skip to main content

Title: Direct wastewater extraction as a simple and effective method for SARS-CoV-2 surveillance and COVID-19 community-level monitoring

Wastewater surveillance has proven to be an effective tool to monitor the transmission and emergence of infectious agents at a community scale. Workflows for wastewater surveillance generally rely on concentration steps to increase the probability of detection of low-abundance targets, but preconcentration can substantially increase the time and cost of analyses while also introducing additional loss of target during processing. To address some of these issues, we conducted a longitudinal study implementing a simplified workflow for SARS-CoV-2 detection from wastewater, using a direct column-based extraction approach. Composite influent wastewater samples were collected weekly for 1 year between June 2020 and June 2021 in Athens-Clarke County, Georgia, USA. Bypassing any concentration step, low volumes (280 µl) of influent wastewater were extracted using a commercial kit, and immediately analyzed by RT-qPCR for the SARS-CoV-2 N1 and N2 gene targets. SARS-CoV-2 viral RNA was detected in 76% (193/254) of influent samples, and the recovery of the surrogate bovine coronavirus was 42% (IQR: 28%, 59%). N1 and N2 assay positivity, viral concentration, and flow-adjusted daily viral load correlated significantly with per-capita case reports of COVID-19 at the county-level (ρ = 0.69–0.82). To compensate for the method’s high limit of detection (approximately 106–107 copies l−1 more » in wastewater), we extracted multiple small-volume replicates of each wastewater sample. With this approach, we detected as few as five cases of COVID-19 per 100 000 individuals. These results indicate that a direct-extraction-based workflow for SARS-CoV-2 wastewater surveillance can provide informative and actionable results.

« less
; ; ; ; ; ; ;
Publication Date:
Journal Name:
FEMS Microbes
Oxford University Press
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract 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 concentrationmore »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.« less
  2. Not Known (Ed.)
    Several treatment plants were sampled for raw influent, primary clarifier sludge, return activated sludge (RAS), and anaerobically digested sludge throughout nine weeks during the summer of the COVID-19 pandemic. Primary clarifier sludge had a significantly higher number of SARS-CoV-2 gene copy number per liter (GC/L) than other sludge samples, within a range from 1.0x105 to 1.0x106 GC/L. Gene copy numbers in raw influent significantly correlated with gene copy numbers in RAS in Silver Creek (p-value = 0.007, R2 = 0.681) and East Canyon (p-value = 0.009, R2 = 0.775) WRFs; both of which lack primary clarifiers or industrial pretreatment processes. This data indicates that SARS-CoV-2 gene copies tend to partition into primary clarifier sludges, at which point a significant portion of them are removed through sedimentation. Furthermore, it was found that East Canyon WRF gene copy numbers in influent were a significant predictor of daily cases (p-value = 0.0322, R2 = 0.561), and gene copy numbers in RAS were a significant predictor of weekly cases (p-value = 0.0597, R2 = 0.449). However, gene copy numbers found in primary sludge samples from other plants significantly predicted the number of COVID-19 cases for the following week (t = 2.279) and the weekmore »after that (t = 2.122). These data indicate that SARS-CoV-2 extracted from WRF biosolids may better suit epidemiological monitoring that exhibits a time lag. It also supports the observation that primary sludge removes a significant portion of SARS-CoV-2 marker genes. In its absence, RAS can also be used to predict the number of COVID-19 cases due to direct flow through from influent. This research represents the first of its kind to thoroughly examine SARS-CoV-2 gene copy numbers in biosolids throughout the wastewater treatment process and the relationship between primary, return activated, and anaerobically digested sludge and reported positive COVID-19 cases.« less
  3. Abstract

    SARS-CoV-2 wastewater surveillance (WWS) at wastewater treatment plants (WWTPs) can reveal sewered community COVID-19 prevalence. For unsewered areas using septic tank systems (STSs) or holding tanks, how to conduct WWS remains unexplored. Here, two large STSs serving Zuma Beach (Malibu, CA) were studied. Supernatant and sludge SARS-CoV-2 concentrations from the directly-sampled STSs parameterized a dynamic solid–liquid separation, a mass balance-based model for estimating the infection rate of users. Pumped septage before hauling and upon WWTP disposal was also sampled and assessed. Most (96%) STS sludge samples contained SARS-CoV-2 N1 and N2 genes, with concentrations exceeding the supernatant and increasing with depth while correlating with total suspended solids (TSS). The trucked septage contained N1 and N2 copies which decayed (coefficients: 0.09–0.29 h−1) but remained detectable. Over approximately 5 months starting in December 2020, modeled COVID-19 prevalence estimations among users ranged from 8 to 18%, mirroring a larger metropolitan area for the first 2 months. The approaches herein can inform public health intervention and augment conventional WWS in that: (1) user infection rates for communal holding tanks are estimable and (2) pumped and hauled septage can be assayed to infer where disease is spreading in unsewered areas.

