Tracking SARS-CoV-2 variants of concern in wastewater: an assessment of nine computational tools using simulated genomic data.

Wastewater-based surveillance (WBS) is an important epidemiological and public health tool for tracking pathogens across the scale of a building, neighbourhood, city, or region. WBS gained widespread adoption globally during the SARS-CoV-2 pandemic for estimating community infection levels by qPCR. Sequencing pathogen genes or genomes from wastewater adds information about pathogen genetic diversity, which can be used to identify viral lineages (including variants of concern) that are circulating in a local population. Capturing the genetic diversity by WBS sequencing is not trivial, as wastewater samples often contain a diverse mixture of viral lineages with real mutations and sequencing errors, which must be deconvoluted computationally from short sequencing reads. In this study we assess nine different computational tools that have recently been developed to address this challenge. We simulated 100 wastewater sequence samples consisting of SARS-CoV-2 BA.1, BA.2, and Delta lineages, in various mixtures, as well as a Delta-Omicron recombinant and a synthetic 'novel' lineage. Most tools performed well in identifying the true lineages present and estimating their relative abundances and were generally robust to variation in sequencing depth and read length. While many tools identified lineages present down to 1 % frequency, results were more reliable above a 5 % threshold. The presence of an unknown synthetic lineage, which represents an unclassified SARS-CoV-2 lineage, increases the error in relative abundance estimates of other lineages, but the magnitude of this effect was small for most tools. The tools also varied in how they labelled novel synthetic lineages and recombinants. While our simulated dataset represents just one of many possible use cases for these methods, we hope it helps users understand potential sources of error or bias in wastewater sequencing analysis and to appreciate the commonalities and differences across methods.

Rapid Assessment of SARS-CoV-2 Variant-Associated Mutations in Wastewater Using Real-Time RT-PCR.

Within months of the COVID-19 pandemic being declared on March 20, 2020, novel, more infectious variants of SARS-CoV-2 began to be detected in geospatially distinct regions of the world. With international travel being a lead cause of spread of the disease, the importance of rapidly identifying variants entering a country is critical. In this study, we utilized wastewater-based epidemiology (WBE) to monitor the presence of variants in wastewater generated in managed COVID-19 quarantine facilities for international air passengers entering the United Kingdom. Specifically, we developed multiplex reverse transcription quantitative PCR (RT-qPCR) assays for the identification of defining mutations associated with Beta (K417N), Gamma (K417T), Delta (156/157DEL), and Kappa (E154K) variants which were globally prevalent at the time of sampling (April to July 2021). The assays sporadically detected mutations associated with the Beta, Gamma, and Kappa variants in 0.7%, 2.3%, and 0.4% of all samples, respectively. The Delta variant was identified in 13.3% of samples, with peak detection rates and concentrations observed in May 2021 (24%), concurrent with its emergence in the United Kingdom. The RT-qPCR results correlated well with those from sequencing, suggesting that PCR-based detection is a good predictor for variant presence; although, inadequate probe binding may lead to false positive or negative results. Our findings suggest that WBE coupled with RT-qPCR may be used as a rapid, initial assessment to identify emerging variants at international borders and mass quarantining facilities. IMPORTANCE With the global spread of COVID-19, it is essential to identify emerging variants which may be more harmful or able to escape vaccines rapidly. To date, the gold standard to assess variants circulating in communities has been the sequencing of the S gene or the whole genome of SARS-CoV-2; however, that approach is time-consuming and expensive. In this study, we developed two duplex RT-qPCR assays to detect and quantify defining mutations associated with the Beta, Gamma, Delta, and Kappa variants. The assays were validated using RNA extracts derived from wastewater samples taken at quarantine facilities. The results showed good correlation with the results of sequencing and demonstrated the emergence of the Delta variant in the United Kingdom in May 2021. The assays developed here enable the assessment of variant-specific mutations within 2 h after the RNA extract was generated which is essential for outbreak rapid response.

