Evaluation of SARS-CoV-2 RNA Presence in Treated and Untreated Hospital Sewage.

Wastewater-based epidemiology (WBE) is a potential approach for determining the viral prevalence in a community. In the wake of the COVID-19 pandemic, researchers have begun to pay close attention to the presence of SARS-COV-2 RNA in various wastewaters. The potential for detecting SARS-CoV-2 RNA in hospital sewage could make it an invaluable resource for epidemiological studies. In this regard, two specialized hospitals dedicated to COVID-19 patients were chosen for this investigation. Both hospitals utilize the same wastewater treatment systems. The influent and effluents of the two hospitals were sampled in May and June of 2021, and the samples were evaluated for their chemical properties. According to the findings of this study, the wastewater qualities of the two studied hospitals were within the standard ranges. The sewage samples were concentrated using ultrafiltration and PEG precipitation techniques. The E and S genes were studied with RT-qPCR commercial kits. We found E gene of SARS-CoV-2 in 83.3% (5/6) and 66.6% (4/6) of wastewater samples from hospital 1 and hospital 2, respectively, using ultrafiltration concentration method. Wastewater samples taken after chlorine treatment accounted for 16.6% of all positive results. In addition, due to the small sample size, there was no significant correlation (p > 0.05) between the presence of SARS-CoV-2 in wastewater and the number of COVID-19 cases. Hospitals may be a source of SARS-CoV-2 pollution, thus it is important to monitor and enhance wastewater treatment systems to prevent the spread of the virus and safeguard the surrounding environment.

Expanding the Pathogen Panel in Wastewater Epidemiology to Influenza and Norovirus.

Since the start of the 2019 pandemic, wastewater-based epidemiology (WBE) has proven to be a valuable tool for monitoring the prevalence of SARS-CoV-2. With methods and infrastructure being settled, it is time to expand the potential of this tool to a wider range of pathogens. We used over 500 archived RNA extracts from a WBE program for SARS-CoV-2 surveillance to monitor wastewater from 11 treatment plants for the presence of influenza and norovirus twice a week during the winter season of 2021/2022. Extracts were analyzed via digital PCR for influenza A, influenza B, norovirus GI, and norovirus GII. Resulting viral loads were normalized on the basis of NH4-N. Our results show a good applicability of ammonia-normalization to compare different wastewater treatment plants. Extracts originally prepared for SARS-CoV-2 surveillance contained sufficient genomic material to monitor influenza A, norovirus GI, and GII. Viral loads of influenza A and norovirus GII in wastewater correlated with numbers from infected inpatients. Further, SARS-CoV-2 related non-pharmaceutical interventions affected subsequent changes in viral loads of both pathogens. In conclusion, the expansion of existing WBE surveillance programs to include additional pathogens besides SARS-CoV-2 offers a valuable and cost-efficient possibility to gain public health information.

A rapid, high-throughput, and sensitive PEG-precipitation method for SARS-CoV-2 wastewater surveillance.

The effective application of wastewater surveillance is dependent on testing capacity and sensitivity to obtain high spatial resolution testing results for a timely targeted public health response. To achieve this purpose, the development of rapid, high-throughput, and sensitive virus concentration methods is urgently needed. Various protocols have been developed and implemented in wastewater surveillance networks so far, however, most of them lack the ability to scale up testing capacity or cannot achieve sufficient sensitivity for detecting SARS-CoV-2 RNA at low prevalence. In the present study, using positive raw wastewater in Hong Kong, a PEG precipitation-based three-step centrifugation method was developed, including low-speed centrifugation for large particles removal and the recovery of viral nucleic acid, and medium-speed centrifugation for the concentration of viral nucleic acid. This method could process over 100 samples by two persons per day to reach the process limit of detection (PLoD) of 3286 copies/L wastewater. Additionally, it was found that the testing capacity could be further increased by decreasing incubation and centrifugation time without significantly influencing the method sensitivity. The entire procedure uses ubiquitous reagents and instruments found in most laboratories to obtain robust testing results. This high-throughput, cost-effective, and sensitive tool will promote the establishment of nearly real-time wastewater surveillance networks for valuable public health information.

Development of an efficient wastewater testing protocol for high-throughput country-wide SARS-CoV-2 monitoring.

