key: cord-0712465-zfz8irdg
authors: Hemalatha, Manupati; Kiran, Uday; Kuncha, Santosh Kumar; Kopperi, Harishankar; Gokulan, C. G.; Mohan, S. Venkata; Mishra, Rakesh K.
title: Surveillance of SARS-CoV-2 spread using wastewater-based epidemiology: Comprehensive study
date: 2021-01-06
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2020.144704
sha: 70dc4712887d7f9e89716e034af70e0a77fdd53f
doc_id: 712465
cord_uid: zfz8irdg

SARS-CoV-2 pandemic is having a devastating effect on human lives. Recent reports have shown that majority of the individuals recovered from COVID-19 have serious health complications, which is going to be a huge economic burden globally. Given the wide-spread transmission of SARS-CoV-2 it is almost impossible to test every individual in densely populated countries. Recent reports have shown that sewage-based surveillance can be used as holistic approach to understand the spread of the pandemic within a population or area. Here we have estimated the spread of SARS-CoV-2 in the city of Hyderabad, India, which is a home for nearly 10 million people. The sewage samples were collected from all the major sewage treatment plants (STPs) and were processed for detecting the viral genome using the standard RT-PCR method. Interestingly, inlet samples of STPs were positive for SARS-CoV-2, while the outlets were negative, which indicates that the standard sewage treatment methods are efficient in eliminating the SARS-CoV-2 viral particles. Based on the detected viral gene copies per litre and viral particle shedding per individual, the total number of individuals exposed to SARS-CoV-2 was estimated. Through this study we suggest that sewage-based surveillance is an effective approach to study the infection dynamics, which helps in efficient management of the SARS-CoV-2 spread.

The surveillance of disease prevalence during pandemic like Coronavirus Disease-19 (COVID-19) is a crucial task considering the spreading rate and high population in different parts of the world. The massive testing of the population to contain the spread of the virus is a challenge. Moreover, the problem is further compounded because a majority of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infected individuals are asymptomatic.

Emerging studies have shown the aftereffects of COVID-19 is going to be huge economic burden globally and therefore pressing the importance of not only managing the infected individuals but also to keep a check on the spread (McKibbin and Fernando 2020).

Asymptomatic and symptomatic infections result in significant uncertainty in the estimated extent of SARS-CoV-2 infection . Considering the present testing capacity and cost incurred, it is impractical to test all the individuals. Thus, there is a need for alternative strategies to assess the disease spread and therefore efficiently allocate resources for disease management. 

The detection of SARS-CoV-2genetic material in domestic sewage was performed by collecting samples from different sewage treatment plants (STPs) in Hyderabad Metropolitan City, India. Hyderabad (17.37°N 78.48°E) is fifth-largest urban economy in India and is a capital for Telangana state occupies ~625 square kilometers. It is the fourth-most populous city in India with 10 million residents in the metropolitan region. The raw sewage samples were collected from inlet and outlet points from the STPs with a total coverage of 712 million litres per day (MLD), that receive wastewater from all parts of the city (80% coverage of the existing STPs).

The sewage samples were collected from 8 th July 2020 to 6 th August 2020 (Table 1) taking all the safety measures as per the standard operating procedure (SOP) designed for this purpose.

A total of 30 samples were collected from 14 inlets (equalization tanks outlet) and 14 treated wastewaters (outlets of secondary clarifiers (SC)) of 10 STPs and 2 samples from a gated community (outlet of collection tank prior to disposing to drains). A 10 MLD STP was selected for a time course study to understand the weekly variation in the viral load, where weekly samples were collected and analysed. The basis for sample collection from equalization tank/secondary clarifier of STP is that it would provide a composite sample accounting for a period of retention time (1-5 h) . Grab sampling protocol (Rimoldi et al., 2020) was employed for sampling one litre of sewage in a disposable bottle (plastic) of 1 liter capacity with 20 ml of 0.1% hypochlorite/liter to inactivate the pathogens. After sampling the surface of the sample container is disinfected with hypochlorite and sealed in multi-layered plastic covers, labelled and transported (2-4 °C) immediately to lab prior to storing at 4°C J o u r n a l P r e -p r o o f until further processing. All the samples were processed within 12 hours of the sampling event unless mentioned otherwise. During sampling, care was taken to follow all biosafety protocols. All the sampling activities were performed during the daytime when peak load was available (8 am to 4 pm), on the days with no report of rainfall events during last 24 hours. 

Collected samples were subjected to gravity filtration with 1 mm thick blotting sheets to remove the debris or larger particles followed by filtration using 0.2 µm filtration units (Nalgene® vacuum filtration system; Thermofisher Scientific) to remove bacteria. The filtrate was collected in 1000 mL sterile wide-mouth bottles (Borosil). 100 mL of the total filtrate was concentrated to ~600 µl using 15 mL 30 kDa Amicon® Ultra-15 (Merck Millipore) by centrifugation at 4000 rpm (4 °C ; 10 min). The concentrated samples were further processed for RNA isolation. Sample filtration, concentration and processing till detection were performed in a Biosafety level 2 (BSL-2) facility. All the materials after use were discarded in biosafety bags followed by decontamination.

