The ongoing global pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a Public Health Emergency of International Concern, ...which was officially declared by the World Health Organization. SARS-CoV-2 is a member of the family Coronaviridae that consists of a group of enveloped viruses with single-stranded RNA genome, which cause diseases ranging from common colds to acute respiratory distress syndrome. Although the major transmission routes of SARS-CoV-2 are inhalation of aerosol/droplet and person-to-person contact, currently available evidence indicates that the viral RNA is present in wastewater, suggesting the need to better understand wastewater as potential sources of epidemiological data and human health risks. Here, we review the current knowledge related to the potential of wastewater surveillance to understand the epidemiology of COVID-19, methodologies for the detection and quantification of SARS-CoV-2 in wastewater, and information relevant for human health risk assessment of SARS-CoV-2. There has been growing evidence of gastrointestinal symptoms caused by SARS-CoV-2 infections and the presence of viral RNA not only in feces of infected individuals but also in wastewater. One of the major challenges in SARS-CoV-2 detection/quantification in wastewater samples is the lack of an optimized and standardized protocol. Currently available data are also limited for conducting a quantitative microbial risk assessment (QMRA) for SARS-CoV-2 exposure pathways. However, modeling-based approaches have a potential role to play in reducing the impact of the ongoing COVID-19 outbreak. Furthermore, QMRA parameters obtained from previous studies on relevant respiratory viruses help to inform risk assessments of SARS-CoV-2. Our understanding on the potential role of wastewater in SARS-CoV-2 transmission is largely limited by knowledge gaps in its occurrence, persistence, and removal in wastewater. There is an urgent need for further research to establish methodologies for wastewater surveillance and understand the implications of the presence of SARS-CoV-2 in wastewater.
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•Presence of SARS-CoV-2 RNA in wastewater has been reported.•SARS-CoV-2 RNA in wastewater can be used to monitor COVID-19 in a community.•Effective concentration method is needed for recovery of SARS-CoV-2 from wastewater.•Surrogate coronavirus data help to predict survival of SARS-CoV-2 in wastewater.•Data on the infectivity of SARS-CoV-2 in wastewater for risk assessment are limited.
We investigated the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater samples in southern Louisiana, USA. Untreated and treated wastewater samples were ...collected on five occasions over a four-month period from January to April 2020. The wastewater samples were concentrated via ultrafiltration (Method A), and an adsorption–elution method using electronegative membranes (Method B). SARS-CoV-2 RNA was detected in 2 out of 15 wastewater samples using two reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays (CDC N1 and N2). None of the secondary treated and final effluent samples tested positive for SARS-CoV-2 RNA. To our knowledge, this is the first study reporting the detection of SARS-CoV-2 RNA in wastewater in North America, including the USA. However, concentration methods and RT-qPCR assays need to be refined and validated to increase the sensitivity of SARS-CoV-2 RNA detection in wastewater.
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•First study in Louisiana, USA reporting the detection of SARS-CoV-2 RNA in wastewater using ultrafiltration.•Two out of seven untreated wastewater samples tested positive for SARS-CoV-2 RNA.•None of the secondary treated and final effluent samples tested positive.•Concentration methods and RT-qPCR assays applied for SARS-CoV-2 RNA detection need further refinement.
This study investigated the magnitude of wet weather overflow (WWO)-driven sewage pollution in an urban lake (Lake Parramatta) located in Sydney, New South Wales, Australia. Water samples were ...collected during a dry weather period and after two storm events, and tested for a range of novel and established sewage- Bacteroides HF183, crAssphage CPQ_056 and pepper mild mottle virus (PMMoV) and animal feces-associated (Bacteroides BacCan-UCD, cowM2 and Helicobacter spp. associated GFD) microbial source tracking marker genes along with the enumeration of culturable fecal indicator bacteria (FIB), namely Escherichia coli (E. coli) and Enterococcus spp. The magnitude of general and source-specific fecal pollution was low in water samples collected during dry weather compared to storm events. The levels of HF183, crAssphage and PMMoV in water samples collected during storm events were as high as 6.39, 6.33 and 5.27 log
GC/L of water, respectively. Moderate to strong positive correlations were observed among the quantitative occurrence of sewage-associated marker genes. The concentrations of HF183 and PMMoV in most storm water samples exceeded the risk benchmark threshold values established in the literature for primary contact recreators. None of the samples tested was positive for the cowM2 (cow) marker gene, while BacCan-UCD (dog) and GFD (avian) animal-associated markers were sporadically detected in water samples collected from both dry weather and storm events. Based on the results, the ongoing advice that swimming should be avoided for several days after storm events appears appropriate. Further research to determine the decay rates of sewage-associated marker genes in relation to each other and enteric viruses would help refine current advice. Microbial source tracking approaches employed in this study provided insights into sources of contamination over currently used FIB.
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.
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•First study that reports the detection of SARS-CoV-2 in wastewater in Australia.•The presence of SARS-CoV-2 was confirmed by sequencing.•A median range of 171 to 1090 infected persons was identified in the catchment.•Further methodological and molecular assay validation will be required.