Assessment of a Program for SARS-CoV-2 Screening and Environmental Monitoring in an Urban Public School District.

Importance: Scalable programs for school-based SARS-CoV-2 testing and surveillance are needed to guide in-person learning practices and inform risk assessments in kindergarten through 12th grade settings. Objectives: To characterize SARS-CoV-2 infections in staff and students in an urban public school setting and evaluate test-based strategies to support ongoing risk assessment and mitigation for kindergarten through 12th grade in-person learning. Design, Setting, and Participants: This pilot quality improvement program engaged 3 schools in Omaha, Nebraska, for weekly saliva polymerase chain reaction testing of staff and students participating in in-person learning over a 5-week period from November 9 to December 11, 2020. Wastewater, air, and surface samples were collected weekly and tested for SARS-CoV-2 RNA to evaluate surrogacy for case detection and interrogate transmission risk of in-building activities. Main Outcomes and Measures: SARS-CoV-2 detection in saliva and environmental samples and risk factors for SARS-CoV-2 infection. Results: A total of 2885 supervised, self-collected saliva samples were tested from 458 asymptomatic staff members (mean [SD] age, 42.9 [12.4] years; 303 women [66.2%]; 25 Black or African American [5.5%], 83 Hispanic [18.1%], 312 White [68.1%], and 35 other or not provided [7.6%]) and 315 students (mean age, 14.2 [0.7] years; 151 female students [48%]; 20 Black or African American [6.3%], 201 Hispanic [63.8%], 75 White [23.8%], and 19 other race or not provided [6.0%]). A total of 46 cases of SARS-CoV-2 (22 students and 24 staff members) were detected, representing an increase in cumulative case detection rates from 1.2% (12 of 1000) to 7.0% (70 of 1000) among students and from 2.1% (21 of 1000) to 5.3% (53 of 1000) among staff compared with conventional reporting mechanisms during the pilot period. SARS-CoV-2 RNA was detected in wastewater samples from all pilot schools as well as in air samples collected from 2 choir rooms. Sequencing of 21 viral genomes in saliva specimens demonstrated minimal clustering associated with 1 school. Geographical analysis of SARS-CoV-2 cases reported district-wide demonstrated higher community risk in zip codes proximal to the pilot schools. Conclusions and Relevance: In this study of staff and students in 3 urban public schools in Omaha, Nebraska, weekly screening of asymptomatic staff and students by saliva polymerase chain reaction testing was associated with increased SARS-CoV-2 case detection, exceeding infection rates reported at the county level. Experiences differed among schools, and virus sequencing and geographical analyses suggested a dynamic interplay of school-based and community-derived transmission risk. Collectively, these findings provide insight into the performance and community value of test-based SARS-CoV-2 screening and surveillance strategies in the kindergarten through 12th grade educational setting.

Protocol for safe, affordable, and reproducible isolation and quantitation of SARS-CoV-2 RNA from wastewater.

The following protocol describes our workflow for processing wastewater with the goal of detecting the genetic signal of SARS-CoV-2. The steps include pasteurization, virus concentration, RNA extraction, and quantification by RT-qPCR. We include auxiliary steps that provide new users with tools and strategies that will help troubleshoot key steps in the process. This protocol is one of the safest, cheapest, and most reproducible approaches for the detection of SARS-CoV-2 RNA in wastewater. Owing to a pasteurization step, it is safe for use in a BSL2 facility. In addition to making the protocol safe for the personnel involved, pasteurization had the added benefit of increasing the SARS-CoV-2 genetic signal. Furthermore, the RNA obtained using this protocol can be sequenced using both Sanger and Illumina sequencing technologies. The protocol was adopted by the New York City Department of Environmental Protection in August 2020 to monitor SARS-CoV-2 prevalence in wastewater in all five boroughs of the city. In the future, this protocol could be used to detect a variety of other clinically relevant viruses in wastewater and serve as a foundation of a wastewater surveillance strategy for monitoring community spread of known and emerging viral pathogens.

