Journal Pre-proof

Defining the methodological approach for wastewater based

epidemiological studies–Surveillance of SARS-CoV-2

Harishankar Kopperi, Athmakuri Tharak, Manupati Hemalatha,

Uday Kiran, C.G. Gokulan, Rakesh K. Mishra, S. Venkata Mohan

PII: S2352-1864(21)00344-8
DOI: https://doi.org/10.1016/j.eti.2021.101696
Reference: ETI 101696

To appear in: Environmental Technology & Innovation

Received date : 5 May 2021

Revised date : 10 June 2021
Accepted date : 13 June 2021

Please cite this article as: H. Kopperi, A. Tharak, M. Hemalatha et al., Defining the methodological
approach for wastewater based epidemiological studies–Surveillance of SARS-CoV-2.
Environmental Technology & Innovation (2021), doi: https://doi.org/10.1016/j.eti.2021.101696.

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Revised Manuscript with Changes Marked

Defining the Methodological Approach for Wastewater Based Epidemiological Studies-

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2 Surveillance of SARS-CoV-2
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9 Harishankar Kopperi1,3†, Athmakuri Tharak1†, Manupati Hemalatha1,3†, Uday Kiran2,3,†,
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12 C. G. Gokulan2†, Rakesh K Mishra2,3,*, S Venkata Mohan1,3,*
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18 Bioengineering and Environmental Sciences Lab, Department of Energy and Environmental
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21 Engineering (DEEE), CSIR-Indian Institute of Chemical Technology (CSIR-IICT),
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23 Hyderabad-500007, India
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28 CSIR-Centre for Cellular and Molecular Biology (CSIR-CCMB), Hyderabad-500007, India,
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31 Academy of Scientific and Innovative Research (AcSIR), Ghaziabad-201002, India
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37 †These authors contributed equally to the work
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44 *Corresponding: svmohan@iict.res.in; vmohan_s@yahoo.com; mishra@ccmb.res.in
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Abstract
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3 Since COVID-19 outbreak, wastewater-based epidemiology (WBE) studies as surveillance

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system is becoming an emerging interest due to its functional advantage as tool for early
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8 warning signal and to catalyze effective disease management strategies based on the
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community diagnosis. An attempt was made in this study to define and establish a
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13 methodological approach for conducting WBE studies in the framework of
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15 identifying/selection of surveillance sites, standardizing sampling policy, designing sampling
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18 protocols to improve sensitivity, adopting safety protocol, and interpreting the data. Data
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20 from hourly sampling indicated a peak in the viral RNA during the morning hours (6-9 am)
21 re-
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23 when the all the domestic activities are maximum. The daily sampling and processing
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25 revealed the dynamic nature of infection spread among the population. The two sampling
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methods viz. grab, and composite showed a good correlation. Overall, this study establishes a
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30 structured protocol for performing WBE studies that could provide useful insights on the
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32 spread of the pandemic at a given point of time. Moreover, this framework could be
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35 extrapolated to monitor several other clinically relevant diseases. Following these guidelines,
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37 it is possible to achieve measurable and reliable SARS-CoV-2 RNA concentrations in
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40 wastewater infrastructure and establish a national surveillance system. The findings could
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42 then be matched and further analysed on a global scale to aid in disease outbreak
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survivability.
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51 Keywords: Urban area, Grab sample; Composite sample; Domestic Sewage treatment plants
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53 (STP); Sampling protocol.
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1. Introduction
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3 The persistence and replication of SARS-CoV-2 in the gastrointestinal (GI) tract and

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shedding through faeces is being established as a transmission route to the environment
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8 settings, which eventually discharges to the wastewater/sewage system (Wang et al., 2020a;
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Wang et al., 2020b; Woelfel et al., 2020; Xiao et al., 2020; Young et al., 2020; Zahedi et al.,
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13 2021; Venkata Mohan et al., 2020). Detection of SARS-CoV-2 genetic material in the
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15 sewage/wastewater documented a significant interest among the research fraternity in the
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18 framework of wastewater based epidemiological (WBE) studies (Venkata Mohan et al., 2020;
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20 Kumar et al., 2020; Hemalatha et al., 2021a & 2021b). Previously, the wastewater functioned
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23 as a surveillance system for poliovirus and Aichi virus (Asghar et al., 2014; Lodder et al.,
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25 2012b). WBE studies also functioned as an important supplement to clinical surveillance in
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polio eradication and have the potential to inform the epidemiology of COVID-19 (Shaw et
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30 al., 2020; Xagoraraki and O’Brien 2020; Hemalatha et al., 2021a; Tharak et al., 2021).
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32 Unlike polio, the COVID-19 vaccine reached the mankind relatively in very short span of
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35 time but with some challenges such as their efficacy among different people and time it takes
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37 to reach entire population. In this scenario, the testing of the massive population to contain
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40 the spread of the virus is a challenge and therefore, an alternative strategy to assess the
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42 disease spread and thereby efficiently manage the disease is critical (Medema et al., 2020;
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Shaw et al., 2020; Venkata Mohan et al., 2020; Xagoraraki and O’Brien 2020; Hemalatha et
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47 al., 2021b; Tharak et al., 2021). Wastewater surveillance is an unbiased tool that helps to
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49 establish an early-warning system that would be able to monitor the occurrence, spread and,
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severity of the infection at a community level and therefore help in early preventative
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54 measures and allocation of resources to potentially affected areas (Torrey et al., 2019;
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57 Brainard et al., 2017; Bibby et al., 2015b; Casanova and Weaver 2015; Bibby and Peccia
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59 2013a), which eventually minimize the outbreak and spread (Venkata Mohan et al., 2020;
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Lednicky et al., 2020; Daughton et al., 2018). Recent reports employed WBE-based
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2 approaches to detect SARS-CoV-2 in domestic/sewage wastewater (Ahmed et al., 2020a;
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of
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5 Hemalatha et al., 2021a; La Rosa et al., 2020a; Medema et al., 2020; Usman et al., 2020; Wu
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7 et al., 2020a). Despite the evidence on the persistence of SARS-CoV-2 RNA in
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10 wastewater/sewage, the virus transmission to the community from wastewater infrastructure