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•After rainfall, untreated sewage from WWOs was present in estuarine waters.•Avian marker gene was consistently present in stormwater samples.•Dog marker was occasionally detected in ...stormwater samples.•Animal fecal contamination does not appear to be an issue in the estuarine waters.
This study investigates the impact of wet weather overflows (WWOs) at three estuarine locations in Sydney, NSW, Australia. WWOs can occur when infiltration of stormwater leads to an excess volume of flow within the sewerage system, resulting in the release of diluted sewage into the environment. Sewage contamination poses a risk to human health due to the presence of pathogens. The magnitude of sewage contamination was monitored using established and novel sewage-associated marker genes, Bacteroides HF183, pepper mild mottle virus (PMMoV), crAssphage CPQ_056, Lachnospiraceae (Lachno3) marker genes along with culturable fecal indicator bacteria (FIB) Escherichia coli (E. coli) and enterococci. Water samples were collected at two water depths (0.5 m below the water surface and 1 m above the bottom surface) during one dry weather and two storm events. Analysis of sewage-associated marker genes showed greater (i.e., 3–5 orders of magnitude) concentrations in water samples collected during the storm events compared to dry weather event. Water samples were also analysed for four animal feces-associated marker genes targeting avian (GFD), dog (BacCan-UCD), cow (cowM2) and horse (HoF597) species to determine the extent of animal fecal contamination. Among the four marker genes, cowM2 and HoF597 could not be detected, while GFD marker gene was consistently present and BacCan-UCD was occasionally detected. Overall results suggested that after rainfall, untreated sewage from WWOs was present at sampling locations. In addition, microbial source tracking (MST) monitoring was able to distinguish the presence of a leaking sewer impacting on the recreational area during dry weather condition. This study demonstrated the capability of the MST monitoring approach to understand sources (sewage or animal) of fecal contamination. This capability will greatly enhance management decisions assisting in the prioritisation of remediation efforts of the sewerage system to improve estuarine bathing water quality and diminish human health risk.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is frequently detected in the feces of infected individuals. While infectious SARS-CoV-2 has not previously been identified in ...wastewater, infectious SARS-CoV-2 has been isolated from the feces of at least one patient, raising concerns about the presence of infectious SARS-CoV-2 in wastewater. The fate and inactivation characteristics of SARS-CoV-2 in water and wastewater are unknown, with current inactivation estimates based on surrogate models. In this study, the persistence of SARS-CoV-2 infectivity and RNA signal was determined in water and wastewater. The times for 90% reduction (T 90) of viable SARS-CoV-2 in wastewater and tap water at room temperature were 1.5 and 1.7 days, respectively. In high-starting titer (105 TCID50 mL–1) experiments, infectious virus persisted for the entire 7-day sampling time course. In wastewater at 50 and 70 °C, the observed T 90 values for infectious SARS-CoV-2 were decreased to 15 and 2 min, respectively. SARS-CoV-2 RNA was found to be significantly more persistent than infectious SARS-CoV-2, indicating that the environmental detection of RNA alone does not substantiate risk of infection.
There is currently a clear benefit for many countries to utilize wastewater-based epidemiology (WBE) as part of ongoing measures to manage the coronavirus disease 2019 (COVID-19) global pandemic. ...Since most wastewater virus concentration methods were developed and validated for nonenveloped viruses, it is imperative to determine the efficiency of the most commonly used methods for the enveloped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Municipal wastewater seeded with a human coronavirus (CoV) surrogate, murine hepatitis virus (MHV), was used to test the efficiency of seven wastewater virus concentration methods: (A–C) adsorption-extraction with three different pre-treatment options, (D–E) centrifugal filter device methods with two different devices, (F) polyethylene glycol (PEG 8000) precipitation, and (G) ultracentrifugation. MHV was quantified by reverse-transcription quantitative polymerase chain reaction and the recovery efficiency was calculated for each method. The mean MHV recoveries ranged from 26.7 to 65.7%. The most efficient methods were adsorption-extraction methods with MgCl2 pre-treatment (Method C), and without pre-treatment (Method B). The third most efficient method used the Amicon® Ultra-15 centrifugal filter device (Method D) and its recovery efficiency was not statistically different from the most efficient methods. The methods with the worst recovery efficiency included the adsorption-extraction method with acidification (A), followed by PEG precipitation (F). Our results suggest that absorption-extraction methods with minimal or without pre-treatment can provide suitably rapid, cost-effective and relatively straightforward recovery of enveloped viruses in wastewater. The MHV is a promising process control for SARS-CoV-2 surveillance and can be used as a quality control measure to support community-level epidemic mitigation and risk assessment.
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•Seven virus concentration methods were evaluated to recover CoV from wastewater.•The mean MHV recoveries ranged from 26.7 to 65.7%.•Adsorption-extraction with MgCl2 pre-treatment most efficiently concentrated MHV.•MHV seems to be an appropriate process control.