Assessment of enteric viruses during a hepatitis outbreak in Detroit MI using wastewater surveillance and metagenomic analysis.

AIMS: This study investigates enteric viruses in wastewater during an outbreak of acute hepatitis caused by hepatitis A virus (HAV) in a large metropolitan area. Emphasis is given to caliciviruses and HAV. METHODS AND RESULTS: Metagenomic analysis was performed to characterize enteric viruses excreted by the population of Detroit MI, during a hepatitis A outbreak that occurred in 2017 and 2018. Additionally, HAV, norovirus GII, and sapovirus were quantified, using qPCR, in 54 untreated wastewater samples collected over the course of 4 months. Correlation analysis was performed to identify associations between the number of disease cases and HAV concentrations in wastewater. HAV obtained the highest relative abundance among other enteric viruses detected in wastewater metagenomes. Metagenomic analysis also detected several other enteric viruses including astrovirus, enterovirus and hepatitis E virus. Average sapovirus concentrations of 1·36 × 106  gc l-1 were significantly greater than norovirus GII concentrations (2·94 × 104  gc l-1 ). Additionally, norovirus GI and GII along with sapovirus GI.1 were detected using metagenomics. HAV loads in wastewater were significantly correlated with the number of disease cases reported 1 week after wastewater sampling. CONCLUSIONS: Surveying untreated wastewater is a promising method for detecting early signs of hepatitis A outbreaks and for routine environmental monitoring of enteric viruses circulating in the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Authors demonstrate the usefulness of metagenomics for genogrouping and enteric viral surveillance.

First confirmed detection of SARS-CoV-2 in untreated wastewater in Australia: A proof of concept for the wastewater surveillance of COVID-19 in the community.

Infection with SARS-CoV-2, the etiologic agent of the ongoing COVID-19 pandemic, is accompanied by the shedding of the virus in stool. Therefore, the quantification of SARS-CoV-2 in wastewater affords the ability to monitor the prevalence of infections among the population via wastewater-based epidemiology (WBE). In the current work, SARS-CoV-2 RNA was concentrated from wastewater in a catchment in Australia and viral RNA copies were enumerated using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) resulting in two positive detections within a six day period from the same wastewater treatment plant (WWTP). The estimated viral RNA copy numbers observed in the wastewater were then used to estimate the number of infected individuals in the catchment via Monte Carlo simulation. Given the uncertainty and variation in the input parameters, the model estimated a median range of 171 to 1,090 infected persons in the catchment, which is in reasonable agreement with clinical observations. This work highlights the viability of WBE for monitoring infectious diseases, such as COVID-19, in communities. The work also draws attention to the need for further methodological and molecular assay validation for enveloped viruses in wastewater.

Quantitative Microbial Risk Assessment for Workers Exposed to Bioaerosol in Wastewater Treatment Plants Aimed at the Choice and Setup of Safety Measures.

Biological risk assessment in occupational settings currently is based on either qualitative or semiquantitative analysis. In this study, a quantitative microbial risk assessment (QMRA) has been applied to estimate the human adenovirus (HAdV) health risk due to bioaerosol exposure in a wastewater treatment plant (WWTP). A stochastic QMRA model was developed considering HAdV as the index pathogen, using its concentrations in different areas and published dose⁻response relationship for inhalation. A sensitivity analysis was employed to examine the impact of input parameters on health risk. The QMRA estimated a higher average risk in sewage influent and biological oxidation tanks (15.64% and 12.73% for an exposure of 3 min). Sensitivity analysis indicated HAdV concentration as a predominant factor in the estimated risk. QMRA results were used to calculate the exposure limits considering four different risk levels (one illness case per 100, 1.000, 10.000, and 100.000 workers): for 3 min exposures, we obtained 565, 170, 54, and 6 GC/m³ of HAdV. We also calculated the maximum time of exposure for each level for different areas. Our findings can be useful to better define the effectiveness of control measures, which would thus reduce the virus concentration or the exposure time.

