key: cord-0781650-pqpzbmlu authors: Bar Or, I.; Yaniv, K.; Shagan, M.; Ozer, E.; Erster, O.; Mendelson, E.; Mannasse, B.; Shirazi, R.; Kramarsky-Winter, E.; Nir, O.; Abu-Ali, H.; Ronen, Z.; Rinott, E.; Lewis, Y. E.; Friedler, E. F.; Paitan, Y.; Bitkover, E.; Berchenko, Y.; Kushmaro, A. title: Regressing SARS-CoV-2 sewage measurements onto COVID-19 burden in the population: a proof-of-concept for quantitative environmental surveillance date: 2020-05-01 journal: nan DOI: 10.1101/2020.04.26.20073569 sha: a6ad1556b611a304026e3ed7cf60c39043ac99e3 doc_id: 781650 cord_uid: pqpzbmlu SARS-CoV-2 is an RNA virus, a member of the coronavirus family of respiratory viruses that includes SARS-CoV-1 and MERS. COVID-19, the clinical syndrome caused by SARS-CoV-2, has evolved into a global pandemic with more than 2,900,000 people infected. It has had an acute and dramatic impact on health care systems, economies, and societies of affected countries within these few months. Widespread testing and tracing efforts are employed in many countries in order to contain and mitigate this pandemic. Recent data has indicated that fecal shedding of SARS-CoV-2 is common, and that the virus can be detected in wastewater. This indicates that wastewater monitoring is a potentially efficient tool for epidemiological surveillance of SARS-CoV-2 infection in large populations at relevant scales. Collecting raw sewage data, representing specific districts, and crosslinking this data with the number of infected people from each location, will enable us to derive and provide quantitative surveillance tools. In particular, this will provide important means to (i) estimate the extent of outbreaks and their spatial distributions, based primarily on in-sewer measurements (ii) manage the early-warning system quantitatively and efficiently (and similarly, verify disease elimination). Here we report the development of a virus concentration method using PEG or alum, providing an important a tool for detection of SARS-CoV-2 RNA in sewage and relating it to the local populations and geographic information. This will provide a proof of concept for the use of sewage associated virus data as a reliable epidemiological tool. Waterborne pathogens, including viruses, bacteria and protozoa can be shed into the urban water cycle via sewers, (Gormley et al. 2017 and 2020) urban runoff, agricultural runoff and wastewater discharges (Arnone and Walling, 2007; La Rosa et al., 2012) . Indeed, it has been reported that there are high concentrations of virus particles in wastewater treatment plants (WWTP), varying from 10 8 to 10 10 viral particles per milliliter (Otawa et al., 2007) . Coronavirus SARS-CoV-2 is a novel RNA virus belonging to a group of viruses that includes amongst others SARS and MERS. SARS-CoV-2 is one of more than 37 coronaviruses in the Coronaviridae family, within the order Nidovirales, and it is currently causing a major pandemic with over 2,900,000 people infected globally. It causes COVID-19, a disease that has daunting effect on health care systems, economies, and societies of affected countries. As a member of the Coronaviridae, which includes viruses known to cause respiratory and/or intestinal infections, SARS-CoV-2 spreads primarily via micro droplets, reflecting its survivorship in humid environments (Chin et al. 2020 regarding polio by analyzing two unique data sets collected during a "natural experiment" All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 May 1, 2020. Here we report a similar study aimed at developing similar quantitative tools for SARS-CoV-2 in wastewater. These results will enable early identification and spatial-based monitoring of future outbreaks, and could be used to confirm virus elimination or to validate the need for more containment efforts. Israel (see Table 2 ) as well as samples of raw sewage from different districts from the Tel Aviv metropolis. Sampling equipment was sanitized and properly sterilized (cooler, sampling bottles, biohazard bags, etc.). In addition, we used automatic samplers at targeted hot-spot areas for 24 hours. Around 200 ml were collected every 30 min for the 24 hours. Samples (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The extracted viral nucleic acids were reverse transcribed and qPCR