key: cord-292045-pnid9dmq
authors: Kumar, Manish; Patel, Arbind Kumar; Shah, Anil V.; Raval, Janvi; Rajpara, Neha; Joshi, Madhvi; Joshi, Chaitanya G.
title: First proof of the capability of wastewater surveillance for COVID-19 in India through detection of genetic material of SARS-CoV-2
date: 2020-07-28
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2020.141326
sha: 
doc_id: 292045
cord_uid: pnid9dmq

Abstract We made the first ever successful effort in India to detect the genetic material of SARS-CoV-2 viruses to understand the capability and application of wastewater-based epidemiology (WBE) surveillance in India. Sampling was carried out on 8 and 27 May 2020 at the Old Pirana Waste Water Treatment Plant (WWTP) at Ahmedabad, Gujarat that receives effluent from Civil Hospital treating COVID-19 patients. All three, i.e. ORF1ab, N and S genes of SARS-CoV-2, were found in the influent with no genes detected in effluent collected on 8 and 27 May 2020. Increase in SARS-CoV-2 genetic loading in the wastewater between 8 and 27 May 2020 samples concurred with corresponding increase in the number of active COVID-19 patients in the city. The number of gene copies was comparable to that reported in untreated wastewaters of Australia, China and Turkey and lower than that of the USA, France and Spain. However, temporal changes in SARS-CoV-2 RNA concentrations need to be substantiated further from the perspectives of daily and short-term changes of SARS-CoV-2 in wastewater through long-term monitoring. The study results SARS-CoV-2 will assist concerned authorities and policymakers to formulate and/or upgrade COVID-19 surveillance to have a more explicit picture of the pandemic curve. While infectivity of SARS-CoV-2 through the excreted viral genetic material in the aquatic environment is still being debated, the presence and detection of genes in wastewater systems makes a strong case for the environmental surveillance of the COVID-19 pandemic.

The current ongoing global Coronavirus disease pandemic, caused by the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread to 216 countries and territories, with 7.7 million of the confirmed cases and more than 425,000 deaths worldwide, as of June 12, 2020 (WHO, 2020) . The active replication of infectious SARS-CoV-2 particles in enterocytes of human intestine due to expression of ACE2 receptor causes shedding of virus in the faeces (Lamers et al. 2020; Qi et al., 2020) . The clinically reported symptoms in COVID-19 patients mainly include cough, difficulty in breathing, fever and diarrhoea (Gao et al., 2020; Kumar et al., 2020a) . However, during a previous study of COVID-19 patients, SARS-CoV-2 RNA was detected in faeces more frequently than gastrointestinal symptoms (17%) such as diarrhoea (Cheung et al., 2020) . These results suggest a large number of asymptomatic individuals along with symptomatic patients, discharge the virus which ultimately reaches sewage treatment plants (Haramoto et al., 2020) . The virus can be shed in faeces for several days, even after the patient stops exhibiting respiratory symptoms . Zheng et al., (2020) , reported detection of SARS-J o u r n a l P r e -p r o o f disease outbreak in a certain catchment by monitoring viral load in the wastewater, as it contains excrement from both symptomatic and asymptomatic individuals (Xagoraraki and O'Brien, 2020; Choi et al. 2018; Yang et al., 2015) . WBE was an effective tool during past outbreak of other enteric viruses, such as poliovirus, hepatitis A and norovirus (Hellmér et al., 2014; Asghar et al., 2014; Kitajima et al., 2020 , Kumar et. al., 2020a , it can be used as an early warning tool for the disease outbreak in a community and used to inform the efficacy of the current public health interventions . WBE data can help estimate actual infected population due to the virus, as it also covers asymptomatic and presymptomatic the patients, which may be underestimated by clinical surveillance (Bivinis et. al., 2020; Tang et al., 2020; Wölfel et al., 2020a; , Kumar et al., 2020a .

