key: cord-350242-4u1iyf0p authors: Yaniv, K.; Shagan, M.; Kramarsky-Winter, E.; Indenbaum, V.; Weil, M.; Elul, M.; Erster, O.; Sela Brown, A.; Mendelson, E.; Mannasse, B.; Shirazi, R.; Lakkakula, S.; Miron, O.; Rinott, E.; Baibich, R. G.; Bigler, I.; Malul, M.; Rishti, R.; Brenner, A.; Lewis, Y. E.; Friedler, E.; Gilboa, Y.; Sabach, S.; Alfiya, Y.; Cheruti, U.; Davidovitch, N.; Bilenko, N.; Moran-Gilad, J.; Berchenko, Y.; Bar-Or, I.; Kushmaro, A. title: City-level SARS-CoV-2 sewage surveillance date: 2020-10-21 journal: nan DOI: 10.1101/2020.10.19.20215244 sha: doc_id: 350242 cord_uid: 4u1iyf0p The COVID-19 pandemic created a global crisis impacting not only healthcare systems, but also world economies and society. Recent data have indicated that fecal shedding of SARS-CoV-2 is common, and that viral RNA can be detected in wastewater. This suggests that wastewater monitoring is a potentially efficient tool for both epidemiological surveillance, and early warning for SARS-CoV-2 circulation at the population level. In this study we sampled an urban wastewater infrastructure in the city of Ashkelon, Israel, during the end of the first COVID-19 wave in May 2020 when the number of infections seemed to be waning. We were able to show varying presence of SARS-CoV-2 RNA in wastewater from several locations in the city during two sampling periods. This was expressed as a new index, Normalized Viral Load (NVL), which can be used in different area scales to define levels of virus activity such as red (high) or green (no), and to follow morbidity in the population at tested area. Our index showed the rise in viral load between the two sampling periods (one week apart) and indicated an increase in morbidity that was evident a month later in the population. Thus, this methodology may provide an early indication for SARS-CoV-2 infection outbreak in a population before an outbreak is clinically apparent. The study was performed in the city of Ashkelon, a Southern coastal city in Israel having a population of approximately 150,000 inhabitants. Ten continuous sewage-sampling units were installed in Ashkelon's wastewater system at nine selected sewer manholes and one at the inlet of the wastewater treatment plant (WWTP). The automated composite samplers were activated during two sampling campaigns, the first during 17-19 of May 2020 and the second on the 25 th of May 2020. Sampling was carried out for 8 hours, from 6:00-14:00 or 07:00-15:00 at each point. The chosen sampling areas included several neighborhoods along 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 October 21, 2020. . https://doi.org/10.1101/2020.10.19.20215244 doi: medRxiv preprint the coast of Ashkelon (tagged as 1 to 4 in Figure 1 ). The sampling points included only household wastewater. They were connected to a central sewer line that was also sampled in designated sewer junction manholes (A to E in Figure 1 ). This central sewer line eventually drains into the municipal WWTP (designated as point H) that treats the city wastewater. For 1 to 4 neighborhoods, the estimated population sizes were 13,500; 5,600; 2,500; 12,500 respectively, based national census data (the Central Bureau of Statistic, https://www.gov.il/en/departments/central_bureau_of_statistics). 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 October 21, 2020. . https://doi.org/10.1101/2020.10.19.20215244 doi: medRxiv preprint Raw sewage samples (1L composite sample) were immediately transferred to the lab under chilled conditions. The samples were kept at -20 o C until processed. Prior to subsampling, the bottles were shaken vigorously and left to rest for 10 minutes. Subsamples of 0.2 mL raw sewage (in duplicate) were then transferred directly into NucleoSpin RNA lysis buffer for RNA extraction (Macherey Nagel, Germany). For creating a standard curve, we used a plasmid containing the full SARS-CoV-2 N gene sequence isolated from Wuhan-Hu-1 (GenBank: NC_045512.2). We prepared standard curves of plasmid log copy number verses Ct value using serial dilutions of the plasmid. We then performed linear regression between the log copy number and the Ct values from the RT-qPCR results. Using the linear equation, we calculated copy number of N1 gene in sewage samples reported in this study. RT-qPCR amplification was performed according to user manual recommendation using CDC's N1 and N2 primers and probe sets and One Step PrimeScript III RT-qPCR mix (TAKARA, Japan). RT-qPCR amplification was executed using Step One Plus real-time PCR system (Applied Biosystems, Thermo Scientific). In parallel to the N1 test, each RNA sample was spiked with N gene plasmid in known concentrations to rule out any amplification inhibition. A second quality control step was carried out by adding MS2 phage to the lysis buffer step as process control for RNA extraction. The analytical LOD (limit of detection) for N gene target was determined to be < 10 target copies per qPCR reaction. Viral copy number per 1L was calculated using the N1 standard curve equation ( Figure S1 ). If Ct value for N1 was ND, then N2 Ct value was used for the calculation. A new index, namely Normalized Viral Load (NVL) was defined. It is expressing the estimated number of SARS-CoV-2 RNA copies per 1,000 people. It is based on quantification of the cumulative number of copies during several hours of composite wastewater sample collection (8 hours in 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 October 21, 2020. . https://doi.org/10.1101/2020. 