Dataset Information

Temporal detection and phylogenetic assessment of SARS-CoV-2 in municipal wastewater.

ABSTRACT: SARS-CoV-2 has recently been detected in feces, which indicates that wastewater may be used to monitor viral prevalence in the community. Here we use RT-qPCR to monitor wastewater for SARS-CoV-2 RNA over a 52-day time course. We show that changes in SARS-CoV-2 RNA concentrations correlate with local COVID-19 epidemiological data (R₂=0.9), though detection in wastewater trails symptom onset dates by 5-8 days. We determine a near complete (98.5%) SARS-CoV-2 genome sequence from the wastewater and use phylogenic analysis to infer viral ancestry. Collectively, this work demonstrates how wastewater can be used as a proxy to monitor viral prevalence in the community and how genome sequencing can be used for high-resolution genotyping of the predominant strains circulating in a community.

SUBMITTER: Nemudryi A

PROVIDER: S-EPMC7276038 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Temporal detection and phylogenetic assessment of SARS-CoV-2 in municipal wastewater.

Nemudryi Artem A Nemudraia Anna A Wiegand Tanner T Surya Kevin K Buyukyoruk Murat M Vanderwood Karl K KK Wilkinson Royce R Wiedenheft Blake B

medRxiv : the preprint server for health sciences 20200705

SARS-CoV-2 has recently been detected in feces, which indicates that wastewater may be used to monitor viral prevalence in the community. Here we use RT-qPCR to monitor wastewater for SARS-CoV-2 RNA over a 52-day time course. We show that changes in SARS-CoV-2 RNA concentrations correlate with local COVID-19 epidemiological data (R<sub>2</sub>=0.9), though detection in wastewater trails symptom onset dates by 5-8 days. We determine a near complete (98.5%) SARS-CoV-2 genome sequence from the wast ...[more]

PMID: 32511611

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Project description:IntroductionCoronavirus SARS-CoV-2 is a causative agent responsible for the current global pandemic situation known as COVID-19. Clinical manifestations of COVID-19 include a wide range of symptoms from mild (i.e., cough, fever, dyspnea) to severe pneumonia-like respiratory symptoms. SARS-CoV-2 has been demonstrated to be detectable in the stool of COVID-19 patients. Waste-based epidemiology (WBE) has been shown as a promising approach for early detection and monitoring of SARS-CoV-2 in the local population performed via collection, isolation, and detection of viral pathogens from environmental sources.MethodsIn order to select the optimal protocol for monitoring the COVID-19 epidemiological situation in region Turiec, Slovakia, we (1) compared methods for SARS-CoV-2 separation and isolation, including virus precipitation by polyethylene glycol (PEG), virus purification via ultrafiltration (Vivaspin®) and subsequent isolation by NucleoSpin RNA Virus kit (Macherey-Nagel), and direct isolation from wastewater (Zymo Environ Water RNA Kit); (2) evaluated the impact of water freezing on SARS- CoV-2 separation, isolation, and detection; (3) evaluated the role of wastewater filtration on virus stability; and (4) determined appropriate methods including reverse transcription-droplet digital PCR (RT-ddPCR) and real-time quantitative polymerase chain reaction (RT-qPCR) (targeting the same genes, i.e., RdRp and gene E) for quantitative detection of SARS-CoV-2 in wastewater samples.Results(1) Usage of Zymo Environ Water RNA Kit provided superior quality of isolated RNA in comparison with both ultracentrifugation and PEG precipitation. (2) Freezing of wastewater samples significantly reduces the RNA yield. (3) Filtering is counterproductive when Zymo Environ Water RNA Kit is used. (4) According to the specificity and sensitivity, the RT-ddPCR outperforms RT-qPCR.DiscussionThe results of our study suggest that WBE is a valuable early warning alert and represents a non-invasive approach to monitor viral pathogens, thus protects public health on a regional and national level. In addition, we have shown that the sensitivity of testing the samples with a nearer detection limit can be improved by selecting the appropriate combination of enrichment, isolation, and detection methods.