Estimation of health risks caused by exposure to enteroviruses from agricultural application of wastewater effluents.

Agricultural reuse of wastewater is a common practice worldwide, especially in arid and semiarid area due to the freshwater scarcity. Wastewater irrigation in the Middle East, one of the most water-stressed regions in the world, could be a key factor for socio-economic development, but the microbial contamination of untreated or partially treated wastewater is a serious public health concern. Potential transmission of enteric viral infections through wastewater reuse in agricultural activities represents a true health risk for exposed individuals. Accordingly, it is important to assess the health risks associated with wastewater reuse. A quantitative microbial risk assessment (QMRA) with Monte-Carlo simulation was used to estimate the annual risk of enterovirus (EV) infection and disease burden for farmers and consumers of wastewater-irrigated lettuce in Iran, a semiarid country in the Middle East region. Risk analysis was performed based on the measured concentrations of EV in effluent of two activated sludge wastewater treatment plants (WWTP). Wastewater effluent sampling was carried out over a nine-month period, and the presence of total and fecal coliforms and EV was determined. Fecal coliform bacteria were found at a high level exceeded the guideline limit for wastewater reuse in agriculture. EVs were detected in 40% of samples with the highest frequency in summer with a mean of 12 and 16 pfu/ml for WWTP-A and B, respectively. Statistical analysis showed no correlation between the concentration of fecal coliforms and EV. The estimated infection risk for EVs was 8.8 × 10-1 and 8.2 × 10-1 per person per year (pppy) for farmers of WWTP-A and -B, respectively which was about 2 log higher than the tolerable infection risk of 2 × 10-3 pppy. The estimated risk for lettuce consumers exhibited a lower level of infection and disease burden but higher than the guideline limits. The median disease burden for consumption of lettuce irrigated with activated sludge effluents was about 10-3 Disability Adjusted Life Years (DALY) pppy which exceeded the WHO guideline threshold of 10-4 DALY pppy. The results of study indicated that the activated sludge effluents require an additional reduction of EVs to achieve the acceptable level of risk for agricultural reuse of wastewater.

Probabilistic quantitative microbial risk assessment model of norovirus from wastewater irrigated vegetables in Ghana using genome copies and fecal indicator ratio conversion for estimating exposure dose.

The need to replace the commonly applied fecal indicator conversions ratio (an assumption of 1:10-5 virus to fecal indicator organism) in Quantitative Microbial Risk Assessment (QMRA) with models based on quantitative data on the virus of interest has gained prominence due to the different physical and environmental factors that might influence the reliability of using indicator organisms in microbial risk assessment. The challenges facing analytical studies on virus enumeration (genome copies or particles) have contributed to the already existing lack of data in QMRA modelling. This study attempts to fit a QMRA model to genome copies of norovirus data. The model estimates the risk of norovirus infection from the intake of vegetables irrigated with wastewater from different sources. The results were compared to the results of a corresponding model using the fecal indicator conversion ratio to estimate the norovirus count. In all scenarios of using different water sources, the application of the fecal indicator conversion ratio underestimated the norovirus disease burden, measured by the Disability Adjusted Life Years (DALYs), when compared to results using the genome copies norovirus data. In some cases the difference was >2 orders of magnitude. All scenarios using genome copies met the 10-4 DALY per person per year for consumption of vegetables irrigated with wastewater, although these results are considered to be highly conservative risk estimates. The fecal indicator conversion ratio model of stream-water and drain-water sources of wastewater achieved the 10-6 DALY per person per year threshold, which tends to indicate an underestimation of health risk when compared to using genome copies for estimating the dose.

Assessment and risk modeling of airborne enteric viruses emitted from wastewater reused for irrigation.

