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      Subsewershed SARS-CoV-2 Wastewater Surveillance and COVID-19 Epidemiology Using Building-Specific Occupancy and Case Data

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          Abstract

          To evaluate the use of wastewater-based surveillance and epidemiology to monitor and predict SARS-CoV-2 virus trends, over the 2020–2021 academic year we collected wastewater samples twice weekly from 17 manholes across Virginia Tech’s main campus. We used data from external door swipe card readers and student isolation/quarantine status to estimate building-specific occupancy and COVID-19 case counts at a daily resolution. After analyzing 673 wastewater samples using reverse transcription quantitative polymerase chain reaction (RT-qPCR), we reanalyzed 329 samples from isolation and nonisolation dormitories and the campus sewage outflow using reverse transcription digital droplet polymerase chain reaction (RT-ddPCR). Population-adjusted viral copy means from isolation dormitory wastewater were 48% and 66% higher than unadjusted viral copy means for N and E genes (1846/100 mL to 2733/100 mL/100 people and 2312/100 mL to 3828/100 mL/100 people, respectively; n = 46). Prespecified analyses with random-effects Poisson regression and dormitory/cluster-robust standard errors showed that the detection of N and E genes were associated with increases of 85% and 99% in the likelihood of COVID-19 cases 8 days later (incident–rate ratio (IRR) = 1.845, p = 0.013 and IRR = 1.994, p = 0.007, respectively; n = 215), and one-log increases in swipe card normalized viral copies (copies/100 mL/100 people) for N and E were associated with increases of 21% and 27% in the likelihood of observing COVID-19 cases 8 days following sample collection (IRR = 1.206, p < 0.001, n = 211 for N; IRR = 1.265, p < 0.001, n = 211 for E). One-log increases in swipe normalized copies were also associated with 40% and 43% increases in the likelihood of observing COVID-19 cases 5 days after sample collection (IRR = 1.403, p = 0.002, n = 212 for N; IRR = 1.426, p < 0.001, n = 212 for E). Our findings highlight the use of building-specific occupancy data and add to the evidence for the potential of wastewater-based epidemiology to predict COVID-19 trends at subsewershed scales.

          Abstract

          Wastewater samples, population estimates, and case outcome data from individual buildings can be used to monitor and predict COVID-19 trends.

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          Presence of SARS-Coronavirus-2 RNA in Sewage and Correlation with Reported COVID-19 Prevalence in the Early Stage of the Epidemic in The Netherlands

          In the current COVID-19 pandemic, a significant proportion of cases shed SARS-Coronavirus-2 (SARS-CoV-2) with their faeces. To determine if SARS-CoV-2 RNA was present in sewage during the emergence of COVID-19 in The Netherlands, sewage samples of six cities and the airport were tested using four qRT-PCR assays, three targeting the nucleocapsid gene (N1–N3) and one the envelope gene (E). No SARS-CoV-2 RNA was detected on February 6, 3 weeks before the first Dutch case was reported. On March 4/5, one or more gene fragments were detected in sewage of three sites, in concentrations of 2.6–30 gene copies per mL. In Amersfoort, N3 was detected in sewage 6 days before the first cases were reported. As the prevalence of COVID-19 in these cities increased in March, the RNA signal detected by each qRT-PCR assay increased, for N1–N3 up to 790–2200 gene copies per mL. This increase correlated significantly with the increase in reported COVID-19 prevalence. The detection of the virus RNA in sewage, even when the COVID-19 prevalence is low, and the correlation between concentration in sewage and reported prevalence of COVID-19, indicate that sewage surveillance could be a sensitive tool to monitor the circulation of the virus in the population.
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            First detection of SARS-CoV-2 in untreated wastewaters in Italy

            Several studies have demonstrated the advantages of environmental surveillance through the monitoring of sewage for the assessment of viruses circulating in a given community (wastewater-based epidemiology, WBE). During the COVID-19 public health emergency, many reports have described the presence of SARS-CoV-2 RNA in stools from COVID-19 patients, and a few studies reported the occurrence of SARS-CoV-2 in wastewaters worldwide. Italy is among the world's worst-affected countries in the COVID-19 pandemic, but so far there are no studies assessing the presence of SARS-CoV-2 in Italian wastewaters. To this aim, twelve influent sewage samples, collected between February and April 2020 from Wastewater Treatment Plants in Milan and Rome, were tested adapting, for concentration, the standard WHO procedure for Poliovirus surveillance. Molecular analysis was undertaken with three nested protocols, including a newly designed SARS-CoV-2 specific primer set. SARS-CoV-2 RNA detection was accomplished in volumes of 250 mL of wastewaters collected in areas of high (Milan) and low (Rome) epidemic circulation, according to clinical data. Overall, 6 out of 12 samples were positive. One of the positive results was obtained in a Milan wastewater sample collected a few days after the first notified Italian case of autochthonous SARS-CoV-2. The study confirms that WBE has the potential to be applied to SARS-CoV-2 as a sensitive tool to study spatial and temporal trends of virus circulation in the population.
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              SARS-CoV-2 in wastewater: potential health risk, but also data source