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12 is yet to be established.
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17 The wastewater/sewage complexity, the dilute nature of biomarker in wastewater and, the
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inability to pinpoint specific locations are some of limitations in establishing quantitative
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predictions of viral RNA load in WBE (Ahmed et al., 2020b; Hart and Halden 2020; Tharak
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et al., 2021). The accuracy of the assessment of viral load among the community is an
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27 important pre-requisite of any WBE Studies (Hemalatha et al., 2021a; Nakamura et al.,
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29 2015). Interpretation with a limited number of positive wastewater sample is challenging
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32 (Asghar et al., 2014; O'Reilly et al., 2020). Setting minimum standards for surveillance sites,
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34 developing a standardized sampling policy, creating laboratory testing protocols to improve
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sensitivity and minimizing the risk of cross-contamination would be necessary for an
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39 informative mode of surveillance (Xagoraraki and O’Brien 2020; Wu et al., 2020b). Several
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41 approaches for sampling were used to increase the volume of a sample which might help to
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44 identify the virus in wastewater which makes intractable to handle samples and process in
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46 laboratories (O'Reilly et al., 2020). The nature, time and frequency of sampling apart from
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49 appropriate sampling station will play a decisive role in comprehensively representing the
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51 community in order to obtain reliable data. The sampling protocol adopted can also be used
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54 to analyze the pattern of viral loads at different time-frequency and finally provide a basis for
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56 regular collection at that particular sampling station. Safety understanding in sample
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collection, handling and processing is needed. Therefore, in this study, a comprehensive
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attempt was made to establish a methodological approach to represent a community with

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2 reference to the prevalence of infection in the framework of WBE studies in terms of
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5 identifying/selection of surveillance sites, standardizing sampling policy, designing sampling
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7 protocols to improve sensitivity, adopting safety protocol, and interpreting the data.
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12 2. Materials and Methods
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15 2.1. Surveillance (sampling) station
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17 The selected sampling station receives domestic wastewater from ~1.8 lakhs people residing
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in the sub-urban areas including Tarnaka, HMT Nagar, Lalaguda, and Nacharam (and partly
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Raghavendra Nagar) in Hyderabad, Telangana (State), India. Based on the available
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population data, the total domestic flow per day was estimated to be 18 million litre per day
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27 (MLD). Domestic wastewater samples were collected at the converging (terminal;
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29 downstream) point of all the lateral drains that further leads to the Sewage Treatment Plant
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32 (STP; 10 MLD capacity; 17.37°N 78.48°E; Nacharam, India) (Fig. 1).
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35 Fig. 1
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37 2.2. Sampling Protocol
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40 2.2.1. Type of Samples
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42 The sampling method (grab/composite samples) play an important role on WBE surveillance
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studies along with population demographics and local epidemiological factors (Alygizakis et
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47 al., 2020). An aggregated sample representing an entire community is more to accessible than
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49 pooled clinical samples (Murakami et al., 2020). Both grab and composite sampling of
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domestic wastewater was employed at the selected sampling station in a defined time
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54 frequency as detailed in Table 1. A discrete grab sampling was employed at a selected
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57 sampling site at multiple time intervals (APHA 2014). Viral load, in general, varies over
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59 time.Therefore, a composite sample was also prepared by pooling together all grab samples
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obtained at various hours of the same day or daily samples obtained throughout the sampling
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2 week (Table 1). The composite sampling depicts the cumulative viral loads over the selected
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5 time-frequency (24 hours or 7 days). The two sampling approaches employed will help
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7 mathematically to evaluate the heterogeneity of viral RNA.
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12 2.2.2. Frequency of sampling
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15 A total number of 14 grab samples were collected for hourly monitoring starting from 5 am
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17 on 05-12-2020 to 4 am on 06-12-2020 (Table 1). A total of 7 wastewater samples was
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collected at 7 am from 05-12-2020 to 11-12-2020 for daily monitoring (Table 1). The time
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period between 5 am to 9 am represent maximum sewage flow at the selected sampling
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station. No rainfall was recorded during the sampling window of 7 days. The two composite
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27 samples were prepared by pooling the hourly and daily collected grab samples individually.
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29 Table 1
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32 2.2.3. Sampling Procedure & Safety Considerations
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34 The grab samples were collected in a clean plastic container (disposable; 1.2 litres)
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containing 20 ml of sodium hypochlorite (0.1%) to inactivate the pathogens (Hemalatha et
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39 al., 2021a & 2021b). Adequate biosafety measures were taken during sampling and
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41 processing of the samples. Samples were collected by wearing PPE kit comprising of gloves,
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44 cover suite, eye safety glasses, N95 protective mask and shoes (Hemalatha et al., 2021a). For
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46 sample collection, the sample container was slightly lowered in the opposite direction of flow
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49 with partial immersion. Grab sample volume of one litre was collected at one time with three
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51 replicates. Sample information was prepared in the form of field sheets (date and time) and
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54 position notified (GPS readings) and point codes, observations. After sampling, the exposed
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56 surface of the container was disinfected (isopropyl alcohol (70%)) and sealed in multi-layered
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plastic covers, labelled, and transported (2-4 °C) to lab and stored at 4°C until further
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processing. Samples were processed within 12 h after sampling for SARS-CoV-2