Reclamation of wastewater (WW) for irrigation, after treatment represents a challenge that could alleviate pressure on water resources and address the increasing demand for agriculture. However, the risks to human health must be assessed, particularly those related to human enteric viruses that resist standard treatments in most wastewater treatment plants (WWTP). The risks associated with exposure to viral bioaerosols near WWTP and near agricultural plots irrigated with WW are poorly documented. The objectives of this study were to 1) better characterize human enteric viruses found in bioaerosols near a "standard WWTP" and over fields irrigated with treated WW and 2) propose a numeric model to assess the health risk to populations located close to the irrigated areas, with particular attention to norovirus, which is responsible for most viral gastroenteritis in France. Water and air samples were collected at various locations in the largest French WW-irrigated site near Clermont-Ferrand, at the WWTP entrance and after treatment, in the air above activated sludge basins, and above fields irrigated with WW. Various enteric viruses were found in the water samples collected both before and after treatment. Norovirus was the most abundant with >10e4 genome copies/l (GC/L) before treatment and ~10e3 GC/L after treatment. Low quantities (<10e3GC/m3) were detected in the air above active sludge pools and irrigated plots. Hepatitis E virus was detected in all sampled compartments. A quantitative microbial risk assessment (QMRA) approach, including a simplified atmospheric dispersion model, allowed assessment of norovirus infection risk. The Bayesian QMRA approach considered wind speed measurements over 21years, and the variability and uncertainty of all measurements throughout the chain up to the risk. The probability of infection within one year for the most exposed WWTP employees was >10e-4 for strong wind speed (≥3m/s) and a constant emission rate of 8e3 GC/m3/s. This probability decreases by 3 log when the distance to the emission source is doubled. This information can aid development of safe water reuse policies in terms of local setback distance and wind conditions for wastewater reuse.

Comparison of Risk Predicted by Multiple Norovirus Dose-Response Models and Implications for Quantitative Microbial Risk Assessment.

The application of quantitative microbial risk assessments (QMRAs) to understand and mitigate risks associated with norovirus is increasingly common as there is a high frequency of outbreaks worldwide. A key component of QMRA is the dose-response analysis, which is the mathematical characterization of the association between dose and outcome. For Norovirus, multiple dose-response models are available that assume either a disaggregated or an aggregated intake dose. This work reviewed the dose-response models currently used in QMRA, and compared predicted risks from waterborne exposures (recreational and drinking) using all available dose-response models. The results found that the majority of published QMRAs of norovirus use the 1 F1 hypergeometric dose-response model with α = 0.04, ß = 0.055. This dose-response model predicted relatively high risk estimates compared to other dose-response models for doses in the range of 1-1,000 genomic equivalent copies. The difference in predicted risk among dose-response models was largest for small doses, which has implications for drinking water QMRAs where the concentration of norovirus is low. Based on the review, a set of best practices was proposed to encourage the careful consideration and reporting of important assumptions in the selection and use of dose-response models in QMRA of norovirus. Finally, in the absence of one best norovirus dose-response model, multiple models should be used to provide a range of predicted outcomes for probability of infection.

Quality of greywater treated in biochar filter and risk assessment of gastroenteritis due to household exposure during maintenance and irrigation.

AIM: This study evaluated treatment of greywater (GW) by a biochar filter in Jordan and assessed the annual risks of infection (Pi-annual ), annual risk of disease (Pd-annual ) and disease burden (in disability-adjusted life years; DALYs) of gastroenteritis caused by Salmonella spp. and rotavirus due to ingestion of GW during system maintenance and consumption of green onions irrigated with treated and nontreated GW. METHODS AND RESULTS: The biochar filter efficiently removed 93% of biochemical oxygen demand (BOD5 ) and 85% of solids, while removal of Escherichia coli was insignificant. Treatment of GW decreased the median Pd-annual due to ingestion of GW from 1·39 × 10-2 to 6·0 × 10-3 for Salmonella spp. but did not affect Pd-annual caused by rotavirus (9·73 × 10-1 to 1·0). Consumption of onions irrigated with treated GW had a median Pd-annual of 1·25 × 10-9 to 1·2 × 10-8 for Salmonella spp. and 4·96 × 10-4 to 4·37 × 10-3 for rotavirus infection, which was 99·9 and 90% lower, respectively, than the risk when consuming onions irrigated with nontreated GW. The highest risks of gastrointestinal disease were thus associated mainly with direct ingestion of GW when maintaining the system. CONCLUSIONS: Garden produce irrigated with GW treated in biochar filter did not display intolerable risks of rotavirus-based gastroenteritis during summer season in the study area given that the produce is harvested 1 to 2 days, and washed, before consumption. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to scientific-based knowledge on the suitability of biochar filters for onsite greywater treatment and confirms the microbial safety of recycling treated greywater for garden irrigation.