              Since the first publications reporting the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in faeces, 1 it became clear that human wastewater might contain the novel coronavirus. From Feb 17, 2020, onwards, we took 24-h 10 L samples once a week from human wastewater collected at Amsterdam Airport Schiphol (Haarlemmermeer, Netherlands) for virus analyses. Samples tested positive for virus RNA by quantitative RT-PCR methodology 4 days after the first cases of coronavirus disease 2019 (COVID-19) were identified in the Netherlands on Feb 27, 2020 (unpublished data). This could be explained by virus excretion from potentially symptomatic, asymptomatic, or presymptomatic individuals passing through the airport. Furthermore, human wastewater sampled near the first Dutch cases in Tilburg, Netherlands, also tested positive for the presence of viral RNA within a week of the first day of disease onset (unpublished data). 2 These findings indicate that wastewater could be a sensitive surveillance system and early warning tool, as was previously shown for poliovirus. 3 To our knowledge, this detection in the Netherlands is the first report of SARS-CoV-2 in wastewater. Whether SARS-CoV-2 is viable under environmental conditions that could facilitate faecal–oral transmission is not yet clear. However, evidence exists of potential community spread, with the virus spreading easily and sustainably in the community in some affected geographic areas such as China. 4 A case has also been reported in the USA in which the individual had not been exposed to anyone known to be infected with SARS-CoV-2 and had not travelled to countries in which the virus is circulating. 5 Potential enteric transmission also has implications for those working with human waste and wastewater, for whom WHO guidance has been developed specifically in relation to COVID-19. Overall, the provision of safe water, sanitation, and hygienic conditions can offer protection from any infectious disease, including COVID-19. Enteric transmission of SARS-CoV-2 is possible and exposure to SARS-CoV-2 in wastewater could pose a health risk. But environmental surveillance of SARS-CoV-2 could serve as a data source, indicating if the virus is circulating in the human population. Previously, this tool has been successfully applied for preclinical identification of Aichi virus. 6 The possibility of faecal–oral transmission of COVID-19 has implications, especially in areas with poor sanitation where diagnostic capacity might be limited, such as Africa. Wastewater surveillance, especially in areas with a scarcity of data, might be informative, as we have previously shown in monitoring antibiotic resistance on a global scale. 7
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                Author and article information

                Journal
                ACS ES T Water
                ACS ES T Water
                ew
                aewcaa
                ACS Es&t Water
                American Chemical Society
                2690-0637
                13 May 2022
                : acsestwater.2c00059
                Affiliations
                []Department of Population Health Sciences, Virginia Tech , Blacksburg, Virginia 24061, United States
                []Department of Civil and Environmental Engineering, Virginia Tech , Blacksburg, Virginia 24061, United States
                [§ ]Department of Statistics, Virginia Tech , Blacksburg, Virginia 24061, United States
                []Hampton Roads Sanitation District , Virginia Beach, Virginia 23455, United States
                []Molecular Diagnostics Laboratory, Fralin Biomedical Research Institute, Virginia Tech , Roanoke, Virginia 24016, United States
                [# ]Integrated Cellular Responses Laboratory, Fralin Biomedical Research Institute at VTC , Roanoke, Virginia 24016, United States
                []Department of Biological Sciences, Virginia Tech , Blacksburg, Virginia 24061, United States
                Author notes
                Author information
                https://orcid.org/0000-0002-9917-8647
                https://orcid.org/0000-0002-8115-7709
                https://orcid.org/0000-0002-3191-6244
                https://orcid.org/0000-0003-2654-5132
                Article
                10.1021/acsestwater.2c00059
                9128018
                37552724
                e0470414-3092-4678-a3b7-c1ec3b857b79
                © 2022 American Chemical Society

                This article is made available via the PMC Open Access Subset for unrestricted RESEARCH re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.

                History
                : 31 January 2022
                : 27 April 2022
                : 25 April 2022
                Funding
                Funded by: National Science Foundation, doi 10.13039/100000001;
                Award ID: 1542100
                Funded by: Institute for Critical Technologies and Applied Science, Virginia Tech, doi 10.13039/100011501;
                Award ID: NA
                Funded by: National Science Foundation, doi 10.13039/100000001;
                Award ID: 2125798
                Funded by: National Science Foundation, doi 10.13039/100000001;
                Award ID: 2004751
                Funded by: National Science Foundation, doi 10.13039/100000001;
                Award ID: 1545756
                Categories
                Article
                Custom metadata
                ew2c00059
                ew2c00059

                wastewater-based surveillance,wastewater-based epidemiology,subsewershed,covid-19,sars-cov-2,digital droplet pcr

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