewater influent from 10 regions in Ontario, Canada, during the transition between Delta and Omicron variants of concern. The Delta and Omicron BA.1/BA.1.1 and BA.2-defining mutations occurring in various frequencies were reported in the consensus and subconsensus sequences of the composite samples

Multiplex RT-qPCR assay (N200) to detect and estimate prevalence of multiple SARS-CoV-2 Variants of Concern in wastewater

Wastewater-based surveillance (WBS) has become an effective tool around the globe for indirect monitoring of COVID-19 in communities. Quantities of viral fragments of SARS-CoV-2 in wastewater are related to numbers of clinical cases of COVID-19 reported within the corresponding sewershed. Variants of Concern (VOCs) have been detected in wastewater by use of reverse transcription quantitative polymerase chain reaction (RT-qPCR) or sequencing. A multiplex RT-qPCR assay to detect and estimate the prevalence of multiple VOCs, including Omicron/Alpha, Beta, Gamma, and Delta, in wastewater RNA extracts was developed and validated. The probe-based multiplex assay, named “N200” focuses on amino acids 199-202, a region of the N gene that contains several mutations that are associated with variants of SARS- CoV-2 within a single amplicon. Each of the probes in the N200 assay are specific to the targeted mutations and worked equally well in single- and multi-plex modes. To estimate prevalence of each VOC, the abundance of the targeted mutation was compared with a non- mutated region within the same amplified region. The N200 assay was applied to monitor frequencies of VOCs in wastewater extracts from six sewersheds in Ontario, Canada collected between December 1, 2021, and January 4, 2022. Using the N200 assay, the replacement of the Delta variant along with the introduction and rapid dominance of the Omicron variant were monitored in near real-time, as they occurred nearly simultaneously at all six locations. The N200 assay is robust and efficient for wastewater surveillance can be adopted into VOC monitoring programs or replace more laborious assays currently being used to monitor SARS- CoV-2 and its VOCs.Ontario Ministry of the Environment, Conservation and Parks||Natural Sciences and Engineering Research Council of Canad

Near real-time determination of B.1.1.7 in proportion to total SARS-CoV-2 viral load in wastewater using an allele-specific primer extension PCR strategy

"The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome corona- virus 2 (SARS-CoV-2) has claimed millions of lives to date. Antigenic drift has resulted in viral variants with putatively greater transmissibility, virulence, or both. Early and near real-time detection of these variants of concern (VOC) and the ability to accurately follow their incidence and prevalence in communities is wanting. Wastewater-based epidemiology (WBE), which uses nucleic acid amplification tests to detect viral fragments, is a reliable proxy of COVID-19 incidence and prevalence, and thus offers the potential to monitor VOC viral load in a given population. Here, we describe and validate a primer extension PCR strategy targeting a signature mutation in the N gene of SARS-CoV-2. This allows quantification of B.1.1.7 versus non-B.1.1.7 allele frequency in wastewater without the need to employ quantitative RT-PCR standard curves. We show that the wastewater B.1.1.7 profile correlates with its clinical counterpart and benefits from a near real-time and facile data collection and reporting pipeline. This assay can be quickly implemented within a current SARS-CoV-2 WBE framework with minimal cost; allowing early and contemporaneous estimates of B.1.1.7 community transmission prior to, or in lieu of, clinical screening and identification. Our study demonstrates that this strategy can provide public health units with an additional and much needed tool to rapidly triangulate VOC incidence/prevalence with high sensitivity and lineage specificity"National Microbiology Laboratory||Water Services at the Cities of Ottawa and Barrie||Ottawa Public Health||Simcoe Muskoka District Health Unit|| Public Health Ontario||Ontario Wastewater Surveillance Initiativ

Wastewater-based surveillance identifies start to the pediatric respiratory syncytial virus season in two cities in Ontario, Canada

IntroductionDetection of community respiratory syncytial virus (RSV) infections informs the timing of immunoprophylaxis programs and hospital preparedness for surging pediatric volumes. In many jurisdictions, this relies upon RSV clinical test positivity and hospitalization (RSVH) trends, which are lagging indicators. Wastewater-based surveillance (WBS) may be a novel strategy to accurately identify the start of the RSV season and guide immunoprophylaxis administration and hospital preparedness.MethodsWe compared citywide wastewater samples and pediatric RSVH in Ottawa and Hamilton between August 1, 2022, and March 5, 2023. 24-h composite wastewater samples were collected daily and 5 days a week at the wastewater treatment facilities in Ottawa and Hamilton, Ontario, Canada, respectively. RSV WBS samples were analyzed in real-time for RSV by RT-qPCR.ResultsRSV WBS measurements in both Ottawa and Hamilton showed a lead time of 12 days when comparing the WBS data set to pediatric RSVH data set (Spearman’s ρ = 0.90). WBS identify early RSV community transmission and declared the start of the RSV season 36 and 12 days in advance of the provincial RSV season start (October 31) for the city of Ottawa and Hamilton, respectively. The differing RSV start dates in the two cities is likely associated with geographical and regional variation in the incidence of RSV between the cities.DiscussionQuantifying RSV in municipal wastewater forecasted a 12-day lead time of the pediatric RSVH surge and an earlier season start date compared to the provincial start date. These findings suggest an important role for RSV WBS to inform regional health system preparedness, reduce RSV burden, and understand variations in community-related illness as novel RSV vaccines and monoclonal antibodies become available