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2 detection.Prior to use, all utilities (including PPE kit) are sealed in bio-hazard bag and
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5 subjected for sterilization before disposal. The unused samples and materials were
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12 2.3. Processing of Samples
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15 All the sample processing and detection experiments were performed in a Biosafety level 2
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17 (BSL-2) laboratories. Initially, collected samples were filtered using 1 mm blotting papers
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(India) to remove the larger debris followed by secondary filtration using 0.2 µm filtration
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units (Nalgene® filtration system(vacuum)) to remove other fine particles and pathogens.
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The filtrate (60 mL) was concentrated to ~600 µl using 30 kDaAmicon® Ultra-15 (15 ml;
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27 Merck Millipore) by ultra-filtration (4 °C; 4000 rpm; 10 min) (Hemalatha et al., 2021a). The
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29 concentrated samples (~600 μL) were aliquoted to 1.5 mL eppendorf vials and 150 μL of the
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2.4. RNA extraction and RT-PCR
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39 The RNA extraction was done using Viral RNA isolation kit (QIAamp, Qiagen) according to
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41 the manufacturer’s protocol. Fosun COVID-19 RT-PCR Detection Kit (Shanghai Fosun Long
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44 March Medical Science Co., Ltd, China) approved by FDA (Food and Drug Administration,
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46 USA) was use to isolate SARS-CoV-2 RNA. The kit consists of primers and probes that
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49 target the envelope protein coding gene (E-gene; ROX), nucleocapsid gene (N-gene; JOE),
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51 and open reading frame1ab (ORF1ab; FAM) of SARS-CoV-2. The RT-PCR
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54 (QuantStudioTM5) was performed as per manufacturer recommendations. The
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56 reaction program includes reverse transcription (50°C for 15 min) and the initial denaturation
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(95°C for 3 minutes) followed by 45 cycles at 95°C for 5 seconds and 60°C for 40 seconds.
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The signals of FAM (ORF1ab), JOE (N gene), ROX (E gene), and CY5 (Internal reference)
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5 kit werealso included in the reaction plates. All the samples were tested in
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7 triplicates. Assessment of RT-PCR kit efficiency, estimation of copy number calculation and
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10 virus recovery from sewage were performed as discussed elsewhere (Hemalatha et al.,

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12 2021a). The filtration units, filter papers and sample bottles were discarded in biosafety bags
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15 followed decontamination.
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2.5. Data Analysis
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The E gene amplified from SARS-CoV-2 RNA was cloned (KpnI and HindIII restriction
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sites) into pcDNA3.1 vector and quantified with dsDNA HS Assay Kit (Qubit™; Invitrogen,
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27 USA) and 4 Fluorometer (Qubit™; Invitrogen, USA). Based on the E gene and vector
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29 sequences, the number of copies per nanogram was calculated
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32 from https://www.ncbi.nlm.nih.gov/nuccore/NC_045512.2?report=fasta&from=26245&to=2
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34 6472 and https://www.addgene.org/browse/sequence_vdb/2093/, respectively. The plasmid
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was diluted from a number of 9.01 log10 to 0.01 log10 copies and performed RT-PCR. The
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39 CT values were plotted against the log copy number and a linear fit equation was obtained
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41 (Hemalatha et al., 2021a). Based on E gene CT values, this calculation is used to calculate the
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44 number of RNA copies in wastewater.
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49 In order to calculate the number of RNA copies per litre of collected domestic wastewater
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51 samples, linear fit equation of the E-gene was employed by which RNA copies per litre
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54 wastewater can be calculated (Hemalatha et al., 2021a). Number of RNA copies was
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56 calculated based on the CT values of E-gene (Equation 1).
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59 ….. (1)
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The number of infected people in the given community was calculated based on the average
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10 Method 1 (Ahmed et al., 2020a)

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13 …… (2)
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18 Faeces excreted/person/day = 128 g (Rose et al., 2015).One positive person sheds 107 RNA
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20 copies/g of faeces (maximum estimate) (Foladori et al., 2020; Bivins et al., 2020).
21 re-
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23 Method 2 (Hellmér et al., 2014).
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26 …… (3)
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31 Number of RNA copies excreted per mL of faeces = 107. Volume of faeces excreted = 120
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33 mL (calculated by considering the density of human faeces is 1.07 g/mL) (Foladori et al.,
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36 2020).
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41 Relative standard deviation (RSD) for CT value of each gene (E-gene, N-gene, ORF1ab) was
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43 calculated usingequation 4, where is the mean of CT value and ‘S’ is the standard deviation.
44
45
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47 …… (4)
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49
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51 Number of individuals who are in active Phase of Infection during the window period was
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53 calculated by considering the infected individuals, window period and infectious period of an
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infected individual.
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8 ……(5)
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10 3. Results and Discussion

