Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance.

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases.

Pathogen performance testing of a natural swimming pool using a cocktail of microbiological surrogates and QMRA-derived management goals.

In recent decades, natural swimming pools (NSPs) have gained popularity in Europe, especially in Germany and Austria. NSPs differ from swimming pools in that they utilize biological treatment processes based on wetland processes with no disinfection residual. However, data are missing on the specific log-reduction performance of NSPs to address enteric virus, bacteria, and parasitic protozoa removal considered necessary to meet the North American risk-based benchmark (<35 illnesses per 1,000 swimming events) set by the USEPA for voluntary swimming. In this study, we examined Canada's first NSP at Borden Park, Edmonton, Canada, to address the following three questions: (1) Given normal faecal shedding rates by bathers, what is the total log reduction (TLR) theoretically needed to meet the EPA benchmark? (2) what is the in-situ performance of the NSP based on spiking suitable microbial surrogates (MS2 coliphage, Enterococcus faecalis, and Saccharomyces cerevisiae [Baker's yeast])? and (3) how much time is required to reach acceptable bather risk levels under different representative volume-turnover rates? A reverse-quantitative microbial risk assessment (QMRA) revealed that of the four reference pathogens selected (Norovirus, Campylobacter, Cryptosporidium, and Giardia), only Norovirus was estimated to exceed the risk benchmark at the 50th, 75th, and 95th percentiles, while Campylobacter was the only other reference pathogen to exceed at the 95th percentile. Log-reduction values (LRVs) were similar to previous reports for bacterial indicators, and novel LRVs were estimated for the other two surrogates. A key finding was that more than 24 h treatment time would be necessary to provide acceptable bather protection following heavy bather use (378 bathers/day for main pool and 26 bathers/day for children's pool), due to the mixing dynamics of the treated water diluting out possible residual pool faecal contamination. The theoretical maximum number of people in the pool per day to be below USEPA's 35 gastro cases in 1,000 swimming events was 113, 47, and 8, at the 50th, 75th, and 95th percentiles. Further, the use of ultra-violet disinfection to the pool return flow had little effect on reducing the treatment time required.

Modelling faecal pathogen flows and health risks in urban Bangladesh: Implications for sanitation decision making.

Faecal-oral infections are a major component of the disease burden in low-income contexts, with inadequate sanitation seen as a contributing factor. However, demonstrating health effects of sanitation interventions - particularly in urban areas - has proved challenging and there is limited empirical evidence to support sanitation decisions that maximise health gains. This study aimed to develop, apply and validate a systems modelling approach to inform sanitation infrastructure and service decision-making in urban environments by examining enteric pathogen inputs, transport and reduction by various sanitation systems, and estimating corresponding exposure and public health impacts. The health effects of eight sanitation options were assessed in a low-income area in Dhaka, Bangladesh, with a focus on five target pathogens (Shigella, Vibrio cholerae, Salmonella Typhi, norovirus GII and Giardia). Relative to the sanitation base case in the study site (24% septic tanks, 5% holding tanks and 71% toilets discharging directly to open drains), comprehensive coverage of septic tanks was estimated to reduce the disease burden in disability-adjusted life years (DALYs) by 48-72%, while complete coverage of communal scale anaerobic baffled reactors was estimated to reduce DALYs by 67-81%. Despite these improvements, a concerning health risk persists with these systems as a result of effluent discharge to open drains, particularly when the systems are poorly managed. Other sanitation options, including use of constructed wetlands and small bore sewerage, demonstrated further reductions in local health risk, though several still exported pathogens into neighbouring areas, simply transferring risk to downstream communities. The study revealed sensitivity to and a requirement for further evidence on log reduction values for different sanitation systems under varying performance conditions, pathogen flows under flooding conditions as well as pathogen shedding and human exposure in typical low-income urban settings. Notwithstanding variability and uncertainties in input parameters, systems modelling can be a feasible and customisable approach to consider the relative health impact of different sanitation options across various contexts, and stands as a valuable tool to guide urban sanitation decision-making.

Screening Level Risk Assessment (SLRA) of human health risks from faecal pathogens associated with a Natural Swimming Pond (NSP).