for the cDNA was performed using mixture containing Fast Start Universal Probe Master, forward and reverse primers including TaqMan probe. Duplicate qPCR amplification was performed in a Step One Plus real-time PCR system. Both positive-control and negative-control assays were performed for quality control. Serial dilutions of plasmid DNA containing the SARS-CoV-2 E gene used to generate the standard curve. This study enabled us to establish novel virus concentration methods (Tables 1 & 2) . These methods were validated using sewage samples from the Dan Panorama Hotel in Tel Aviv, which currently functions as a COVID-19 isolation facility. Results show that the most efficient concentration methods were achieved using PEG and Alum reaching positive values with qPCR Ct of 33 and 33.6 for PEG and Alum, respectively (Table 1) . We further established a proof-of-concept for our ability to detect SARS-CoV-2 RNA from raw sewage samples from different location in Israel (Table 3) (Table 3 ). In conclusion, we present a preliminary study demonstrating a proof-of-concept for the detection of SARS-CoV-2 RNA in sewage. We also present a linear "dose-dependent" curve as a tool for viral surveillance in environmental samples. However, we urge the readers to be cautious in their use of fig. 1 as a basis for their own data since: (i) our previous work (Berchenko et al. 2017 ) showed how different localities should be compared by considering daily sewage production as a measure of the local population size, and (ii) our work is still preliminary and ongoing, therefore further data is still warranted. Furthermore, we present two methods for viral isolation from wastewater by concentration with PEG and/or alum. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 May 1, 2020. Understanding the ecology of SARS-CoV-2 in human waste could lead to environmental applications including efficient monitoring and surveillance of SARS-CoV-2. In the future, this study may also provide tools to for sewage monitoring as an early warning alarm for SARS-CoV-2 outbreaks in the population. (which was not certified by peer review) 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 May 1, 2020. (which was not certified by peer review) 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 May 1, 2020. . https://doi.org/10.1101/2020.04.26.20073569 doi: medRxiv preprint Waterborne pathogens in urban watersheds Estimation of polio infection prevalence from environmental surveillance data Stability of SARS-CoV-2 in different environmental conditions Mining Population Exposure and Community Health via Wastewater-Based Epidemiology Novel coronavirus infection and gastrointestinal tract Pathogen crosstransmission via building sanitary plumbing systems in a full scale pilot test-rig COVID-19: mitigating transmission via wastewater plumbing systems Shin Giek concentration and extraction of viruses from sewage in the context of metagenomic sequencing COVID-19: faecal-oral transmission? Temporal dynamics of norovirus determined through monitoring of municipal wastewater by pyrosequencing and virological surveillance of gastroenteritis cases Clinical Evaluation of Safety and Immunogenicity of PADRE-Cytomegalovirus (CMV) and Tetanus-CMV Fusion Peptide Vaccines With or Without Adjuvant SARS-CoV-2 in wastewater: potential health risk, but also data source. The lancet Presence of SARS-Coronavirus-2 in sewage Editorial Perspectives: 2019 novel coronavirus (SARS-CoV-2): what is its fate in urban water cycle and how can the water research community respond? Abundance, diversity, and dynamics of viruses on microorganisms in activated sludge processes Occurrence of traditional and alternative fecal indicators in tropical urban environments under different land use patterns Concentration and detection of SARS coronavirus in sewage from Xiao Tang Shan Hospital and the 309th Hospital of the Chinese People's Liberation Army SARS-CoV-2 titers in wastewater are higher than expected from clinically confirmed cases Time course quantitative detection of SARS-CoV-2 in Parisian wastewaters correlates with COVID-19 confirmed cases Jinling Tang, Kang Zhang and Sitang Gong. 2020. Characteristics of pediatric SARS-CoV-2 infection and potential evidence for persistent fecal viral shedding