Wastewater-based surveillance has been widely used as a non-intrusive tool to monitor population-level transmission of COVID-19. Although various approaches are available to concentrate viruses from wastewater samples, scalable methods remain limited. Here, we sought to identify and evaluate SARS-CoV-2 virus concentration protocols for high-throughput wastewater testing. A total of twelve protocols for polyethylene glycol (PEG) precipitation and four protocols for ultrafiltration-based approaches were evaluated across two phases. The first phase entailed an initial evaluation using a small sample set, while the second phase further evaluated five protocols using wastewater samples of varying SARS-CoV-2 concentrations. Permutations in the pre-concentration, virus concentration and RNA extraction steps were evaluated. Among PEG-based methods, SARS-CoV-2 virus recovery was optimal with 1) the removal of debris prior to processing, 2) 2 h to 24 h incubation with 8% PEG at 4 °C, 3) 4000 xg or 14,000 xg centrifugation, and 4) a column-based RNA extraction method, yielding virus recovery of 42.4-52.5%. Similarly, the optimal protocol for ultrafiltration included 1) the removal of debris prior to processing, 2) ultrafiltration, and 3) a column-based RNA extraction method, yielding a recovery of 38.2%. This study also revealed that SARS-CoV-2 RNA recovery for samples with higher virus concentration were less sensitive to changes in the PEG method, but permutations in the PEG protocol could significantly impact virus yields when wastewater samples with lower SARS-CoV-2 RNA were used. Although both PEG precipitation and ultrafiltration methods resulted in similar SARS-CoV-2 RNA recoveries, the former method is more cost-effective while the latter method provided operational efficiency as it required a shorter turn-around-time (PEG precipitation, 9-23 h; Ultrafiltration, 5 h). The decision on which method to adopt will thus depend on the use-case for wastewater testing, and the need for cost-effectiveness, sensitivity, operational feasibility and scalability.

Selection of surrogate viruses for process control in detection of SARS-CoV-2 in wastewater.

Since SARS-CoV-2 RNA in wastewater is often present at low concentration or under detection limit, ensuring the reliability of detection processes using appropriate process controls is essential. The objective of this study was to evaluate applicability and limitations of candidate surrogate viruses as process controls under combinations of different virus concentration and RNA extraction methods. Detection efficiency of SARS-CoV-2 spiked in wastewater was compared with those of candidate surrogate viruses of bacteriophage ϕ6, pepper mild mottle virus (PMMoV), F-specific coliphage (F-phage), and murine norovirus (MNV). After inactivated SARS-CoV-2 and ϕ6 were spiked in two different wastewaters, the viruses in solid and liquid fractions of wastewater were concentrated by centrifuge and polyethylene glycol (PEG) precipitation, respectively. Viral RNA was extracted by using QIAamp Viral RNA Mini Kit and 3 other commercially available extraction kits, then quantified by reverse transcription-quantitative PCR using CDCN1 assay. Regardless of extraction kits, SARS-CoV-2 was consistently detected with good efficiency from both liquid (11-200%) and solid fractions (7.1-93%). Among the candidate process controls, PMMoV was widely detected at good efficiencies from both liquid and solid fractions regardless of selection of RNA extraction kits. F-phage and MNV also showed good detection efficiencies in most combinations of wastewater fractions and RNA extraction kits. An enveloped virus ɸ6 was found often undetected or to have very low detection efficiency (0.1-4.2%) even when SARS-CoV-2 spiked in wastewater was detected with good efficiency. Consequently, PMMoV is widely applicable as process control for detection of SARS-CoV-2 either in liquid fractions concentrated by PEG precipitation, or in solid fractions concentrated by centrifuge.

Sensitivity of wastewater-based epidemiology for detection of SARS-CoV-2 RNA in a low prevalence setting.

To assist public health responses to COVID-19, wastewater-based epidemiology (WBE) is being utilised internationally to monitor SARS-CoV-2 infections at the community level. However, questions remain regarding the sensitivity of WBE and its use in low prevalence settings. In this study, we estimated the total number of COVID-19 cases required for detection of SARS-CoV-2 RNA in wastewater. To do this, we leveraged a unique situation where, over a 4-month period, all symptomatic and asymptomatic cases, in a population of approximately 120,000, were precisely known and mainly located in a single managed isolation and quarantine facility (MIQF) building. From 9 July to 6 November 2020, 24-hr composite wastewater samples (n = 113) were collected daily from the sewer outside the MIQF, and from the municipal wastewater treatment plant (WWTP) located 5 km downstream. New daily COVID-19 cases at the MIQF ranged from 0 to 17, and for most of the study period there were no cases outside the MIQF identified. SARS-CoV-2 RNA was detected in 54.0% (61/113) at the WWTP, compared to 95.6% (108/113) at the MIQF. We used logistic regression to estimate the shedding of SARS-CoV-2 RNA into wastewater based on four infectious shedding models. With a total of 5 and 10 COVID-19 infectious cases per 100,000 population (0.005% and 0.01% prevalence) the predicated probability of SARS-CoV-2 RNA detection at the WWTP was estimated to be 28 and 41%, respectively. When a proportional shedding model was used, this increased to 58% and 87% for 5 and 10 cases, respectively. In other words, when 10 individuals were actively shedding SARS-CoV-2 RNA in a catchment of 100,000 individuals, there was a high likelihood of detecting viral RNA in wastewater. SARS-CoV-2 RNA detections at the WWTP were associated with increasing COVID-19 cases. Our results show that WBE provides a reliable and sensitive platform for detecting infections at the community scale, even when case prevalence is low, and can be of use as an early warning system for community outbreaks.

