key: cord-0698871-3c10ecgd authors: Arora, S.; Nag, A.; Sethi, J.; Rajvanshi, J.; Saxena, S.; Shrivastava, S. K.; Gupta, A. B. title: Sewage surveillance for the presence of SARS-CoV-2 genome as a useful wastewater based epidemiology (WBE) tracking tool in India date: 2020-06-20 journal: nan DOI: 10.1101/2020.06.18.20135277 sha: c9ca72cff89022b4735b823463b5757835a5d367 doc_id: 698871 cord_uid: 3c10ecgd The infection with SARS-CoV-2 is reported to be accompanied by the shedding of the virus in stool samples of infected patients. Earlier reports have suggested that COVID-19 agents can be present in the fecal and sewage samples and thus it can be a good indication of the pandemic extent in a community. However, no such studies have been reported in the Indian context so far. Since, several factors like local population physiology, the climatic conditions, sewage composition, and processing of samples could possibly affect the detection of the viral genome, it becomes absolutely necessary to check for the presence of the SARS-CoV-2 in the wastewater samples from wastewater treatment plants (WWTPs) serving different localities of Jaipur city, which has been under red zone (pandemic hotspots) since early April 2020. Samples from different local municipal WWTPs and hospital wastewater samples were collected and wastewater based epidemiology (WBE) studies for the presence of SARS-CoV-2 were carried out using the RT-PCR technique to confirm the presence of different COVID-19 target genes namely S gene, E gene, ORF1ab gene, RdRp gene and N gene in the viral load of wastewater samples. In the present study, the untreated wastewater samples from the municipal WWTPs and hospital wastewater samples showed the presence of SARS-CoV-2 viral genome, which was correlated with the increased number of COVID-19 positive patients from the concerned areas, as per reported in the publically available health data. This is the first study that investigated the presence of SARS-CoV-2 viral genome in wastewater, at higher ambient temperature (above 40{degrees}C), further validating WBE as a potential tool in predicting and mitigating outbreaks. provides the first reported evidence of the presence of SARS-CoV-2 RNA in sewage samples 151 of Jaipur, Rajasthan (India) and these findings demonstrate the applicability of WBE or to the sample and mixed by pulse vortexing for 15 seconds. The mixture was then transferred 200 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 20, 2020. . https://doi.org/10.1101/2020.06.18.20135277 doi: medRxiv preprint to the spin column and centrifuged at 10,000 g followed by sequential washing with three 201 wash buffers provided in the kit followed by centrifugation at 10,000g for 1 minute at each 202 washing step. After complete drying of the spin column, the RNA was eluted out using a 50-203 100 μl elution buffer followed by centrifugation at 12,000 g for 1 minute. reactions began with UNG incubation at 25℃ for 2 minutes, followed by reverse 224 transcription at 53℃ for 10 minutes, activation at 95℃ for 2 minutes and 40 cycles of 225 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 20, 2020. Ramniwas garden WWTP (Site 5) is currently serving the walled city area of Jaipur that 249 includes the major hotspot of the city, the Ramganj area, having maximum reported cases. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 20, 2020. . https://doi.org/10.1101/2020.06.18.20135277 doi: medRxiv preprint Our study reveals that a significant increase in the numbers of positive tested cases was 251 reported within 6-14 days of our first date of sampling (May 4 th , 2020). This increase was 252 even more significant owing to a particular hotspot area of Jaipur city (Central jail), which 253 showed a surge in the number of positive cases within six days of testing of the sample from 254 site 5 (WWTP) that confirmed the presence of the virus. testing. This is critical as even a delay of three days in COVID-19 detection can lead to a 274 potential spread to thousands of people (Mallapaty, 2020). . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 20, 2020. In India and other developing countries, the effluents from wastewater treatment plants find 348 their way to the nearby gardens and agricultural areas for irrigation reuse. In this context, it 349 becomes necessary to validate the presence of the viral genome in the treated effluent 350 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 20, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 20, 2020. . https://doi.org/10.1101/2020.06.18.20135277 doi: medRxiv preprint showed a discrepancy between the samples processed under the two different conditions, as genome before the exponential phase of the epidemic (Wurtzer et al., 2020) , and another to curb the spread of the disease. Therefore, our outcomes support the proposition that WBE 426 could be used as an early warning tool to monitor the status of COVID-19 infection within a 427 community. Additionally, we believe that this environmental surveillance could be used as an 428 instrument to drive the right decisions to reduce the risk of lifting restrictions too early. For 429 instance, a key question is how to reduce the risk of a "second wave" and/or recurring local CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 20, 2020. . https://doi.org/10.1101/2020.06.18.20135277 doi: medRxiv preprint CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 20, 2020. . https://doi.org/10.1101/2020.06.18.20135277 doi: medRxiv preprint CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 20, 2020. . https://doi.org/10.1101/2020.06.18.20135277 doi: medRxiv preprint CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 20, 2020. . https://doi.org/10.1101/2020.06.18.20135277 doi: medRxiv preprint naming-the-coronavirus-disease-(covid-2019)-624 andthe-virus-that-causes-it 625 36. World Health Organization SARS-CoV-2 titers in wastewater are higher than expected from clinically confirmed 634 cases Prolonged presence of SARS-CoV-2 viral RNA in faecal samples Evaluation of lockdown impact on SARS-CoV-2 dynamics through Human intestinal tract serves as an alternative infection route for Middle East respiratory 648 syndrome coronavirus A pneumonia outbreak associated with a new coronavirus of probable bat origin. 651 nature Evidence for gastrointestinal infection of SARS-CoV-2