The Role of Wastewater Testing for SARS-CoV-2 Surveillance

Wastewater testing for SARS-CoV-2 is relatively new; however, it builds on existing public health surveillance infrastructure. There is a limited but growing evidence base for its use, despite notable interpretation challenges. Wastewater testing results have helped to inform public health policy and interventions during the COVID-19 pandemic in Ontario and other jurisdictions. Wastewater testing for SARS-CoV-2 is useful for early detection of outbreaks and surges as well as population-wide surveillance of COVID-19 that is complementary to clinical testing. Further, it offers an efficient means of SARS-CoV-2 surveillance for specific settings such as correctional facilities, shelters, and university residences. Wastewater testing can also be used for the detection and monitoring of variants of concern (VOCs)

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 extant and anticipated reduction of clinical testing as the disease moves into an endemic state, SARS-CoV-2 wastewater surveillance (WWS) is likely to have greater value as an important diagnostic tool to inform public health. As the widespread adoption of WWS is relatively new at the scale employed for COVID-19, interpretation of data, including the relationship to clinical cases, has yet to be standardized. An in-depth analysis of the metrics derived from WWS is required for public health units/agencies to interpret and utilize WWS-acquired data effectively and efficiently. In this study, the SARS-CoV-2 wastewater signal to clinical cases (WC) ratio was investigated across seven different cities in Canada over periods ranging from 8 to 21 months. Significant increases in the WC ratio occurred when clinical testing eligibility was modified to appointment-only testing, identifying a period of insufficient clinical testing in these communities. The WC ratio decreased significantly during the emergence of the Alpha variant of concern (VOC) in a relatively non-immunized community’s wastewater (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’s wastewater (40-60% allelic proportion). Finally, a rapid and 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 vaccine-induced community immunity was high. The WC ratio, used as an additional monitoring metric, complements clinical case counts and wastewater signals as individual metrics in its ability to identify important epidemiological occurrences, adding value to WWS as a diagnostic technology during the COVID-19 pandemic and likely for future pandemics.Ontario's Ministry of Environment, Conservation and Parks||Alberta Healt

Community Surveillance of Omicron in Ontario: Wastewater-based Epidemiology Comes of Age

Wastewater-based surveillance of SARS-CoV-2 RNA has been implemented at building, neighbourhood, and city levels throughout the world. Implementation strategies and analysis methods differ, but they all aim to provide rapid and reliable information about community COVID-19 health states. A viable and sustainable SARS-CoV-2 surveillance network must not only provide reliable and timely information about COVID-19 trends, but also provide for scalability as well as accurate detection of known or unknown emerging variants. Emergence of the SARS-CoV-2 variant of concern Omicron in late Fall 2021 presented an excellent opportunity to benchmark individual and aggregated data outputs of the Ontario Wastewater Surveillance Initiative in Canada; this public health-integrated surveillance network monitors wastewaters from over 10 million people across major population centres of the province. We demonstrate that this coordinated approach provides excellent situational awareness, comparing favourably with traditional clinical surveillance measures. Thus, aggregated datasets compiled from multiple wastewater-based surveillance nodes can provide sufficient sensitivity (i.e., early indication of increasing and decreasing incidence of SARS-CoV-2) and specificity (i.e., allele frequency estimation of emerging variants) with which to make informed public health decisions at regional- and state-levels.Ontario Ministry of the Environment, Conservation and Parks|| Genome Canada and Ontario Genomics (OGI-209)||NSERC (ALLRP 555041-20 to C.O.)||Ontario Clean Water Agenc

Characterization of hydraulic parameters affecting the performance of aerated lagoons by Robert Delatolla.