Concentration techniques tailored for the detection of SARS-CoV-2 genetic material in domestic wastewater and treatment plant sludge: A review.

Upon the outbreak of COVID-19 pandemic, detection and quantification of SARS-CoV-2 genetic material in domestic wastewater have led to an increase in the efforts to define and implement the wastewater-based epidemiology (WBE). This application provides valuable information to define local contamination monitoring, emergence of COVID-19 and its variants and many other aspects to cope with and control the pandemic. WBE surveillance, however, requires several consecutive steps such as sampling, pretreatment and concentration of samples, and detection and quantification of SARS-CoV-2 genetic material in wastewater. In this review paper, the literature regarding to all these applications reviewed considering their advantages, disadvantages as well as their applicability. A specific emphasis was placed on the last step, detection and quantification since it covers the most critical procedure for concentrating the virus before measurement. Evaluation of the existing data indicating ultrafiltration, polyethylene glycol (PEG) precipitation and electronegative membrane filtration (ENMF) were the most promising techniques for concentration. The ongoing studies are proposed to be continued within the context of standard methods. Future research needs are delineated and suggestions are made for details.

Methods Evaluation for Rapid Concentration and Quantification of SARS-CoV-2 in Raw Wastewater Using Droplet Digital and Quantitative RT-PCR.

Wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging public health tool to understand the spread of Coronavirus Disease 2019 (COVID-19) in communities. The performance of different virus concentration methods and PCR methods needs to be evaluated to ascertain their suitability for use in the detection of SARS-CoV-2 in wastewater. We evaluated ultrafiltration and polyethylene glycol (PEG) precipitation methods to concentrate SARS-CoV-2 from sewage in wastewater treatment plants and upstream in the wastewater network (e.g., manholes, lift stations). Recovery of viruses by different concentration methods was determined using Phi6 bacteriophage as a surrogate for enveloped viruses. Additionally, the presence of SARS-CoV-2 in all wastewater samples was determined using reverse transcription quantitative PCR (RT-qPCR) and reverse transcription droplet digital PCR (RT-ddPCR), targeting three genetic markers (N1, N2 and E). Using spiked samples, the Phi6 recoveries were estimated at 2.6-11.6% using ultrafiltration-based methods and 22.2-51.5% using PEG precipitation. There was no significant difference in recovery efficiencies (p < 0.05) between the PEG procedure with and without a 16 h overnight incubation, demonstrating the feasibility of obtaining same day results. The SARS-CoV-2 genetic markers were more often detected by RT-ddPCR than RT-qPCR with higher sensitivity and precision. While all three SARS-CoV-2 genetic markers were detected using RT-ddPCR, the levels of E gene were almost below the limit of detection using RT-qPCR. Collectively, our study suggested PEG precipitation is an effective low-cost procedure which allows a large number of samples to be processed simultaneously in a routine wastewater monitoring for SARS-CoV-2. RT-ddPCR can be implemented for the absolute quantification of SARS-CoV-2 genetic markers in different wastewater matrices.

Detection of SARS-CoV-2 in wastewater in Japan during a COVID-19 outbreak.

The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater samples has been documented in several countries. Wastewater-based epidemiology (WBE) is potentially effective for early warning of a COVID-19 outbreak. In this study, presence of SARS-CoV-2 RNA in wastewater samples was investigated and was compared with the number of the confirmed COVID-19 cases in the study area during COVID-19 outbreak in Japan. In total, 45 influent wastewater samples were collected from five wastewater treatment plants in Ishikawa and Toyama prefectures in Japan. During the study period, the numbers of confirmed COVID-19 cases in these prefectures increased from 0.3 and 0 to >20 per 100,000 people. SARS-CoV-2 ribonucleic acid (RNA) in the samples was detected using several PCR-based assays. Of the 45 samples, 21 were positive for SARS-CoV-2 according to at least one of the three quantitative RT-PCR assays. The detection frequency increased when the number of total confirmed SARS-CoV-2 cases in 100,000 people exceeded 10 in each prefecture; however, SARS-CoV-2 could also be detected at a low frequency even when the number was below 1.0. SARS-CoV-2 in wastewater could be detected in the early stage of the epidemic, even if the number of confirmed cases potentially underestimates the actual numbers of cases. This suggests that WBE approach can potentially act as an early warning of COVID-19 outbreaks in Japan.

Concentration of infectious SARS-CoV-2 by polyethylene glycol precipitation.

The development of medical countermeasures against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires robust viral assays. Here we have adapted a protocol for polyethylene glycol (PEG)-mediated precipitation of SARS-CoV-2 stocks without the need for ultracentrifugation. Virus precipitation resulted in a ∼1.5 log10 increase in SARS-CoV-2 titres of virus prepared in VeroE6 cells and enabled the infection of several immortalized human cell lines (Caco-2 and Calu-3) at a high multiplicity of infection not practically achievable without virus concentration. This protocol underscores the utility of PEG-mediated precipitation for SARS-CoV-2 and provides a resource for a range of coronavirus research areas.