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13 3.1. Hourly Sampling - Detection of Viral Load
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15 SARS-CoV-2 genetic material was detected in all the hourly samples (n=14) with temporal
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18
variation in the viral load. Dynamic detection for viral genome observed in the domestic
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20 sewage indicated the spread of SARS-CoV-2 among the selected community. CT value based
21 re-
22 on the average of each gene showed 28.62±1.34% for E-Gene, 27.32±1.38% for N-gene and
23
24
25 27.43±1.35% for ORF1ab in the time frame window of 24 hours (Fig. 2a; Table 2). RNA
26
27 copy number calculated based on E- gene (Hemalatha et al., 2021a) showed a24houraverage
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30 of 22,871 RNA copies/L in the hourly monitored samples (Fig. 2b; Table 2). From 5 am to 9
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32 am, the average RNA copies recorded was 45,456 RNA copies /L which was relatively
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34
35
higher compared to the average of 14 hours window (10 am to 1 am; 13,387 RNA copies/L).
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37 Fig. 2
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39 Higher RNA copies were observed in the samples collected at 6 am (53531 RNA copies/L)
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42 and 9 am (98522 RNA copies/L). Between 10 am and 1 am (nearly 14 hours) the viral loads
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44 got stabilized between 9,340 and 17,556 RNA copies/L (Table 2). The lowest viral load of
45
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47 5,880 RNA copies/L was recorded with the sample collected at 4 am (Table 2). Individual
48
49 CT values of three genes also followed the trends with the average CT and RNA copies. E
50
51
gene CT value varied between 26.31±5.74% and 29.67±1.26%. Similarly, the CT values of N
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54 gene and ORF1 ab was observed between 25.95±3.47% to 28.07±0.88% and 24.47±1.14% to
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28.48±2.06%, respectively (Table 2). Higher viral load (genetic material) was observed in
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59 early hours i.e., 6 am to 9 am, where the peak domestic activity will happen normally during
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the 24 hours window. Higher detection of viral genome in morning hours might be attributed
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2 to the shedding through faeces, which represent the majority of the population in the
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of
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5 community. Several factors like sampling frequency, variation in sampling method, the
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7 concentration of disinfectant added, the flow rate of sewage, storage condition, and
8
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10 downstream process could affect the RNA copy numbers (Ahmed et al., 2020a; 2020b).

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13
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15 Table 2
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18 3.2. Understanding the dynamics of SARS-CoV-2 RNA load on daily basis
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21 Based on the results obtained from the hourly monitoring, sampling time of 7 am was
re-
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23 commonly chosen for daily monitoring to obtain the average distribution of the viral load.
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26 SARS-CoV-2 genetic material was detected in all the seven daily monitored samples
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28 collected during the window period of one week (05-12-2020 to 11-12-2020) (Table 2; Fig
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31 3). Detection of the viral genetic material over 7 days window period indicated the presence
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33 of SARS-CoV-2 RNA in domestic wastewater.
34
35
36
Fig. 3
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39
40
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41 E-gene CT values varied between 27.75±0.26% and 31.37±2.80%with an average of

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44
29.12±0.96%, N-gene between 26.03±1.00% and 30.35±2.26% with an average of
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46 27.74±1.60% and ORF1ab between 26.49±0.21% and 29.60±0.20% with an average of
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48 27.74±1.16% (Fig. 3a; Table 3). Progressive increase in CT values followed by decrement
49
50
51 was observed in 7 days of sampling. The RNA copies/L ranged from 2,836 to 35,895 with an
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53 average of 17,982 (Fig. 3b; Table 3).
54
55
56 Table 3
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59 3.3. Composite Sample Analysis
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To understand the consistency of sampling procedure two composite samples (hourly and
1
2 daily) wereanalysedduring the study period (Fig. 4; Table 4). The hourly composite sample
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of
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5 was prepared by poolingall the hourly collected samples and similarly for daily composite
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7 samples (uniform sample volume). Hourly composite CTofE-gene, N-gene and ORF1ab was
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9
10 detected to be 28.80±1.10%, 27.04±1.43% and 27.56±3.20% respectively, which is

pro
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12 correlating well with the CT values of the hourly sample (Fig. 4; Table 4). Daily composite
13
14
15 CT of E-gene, N-gene and ORF1ab was detected to be 28.62±0.60%, 27.25±1.38% and
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17 26.99±2.58% respectively. which is correlating well with the CT values of the daily sample
18
19
(Fig. 4; Table 4). Quantitatively, RNA copies present in the hourly and daily composite
20
21
samples was observed to be 19,503 RNA copies/L and 17,191 RNA copies/L, respectively
22
23
24
25
re-
(Fig. 4; Table 4), which correlates well with the average RNA copies of hourly and daily
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27 monitored samples 22,871 RNA copies/L and 17,982 RNA copies/L, respectively.
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29 Correlation between the composite and cumulative RNA copies represents the efficiency of
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31
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32 sampling frequency. Variation in RNA copies might be due to several environmental factors
33
34 at a given time such as temperature, humidity, presence of organic content, detergents,
35
36
37
oxidising chemicals affect the persistence of the virus (Paul et al., 2021; Hemalatha et al.,
38
39 2021a; La Rosa et a., 2020b)
40
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41 Fig. 4
42
43
44 Table 4
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46
47
48
3.4. Epidemiological status of the community
49
50
51 To estimate the community spread of the virus, the average RNA copies of hourly average,
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54 daily average, and composites (hourly and daily) representing 7 days window of sampling
55
56 period was taken into consideration along with the capacity of sewage generated in the
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selected community (Table 5). Both hourly and daily samples showed variability in the viral
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load with the time scale. The estimated number of infected individuals would include those in
1
2 the early as well as later stages of infection and shedding viral particles in their faecal
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of
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5 matter. This period was chosen based on the reports on persistence of SARS-CoV-2 RNA
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7 material for up to 47 days after the infection (Wu et al., 2020a; 2020b). Independent reports
8
9
10 highlighted the replication of SARS-CoV-2 in GI tract and the prolonged shedding viral