Natural swimming ponds (NSPs) are artificially created bodies of water intended for human recreation, characterised by the substitution of chemical disinfection with natural biological processes for water purification. NSPs are growing in popularity, however little is known regarding the public health risks. A screening level risk assessment was undertaken as an initial step in assessing the first Canadian public NSP located in Edmonton, Alberta. Risk of enteric pathogens originating from pool bathers was assessed under normal conditions and following accidental faecal release events. The performance of the natural treatment train for health protection was quantified with and without the addition of UV disinfection of naturally-treated water, and compared to the US EPA benchmark to provide a reference point to consider acceptability. Estimated levels of pathogen contamination of the pond were dependant upon the discrete number of shedders present, which in turn depended upon the prevalence of infection in the population. Overall performance of the natural disinfection system was dependant upon the filtration rate of the natural treatment system or turnover time. Addition of UV disinfection reduced the uncertainty around the removal efficacy, and mitigated the impact of larger shedding events, however the impact of UV disinfection on the natural treatment biome is unknown. Further information is needed on the performance of natural barriers for pathogen removal, and therefore challenge studies are recommended. Given the identified risks, the pool is posted that there is risk from accidental faecal releases, as in any natural water body with swimmers. Screening level risk assessment was a valuable first step in understanding the processes driving the system and in identifying important data gaps.

Implementation of environmental surveillance for SARS-CoV-2 virus to support public health decisions: Opportunities and challenges.

Analysing wastewater can be used to track infectious disease agents that are shed via stool and urine. Sewage surveillance of SARS-CoV-2 has been suggested as a tool to determine the extent of COVID-19 in cities and serve as an early warning for (re-)emergence of SARS-CoV-2 circulation in communities. The focus of this review is on the strength of evidence, opportunities and challenges for the application of sewage surveillance to inform public health decision making. Considerations for undertaking sampling programs are reviewed including sampling sites, strategies, sample transport, storage and quantification methods; together with the approach and evidence base for quantifying prevalence of infection from measured wastewater concentration. Published SARS-CoV-2 sewage surveillance studies (11 peer reviewed and 10 preprints) were reviewed to demonstrate the current status of implementation to support public health decisions. Although being very promising, a number of areas were identified requiring additional research to further strengthen this approach and take full advantage of its potential. In particular, design of adequate sampling strategies, spatial and temporal resolution of sampling, sample storage, replicate sampling and analysis, controls for the molecular methods used for the quantification of SARS-CoV-2 RNA in wastewater. The use of appropriate prevalence data and methods to correlate or even translate SARS-CoV-2 concentrations in wastewater to prevalence of virus shedders in the population is discussed.

Utility of QMRA to compare health risks associated with alternative urban sewer overflow management strategies.

Wet weather sewer overflows pose potential short-term public health risks. With increasing populations, aging infrastructure and climate change, utilities are challenged with managing sewerage infrastructure to provide optimum outcomes. This study compared how modelled public health risk profiles could change under alternative sewer overflow management strategies during 12 and 24-month rainfall-runoff events. Specifically, existing conditions were compared with both a 'business-as-usual' (BAU) sewer upgrade and a more holistic 'effects-based planning' (EBP) approach based on pumped wet weather sewage overflows directed to a local receiving waterway. Options were compared based on their efficacy to reduce manhole overflows, recreational waterway guideline exceedances and downstream recreational waterway health risks estimated through a screening-level Quantitative Microbial Risk Assessment (QMRA). Results indicated that the two management strategies would be equally effective in reducing the frequency, duration and volume of manhole sewer overflows, eliminating them in the 12-month scenarios and reducing them from >5000 m3 for the 24-month baseline scenario, to 23 and 35 m3 for BAU and EBP, respectively. Baseline, BAU and EBP scenarios produced similar hours of enterococci guideline exceedances, ranging from 1 to 4 h difference. The QMRA produced similar health risk profiles for downstream recreational waterway users for all design events, suggesting that sewer overflows are not the primary driver of public health risks during and immediately following high rainfall events. As such, QMRA provided evidence that an EBP strategy may be used to manage wet weather sewer overflows in lieu of an expensive BAU upgrade, without exacerbating the public health of downstream waterway users. Further investigation of the broader environmental health impacts of implementing this type of innovative approach is warranted. Nonetheless, this work highlights the value of integrating QMRA with other modelling approaches to guide and inform sewer overflow management.