Detection of, SARS-CoV-2 RNA in wastewater has been reported in Australia, China, France, Israel, Italy, Japan, Netherlands, Spain and the US (Ahmed et al., 2020a,b; Bar-or et al., 2020; Haramoto et al., 2020; La Rosa et al., 2020; Medema et al., 2020; Nemudryi et al., 2020; Randazzo et al., 2020; Rimoldi et al., 2020; Wurtzer et al., 2020 , Kumar et al., 2020c . According to some of these studies, after the number of confirmed cases reached to 1-100 per million population, SARS-CoV-2 RNA was detected in wastewater (Ahmed et al., 2020a; Bar-or et al., 2020; Medema et al., 2020; Nemudryi et al., 2020; Wurtzer et al., 2020 , Kumar et al., 2020d . To date of submission of this work, there is no study reporting detection of SARS-CoV-2 in wastewater in India. As of June 12, 2020, the number of confirmed cases in India was 223 per million population. The first case of COVID-19 in India was reported on January 30, 2020 and the number of confirmed cases has reached more than 300,000 as of June 12, 2020 (Ministry of Health and Family Welfare, India). The state of Gujarat has reported >22,500 confirmed cases of COVID-19, as of June 12, J o u r n a l P r e -p r o o f 2020, with >12,000 confirmed cases in Ahmedabad city (Ministry of Health and Family Welfare, India).

To further understand the capability and potential application of WBE surveillance, we made the first successful detection of genetic material of the SARS-CoV-2 virus in India. We also analysed the temporal variation in genetic material loadings in the same wastewater treatment plant during a lockdown period in India. Finally, we evaluated the effect of traditional treatment systems on SARS-CoV-2 genetic material and aim to assist concerned authorities and policymakers to formulate and/or upgrade COVID-19 surveillance to include an explicit picture of the pandemic curve.

Wastewater samples were collected on 8 and 27 May, 2020 from the Old Pirana Waste Water Treatment Plant (WWTP) at Ahmedabad, Gujarat which is the largest wastewater treatment plant in Asia with a capacity of >180 m 3 /day. The WWTP is equipped with an Upflow Anaerobic Sludge Blanket (UASB) as an advanced process to treat the wastewater.

This WWTP is designed to produce treated water with pH, biological oxygen demand (BOD), total suspended solids (TSS), and chemical oxygen demand of 7-8.5, <20 mg.L -1 , <30 mg.L -1 and <100 mg.L -1 respectively. The sampling location for this study was selected based on the fact that Pirana WWTP receives the sewage waste of a government civil hospital treating COVID-19 patient. To ensure accuracy and precision, duplicated analyses of the samples were also performed for a raw wastewater, in which the reproducibility was fairly high (average C T difference of 1.2). Several blanks were prepared and run to check the cross-contamination, and sensitivity of the protocol, extraction and instrumentation. All analyses were conducted at the Indian Council of Medical Research (ICMR), New Delhi, an approved facility of the Gujarat Biotechnology Research Centre (GBRC).

Viral RNAs were isolated from sewage samples using following steps: precipitation of viral particles; viral RNA isolation and quality checking.

The sewage samples (50 mL) were centrifuged at 4500×g (Model: Sorvall ST 40R ,Thermo Scientific) for 30 min followed by filtration of supernatant using 0.22 micron filters (Mixed cellulose esters syringe filter, Himedia). Each sewage filtrate was then concentrated using the poly ethylene glycol (PEG) methods. For this method, PEG 9000 (80 g/L) and NaCl (17.5 g/L)

were mixed with 25 ml filtrate and this was incubated overnight at 17°C and 100 rpm (Model:

Incu-Shaker TM 10LR, Benchmark). The following day the mixture was centrifuged at 13000×g (Model: Kubota 6500, Kubota Corporation) for 90 mins. After centrifugation, the supernatant was discarded and the pellet resuspended in 300 µL RNase-free water. This was further used as a sample for RNA isolation.

RNA isolation was carried out using a commercially available kit (NucleoSpin ® RNA Virus, Macherey-Nagel GmbH & Co. KG, Germany). Concentrated viral particles (200 µL) were mixed with 10 µL MS2 phage, 20 µL Proteinase K (20mg/mL) solution and 600 µL of RAV1 buffer containing carrier RNA. Here, MS2 phage was taken as a molecular process inhibition control (MPC; Haramoto et al 2018) for evaluating the efficiency of nucleic acid extraction and PCR inhibition. It is to be noted that MS2 may naturally occur in wastewater, it is therefore there is possibility that recovered MS2 may consist both the spiked and background viral content. Further steps were carried out as instructed in the product manual (Macherey-Nagel GmbH & Co. KG). Final elution was carried out with 30 µL of elution buffer (provided by kit). RNA concentrations were determined using a Qubit 4 Fluorometer (Invitrogen).