The low capital and operating costs of aerated lagoons has lead to their extensive use in rural areas as a method of wastewater treatment. Although the performance of the system depends directly on the hydraulic mixing, there is currently no consensus on the key hydraulic parameters that influence the mixing of these lagoons. Tracer studies were performed on the St-Hermas aerated lagoon and a dynamically similar laboratory model to determine the effects of the water flow rate and the aeration rate on the performance of these aerated lagoons, to evaluate the method of dynamic similarity used to design the laboratory model and to evaluate the accuracy of predictive empirical dispersion models. The results were analysed using flow visualisation, simple observational and mathematical Retention Time Distribution (RTD) techniques and various hydraulic models. From these results it is concluded that the water flow rate and the aeration rate both significantly affect the hydraulic mixing of aerated lagoons, with the water flow rate being the dominant parameter. Furthermore, it is proven that the method of dynamic similarity used in this research is valid and that the only dispersion model that predicted reasonably accurate results was Arcievala (1981). An additional finding during the course of this research indicates that tracer study E-curves must be developed to completion in order to ensure conservation of mass and accurate analytical results

Nitrifying biofilms at cold temperatures: kinetics and in-situ characterization

The biological process of nitrification is currently the most economical and commonly employed means of removing ammonia from wastewater. Although nitrification is cost-effective and has been used in many treatment facilities in North America, the temperature sensitivity of suspended growth nitrifying bacteria is of considerable concern when designing wastewater treatment facilities in many North American cities. Notwithstanding the limited ability of suspended growth treatment systems to achieve nitrification at low temperature, there is evidence that attached growth nitrification processes have the potential to consistently achieve ammonia removal at low temperatures. The objective of this dissertation is to investigate the effect of prolonged exposure to cold temperatures on the kinetics of attached growth nitrification in wastewater treatment facilities as well as the subsequent consequences on the nitrifying bacterial community. Methodological techniques to characterize the weight, nitrogen content and volume of the biofilm along with the volume of nitrifying biomass were developed that minimize loss of mass and manipulation of the biofilm samples. Subsequently, laboratory experiments were performed on attached growth nitrifiers at 4°C. In addition, field experiments were conducted on attached growth laboratory-scale and pilot-scale treatment systems at 4°C. The laboratory experiments demonstrated significant rates of attached growth nitrification at 4°C for the approximate span of a cold climate, North American winter. Specifically, significant nitrification rates were confirmed after 115 days at 4°C. A pronounced kinetic reduction of the rate of nitrificatiÀ présent, le processus biologique de nitrification est le moyen le plus économique d'enlever l'ammoniaque des eaux usées. Bien que cette méthode soit utilisée dans des installations de traitement à travers l'Amérique du Nord, la sensibilité des bactéries à la température est problématique pour la conception de certaines nouvelles installations. Même si les bactéries ont une plus faible capacité de nitrification à basse température, la recherche suggère que cette méthode pourrait enlever l'ammoniaque systématiquement même à basse température. L'objectif de cette thèse était d'investiguer l'effet d'une exposition prolongée à de basses températures sur la cinétique de nitrification de bactéries fixées dans une installation de traitement, ainsi que les effets subséquents sur la communauté de bactéries. Des techniques méthodologiques pour caractériser le poids, la teneur en azote, le volume de biofilm ainsi que le volume de bactéries nitrifiantes ont été développées qui minimisent la perte de masse et la manipulation des prélèvements de biofilm. Ensuite, des expériences ont été effectuées avec une biomasse nitrifiante fixée à 4°C. De plus, des expériences de terrain ont été accomplies avec des bactéries fixées à 4°C avec des unités pilote et de laboratoire. Les expériences ont démontré des taux significatifs de croissance des bactéries fixées et de nitrification à 4°C pendant une durée correspondant à un hiver froid nord-américain. En particulier, des taux significatifs de nitrification ont été confirmés après 115 jours à 4°C. Le taux a baissé immédiatement après la période d'acclimatation et aprè

Performance and Kinetics of a Pond-Constructed Wetland System Treating Beef Manure Pile and Exercise Yard Runoff in Eastern Ontario

Runoff from manure piles and permanent outdoor confinement areas are common sources of pollution from livestock operations, which must be properly managed in accordance with local environmental regulations. Seven months of performance data from a pond-free water surface constructed wetland system receiving cattle feedlot runoff in Eastern Ontario, Canada, were used to evaluate performance and quantify first-order reaction rate constants (k) for organic matter and nutrients. Kinetic values were shown to be strongly temperature dependent and to adequately represent average treatment over the operating season; however, k values varied significantly on shorter time scales due to the event-based nature of the treatment system. Plant (duckweed) uptake of soluble phosphorus and ammonia in the pond was shown to be a predominant mechanism of nutrient removal. The pond-wetland system was effective at reducing biological oxygen demand (BOD5) by 67%, total phosphorus (TP) by 45%, and total nitrogen (TN) by 64%, providing a cost-effective and appropriate solution for beef producers