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To quantify SARS-CoV-2 RNA in sewage samples, a total of 300 µl concentrate was used for RNA extraction using QIAamp Viral RNA isolation kit (Qiagen, Germany) by following manufacturer's protocol. The isolated RNA samples were tested for presence of SARS-CoV- 

To assess the performance and efficiency of the qRT-PCR kit used in this work, 2.14*10 7 pfu/mL viral culture was inactivated at 55 0 C for 30 min and provided to us by Dr. H H Krishnan, CSIR-CCMB. RNA was isolated from the heat-inactivated SARS-CoV-2 which was followed by the preparation of log 10 dilutions of the RNA. RT-PCR was performed in J o u r n a l P r e -p r o o f triplicates for each dilution. The R 2 values obtained from linear regression and efficiency was calculated as described (Ginzinger, 2002 ).

To calculate the number of RNA copies, present in the wastewater samples, the envelope protein coding gene (E gene) amplified from the SARS-CoV-2 RNA was cloned into the vector pcDNA3.1 between KpnI and HindIII restriction sites. The cloned plasmid was then quantified using Qubit™ dsDNA HS Assay Kit (Invitrogen, USA) and Qubit™ 4

Fluorometer (Invitrogen, USA). The copy number per nanogram was calculated using the E gene and vector sequences retrieved from https://www.ncbi.nlm.nih.gov/nuccore/NC_045512.2?report=fasta&from=26245&to=26472

and https://www.addgene.org/browse/sequence_vdb/2093/, respectively. The plasmid was serially diluted from 9.01 log 10 copies to 0.01 log 10 copies and the RT-PCR reaction was performed as mentioned in section 2.5 in triplicates. The C T values were plotted against the log copy number and a linear fit equation was obtained (Supplementary Table 3 ;

To find out the recovery of SARS-CoV-2 from sewage samples, 1 ml of 2.14x10 7 pfu/ml SARS-CoV-2 virus (heat-inactivated) was added to 100 ml of sewage water and four log 10 dilutions were prepared (with sewage water). As a control, similar dilutions were made using milliQ water (MQ). The RNA was isolated from all the samples and RT-PCR was performed in triplicates.

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To identify the viral copy number present in the wastewater samples, the linear fit equation showing higher C T (Fig. 3) . This indicates the presence of viral genome even after 3 weeks when the samples are stored at 4 0 C, however, it is best to analysed the samples before 24 hours for all practical purpose. Table 2 ). We also surveyed samples collected from a gated residential community where confirmed positive cases were reported during the sample collection period and observed the presence of SARS-CoV-2 RNA in the samples in trace amounts (Table 5 ). Table 5 3

One of the STPs (10 MLD), was sampled at different time to assess the dynamics of disease spread with time. We observed a highly fluctuating pattern of viral RNA presence with time, from as low as 661 copies per L wastewater (on 14-07-2020) to as high as 24469 copies per L wastewater (on 29-07-2020) ( Table 6; Supplementary Table 3 (Table 7 and 8) . Owing to the uncertainty and difference in the number of viral particles excreted by infected individuals, we calculated the possible number of infected people for three different shedding rates within the reported range (10 5 , 10 6 , and 10 7 copies/mL faeces) ( Table 8) . Results indicate that the number of infected people might be anywhere between thirty thousand and three million during the study period (Table 8) . Based on the recent learning from SARS-CoV-2, it is evident that screening large population to contain the spread is an inconceivable task and it is more complex in urban areas with high population density. As the SARS-CoV-2 colonise the GI tract and is released through faeces This study provides a concrete evidence for the application of WBE as a potential method for disease as well as environmental surveillance. These results will be an immense resource for the healthcare and associated departments to vigilantly allocate the necessary resources to J o u r n a l P r e -p r o o f manage existing cases as well as to carefully contain the disease spread. Along with clinical data, WBE could provide critical monitoring of SARS-CoV-2 transmission within a community including the beginning, tapering, or reemergence of an epidemic (Bivins et al., 2020 . Hence, sewage-based surveillance provides a holistic approach to manage the pandemic and also to monitor for future outbreaks, if any. The current study also offers a framework to monitor other pathogens to avoid future epidemic. Overall, this study provides a simplistic framework for WBE studies with basic resources available in majority of the labs towards sustainable environmental Surveillance. We strongly recommend the scientific community and the healthcare agencies to pursue similar studies periodically, for allocating resources appropriately to fight the pandemic.

The authors declare no conflict of interest. J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f 

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