Temporal detection and phylogenetic assessment of SARS-CoV-2 in municipal wastewater.
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ABSTRACT:
SUBMITTER: Nemudryi A
PROVIDER: S-EPMC7276038 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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 74-day time course. We show that changes in SARS-CoV-2 RNA concentrations follow symptom onset gathered by retrospective interview of patients but precedes clinical test results. In addition, we determine a nearly complete (98.5%) SARS-CoV-2 genome sequence from wastewater and use phylogenetic 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 genotyping viral strains circulating in a community.
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Project description:A multiplex reverse transcription quantitative PCR (RT-qPCR)-based method was designed for the simultaneous detection of different SARS-CoV-2 genes. In this study, we used three target genes encoding for the nucleocapsid 1 and 3 (N1, N3), and the spike (S) proteins, all commonly used in the detection of SARS-CoV-2 in human and environmental samples. The performance of the multiplex assay, compared to the single assay was assessed for the standard calibration curve, required for absolute quantification, and then, for the real environmental samples to detect SARS-CoV-2. For this latter, four environmental samples were collected at a local wastewater treatment plant (WWTP). The results showed that the cycle threshold (Ct) values of the multiplex were comparable to the values obtained by the singleplex PCR. The amplification of the three target genes indicated the presence of SARS-CoV-2 in the four water samples with an increasing trend in February and these results were confirmed in the multiplex approach, showing the robustness of this method and its applicability for the relative abundance analysis among the samples. Overall, both the laboratory and field work results demonstrated that the multiplex PCR assay developed in this study could provide a method for SARS-CoV-2 detection as robust as the single qPCR, but faster and cost-effective, reducing by three times the number of reactions, and consequently the handling time and reagents.
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Project description:The current pandemic disease coronavirus (COVID-19) has not only become a worldwide health emergency, but also devoured the global economy. Despite appreciable research, identification of targeted populations for testing and tracking the spread of COVID-19 at a larger scale is an intimidating challenge. There is a need to quickly identify the infected individual or community to check the spread. The diagnostic testing done at large-scale for individuals has limitations as it cannot provide information at a swift pace in large populations, which is pivotal to contain the spread at the early stage of its breakouts. Recently, scientists are exploring the presence of SARS-CoV-2 RNA in the faeces discharged in municipal wastewater. Wastewater sampling could be a potential tool to expedite the early identification of infected communities by detecting the biomarkers from the virus. However, it needs a targeted approach to choose optimized locations for wastewater sampling. The present study proposes a novel fuzzy based Bayesian model to identify targeted populations and optimized locations with a maximum probability of detecting SARS-CoV-2 RNA in wastewater networks. Consequently, real time monitoring of SARS-CoV-2 RNA in wastewater using autosamplers or biosensors could be deployed efficiently. Fourteen criteria such as population density, patients with comorbidity, quarantine and hospital facilities, etc. are analysed using the data of 14 lac individuals infected by COVID-19 in the USA. The uniqueness of the proposed model is its ability to deal with the uncertainty associated with the data and decision maker's opinions using fuzzy logic, which is fused with Bayesian approach. The evidence-based virus detection in wastewater not only facilitates focused testing, but also provides potential communities for vaccine distribution. Consequently, governments can reduce lockdown periods, thereby relieving human stress and boosting economic growth.
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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 ?6, 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 ?6 recovery to the detection of SARS-CoV-2 RNA. Among the tested combinations, PEG+TRIzol provided the highest ?6 recovery ratio of 29.8% to 49.8% (geometric mean). UF + QIAamp Viral RNA Mini Kit provided the second highest ?6 recovery of 6.4% to 35.8%. The comparable ?6 recovery was observed for UF + TRIzol (13.8-30.0%). PEG + QIAamp Viral RNA Mini Kit provided only 1.4% to 3.0% of ?6 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 ?6 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 ?6 recovery and allowed for the detection of SARS-CoV-2 RNA from raw sewage.
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Project description:Respiratory infections, like the current COVID-19 pandemic, target epithelial cells in the respiratory tract. Alveolar macrophages (AMs) are tissue-resident macrophages located within the lung. They play a key role in the early phases of an immune response to respiratory viruses. AMs are likely the first immune cells to encounter SARS-CoV-2 during an infection and their reaction to the virus will have a profound impact on the outcome of the infection. Interferons (IFNs) are antiviral cytokines and among the first cytokines produced upon viral infection. In this study, AMs from non-infectious donors are challenged with SARS-CoV-2. We demonstrate that challenged AMs are incapable of sensing SARS-CoV-2 and of producing an IFN response in contrast to other respiratory viruses, like influenza A virus and Sendai virus, which trigger a robust IFN response. The absence of IFN production in AMs upon challenge with SARS-CoV2 could explain the initial asymptotic phase observed during COVID-19 and argues against AMs being the sources of proinflammatory cytokines later during infection.
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Project description:SARS-CoV-2 has been transmitted and outbreak took place in India during the last week before nationwide 1st lockdown took place. Urban areas found more vulnerable and reported nearly 65% of cases during every phase of lockdown. Mumbai, among four metropolitan cities found huge number of containment zones with nearly 30% of SARS-CoV-2 cases indicating clustering of cases. Most of the containment zones of SARS-CoV-2 cases in Kolkata Municipal Corporation found a significant relation with slum areas. The study primarily tries considering the nature of SARS-CoV-2 cases in different urban centres with the help of cartographic techniques. AHP method has been used to determine the factors responsible for such concentration of SARS-CoV-2 cases with vulnerability assessment (exposure, sensitivity and resilience) and risks. Before nationwide lockdown starts, the share of urban centres found 25% which has been transformed into nearly 60% at the end of 3rd phase of lockdown. Growth rate of SARS-CoV-2 cases found very high for Chennai and Thane with less number of doubling time to nation. Slum concentration and containment density shows a higher degree of correlation in Kolkata Municipal Corporation. Risk map also shows the concentration of cases in central and north Kolkata with higher degree of diseases exposure and sensitivity. Control measures must be taken by the central and state Government to minimise the transmission rate of SARS-CoV-2 mainly urban areas. As urban area contributing a higher share of SARS-CoV-2 cases, a proper management plan must be enforce. Electronic supplementary material The online version of this article (10.1007/s41324-020-00354-6) contains supplementary material, which is available to authorized users.
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