key: cord-0840075-orr25tvh authors: Kopperi, H.; Tharak, A.; Hemalatha, M.; Kiran, U.; Gokulan, C. G.; Mishra, R. K.; Venkata Mohan, S. title: Methodological Approach for Wastewater Based Epidemiological Studies for SARS-CoV-2 date: 2021-02-22 journal: nan DOI: 10.1101/2021.02.17.21251905 sha: c56a4206fc5ae0ab19362ebcc68d34bfc609d08f doc_id: 840075 cord_uid: orr25tvh Post COVID-19 outbreak, wastewater-based epidemiology (WBE) studies as surveillance system is becoming an emerging interest due to its functional advantage as tool for early warning signal and to catalyze effective disease management strategies based on the community diagnosis. A comprehensive attempt was made in this study to define a methodological approach for conducting WBE studies in the framework of identifying/selection of surveillance sites, standardizing sampling policy, designing sampling protocols to improve sensitivity, adopting safety protocol, and interpreting the data. The methodology was applied to a community and studied its epidemiological status with reference to occurrence, persistence, and variation of SARS-CoV-2 genome load in wastewater system to understand the prevalence of infection. Hourly and daily grab samples were analyzed and compared with the composite samples over a surveillance window of 7 days. Based on the SARS-CoV-2 RNA copies/L, faeces shedding, and volume of sewage generated the infected individuals and the population who are in active phase in the studied community was estimated. The persistence and replication of SARS-CoV-2 in the gastrointestinal (GI) tract and shedding through faeces is being established as a transmission route to the environment settings, which eventually discharges to the wastewater/sewage system (Wang et (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. sampling station will play a decisive role in comprehensively representing the community in order to obtain reliable data. The sampling protocol adopted can also be used to analyze the pattern of viral loads at different time-frequency and finally provide a basis for regular collection at that particular sampling station. Safety understanding in sample collection, handling and processing is needed. Therefore, in this study, a comprehensive attempt was made to establish a methodological approach to represent a community with reference to the prevalence of infection in the framework of WBE studies in terms of identifying/selection of 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. surveillance sites, standardizing sampling policy, designing sampling protocols to improve sensitivity, adopting safety protocol, and interpreting the data. The selected sampling station receives domestic wastewater from ~1.8 lakhs people residing in the sub-urban areas including Tarnaka, HMT Nagar, Lalaguda, and Nacharam (and partly Raghavendra Nagar) in Hyderabad, Telangana (State), India. Based on the available population data, the total domestic flow per day was estimated to be 18 million litre per day 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. ). An aggregated sample representing an entire community is more to accessible than pooled clinical samples (Murakami et al., 2020) . Both grab and composite sampling of domestic wastewater was employed at the selected sampling station in a defined time frequency as detailed in Table 1 . A discrete grab sampling was employed at a selected sampling site at multiple time intervals. Viral load, in general, varies over time. Therefore, a composite sample was also prepared by pooling together all grab samples obtained at various hours of the same day or daily samples obtained throughout the sampling week ( Table 1 ). The composite sampling depicts the cumulative viral loads over the selected time-frequency (24 hours or 7 days). The two sampling approaches employed will help mathematically to evaluate the heterogeneity of viral RNA. A total number of 14 grab samples were collected for hourly monitoring starting from 5 am on 05-12-2020 to 4 am on 06-12-2020 (Table 1) . A total of 7 wastewater samples was collected at 7 am from 05-12-2020 to 11-12-2020 for daily monitoring ( Table 1 ). The time period between 5 am to 9 am represent maximum sewage flow at the selected sampling station. No rainfall was recorded during the sampling window of 7 days. The two composite samples were prepared by pooling the hourly and daily collected grab samples individually. 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. 7 am 06-12-2020 07-12-2020 08-12-2020 09-12-2020 10-12-2020 11-12-2020 The grab samples were collected in a clean plastic container (disposable; 1. replicates. Sample information was prepared in the form of field sheets (date and time) and position notified (GPS readings) and point codes, observations. After sampling, the exposed surface of the container was disinfected (isopropyl alcohol (70%)) and sealed in multi-layered 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 February 22, 2021. ; https://doi.org/10.1101/2021.02.17.21251905 doi: medRxiv preprint plastic covers, labelled, and transported (2-4 °C) to lab and stored at 4°C until further processing. Samples were processed within 12 h after sampling for SARS-CoV-2 detection. Prior to use, all utilities (including PPE kit) are sealed in bio-hazard bag and subjected for sterilization before disposal. The unused samples and materials were inactivated/disinfected before disposal. All the sample processing and detection experiments were performed in a Biosafety level 2 (BSL-2) laboratories as outlined by Hemalatha et al., (2021) . Initially, collected samples were filtered using 1 mm filter papers to remove the larger debris followed by secondary filtration using 0.2 µm filtration units (Nalgene® filtration system(vacuum)) to remove other fine particles and pathogens. The filtrate (60 mL) was concentrated to ~600 µl using 30 kDa Amicon® Ultra-15 (15 ml; Merck