  4. Swanson, Michele S. (Ed.)
    ABSTRACT Wastewater surveillance (WS), when coupled with advanced molecular techniques, offers near real-time monitoring of community-wide transmission of SARS-CoV-2 and allows assessing and mitigating COVID-19 outbreaks, by evaluating the total microbial assemblage in a community. Composite wastewater samples (24 h) were collected weekly from a manhole between December 2020 and November 2021 in Maryland, USA. RT-qPCR results showed concentrations of SARS-CoV-2 RNA recovered from wastewater samples reflected incidence of COVID-19 cases. When a drastic increase in COVID-19 was detected in February 2021, samples were selected for microbiome analysis (DNA metagenomics, RNA metatranscriptomics, and targeted SARS-CoV-2 sequencing). Targeted SARS-CoV-2 sequencing allowed for detection of important genetic mutations, such as spike: K417N, D614G, P681H, T716I, S982A, and D1118H, commonly associated with increased cell entry and reinfection. Microbiome analysis (DNA and RNA) provided important insight with respect to human health-related factors, including detection of pathogens and their virulence/antibiotic resistance genes. Specific microbial species comprising the wastewater microbiome correlated with incidence of SARS-CoV-2 RNA, suggesting potential association with SARS-CoV-2 infection. Climatic conditions, namely, temperature, were related to incidence of COVID-19 and detection of SARS-CoV-2 in wastewater, having been monitored as part of an environmental risk score assessment carried out in this study. In summary, themore »wastewater microbiome provides useful public health information, and hence, a valuable tool to proactively detect and characterize pathogenic agents circulating in a community. In effect, metagenomics of wastewater can serve as an early warning system for communicable diseases, by providing a larger source of information for health departments and public officials. IMPORTANCE Traditionally, testing for COVID-19 is done by detecting SARS-CoV-2 in samples collected from nasal swabs and/or saliva. However, SARS-CoV-2 can also be detected in feces of infected individuals. Therefore, wastewater samples can be used to test all individuals of a community contributing to the sewage collection system, i.e., the infrastructure, such as gravity pipes, manholes, tanks, lift stations, control structures, and force mains, that collects used water from residential and commercial sources and conveys the flow to a wastewater treatment plant. Here, we profile community wastewater collected from a manhole, detect presence of SARS-CoV-2, identify genetic mutations of SARS-CoV-2, and perform COVID-19 risk score assessment of the study area. Using metagenomics analysis, we also detect other microorganisms (bacteria, fungi, protists, and viruses) present in the samples. Results show that by analyzing all microorganisms present in wastewater, pathogens circulating in a community can provide an early warning for contagious diseases.« less
  5. In response to COVID-19, the international water community rapidly developed methods to quantify the SARS-CoV-2 genetic signal in untreated wastewater. Wastewater surveillance using such methods has the potential to complement clinical testing in assessing community health. This interlaboratory assessment evaluated the reproducibility and sensitivity of 36 standard operating procedures (SOPs), divided into eight method groups based on sample concentration approach and whether solids were removed. Two raw wastewater samples were collected in August 2020, amended with a matrix spike (betacoronavirus OC43), and distributed to 32 laboratories across the U.S. Replicate samples analyzed in accordance with the project's quality assurance plan showed high reproducibility across the 36 SOPs: 80% of the recovery-corrected results fell within a band of ±1.15 log 10 genome copies per L with higher reproducibility observed within a single SOP (standard deviation of 0.13 log 10 ). The inclusion of a solids removal step and the selection of a concentration method did not show a clear, systematic impact on the recovery-corrected results. Other methodological variations ( e.g. , pasteurization, primer set selection, and use of RT-qPCR or RT-dPCR platforms) generally resulted in small differences compared to other sources of variability. These findings suggest that a variety of methods are capablemore »of producing reproducible results, though the same SOP or laboratory should be selected to track SARS-CoV-2 trends at a given facility. The methods showed a 7 log 10 range of recovery efficiency and limit of detection highlighting the importance of recovery correction and the need to consider method sensitivity when selecting methods for wastewater surveillance.« less