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.
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.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus which causes coronavirus disease (COVID-19), has spread rapidly across the globe infecting millions of people and causing ...significant health and economic impacts. Authorities are exploring complimentary approaches to monitor this infectious disease at the community level. Wastewater-based epidemiology (WBE) approaches to detect SARS-CoV-2 RNA in municipal wastewater are being implemented worldwide as an environmental surveillance approach to inform health authority decision-making. Owing to the extended excretion of SARS-CoV-2 RNA in stool, WBE can surveil large populated areas with a longer detection window providing unique information on the presence of pre-symptomatic and asymptomatic cases that are unlikely to be screened by clinical testing. Herein, we analysed SARS-CoV-2 RNA in 24-h composite wastewater samples (n = 63) from three wastewater treatment plants (WWTPs) in Brisbane, Queensland, Australia from 24th of February to 1st of May 2020. A total of 21 samples were positive for SARS-CoV-2, ranging from 135 to 11,992 gene copies (GC)/100 mL of wastewater. Detections were made in a Southern Brisbane WWTP in late February 2020, up to three weeks before the first clininal case was reported there. Wastewater samples were generally positive during the period with highest caseload data. The positive SARS-CoV-2 RNA detection in wastewater while there were limited clinical reported cases demonstrates the potential of WBE as an early warning system to identify hotspots and target localised public health responses, such as increased individual testing and the provision of health warnings.
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•SARS-CoV-2 RNA was detected in wastewater when clinical cases were low.•The decline of the RNA occurrence in wastewater aligned with the tapering of the epidemic.•SARS-CoV-2 RNA in wastewater showed no correlation with daily cases.•Early detection of SARS-CoV-2 in wastewater is useful for COVID-19 management.
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.
The exploration and proposed mining of sulfide massive deposits in deep-sea environments and increased use deep-sea tailings placement (DSTP) in coastal zones has highlighted the need to better ...understand the fate and effects of mine-derived materials in marine environments. Metal sulfide ores contain high concentrations of metal(loid)s, of which a large portion exist in highly mineralized or sulfidised forms and are predicted to exhibit low bioavailability. In this study, sediments were spiked with a range of natural sulfide minerals (including chalcopyrite, chalcocite, galena, sphalerite) to assess the bioavailability and toxicity to benthic invertebrates (bivalve survival and amphipod survival and reproduction). The metal sulfide phases were considerably less bioavailable than metal contaminants introduced to sediment in dissolved forms, or in urban estuarine sediments contaminated with mixtures of metal(loid)s. Compared to total concentrations, the dilute-acid extractable metal(loid) (AEM) concentrations, which are intended to represent the more oxidized and labile forms, were more effective for predicting the toxicity of the sulfide mineral contaminated sediments. The study indicates that sediment quality guidelines based on AEM concentrations provide a useful tool for assessing and monitoring the risk posed by sediments impacted by mine-derived materials in marine environments.
Digital polymerase chain reaction (dPCR) is emerging as a reliable platform for quantifying microorganisms in the field of water microbiology. This paper reviews the fundamental principles of dPCR ...and its application for health-related water microbiology. The relevant literature indicates increasing adoption of dPCR for measuring fecal indicator bacteria, microbial source tracking marker genes, and pathogens in various aquatic environments. The adoption of dPCR has accelerated recently due to increasing use for wastewater surveillance of Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) - the virus that causes Coronavirus Disease 2019 (COVID-19). The collective experience in the scientific literature indicates that well-optimized dPCR assays can quantify genetic material from microorganisms without the need for a calibration curve and often with superior analytical performance (i.e., greater sensitivity, precision, and reproducibility) than quantitative polymerase chain reaction (qPCR). Nonetheless, dPCR should not be viewed as a panacea for the fundamental uncertainties and limitations associated with measuring microorganisms in water microbiology. With dPCR platforms, the sample analysis cost and processing time are typically greater than qPCR. However, if improved analytical performance (i.e., sensitivity and accuracy) is critical, dPCR can be an alternative option for quantifying microorganisms, including pathogens, in aquatic environments.