Setback distances between small biological wastewater treatment systems and drinking water wells against virus contamination in alluvial aquifers.

Contamination of groundwater by pathogenic viruses from small biological wastewater treatment system discharges in remote areas is a major concern. To protect drinking water wells against virus contamination, safe setback distances are required between wastewater disposal fields and water supply wells. In this study, setback distances are calculated for alluvial sand and gravel aquifers for different vadose zone and aquifer thicknesses and horizontal groundwater gradients. This study applies to individual households and small settlements (1-20 persons) in decentralized locations without access to receiving surface waters but with the legal obligation of biological wastewater treatment. The calculations are based on Monte Carlo simulations using an analytical model that couples vertical unsaturated and horizontal saturated flow with virus transport. Hydraulic conductivities and water retention curves were selected from reported distribution functions depending on the type of subsurface media. The enteric virus concentration in effluent discharge was calculated based on reported ranges of enteric virus concentration in faeces, virus infectivity, suspension factor, and virus reduction by mechanical-biological wastewater treatment. To meet the risk target of <10-4infections/person/year, a 12 log10 reduction was required, using a linear dose-response relationship for the total amount of enteric viruses, at very low exposure concentrations. The results of this study suggest that the horizontal setback distances vary widely ranging 39 to 144m in sand aquifers, 66-289m in gravel aquifers and 1-2.5km in coarse gravel aquifers. It also varies for the same aquifers, depending on the thickness of the vadose zones and the groundwater gradient. For vulnerable fast-flow alluvial aquifers like coarse gravels, the calculated setback distances were too large to achieve practically. Therefore, for this category of aquifer, a high level of treatment is recommended before the effluent is discharged to the ground surface.

Risk management of viral infectious diseases in wastewater reclamation and reuse: Review.

Inappropriate usage of reclaimed wastewater has caused outbreaks of viral infectious diseases worldwide. International and domestic guidelines for wastewater reuse stipulate that virus infection risks are to be regulated by the multiple-barrier system, in which a wastewater treatment process composed of sequential treatment units is designed based on the pre-determined virus removal efficiency of each unit. The objectives of this review were to calculate representative values of virus removal efficiency in wastewater treatment units based on published datasets, and to identify research topics that should be further addressed for improving implementation of the multiple-barrier system. The removal efficiencies of human noroviruses, rotaviruses and enteroviruses in membrane bioreactor (MBR) and conventional activated sludge (CAS) processes were obtained by a systematic review protocol and a meta-analysis approach. The log10 reduction (LR) of norovirus GII and enterovirus in MBR were 3.35 (95% confidence interval: 2.39, 4.30) and 2.71 (1.52, 3.89), respectively. The LR values of rotavirus, norovirus GI and GII in CAS processes were 0.87 (0.20, 1.53), 1.48 (0.96, 2.00) and 1.35 (0.52, 2.18), respectively. The systematic review process eliminated a substantial number of articles about virus removal in wastewater treatment because of the lack of information required for the meta-analysis. It is recommended that future publications should explicitly describe their treatment of left-censored datasets. Indicators, surrogates and methodologies appropriate for validating virus removal performance during daily operation of wastewater reclamation systems also need to be identified.