Millipore) by ultra-filtration (4 °C; 4000 rpm; 10 min). The concentrated samples (~600 μ L) were aliquoted to 1.5 mL eppendorf vials and 150 μ L of the sample was subjected for RNA extraction. The RNA extraction was done using Viral RNA isolation kit (QIAamp, Qiagen) according to the manufacturer's protocol. (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 February 22, 2021. ; https://doi.org/10.1101/2021.02.17.21251905 doi: medRxiv preprint during the cycling stage was monitored. Positive and negative controls provided along with kit were also included in the reaction plates. All the samples were tested in triplicates. Assessment of RT-PCR kit efficiency, estimation of copy number calculation and virus recovery from sewage were performed as discussed elsewhere . The filtration units, filter papers and sample bottles were discarded in biosafety bags followed decontamination. In order to calculate the number of RNA copies per litre of collected domestic wastewater 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. SARS-CoV-2 genetic material was detected in all the hourly samples (n=14) with temporal variation in the viral load. Dynamic detection for viral genome was observed in the domestic Fig 2b) . From 5 am to 9 am, the average RNA copies recorded was 45,456 RNA copies /L which was relatively higher compared to the average of 14 hours window (10 am to 1 am; 13,387 RNA copies/L). (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. respectively. Higher viral load (genetic material) was observed in early hours i.e., 6 am to 9 am, where the peak domestic activity will happen normally during the 24 hours Based on the results obtained from the hourly monitoring, sampling time of 7 am was commonly chosen for daily monitoring to obtain the average distribution of the viral load. 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 February 22, 2021. ; https://doi.org/10.1101/2021.02.17.21251905 doi: medRxiv preprint SARS-CoV-2 genetic material was detected in all the seven daily monitored samples collected during the window period of one week (05-12-2020 to 11-12-2020) ( Table 2 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. To understand the consistency of sampling procedure two composite samples (hourly and daily) were analysed during the study period ( Fig. 4; Table 3 ). The hourly composite sample was prepared by pooling all the hourly collected samples and similarly for daily composite samples (uniform sample volume). Hourly composite C T of E-gene, N-gene and ORF1ab was detected to be 28.80±1.10%, 27.04±1.43% and 27.56±3.20% respectively. which is correlating well with the C T values of the hourly sample (Table 2; Fig 4) . Daily composite C T of E-gene, N-gene and ORF1ab was detected to be 28.62±0.60%, 27.25±1.38% and 26.99±2.58% respectively. which is correlating well with the C T values of the daily sample ( Fig 4) . Quantitatively, RNA copies present in the hourly and daily composite samples was observed to be 19,503 RNA copies/L and 17,191 RNA copies/L, respectively, which correlates well with the average RNA copies of hourly and daily monitored samples 22,871 RNA copies/L and 17,982 RNA copies/L, respectively. Correlation between the composite and cumulative RNA copies represents the efficiency of sampling frequency. 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. 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 February 22, 2021. ; 1 6 To estimate the community spread of the virus, the average RNA copies of hourly average, daily average, and composites (hourly and daily) representing 7 days window of sampling period was taken into consideration along with the capacity of sewage generated in the selected community (Table 5) During the current pandemic, various mutation of SARS-CoV-2 may be evolved and WBE can also facilitate to detect the mutants by employing metagenomic analysis. Overall, this study provides a methodological framework for WBE studies towards viral surveillance in wastewater/sewage infrastructure to precisely represent a selected community with a defined window period in terms of identifying/selection of surveillance sites, standardizing sampling policy, designing sampling protocols to improve sensitivity, adopting safety protocol, and interpreting the data. Wastewater/sewage-based surveillance can help to understand the occurrence and spread of the pandemic in the selected population or area in a more compressive approach by adopting a well-defined sampling protocol. Past experience with viral outbreaks there is more chances of epidemic/pandemic outbreaks in future due to the persistent anthropogenic induce ecological disturbances. In the environmental monitoring system, induction of viral/pathogen surveillance as a regular parameter along with routine monitoring of wastewater quality parameter will form an important basis to understand the early warning of the outbreaks. It is strongly recommended that healthcare/environmental agencies include WBE studies periodically to fight present and future pandemic outbreaks. Apart from an early warning signal for predicting the outbreaks, WBE also 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 February 22, 2021. ; https://doi.org/10.1101/2021.02.17.21251905 doi: medRxiv preprint 0 supports clinical scrutiny along with the disease detection and management systems. The current study also offers a methodological approach to monitor other pathogens. 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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity The work was supported by Council of Scientific and Industrial Research (CSIR), New Delhi, India in the form of project entitled 'Testing for COVID-19 in wastewater as a community surveillance measure (6/1/COVID-19/2020/IMD)'. UK thanks UGC, CGG and MH thank CSIR for the financial support received. KH, AT, MH and SVM acknowledge the Director, CSIR-IICT for the support.