10.19.20215244 doi: medRxiv preprint this study) in a specific location, within a steady timeframe of a day (see equation1). It can be fitted to various scales such as a street, a quarter, a small town, or a large city. Sampling location, duration, and method (preferably composite) should be unified to enable reliable use of the NVL. If the sampling is extended to 24 hours, then the NVL can be expressed as number of SARS-CoV-2 RNA copies per 1,000 people per day. Nevertheless, even sampling of several hours during a steady timeframe can serve as a reliable relative index to be assessed chronologically. This index can assist define levels of virus activity in a semiquantitative fashion (e.g. using a 'traffic-light' scheme), and to follow morbidity of the population in the tested area. . Ashkelon was chosen for surveillance of SARS-CoV-2 in sewage due to a relatively low COVID-19 prevalence during the time of study following a national lockdown period during Table S1 ). In this study we carried out two sampling campaigns during May 2020, a period with limited reports of COVID-19 in the city and with a very low prevalence of COVID-19 in all 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 October 21, 2020. sampling is applied only to the WWTP or larger geographical areas, especially when the prevalence is low and/or highly localized. 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. (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 October 21, 2020. In the second sampling campaign all sampling points were positive, with NVL between 5.9·10 11 to 2.77·10 13 (copy number/1000 person). Unfortunately, due to technical problem point 1 sample is missing from the second campaign. Interestingly, the viral load in point A increased by an order of magnitude from NVL of 3.63·10 12 to 2.77·10 13 (copy number/1000 person) between the two sampling campaigns. The low morbidity in the city reported during May, particularly at the end of the month, might have been an under estimation of COVID-19 cases due to limited testing efforts and due to asymptomatic cases (and therefore untested per testing guidelines at the time of study) . The presence of the virus in the sewage despite the low prevalence reported in May attests to the sensitivity of the method. Indeed, a resurgence in COVID-19 cases became evident in the city's population in June-July 2020. Understanding the dynamics of SARS-CoV-2 in human excreta could lead to efficient monitoring and surveillance of this virus as well as to underpin the important of such environmental surveillance 9 . Our results also demonstrate the need for an inner-city sewer sampling design to overcome dilution effect that can eventually affect the sensitivity and validity of this surveillance method. Such an inner-city sewer study may assist in geographical localization of disease hotspots. Multiple sampling efforts within a city could 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 October 21, 2020. . 1 1 allow assessment of virus spread in each area and assess the proportion of areas with virus activity in a city, thus predicting imminent resurgences. In conclusion, we present a proof-of-concept study demonstrating the feasibility of SARS-CoV-2 RNA detection in raw sewage originating from the city sewer system that reflects virus circulation in the assessed area. This approach should be further studied and validated as an early warning for SARS-CoV-2 resurgence in urban settings and as a national decision support tool 10, 11 . 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. Raw sewage (1L composite sample) was irradiated for two hours using LP-UV lamp (7 mW/cm2, VL_230.G, Lamp part number T-30.C, Cole Parmer Instrument Co., USA.). During the irradiation process, samples were continuously mixed in order to assure that the samples are properly inactivated. Samples were filtered through 90 mm GF/A glass fiber filter (Whatman, UK). Filtered samples were analyzed for total organic carbon (TOC) and total nitrogen (TN), using total organic carbon analyzer (TOC-V CPH, equipped with total nitrogen measuring unit, TNM-1; Shimadzu, Japan). 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 October 21, 2020. . 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 October 21, 2020. . https://doi.org/10.1101/2020.10.19.20215244 doi: medRxiv preprint Intensified Environmental Surveillance Supporting the Response to Wild Poliovirus Type 1 Silent Circulation in Israel Global Monitoring of Antimicrobial Resistance Based on Metagenomics Analyses of Urban Sewage Faecal Indicators and Human-Specific Microbial Source-Tracking Markers in Sewage Clinical Features of Patients Infected with 2019 Novel Coronavirus in First Case of 2019 Novel Coronavirus in the United States Epidemiological and Clinical Characteristics of 99 Cases of 2019 Novel Coronavirus Pneumonia in Wuhan, China: A Descriptive Study SARS-CoV-2 Titers in Wastewater Foreshadow Dynamics and Clinical Presentation of New COVID-19 Cases Regressing SARS-CoV-2 Sewage Measurements onto COVID-19 Burden in the Population: A Proof-of-Concept for Quantitative Environmental Surveillance SARS-CoV-2 in Environmental Perspective: Occurrence, Persistence, Surveillance, Inactivation and Challenges 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 Making Waves: Wastewater Surveillance of SARS-CoV-2 for Population-Based Health Management