pro
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12 material through faeces during and after active infectious phases (Ahmed et al., 2020a;
13
14
15 Holshue et al., 2020; Kitajima et al., 2020; La Rosa et al., 2020a;Woelfel et al., 2020;
16
17 Wurtzer et al., 2020; Wu et al., 2020b). The number of infected individuals were estimated
18
19
based on shedding range of 106 and 107 RNA copies/mL faeces (Foladori et al., 2020)
20
21
employing the average RNA copies obtained from this study to avoid the
22
23
24
25
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ambiguity/discrepancy in the number of viral particles excreted by infected individuals. With
26
27 reference to the studied community, the estimated infected individuals during the sampled
28
29 window period are between 282 (107 RNA copies/mL faeces) and 2817 (106 RNA copies/mL
30
31
lP
32 faeces) and the number of individuals in the active phase of infection might be between 113
33
34 to 1127 with a wastewater flow rate of 18 MLD (Table 5). Based on this number the infected
35
36
37
individuals for the metropolitan Hyderabad city were also calculated with total sewage
38
39 generation of 1800 MLD
40
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41 (https://numerical.co.in/numerons/collection/5e8fd4f6f3c42b5803c09a3b).
42
43
44
45
46 The number of infected individuals during the sampling window was estimated between
47
48
49 28,200 and 2,81,700 with a active phase of infection between 11,280 and 1,12,680 (Table 5).
50
51 The loss of 0.02 to 3000 RNA copies/mL was reported during the transit of faeces from point
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54 of excretion to the drain (Foladori et al., 2020) which could influence the overall estimation
55
56 of infected individuals. The obtained figures might include pre-symptomatic, post-
57
58
symptomatic, asymptomatic, and symptomatic patients of which asymptomatic individuals
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might be the major contributors based on the community behaviour and serological
1
2 data. WBE can quantify the scale of infection prevailing in the selected community with a
3

of
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5 benefit of detection for the individuals who have not been tested, asymptomatic, potentially
6
7 symptomatic, pre-symptomatic, or only have mild symptoms (Hata and Honda et al., 2020;
8
9
10 Hemalatha et al., 2021a; Medema et al., 2020; Mallapaty 2020; Naddeo and Liu 2020; Qu et

pro
11
12 al., 2020; Venkata Mohan et al., 2020; Lodder et al., 2012b). Asymptomatic and symptomatic
13
14
15 cases also result in significant uncertainty in the estimated extent of SARS-CoV-2 infection.
16
17 However, this kind of estimation using the CT value and RNA copies could help to predict the
18
19
near precise number of infected individuals in a selected community/area.
20
21
Table 5
22
23
24
25
re-
26
27 3.5. Benefits and Challenges of WBE
28
29
30
31
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32 WBE provides a cost-efficient way of surveillance due to representative sampling of a
33
34 population that allows broad surveillance that could detect crucial changes in the status of the
35
36
37
infection without selection bias (Alygizakis et al., 2020). Surveillance for SARS-CoV-2 RNA
38
39 in wastewater via WBE provides an additional monitoring tool, devoting to community-level
40
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41 screening and control measures in some cases, as these measurements have preceded disease
42
43
44 cases in some cases. Detections of SARS-CoV-2 RNA employing different methods have
45
46 been reported worldwide, illustrating the technical feasibility of routine monitoring (Table 6).
47
48
49 WBS can be used as a complementary and possibly early tool to detect pathogens in a
50
51 community, considering that specific and representative sampling points in areas with and
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54 without sewerage systems are chosen (Venkata Mohan et al., 2020; Hemalatha et al., 2021b).
55
56 Similarly, WBS can be used as a complementary tool to help in the fight against the spread of
57
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COVID-19. WBS can be a key tool to track SARS-CoV-2 circulation in low-developed areas
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(Calabria de Araujo et al., 2021). It is possible to estimate the number of infected people in a
1
2 specific sewage catchment area if the virus concentration in the wastewater per day and the
3

of
4
5 virus concentration in the faeces of an infected person per day are known (Qu et al., 2020).
6
7 WBE provides the categorization of the selected community into the zones based on the
8
9
10 severity of viral load in the sewage (Tharak et al., 2021). WBE predicts infection dynamics

pro
11
12 including the symptomatic, asymptomatic and pre/post symptomatic characteristics (Bai et
13
14
15 al., 2020). It is possible to predict the raise in infection rate before 2-5 weeks of actual
16
17 manifestation in the population (Wu et al., 2020b).
18
19
20
21
One of the major challenges on WBS is the use of different protocols (Wu et al., 2020a).
22
23
24
25
re-
Strategies for WBS are unique and there is standard protocol for SARS-CoV-2 surveillance
26
27 from wastewaters (Calabria de Araujo et al., 2021) and to estimate the number of infected
28
29 people in a community from the quantification of SARS-CoV-2 in wastewater (Wu et al.,
30
31
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32 2020a). The SARS-CoV-2 RNA concentration (copies/L) measured in the sewage may be
33
34 dependent on excrete from the infected person, immunity level, locality and age groups
35
36
37
(Hemalatha et al., 2021a; Ahmed et al., 2020; Foladori et al., 2020; Bivins et al., 2020;
38
39 Hellmér et al., 2014). Advanced detection tools (RT-qPCR) for rapid, efficient identification
40
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41 may require standard laboratory equipment, being unfeasible for many countries (Medama et
42
43
44 al., 2020; Wurtzer et al., 2020; Ahmed et al., 2020). The persistence of virus in sewage
45
46 influenced by factors such as temperature, population, drain flow rate, infected individuals,
47
48
49 time and day of sampling, dilution effect, community soci-economic conditions, extent of
50
51 sanitation and facility, humidity, rainfall event, etc (Calabria de Araujo et al., 2021). The
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54 supplemental quality control data and standardisation in sampling design, sample processing,
55
56 data interpretation, and reporting are mandated to reliably interpret data generated by these
57
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59
60
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efforts for informing public health interventions (Calabria de Araujo et al., 2021). WBE also
1
2 can also can detect different viral variants currently circulating in the population.
3