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•dPCR application for water microbiology is accelerating.•dPCR may improve analytical performance for microbial targets in complex aqueous matrices.•Increased costs, processing time, and need for specialized instruments constrain widespread adoption of dPCR.•dPCR relies on fundamental assumptions and should not be viewed as a panacea for water microbiology.
•Wastewater PLOD values were determined for six SARS-CoV-2 assays.•The US CDC N1 RT-dPCR and RT-qPCR assays were the most sensitive assays.•The US CDC N2 and E_Sarbeco assays were the least sensitive ...assays.•At seeding levels > 2.32 × 104 GC/50 mL, consistent amplification was observed.•At seeding levels < 2.32 × 103 GC/50 mL, inconsistent amplification was observed.
Effective wastewater surveillance of SARS-CoV-2 RNA requires the rigorous characterization of the limit of detection resulting from the entire sampling process - the process limit of detection (PLOD). Yet to date, no studies have gone beyond quantifying the assay limit of detection (ALOD) for RT-qPCR or RT-dPCR assays. While the ALOD is the lowest number of gene copies (GC) associated with a 95% probability of detection in a single PCR reaction, the PLOD represents the sensitivity of the method after considering the efficiency of all processing steps (e.g., sample handling, concentration, nucleic acid extraction, and PCR assays) to determine the number of GC in the wastewater sample matrix with a specific probability of detection. The primary objective of this study was to estimate the PLOD resulting from the combination of primary concentration and extraction with six SARS-CoV-2 assays: five RT-qPCR assays (US CDC N1 and N2, China CDC N and ORF1ab (CCDC N and CCDC ORF1ab), and E_Sarbeco RT-qPCR, and one RT-dPCR assay (US CDC N1 RT-dPCR) using two models (exponential survival and cumulative Gaussian). An adsorption extraction (AE) concentration method (i.e., virus adsorption on membrane and the RNA extraction from the membrane) was used to concentrate gamma-irradiated SARS-CoV-2 seeded into 36 wastewater samples. Overall, the US CDC N1 RT-dPCR and RT-qPCR assays had the lowest ALODs (< 10 GC/reaction) and PLODs (<3,954 GC/50 mL; 95% probability of detection) regardless of the seeding level and model used. Nevertheless, consistent amplification and detection rates decreased when seeding levels were < 2.32 × 103 GC/50 mL even for US CDC N1 RT-qPCR and RT-dPCR assays. Consequently, when SARS-CoV-2 RNA concentrations are expected to be low, it may be necessary to improve the positive detection rates of wastewater surveillance by analyzing additional field and RT-PCR replicates. To the best of our knowledge, this is the first study to assess the SARS-CoV-2 PLOD for wastewater and provides important insights on the analytical limitations for trace detection of SARS-CoV-2 RNA in wastewater.
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•Only 26% of published RT-qPCR assays used for wastewater surveillance reported all standard curve parameters.•Many of the reported standard curve parameters are outside the bounds of expected ...performance for RT-qPCR.•Variation is further exacerbated by use of a plasmid control material without linearization.•At a minimum 100% reporting of standard curve parameters should be achieved.•Editors and reviewers for wastewater surveillance should adopt MIQE guidelines.
Due to the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has become an important tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within communities. In particular, reverse transcription-quantitative PCR (RT-qPCR) has been used to generate large datasets aimed at detecting and quantifying SARS-CoV-2 RNA in wastewater. Although RT-qPCR is rapid and sensitive, there is no standard method yet, there are no certified quantification standards, and experiments are conducted using different assays, reagents, instruments, and data analysis protocols. These variations can induce errors in quantitative data reports, thereby potentially misleading interpretations, and conclusions. We review the SARS-CoV-2 wastewater surveillance literature focusing on variability of RT-qPCR data as revealed by inconsistent standard curves and associated parameters. We find that variation in these parameters and deviations from best practices, as described in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines suggest a frequent lack of reproducibility and reliability in quantitative measurements of SARS-CoV-2 RNA in wastewater.