Hepatitis A and E Viruses in Wastewaters, in River Waters, and in Bivalve Molluscs in Italy.

Several studies have reported the detection of hepatitis A (HAV) and E (HEV) virus in sewage waters, indicating a possibility of contamination of aquatic environments. The objective of the present study was to assess the occurrence of HAV and HEV in different water environments, following the route of contamination from raw sewage through treated effluent to the surface waters receiving wastewater discharges . Bivalve molluscan shellfish samples were also analyzed, as sentinel of marine pollution. Samples were tested by RT-PCR nested type in the VP1/2A junction for HAV, and in the ORF1 and ORF2 regions for HEV. Hepatitis A RNA was detected in 12 water samples: 7/21 (33.3%) raw sewage samples, 3/21 (14.3%) treated sewage samples, and 2/27 (7.4%) river water samples. Five sequences were classified as genotype IA, while the remaining 7 sequences belonged to genotype IB. In bivalves, HAV was detected in 13/56 samples (23.2%), 12 genotype IB and one genotype IA. Whether the presence of HAV in the matrices tested indicates the potential for waterborne and foodborne transmission is unknown, since infectivity of the virus was not demonstrated. HEV was detected in one raw sewage sample and in one river sample, both belonging to genotype 3. Sequences were similar to sequences detected previously in Italy in patients with autochthonous HEV (no travel history) and in animals (swine). To our knowledge, this is the first detection of HEV in river waters in Italy, suggesting that surface water can be a potential source for exposure .

Assessment of public health risk associated with viral contamination in harvested urban stormwater for domestic applications.

Capturing stormwater is becoming a new standard for sustainable urban stormwater management, which can be used to supplement water supply portfolios in water-stressed cities. The key advantage of harvesting stormwater is to use low impact development (LID) systems for treatment to meet water quality requirement for non-potable uses. However, the lack of scientific studies to validate the safety of such practice has limited its adoption. Microbial hazards in stormwater, especially human viruses, represent the primary public health threat. Using adenovirus and norovirus as target pathogens, we investigated the viral health risk associated with a generic scenario of urban stormwater harvesting practice and its application for three non-potable uses: 1) toilet flushing, 2) showering, and 3) food-crop irrigation. The Quantitative Microbial Risk Assessment (QMRA) results showed that food-crop irrigation has the highest annual viral infection risk (median range: 6.8×10(-4)-9.7×10(-1) per-person-per-year or pppy), followed by showering (3.6×10(-7)-4.3×10(-2)pppy), and toilet flushing (1.1×10(-7)-1.3×10(-4)pppy). Disease burden of each stormwater use was ranked in the same order as its viral infection risk: food-crop irrigation>showering>toilet flushing. The median and 95th percentile risk values of toilet-flushing using treated stormwater are below U.S. EPA annual risk benchmark of ≤10(-4)pppy, whereas the disease burdens of both toilet-flushing and showering are within the WHO recommended disease burdens of ≤10(-6)DALYspppy. However, the acceptability of showering risk interpreted based on the U.S. EPA and WHO benchmarks is in disagreement. These results confirm the safety of stormwater application in toilet flushing, but call for further research to fill the data gaps in risk modeling as well as risk benchmarks.

Spatial and hydrologic variation of Bacteroidales, adenovirus and enterovirus in a semi-arid, wastewater effluent-impacted watershed.