Faecal Pathogen Flows and Their Public Health Risks in Urban Environments: A Proposed Approach to Inform Sanitation Planning.

Public health benefits are often a key political driver of urban sanitation investment in developing countries, however, pathogen flows are rarely taken systematically into account in sanitation investment choices. While several tools and approaches on sanitation and health risks have recently been developed, this research identified gaps in their ability to predict faecal pathogen flows, to relate exposure risks to the existing sanitation services, and to compare expected impacts of improvements. This paper outlines a conceptual approach that links faecal waste discharge patterns with potential pathogen exposure pathways to quantitatively compare urban sanitation improvement options. An illustrative application of the approach is presented, using a spreadsheet-based model to compare the relative effect on disability-adjusted life years of six sanitation improvement options for a hypothetical urban situation. The approach includes consideration of the persistence or removal of different pathogen classes in different environments; recognition of multiple interconnected sludge and effluent pathways, and of multiple potential sites for exposure; and use of quantitative microbial risk assessment to support prediction of relative health risks for each option. This research provides a step forward in applying current knowledge to better consider public health, alongside environmental and other objectives, in urban sanitation decision making. Further empirical research in specific locations is now required to refine the approach and address data gaps.

A theoretical approach to using faecal indicator data to model norovirus concentration in surface water for QMRA: Glomma River, Norway.

Monitoring of faecal indicator organisms, such as Escherichia coli, in environmental and drinking waters is inadequate for the protection public health, primarily due to the poor relationship between E. coli and the occurrence of human pathogens, especially viruses, in environmental samples. Nevertheless, measurements of faecal indicator organisms within the risk based approach, can provide valuable information related to the magnitude and variability of faecal contamination, and hence provide insight into the expected level of potential pathogen contamination. In this study, a modelling approach is presented that estimates the concentration of norovirus in surface water relying on indicator monitoring data, combined with specific assumptions regarding the source of faecal contamination. The model is applied to a case study on drinking water treatment intake from the Glomma River in Norway. Norovirus concentrations were estimated in two sewage sources discharging into the river upstream of the drinking water offtake, and at the source water intake itself. The characteristics of the assumed source of faecal contamination, including the norovirus prevalence in the community, the size of the contributing population and the relative treatment efficacy for indicators and pathogens in the sewage treatment plant, influenced the magnitude and variability in the estimated norovirus concentration in surface waters. The modelling exercise presented is not intended to replace pathogen enumeration from environmental samples, but rather is proposed as a complement to better understand the sources and drivers of viruses in surface waters. The approach has the potential to inform sampling regimes by identifying when the best time would be to collect environmental samples; fill in the gaps between sparse datasets; and potentially extrapolate existing datasets in order to model rarer events such as an outbreak in the contributing population. In addition, and perhaps most universally, in the absence of pathogen data, this approach can be used as a first step to predict the source water pathogen concentration under different contamination scenarios for the purpose of quantifying microbial risks.

Evaluation of three full-scale stormwater treatment systems with respect to water yield, pathogen removal efficacy and human health risk from faecal pathogens.

In this study, three full-scale, operational stormwater harvesting systems located in Melbourne, Australia were evaluated with respect to water yields; pathogen removal performance by analysis of native surrogate data (Escherichiacoli, somatic coliphages and Clostridium perfringens); and potential human health risk associated with exposures to faecal pathogens using Quantitative Microbial Risk Assessment (QMRA). The water yield assessment confirmed variation between design and measured yields. Faecal contamination of urban stormwater was site specific and variable. Different treatment removal performance was observed between each of the microbial surrogates and varied between event and baseline conditions, with negligible removal of viruses during event conditions. Open storages that provide a habitat for waterfowl may lead to elevated risk due to the potential for zoonotic transmission. Nevertheless, in the Australian urban setting studied, the potential for human faecal contamination of the separated stormwater system was a critical driver of risk. If the integrity of the sewerage system can be ensured, then predicted health risks are dramatically reduced.