Project description:The primary concentration and molecular process are critical to implement wastewater-based epidemiology for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the previously developed methods were optimized for nonenveloped viruses. Few studies evaluated if the methods are applicable to the efficient recovery of enveloped viruses from various types of raw sewage. This study aims (1) to compare the whole process recovery of Pseudomonas phage φ6, a surrogate for enveloped viruses, among combinations of primary concentration [ultrafiltration (UF), electronegative membrane vortex (EMV), and polyethylene glycol precipitation (PEG)] and RNA extraction methods (spin column-based method using QIAamp Viral RNA Mini Kit and acid guanidinium thiocyanate-phenol-chloroform extraction using TRIzol reagent) for three types of raw sewage and (2) to test the applicability of the method providing the highest φ6 recovery to the detection of SARS-CoV-2 RNA. Among the tested combinations, PEG+TRIzol provided the highest φ6 recovery ratio of 29.8% to 49.8% (geometric mean). UF + QIAamp Viral RNA Mini Kit provided the second highest φ6 recovery of 6.4% to 35.8%. The comparable φ6 recovery was observed for UF + TRIzol (13.8-30.0%). PEG + QIAamp Viral RNA Mini Kit provided only 1.4% to 3.0% of φ6 recovery, while coliphage MS2, a surrogate for nonenveloped viruses, was recovered comparably with PEG + TRIzol. This indicated that the nonenveloped surrogate (MS2) did not necessarily validate the efficient recovery for enveloped viruses. EMV + QIAamp Viral RNA Mini Kit provided significantly different φ6 recovery (1.6-21%) among the types of raw sewage. Then, the applicability of modified PEG + TRIzol was examined for the raw sewage collected in Tokyo, Japan. Of the 12 grab samples, 4 were positive for SARS-CoV-2 CDC N1 and N3 assay. Consequently, PEG + TRIzol provided the highest φ6 recovery and allowed for the detection of SARS-CoV-2 RNA from raw sewage.

Project description:Wastewater-based genomic surveillance of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus shows promise to complement genomic epidemiology efforts. Multiplex tiling PCR is a desirable approach for targeted genome sequencing of SARS-CoV-2 in wastewater due to its low cost and rapid turnaround time. However, it is not clear how different multiplex tiling PCR primer schemes or wastewater sample matrices impact the resulting SARS-CoV-2 genome coverage. The objective of this work was to assess the performance of three different multiplex primer schemes, consisting of 150-bp, 400-bp, and 1,200-bp amplicons, as well as two wastewater sample matrices, influent wastewater and primary sludge, for targeted genome sequencing of SARS-CoV-2. Wastewater samples were collected weekly from five municipal wastewater treatment plants (WWTPs) in the Metro Vancouver region of British Columbia, Canada during a period of increased coronavirus disease 19 (COVID-19) case counts from February to April 2021. RNA extracted from clarified influent wastewater provided significantly higher genome coverage (breadth and median depth) than primary sludge samples across all primer schemes. Shorter amplicons appeared to be more resilient to sample RNA degradation but were hindered by greater primer pool complexity in the 150-bp scheme. The identified optimal primer scheme (400 bp) and sample matrix (influent) were capable of detecting the emergence of mutations associated with genomic variants of concern, for which the daily wastewater load significantly correlated with clinical case counts. Taken together, these results provide guidance on best practices for implementing wastewater-based genomic surveillance and demonstrate its ability to inform epidemiology efforts by detecting genomic variants of concern circulating within a geographic region. IMPORTANCE Monitoring the genomic characteristics of the SARS-CoV-2 virus circulating in a population can shed important insights into epidemiological aspects of the COVID-19 outbreak. Sequencing every clinical patient sample in a highly populous area is a difficult feat, and thus sequencing SARS-CoV-2 RNA in municipal wastewater offers great promise to augment genomic surveillance by characterizing a pooled population sample matrix, particularly during an escalating outbreak. Here, we assess different approaches and sample matrices for rapid targeted genome sequencing of SARS-CoV-2 in municipal wastewater. We demonstrate that the optimal approach is capable of detecting the emergence of SARS-CoV-2 genomic variants of concern, with strong correlations to clinical case data in the province of British Columbia. These results provide guidance on best practices on, as well as further support for, the application of wastewater genomic surveillance as a tool to augment current genomic epidemiology efforts.

Dataset Information

Temporal detection and phylogenetic assessment of SARS-CoV-2 in municipal wastewater.

Publications

Temporal detection and phylogenetic assessment of SARS-CoV-2 in municipal wastewater.

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