of
4
5 Table 6
6
7
8
9
10 3.6. Way forward

pro
11
12 Based on the outcome from this study, the following points can be considered for planning
13
14
15 WBE (a) Sample/sampling station(s) should be a true representative of the selected
16
17 community to be studied; (b) Sampling station(s) should be selected at the downstream
18
19
converging point of discharge line if flowing water is being analysed. Inlet discharge point to
20
21
STP can be considered for sampling which comprehensively indicates the infection of the
22
23
24
25
re-
community/area under the STP coverage; (c) Combined grab and composite sampling
26
27 strategies can be adopted with a define sampling frequency; (d) Sampling frequency should
28
29 be extended for not less than 24 h for hourly sampling and for not less than 7 days for daily
30
31
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32 sampling to get a true average representation of the community. For daily sampling, the time
33
34 of sample should be carefully chosen to represent average values and (e) Estimation of the
35
36
37
viral infection of the study area can be calculated based on the population and its wastewater
38
39 discharge data. Along with this, in this study, the estimate of infected individuals was based
40
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41 on the CT values of E-gene specific to SARS-CoV-2, as it was amplified and cloned in a

42
43
44 vector as reported by Hemalatha and co-workers (2021a). In a similar way to get infected
45
46 population one can also amplify and clone the respective gene(s) specific to SARS-CoV-2 as
47
48
49 per the targets available in the testing kits.
50
51
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54 WBE ability to detect low levels of viruses especially at early stages of an outbreak or when
55
56 infection levels are decreasing (following the interventions) is very important (Kitajima et al.,
57
58
2020). Wastewater sampling in cities requires sewer network maps to understand the
59
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population being represented. Outside of dense populations, there are fewer sewer networks
1
2 that restricts the representative wastewater sampling (Hamisu et al., 2020). Interpretation of
3

of
4
5 positive wastewater samples along with clinical investigation will yield more information
6
7 (Asghar et al., 2014; O'Reilly et al., 2020). WBE studies functioned as an important
8
9
10 supplement to clinical surveillance in polio eradication (Xagoraraki and O’Brien 2020; Shaw

pro
11
12 et al., 2020).
13
14
15
16
17 Given the wide-spread transmission of SARS-CoV-2 it is almost impossible to test every
18
19
individual. Sewage based surveillance is a holistic and effective approach to study the
20
21
infection dynamics, which helps in the efficient management of the SARS-CoV-2 spread. A
22
23
24
25
re-
clear connection between the RT-PCR quantitative data and the recorded clinical data can be
26
27 correlated to reduce ambiguity viral-wastewater surveillance (Xiao et al., 2020; O'Reilly et
28
29 al., 2020). Longitudinal sampling from the same location along with metagenomics can
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31
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32 provide an additional illustration on the status (Torrey et al., 2019). Associated with clinical
33
34 data, WBE could provide information on SARS-CoV-2 transmission within a community
35
36
37
including the beginning, tapering, or reemergence of an epidemic(Lodder et al., 2012;
38
39 Naddeo and Liu 2020). During the current pandemic, various mutation of SARS-CoV-2 may
40
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41 get be evolved and WBE can also facilitate to detect the mutants by employing metagenomic
42
43
44 analysis.
45
46
47
48
49 4. Conclusion
50
51
Overall, this study provides a methodological framework for WBE studies towards viral
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54 surveillance in wastewater/sewage infrastructure to precisely represent a selected community
55
56
57 with a defined window period in terms of identifying/selection of surveillance sites,
58
59 standardizing sampling policy, designing sampling protocols to improve sensitivity, adopting
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safety protocol, and interpreting the data. The current study focuses on a methodological
1
2 approach for sampling wastewater in order to detect SARS-CoV-2 RNA, as well as available
3

of
4
5 information on sample concentration, extraction, and detection methods. Wastewater/sewage-
6
7 based surveillance can help to understand the occurrence and spread of the pandemic in the
8
9
10 selected population or area in a more compressive approach by adopting a well-defined

pro
11
12 sampling protocol. Past experience with viral outbreaks there is more chances of
13
14
15 epidemic/pandemic outbreaks in future due to the persistent anthropogenic induce ecological
16
17 disturbances. In the environmental monitoring system, induction of viral/pathogen
18
19
surveillance as a regular parameter along with routine monitoring of wastewater quality
20
21
parameter will form an important basis to understand the early warning of the outbreaks. It is
22
23
24
25
re-
strongly recommended that healthcare/environmental agencies include WBE studies
26
27 periodically to fight present and future pandemic outbreaks. Apart from an early warning
28
29 signal for predicting the outbreaks. This works highlights potential areas for standardisation,
30
31
lP
32 such as sampling time and frequency in relation to peak faecal loading times; the
33
34 implementation of relevant information on sample collection type and points, sample volume
35
36
37
collected, transport and storage conditions, extraction process and Real-Time PCR analysis.
38
39 WBE also supports clinical scrutiny along with the disease detection and management
40
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41 systems. The current study also offers a methodological approach to monitor other
42
43
44 pathogens. A policy in the framework of public health by appending to the environmental
45
46 systems will eventually help to safeguard the community with future outbreak.
47
48
49
50
51 Authorship contribution statement
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54 Harishankar Kopperi: Methodology, Investigation, Writing – original draft. Athmakuri
55
56 Tharak: Methodology, Investigation, Data curation, Writing – original draft. Manupati
57
58
Hemalatha: Methodology, Investigation, Data curation, Writing – original draft. Uday Kiran:
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Methodology, Investigation, Datacuration, Writing – original draft. C.G. Gokulan:

1
2 Methodology, Investigation, Formal analysis, Writing – original draft. Rakesh K. Mishra:
3

of
4
5 Conceptualization, Supervision, Funding acquisition, Validation, Writing – review & editing.
6
7 and S. Venkata Mohan: Conceptualization, Methodology, Supervision, Validation, Writing –
8
9
10 original draft, Writing – review & editing.

pro
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12 Declaration of competing interest
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14
15 The authors declare that they have no known competing financial interests or personal
16
17 relationships that could have appeared to influence the work reported in this paper.
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19
20
21
Acknowledgments
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25
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The work was supported by Council of Scientific and Industrial Research (CSIR), New
26
27 Delhi, India in the form of project entitled ‘Testing for COVID-19 in wastewater as a
28
29 community surveillance measure (6/1/COVID-19/2020/IMD)’. UK thanks UGC, CGG and
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32 MH thank CSIR for the financial support received. KH, AT, MH and SVM acknowledge the
33
34 Director, CSIR-IICT (Manuscript No. IICT/Pubs./2019/292) for the support.
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Figure Click here to access/download;Figure;Figures.doc

Figures

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Figure 1: Sampling point selected for daily and hourly monitoring at the inlet of the STP in

Nacharam, Hyderabad. (Map source: Google Map).

Figure 2: a) CT values of E gene, N gene, and ORF1ab; b) RNA copies calculated based on

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linear fit equation of E-gene of samples collected hourly from selected point (All values

represent, +SD).

Figure 3: a) CT values of E gene, N gene, and ORF1ab; b) RNA copies calculated based on

linear fit equation of E-gene of samples collected hourly from selected point (All values

represent, +SD).
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Figure 4: a) CT values of E gene, N gene, and ORF1ab and RNA copies of hourly, daily

average and composite samples (All values represent, +SD).

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Fig. 1
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 RNA Copies/L
CT Value

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Table Click here to access/download;Table;modified Tables.doc

List of Tables

Table 1: Details of Sampling with reference to time of hourly and daily samples.

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Sample Date Time
5 am
6 am
7 am

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8 am
9 am
10 am
Hourly Sample 05-12-2020
12 pm
2 pm
4 pm
6 pm
8 pm
re- 11 pm
1 am
06-12-2020
4 am
05-12-2020
06-12-2020
07-12-2020
Daily Sampling 08-12-2020 7 am
09-12-2020
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10-12-2020
11-12-2020
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Table 2: SARS-CoV-2 RNA load with hourly domestic sewage samples

RNA copies/L

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Date Time E gene* N gene* ORF1ab* ***
(using eq. 1)
05-12-2020 5 am 28.43±0.78% 26.63±0.40% 26.98±3.92% 22,282
6 am 27.18±3.95% 25.96±2.90% 24.47±1.14% 53,531
7 am 28.05±0.39% 26.88±2.38% 26.85±3.26% 29,086

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8 am 28.17±1.20% 26.10±0.34% 27.07±0.75% 26,738
9 am 26.31±5.74% 25.95±3.47% 27.80±0.85% 98,522
10 am 29.48±0.98% 28.34±1.22% 28.48±2.06% 10,671
12 noon 29.17±0.15% 27.18±0.65% 27.63±0.10% 13,262
2 pm 29.07±0.77% 27.13±0.55% 27.24±0.29% 14,226
4 pm 28.77±1.08% 27.55±1.75% 27.66±0.66% 17,556
6 pm 28.79±0.43% 27.92±0.82% 27.38±0.60% 17,312
8 pm 29.67±1.26% 27.88±0.29% 28.02±1.25% 9,340
11 pm 29.26±0.27% 28.07±0.88%
re- 27.80±0.79% 12,451
06-12-2020 1 am 29.28±1.09% 27.62±0.64% 27.74±1.38% 12,278
4 am 30.33±0.65% 29.28±3.09% 28.88±1.83% 5,880
Average 28.62±1.34% 27.32±1.38% 27.43±1.35% 18,292
*Represent +RSD; ***RNA copies (based on E gene) were calculated based on the
linear fit equation
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Table 3: SARS-CoV-2 RNA CT values detected for daily sample.

RNA
copies/L

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Date Time E gene* N gene* ORF1ab*
***
(using eq. 1)
05-12-2020 7am 28.05±0.39% 26.88±2.38% 26.85±3.26% 29,086
06-12-2020 7am 29.20±1.28% 27.25±0.74% 27.81±1.13% 12,986
07-12-2020 7am 31.37±2.80% 30.35±2.26% 29.60±0.20% 2,836

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08-12-2020 7am 30.24±1.33% 29.29±3.58% 28.67±2.51% 6,263
09-12-2020 7am 28.72±0.44% 27.56±0.53% 27.59±0.30% 18,182
10-12-2020 7am 27.75±0.26% 26.03±1.00% 26.49±0.21% 35,895
11-12-2020 7am 28.54±0.21% 26.84±0.73% 27.19±0.49% 20,628
Average 29.12±0.96% 27.74±1.60% 27.74±1.16% 17,982
*Represent +RSD; ***RNA copies (based on E gene) were calculated based on the linear fit equation.

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Table 4: SARS-CoV-2 RNA load with hourly and daily composite domestic sewage
samples
RNA

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copies/L
Sample E gene* N gene* ORF1ab*
***
(using Eq. 1)
Hourly Average 28.71±1.25% 27.32±1.79% 27.43±1.35% 22,871
Hourly Composite 28.80±1.10% 27.04±1.43% 27.56±3.20% 19,503

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Daily average 29.12±1.00% 27.74±1.62% 27.74±1.15% 17,982
Daily Composite 28.62±0.60% 27.25±1.38% 26.99±2.58% 17,191
Average 28.81±0.99% 27.34±1.56% 27.43±2.07% 19,387
*Represent +RSD; ***RNA copies (based on E gene) were calculated based on the linear fit equation.