Bacteroidales and viruses were contemporaneously measured during dry and wet weather conditions at a watershed-scale in a semi-arid watershed impacted by a mixture of agricultural runoff, municipal wastewater effluent and municipal runoff. The results highlight the presence of municipal wastewater effluent as a confounding factor for microbial source tracking (MST) studies, and thus data were segregated into groups based on whether they were impacted by wastewater effluent. In semi-arid environments such as the Calleguas Creek watershed, located in southern California, the relative contribution of municipal wastewater effluent is dependent on hydrology as storm events lead to conditions where agricultural and municipal stormwater dominate receiving waters (rather than municipal wastewater, which is the case during dry weather). As such, the approach to data segregation was dependent on hydrology/storm conditions. Storm events led to significant increases in ruminant- and dog-associated Bacteroidales concentrations, indicating that overland transport connects strong non-human fecal sources with surface waters. Because the dataset had a large number of non-detect samples, data handling included the Kaplan-Meir estimator and data were presented graphically in a manner that reflects the potential effect of detection limits. In surface water samples with virus detections, Escherichia coli concentrations were often below (in compliance with) the recreational water quality criteria. In fact, sites downstream of direct inputs of municipal wastewater effluent exhibited the lowest concentrations of E. coli, but the highest concentrations of human-associated Bacteroidales and highest detection rates of human viruses. The toolkit, comprised of the four Bacteroidales assays and human virus assays used, can be successfully applied to inform watershed managers seeking to comply with recreational water quality criteria. However, care should be taken when analyzing data to account for the effect of non-detect samples, sources with differing microbial viability, and diverging hydrologic conditions.

Assessment of virus removal by managed aquifer recharge at three full-scale operations.

Managed aquifer recharge (MAR) systems such as riverbank filtration and soil-aquifer treatment all involve the use of natural subsurface systems to improve the quality of recharged water (i.e. surface water, stormwater, reclaimed water) before reuse. During MAR, water is either infiltrated via basins, subsurface injected or abstracted from wells adjacent to rivers. The goal of this study was to assess the removal of selected enteric viruses and a potential surrogate for virus removal at three full-scale MAR systems located in different regions of the United States (Arizona, Colorado, and California). Samples of source water (i.e., river water receiving treated wastewater and reclaimed water) before recharge and recovered groundwater at all three sites were tested for adenoviruses, enteroviruses, Aichi viruses and pepper mild mottle virus (PMMoV) by quantitative polymerase chain reaction (qPCR). Samples of groundwater positive for any virus were also tested for the presence of infectious virus by cell culture. PMMoV was the most commonly detected virus in the groundwater samples. Infectious enteric viruses (reovirus) were only detected in one groundwater sample with a subsurface residence time of 5 days. The results suggested that in groundwater with a residence time of greater than 14 days all of the viruses are removed below detection indicating a 1 to greater than 5 log removal depending upon the type of virus. Given its behavior, PMMoV may be suitable to serve as a conservative tracer of enteric virus removal in managed aquifer treatment systems.

Qualitative and Quantitative Assessment of Hepatitis A Virus in Wastewaters in Tunisia.

Hepatitis A causes substantial morbidity in both industrialized and non-industrialized countries and represents an important health problem in several southern Mediterranean countries. The objectives of the study were as follows: (a) to assess the occurrence of hepatitis A virus (HAV) in Tunisia through the monitoring of urban wastewaters collected at wastewater treatment plants (WTPs); (b) to characterize environmental strains; and (c) to estimate the viral load in raw and treated sewages, in order to evaluate the potential impact on superficial waters receiving discharges. A total of 150 raw and treated wastewaters were collected from three WTPs and analyzed by both qualitative (RT-PCR/nested) and quantitative (qRT-PCR) methods. Of these, 100 (66%) were found to be positive for HAV by the qualitative assay: 68.3% in influents and 64.7% in effluents. The vast majority of HAV sequences belonged to sub-genotype IA, with 11 different strains detected found to be identical to clinical strains isolated from Tunisian patients with acute hepatitis. Five unique variants were also detected, not previously reported in clinical cases. Only two IB strains were found, confirming the rarity of this sub-genotype in this country. The results of the present study indicate a wide circulation of the pathogen in the population, most probably in the form of asymptomatic infections, a finding consistent with the classification of the country as having intermediate/high endemicity. Quantitative data showed high viral loads in influents (3.5E+05 genome copies/liter, mean value) as well as effluents (2.5E+05 genome copies/liter, mean value), suggesting that contaminated water could be a critical element in transmission.