Variability in the recovery of a virus concentration procedure in water: Implications for QMRA.

Methods for analysing water for viruses are known to have variable and relatively poor recovery efficiencies. Quantitative method recovery data are needed to correct virus enumeration results so that estimates of virus concentrations in surface waters for QMRA are not too low. Obtaining quantitative data representing method recoveries for different pathogenic viruses is a significant challenge. In this study, we investigated the use of mengovirus process control data for quantifying recovery efficiency of human adenovirus (AdV) and noroviruses GI (NoVGI) and GII (NoVGII) from surface waters. Samples were collected from the inlet to a drinking water treatment plant on the Glomma River, Norway. Performance of the sample concentration procedure was quantified by comparing the virus concentrations found in concentrated and unconcentrated samples. The mean recovery of viruses (1.2%, 0.31%, 0.15% and 0.053% for mengovirus (n = 86), AdV (n = 20), NoVGI (n = 33) and NoVGII (n = 21) respectively) estimated in this study were lower than expected, and the between sample variability in estimated recovery was very high, spanning around 6 orders of magnitude for mengovirus. Within-sample correlation between the estimated recovery of mengovirus and human viruses was poor, and therefore sample specific mengovirus data could not be used to correct all human virus concentrations. Instead beta distributions were fitted to human virus-specific recovery estimates. The magnitude and variability of virus concentration when corrected for the variable recovery efficiency was orders of magnitude higher than the uncorrected concentration. Better estimates of virus concentration could be achieved if a sample-specific spiking control could be developed that mimicked closely the behaviour of human viruses in environmental samples.

Microbial risk assessment of drinking water based on hydrodynamic modelling of pathogen concentrations in source water.

Norovirus contamination of drinking water sources is an important cause of waterborne disease outbreaks. Knowledge on pathogen concentrations in source water is needed to assess the ability of a drinking water treatment plant (DWTP) to provide safe drinking water. However, pathogen enumeration in source water samples is often not sufficient to describe the source water quality. In this study, the norovirus concentrations were characterised at the contamination source, i.e. in sewage discharges. Then, the transport of norovirus within the water source (the river Göta älv in Sweden) under different loading conditions was simulated using a hydrodynamic model. Based on the estimated concentrations in source water, the required reduction of norovirus at the DWTP was calculated using quantitative microbial risk assessment (QMRA). The required reduction was compared with the estimated treatment performance at the DWTP. The average estimated concentration in source water varied between 4.8×10(2) and 7.5×10(3) genome equivalents L(-1); and the average required reduction by treatment was between 7.6 and 8.8 Log10. The treatment performance at the DWTP was estimated to be adequate to deal with all tested loading conditions, but was heavily dependent on chlorine disinfection, with the risk of poor reduction by conventional treatment and slow sand filtration. To our knowledge, this is the first article to employ discharge-based QMRA, combined with hydrodynamic modelling, in the context of drinking water.

Incorporating method recovery uncertainties in stochastic estimates of raw water protozoan concentrations for QMRA.

The impact of incorporating recovery data on protozoan concentration estimates was investigated for Cryptosporidium and Giardia using a large dataset (n=99) of [oo]cyst assay results with paired recovery estimates. Stochastic [oo]cyst concentration was estimated using three approaches: I-no availability/consideration of recovery, II-limited recovery data, where sample recovery was considered as an independent random variable, and III-every [oo]cyst assay result was adjusted for a concurrently derived recovery estimate. Critically, Approach I underestimated [oo]cyst concentrations by about 100% compared to Approaches II and III, which were similar. The impact of dataset size on statistical uncertainty about the concentration estimate for Approach II was investigated; little improvement in parameter uncertainty was achieved beyond n=20. It is suggested that recovery data be incorporated into source water concentration estimates, especially when used to infer health risks to consumers, so as not to underestimate the risk. Where none is available, conservatively low recoveries should be assumed. When designing monitoring programmes, recovery data should be collected as a pair with [oo]cyst count data for an initial period at least, so that site-specific relationships between those parameters may be ascertained and incorporated into source water concentration estimates.