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Table 5. Infection estimating through the number of people infected (symptomatic,

asymptomatic, and recovered) during the sampling window on average basis of
sampling

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107 RNA copies/mL faeces 106 RNA copies/mL faeces
Capacity
Sample Per person Per person
of the Method Method Method Method
Name contribution contribution
Sewage 1 2 1 2
to Sewage to Sewage
(MLD)

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Hourly
342 365 3,418 3,646
Average
Daily
253 270 2,529 2,697
Average
18 67
Hourly
274 293 6.70 2,743 2,925
Composite
Daily
242 258 2,417 2,579
Composite
Estimate of infected individuals (for
273 291 2,726 2,908
Study Area with 18 MLD)
Average estimate of infected individuals
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Estimate of the population in active phase of
282 2,817

the infection during the window period of 35 113 1,127

days
For 1800 MLD (on total Sewage
27,300 29,100 2,72,600 2,90,800
Generation of Hyderabad city)
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Average estimate of infected individuals 28,200 2,81,700
Estimate of the population in active phase of
the infection during the window period of 35 11,280 1,12,680
days
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Table 6: Various sampling methods adopted and detection assay for estimation of virus
in sewage wastewater.

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Virus Sampling Detection assay Reference
and Primers
SARS-CoV-1 Grab samples RT-qPCR Wang et al.,
Sewage water from SARS patients 2005c

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Beijing, China
Human Grab samples PCR (In-silico) Bibby and
Coronavirus Sewage sludge and influent and Peccia., 2013a
229E; Human effluent sludge
Coronavirus U.S.A
HKU1
Coronaviridae Grab samples (1 L) qPCR Alexyuk et al.,
(Virome); Lake samples (1.0 m depth) 2017a
Alphacoronavirus; Lake Balkhash, Kazakhstanre-
Betacoronavirus
Grab and composite sampling (24 h; 1 This study
L)
Domestic wastewater (sewage) RT-PCR
Hyderabad, India ORF1ab, E and N
Grab Samples (1 L) Hemalatha et al.,
STP inlet and outlet samples 2021a
Hyderabad, India.
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Grab samples (100 mL) Tharak et al.,
Domestic wastewater from Drains 2021
Hyderabad, India
Grab samples (100 mL) RT-PCR Kumar et al.,
Domestic wastewater from Drains ORF1ab, N and 2020
Ahmedabad, India. S genes)
Grab and composite sampling (24 h; RT-PCR; Ahmed et al.,
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100-200 mL) N_Sarbeco; 2020a; Ahmed

Untreated wastewater, Australia NIID_2019- et al., 2020b
SARS-CoV-2 nCOV_N
Composite sample (10 h) RT-PCR; CDC Prado et al.,
Untreated sewage wastewater from N2 2020
Rio de Janeiro, Brazil
Grab samples RT-PCR; SARS- Wang et al.,
Hospital septic tank influent Cov-2 nucleic 2020a;
Zhejiang University, China acid detection kit
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Grab samples (2 L) RT-PCR; CCDC- Zhang et al.,

Inlets and outlet of pre-processing ORF1, CCDC-N 2020
disinfection pool af STP gene
China
Composite sample (24 h; 500 mL) RT-PCR Mlejnkova et al.,
Untreated wastewater EliGene® 2020
Czech Republic COVID19
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BASIC A RT kit
Composite samples (24 h; 45 mL) RT-PCR Westhaus et
Untreated and treated wastewater M gene RdRP al.,2020

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Federal State of North Rhine- gene
Westphalia, Germany
Grab and composite samples (24 h; RT-PCR Lodder et al.,
250 mL) ORF1ab 2020a
Untreated wastewater Spike protein

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Italy RdRP
Grab samples (200-500 mL) RT-qPCR Haramoto et al.,
Influent and secondary treated N_Sarbeco 2020
wastewater Yamanashi Prefecture, NIID_2019-
Japan nCOV_N
CDC N1
CDC N2
Grab sampling (200 mL) RT-qPCR Randazzo et al.,
Influent, Secondary, Tertiary effluent, CDC N1, N2, N3 2020
Region of Murcia, Spain re- CDC N1, N2, N3
CDC N1, N2

Composite sampling (24 h; 250 mL) RT-qPCR Medema et al.,

Untreated Wastewater CDC N1 2020
Netherlands CDC N2
CDC N3
E_Sarbeco
Composite sample (24 h; 40 mL) RT-qPCR Wu et al., 2020b
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Untreated wastewater CDC N1
Massachusetts, USA CDC N2
CDC N3
Grab and composite samples (24 h, RT-qPCR Sherchan et al.,
100-750 mL) CDC N1 2020
Untreated and Secondary treated CDC N2
wastewater
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Southern Louisiana, USA

Grab sampling (40-70 L) RT-qPCR Miyani et al.,
Untreated wastewater CDC N1 2020
Southeast Michigan, USA
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Highlights

 Designed methodological approach for sewage surveillance of COVID-19

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 Hourly and daily sewage sampling were employed

 Viral RNA copies in sewage were assessed based on CT values obtained

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 Infected population in a community based on RNA copies in sewage

 Framework for wastewater-based epidemiology (WBE)

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Graphical Abstract

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Declaration of Interest Statement

 The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

☐ The authors declare the following financial interests/personal relationships which may be

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considered as potential competing interests:

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