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To support public-health-related disease surveillance and monitoring, it is crucial to concentrate both enveloped and non-enveloped viruses from domestic wastewater. To date, most concentration ...methods were developed for non-enveloped viruses, and limited studies have directly compared the recovery efficiency of both types of viruses. In this study, the effectiveness of two different concentration methods (Concentrating pipette (CP) method and an adsorption-extraction (AE) method amended with MgCl2) were evaluated for untreated wastewater matrices using three different viruses (SARS-CoV-2 (seeded), human adenovirus 40/41 (HAdV 40/41), and enterovirus (EV)) and a wastewater-associated bacterial marker gene targeting Lachnospiraceae (Lachno3). For SARS-CoV-2, the estimated mean recovery efficiencies were significantly greater by as much as 5.46 times, using the CP method than the AE method amended with MgCl2. SARS-CoV-2 RNA recovery was greater for samples with higher titer seeds regardless of the method, and the estimated mean recovery efficiencies using the CP method were 25.1 ± 11% across ten WWTPs when wastewater samples were seeded with 5 × 104 gene copies (GC) of SARS-CoV-2. Meanwhile, the AE method yielded significantly greater concentrations of indigenous HAdV 40/41 and Lachno3 from wastewater compared to the CP method. Finally, no significant differences in indigenous EV concentrations were identified in comparing the AE and CP methods. These data indicate that the most effective concentration method varies by microbial analyte and that the priorities of the surveillance or monitoring program should be considered when choosing the concentration method.
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•Concentration methods varied by microbial target.•Concentrating pipette yielded better recovery for SARS-CoV-2 than adsorption-extraction method.•SARS-CoV-2 RNA recovery was greater for samples with higher titer seeds.•Laboratories must empirically validate methods for water matrix and microbial target.
For the purpose of sediment quality assessment, the prediction of toxicity risk-levels for aquatic organisms based on simple environmental measurements is desirable. One commonly used approach is the ...comparison of total contaminant concentrations with corresponding water and sediment quality guideline values, serving as a Line of Evidence (LoE) based on chemistry-toxicity effects relationships. However, the accuracy of toxicity predictions can be improved by considering the factors that modify contaminant bioavailability. In this study we used paired chemistry-ecotoxicity data sets for sediments to evaluate the improvement in toxicity risk predictions using bioavailability-modified guidelines. The sediments were predominantly contaminated with metals, and measurements of sediment particle size, total organic carbon (TOC) and acid volatile sulfide (AVS) were used to modify hazard quotients (HQ). To further assess the predictive efficacy of the bioavailability-modified guideline models, sediments with differing contamination levels were tested for toxicity to a benthic amphipod's reproduction. To account for differences between laboratory exposure and field exposure scenarios, where the latter creates greater dilution, both static-renewal and flow-through test procedures were employed, and flow-through resulted in lower dissolved metal concentrations in the overlying waters. We also investigated how lower AVS concentration by oxidation modified the toxicity. This study reaffirmed that consideration of factors that influence contaminant bioavailability improves toxicity risk predictions, however the improvements may be modest. The sediment particle size data had the greatest influence on the modified HQ, indicating that higher percentage of fine particle size (<63 μm) contributed most to a lower predicted toxicity. The comparison of the static-renewal and flow-through test results continue to raise important questions about the relevance of static or static-renewal toxicity test results for risk assessment decisions, as both these test designs may cause unrealistically high contributions of dissolved metals in overlying waters to toxicity. Overall, this study underscores the value of incorporating outcomes from simple and routine sediment analysis (e.g., particle size, TOC, and consideration of AVS) to enhance the predictive efficacy of toxicity risk assessments in the context of sediment quality risk assessment.
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•Sediment particle size had a greater influence than TOC and AVS on toxicity risk of metals.•Bioavailability-modified guideline predicted toxicity risk correctly more frequently.•Translating toxicity risk predictions from laboratory to field has considerable uncertainty.•High dissolved metal concentrations in laboratory are not environmentally realistic.
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