The HF183 marker gene, derived from the 16S rRNA gene of Bacteroides dorei, has been widely used to identify sewage pollution in environmental waters. CrAssphages are recently discovered DNA ...bacteriophages that are highly abundant in untreated sewage and have shown promises for tracking sewage contamination in environmental waters. In this paper, we report the development of a duplex quantitative PCR (qPCR) assay for simultaneous quantification of HF183 and crAssphage CPQ_056 marker genes in untreated sewage and sewage impacted stormwater. Same primer and probe sequences were used in the duplex qPCR assay as used in published simplex qPCR assays. The performance characteristics of the duplex qPCR assay were similar to its simplex counterparts. We validated the performance of the duplex assay in a collaborative laboratory study with the aim to evaluate reproducibility, sensitivity and concordance for field study. The concordance values between the simplex vs. duplex qPCR assays for HF183 and crAssphage CPQ_056 marker genes ranged from 96.7 to 100% and the mean concentrations of HF183 and CPQ_056 in environmental water samples were remarkably similar or in some cases slightly greater for the duplex qPCR assay suggesting the reliability of this assay for monitoring HF183 and CPQ_056 simultaneously. The newly developed duplex qPCR assay will be a valuable addition to the MST toolbox for sewage pollution monitoring and would allow rapid and comparative sample analysis.
•Duplex qPCR assay can quantify HF183 and crAssphage CPQ_056 marker genes.•Duplex and simplex qPCR assays showed 100% concordance for sewage samples.•Duplex and simplex qPCR assays were highly correlated.
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•EPISENS-M consists of RNA extraction from membrane, RT-preamplification, and qPCR.•EPISENS-M enables sensitive detection of SARS-CoV-2 RNA from wastewater.•SARS-CoV-2 RNA ...concentrations in wastewater correlated well with the reported cases.•A mathematical model was developed for predicting reported COVID-19 cases via WBE.•EPISENS-M with the mathematical model enables reliable prediction of reported cases.
Wastewater-based epidemiology (WBE) has the potential to predict COVID-19 cases; however, reliable methods for tracking SARS-CoV-2 RNA concentrations (CRNA) in wastewater are lacking. In the present study, we developed a highly sensitive method (EPISENS-M) employing adsorption-extraction, followed by one-step RT-Preamp and qPCR. The EPISENS-M allowed SARS-CoV-2 RNA detection from wastewater at 50 % detection rate when newly reported COVID-19 cases exceed 0.69/100,000 inhabitants in a sewer catchment. Using the EPISENS-M, a longitudinal WBE study was conducted between 28 May 2020 and 16 June 2022 in Sapporo City, Japan, revealing a strong correlation (Pearson’s r = 0.94) between CRNA and the newly COVID-19 cases reported by intensive clinical surveillance. Based on this dataset, a mathematical model was developed based on viral shedding dynamics to estimate the newly reported cases using CRNA data and recent clinical data prior to sampling day. This developed model succeeded in predicting the cumulative number of newly reported cases after 5 days of sampling day within a factor of √2 and 2 with a precision of 36 % (16/44) and 64 % (28/44), respectively. By applying this model framework, another estimation mode was developed without the recent clinical data, which successfully predicted the number of COVID-19 cases for the succeeding 5 days within a factor of √2 and 2 with a precision of 39 % (17/44) and 66 % (29/44), respectively. These results demonstrated that the EPISENS-M method combined with the mathematical model can be a powerful tool for predicting COVID-19 cases, especially in the absence of intensive clinical surveillance.
Wastewater-based epidemiology (WBE) demonstrates potential for COVID-19 community transmission monitoring; however, data on the stability of SARS-CoV-2 RNA in wastewater are needed to interpret WBE ...results. The decay rates of RNA from SARS-CoV-2 and a potential surrogate, murine hepatitis virus (MHV), were investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in untreated wastewater, autoclaved wastewater, and dechlorinated tap water stored at 4, 15, 25, and 37 °C. Temperature, followed by matrix type, most greatly influenced SARS-CoV-2 RNA first-order decay rates (k). The average T90 (time required for 1-log10 reduction) of SARS-CoV-2 RNA ranged from 8.04 to 27.8 days in untreated wastewater, 5.71 to 43.2 days in autoclaved wastewater, and 9.40 to 58.6 days in tap water. The average T90 for RNA of MHV at 4 to 37 °C ranged from 7.44 to 56.6 days in untreated wastewater, 5.58–43.1 days in autoclaved wastewater, and 10.9 to 43.9 days in tap water. There was no statistically significant difference between RNA decay of SARS-CoV-2 and MHV; thus, MHV is suggested as a suitable persistence surrogate. Decay rate constants for all temperatures were comparable across all matrices for both viral RNAs, except in untreated wastewater for SARS-CoV-2, which showed less sensitivity to elevated temperatures. Therefore, SARS-CoV-2 RNA is likely to persist long enough in untreated wastewater to permit reliable detection for WBE application.
•Temperature most greatly influenced SARS-CoV-2 RNA first-order decay rates.•SARS-CoV-2 and MHV RNA decay characteristics were similar.•MHV is suggested as suitable persistence surrogate.•SARS-CoV-2 RNA is likely to persist long enough in wastewater to permit detection.
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