Comparative analysis of rapid concentration methods for the recovery of SARS-CoV-2 and quantification of human enteric viruses and a sewage-associated marker gene in untreated wastewater.

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.

Decay of SARS-CoV-2 and surrogate murine hepatitis virus RNA in untreated wastewater to inform application in wastewater-based epidemiology.

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.

Detection of SARS-CoV-2 RNA in commercial passenger aircraft and cruise ship wastewater: a surveillance tool for assessing the presence of COVID-19 infected travellers.

BACKGROUND: Wastewater-based epidemiology (WBE) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be an important source of information for coronavirus disease 2019 (COVID-19) management during and after the pandemic. Currently, governments and transportation industries around the world are developing strategies to minimize SARS-CoV-2 transmission associated with resuming activity. This study investigated the possible use of SARS-CoV-2 RNA wastewater surveillance from airline and cruise ship sanitation systems and its potential use as a COVID-19 public health management tool. METHODS: Aircraft and cruise ship wastewater samples (n = 21) were tested for SARS-CoV-2 using two virus concentration methods, adsorption-extraction by electronegative membrane (n = 13) and ultrafiltration by Amicon (n = 8), and five assays using reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and RT-droplet digital PCR (RT-ddPCR). Representative qPCR amplicons from positive samples were sequenced to confirm assay specificity. RESULTS: SARS-CoV-2 RNA was detected in samples from both aircraft and cruise ship wastewater; however concentrations were near the assay limit of detection. The analysis of multiple replicate samples and use of multiple RT-qPCR and/or RT-ddPCR assays increased detection sensitivity and minimized false-negative results. Representative qPCR amplicons were confirmed for the correct PCR product by sequencing. However, differences in sensitivity were observed among molecular assays and concentration methods. CONCLUSIONS: The study indicates that surveillance of wastewater from large transport vessels with their own sanitation systems has potential as a complementary data source to prioritize clinical testing and contact tracing among disembarking passengers. Importantly, sampling methods and molecular assays must be further optimized to maximize detection sensitivity. The potential for false negatives by both wastewater testing and clinical swab testing suggests that the two strategies could be employed together to maximize the probability of detecting SARS-CoV-2 infections amongst passengers.

Comparison of virus concentration methods for the RT-qPCR-based recovery of murine hepatitis virus, a surrogate for SARS-CoV-2 from untreated wastewater.

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.