J o u r n a l P r e -p r o o f RNAs were analysed for the detection of ORF1ab, N gene and S gene of SARS-CoV-2 and MS2 (internal process control) by RT-PCR using TaqPath TM Covid-19 RT-PCR Kit (Applied Biosystems). Amplification was performed in a 25 μL reaction mixture containing 7 μL extracted nucleic acids of each samples. Positive control (2 μL) (TaqPath™ COVID-19 Control) and purified negative control (5 μL) were used in case of positive and negative control respectively. Nuclease free water was used as no template control in this study. Further procedures were carried out as described in product manual. RT-PCR experiment consisted of UNG incubation at 25°C for 2 min, reverse transcription at 53 °C for 10 min and activation at 95 °C for 2 min, followed by 40 cycles, each involving denaturation at 95 °C for 3s followed by annealing/extention at 60 °C for 30s. The reactions were performed in Applied Biosystems™ 7500 Fast Real Time PCR system (Applied Biosystem), and interpreted as instructed in the manual.

Although there is no direct correlation of the C T value to copy numbers as the kit used for the detection is qualitative assay yet we put an effort to calculate number of gene copies present in a unit volume of the sample. For this the well-established principle of 3.3 C T change corresponds to 10-fold change has been used. More precisely, 500 copies of SARS-CoV-2 genes were taken as positive control with C T of average 26 for all the three genes i.e. ORF1ab, N and S, which were then extrapolated to compare it with sample C T values and derive approximate copies of genes in the wastewater sample. The amount of RNA used as template was multiplied with the enrichment factor to derive an estimated copy numbers for each wastewater sample. Table 1 shows the number of active cases for the city i.e. Ahmedabad and India, obtained by deducting recovered cases from total confirmed cases since 17 March, 2020.

Consistency between abundance of SARS-CoV-2 genetic materials and number of confirmed cases was observed in the previous reports in Australia, France, Italy, Spain and Japan Further, referring to the limitations of the present study owing to lockdown scenario, we recommend that although based MPC analysis, the efficiency of RNA extraction and RT-PCR is considered high for all the wastewater samples collected for this study, the efficiency of PEG method could have been better established. Further, based on indigenous F-phage analysis Hata et al., (2020) reported a high efficiency of PEG method in Japanese wastewater, yet an evaluation of sample concentration efficiency, using the whole process control (WPC) together with MPC is recommended (Haramoto et al 2018) . We recommend longer monitoring with several replicated analyses to evaluate the correlation as well as uncertainties involving RT-PCR (Stuart et al 2014) and then replace the semi-quantitative method employed in this study with precise copy calculations using suitable methods.

Nevertheless, the bottom line is that the patterns of obtained C T values suggest successful detection of SARS-CoV-2 RNA from the wastewater samples, their increasing abundance together with an increase of COVID-19 confirmed cases, and their reduction by UASB treatment and aeration pond. In summary, results demonstrated the capability of wastewater-based epidemiology in Indian settings and strongly advocates that despite the lack of quality sewer infrastructure or other wastewater collection issues, WBE can be applicable and thus we strongly implementing environmental surveillance of the CVOID-19 pandemic in India, starting with major cities.

While the world is providing high resolution proofs of the WBE concept, India needed indigenous proof of concept and its applicability. In this context, we achieved two major outcomes: i) for the first time in India and top 10 efforts in the world, we isolated SARS-CoV-2 genetic material and detected it during a lockdown period owing to good coordination among the government organizations; ii) temporal variation in Ct value demonstrated the capability of WBE surveillance in India; and iii) for the third time in the world treated water was analysed for the presence and confirmation of SARS-CoV-2 genetic material. The results were of good resolution and provided significant indication of temporal variation in COVID-19 patient loadings. However, owing to limited samples analysed in this preliminary study, even though the case numbers align with increased RNA concentrations in wastewater, the temporal changes in SARS-CoV-2 RNA concentrations needs to be further investigated from the several perspectives of daily, short-term and long-term changes. Our results demonstrated that a conventional treatment plant is capable of removing genetic materials J o u r n a l P r e -p r o o f of SARS-CoV-2, however there may not be the complete elimination. In a country like India where sewer systems are not complete and only a part of the waste is received at WWTPs, it is essential to study each treatment stage to determine the effectiveness of treatment. This will help reduce the commonly perceived fear of the commons pertaining to the effectiveness of treatment plants as well as transmission through wastewater.

The authors declare no competing financial interest.

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We acknowledge the unimaginable support from every corner as naming all of them involved or helped during analyses or sampling or protocol development would be difficult. Special 
