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.

For the first time, we present, i) an account of decay in the genetic material loading of SARS-CoV-2 during Upflow Anaerobic Sludge Blanket (UASB) treatment of wastewater, and ii) comparative evaluation of polyethylene glycol (PEG), and filtration as virus concentration methods from wastewater for the quantification of SARS-CoV-2 genes. The objectives were achieved through tracking of SARS-CoV-2 genetic loadings i.e. ORF1ab, N and S protein genes on 8th and 27th May 2020 along the wastewater treatment plant (106 million liters per day) equipped with UASB system in Ahmedabad, India. PEG method performed better in removing materials inhibiting RT-qPCR for SARS-CoV-2 gene detection from the samples, as evident from constant and lower C T values of control (MS2). Using the PEG method, we found a reduction >1.3 log 10 in SARS-CoV-2 RNA abundance during UASB treatment, and the RNA was not detected at all in the final effluent. The study implies that i) conventional wastewater treatment systems is effective in SARS-CoV-2 RNA removal, and ii) UASB system significantly reduces SARS-CoV-2 genetic loadings. Finally, PEG method is recommended for better sensitivity and inhibition removal during SARS-CoV-2 RNA quantification in wastewater.

we made the first-ever successful effort from India to detect the genetic material of SARS-CoV-2 viruses to understand the capability and application of WBE surveillance in India. Sampling was carried out on 8 and 27 May 2020 from Old Pirana Waste Water Treatment Plant (WWTP) at Ahmedabad, Gujarat with 106 million liters per day (MLD) capacity receiving effluent of Civil Hospital treating COVID-19 patient. All three i.e. ORF1ab, N and S genes of SARS-CoV-2 were discerned in the influents with no gene spotted in the effluent collected on 8 and 27 May 2020. Temporal difference between 8 and 27 May 2020 samples was of 10x in gene copy loading with a corresponding change of 2x in the number active COVID-19 patient in the city. Number of gene copies was found comparable to that reported in the untreated wastewaters of Australia, China, and Turkey and lower than that of the USA, France, and Spain. This study, being the first from India and probably among the first ten reports in the world of gene detection of SARS-CoV-2 in the environmental samples, aims to assist concerned authorities and policymakers to formulate and/or upgrade the COVID-19 surveillance to have an explicit picture of the phase of the pandemic. While infectious SARS-CoV-2 has yet to be identified in the aquatic environment, the virus potentially enters the wastewater stream from patient excretions and thus can be a great tool for pandemic monitoring.

Wastewater-based Epidemiological (WBE) surveillance offers a promising approach to assess the pandemic situation covering pre-symptomatic and asymptomatic cases in highly populated area under limited clinical tests. In the present study, we analysed SARS-CoV-2 RNA in the influent wastewater samples ( n = 43) from four wastewater treatment plants (WWTPs) in Gandhinagar, India, during August 7 th to September 30 th , 2020. A total of 40 samples out of 43 were found positive i.e. having at least two genes of SARS-CoV-2. The average Ct values for S, N, and ORF 1ab genes were 32.66, 33.03, and 33.95, respectively. Monthly variation depicted a substantial rise in the average copies of N (∼120%) and ORF 1ab (∼38%) genes in the month of September as compared to August, while S-gene copies declined by 58% in September 2020. The SARS-CoV-2 genome concentration was higher in the month of September (∼924.5 copies/L) than August (∼897.5 copies/L), corresponding to a ∼ 2.2-fold rise in the number of confirmed cases during the study period. Further, the percentage change in genome concentration level on a particular date was found in the lead of 1-2 weeks of time with respect to the official confirmed cases registered based on clinical tests on a temporal scale. The results profoundly unravel the potential of WBE surveillance to predict the fluctuation of COVID-19 cases to provide an early warning. Our study explicitly suggests that it is the need of hour that the wastewater surveillance must be included as an integral part of COVID-19 pandemic monitoring which can not only help the water authorities to identify the hotspots within a city but can provide up to 2 weeks of time lead for better tuning the management interventions.

Following the proven concept, capabilities, and limitations of detecting the RNA of Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) in wastewater, it is pertinent to understand the utility of wastewater surveillance data on various scale. In the present work, we put forward the first wastewater surveillance-based city zonation for effective COVID-19 pandemic preparedness. A three-month data of Surveillance of Wastewater for Early Epidemic Prediction (SWEEP) was generated for the world heritage city of Ahmedabad, Gujarat, India. In this expedition, one hundred sixteen wastewater samples were analyzed to detect SARS-CoV-2 RNA, from September 3rd to November 26th, 2020. A total of 111 samples were detected with at least two out of three SARS-CoV-2 genes (N, ORF 1ab, and S). Monthly variation depicted a significant decline in all three gene copies in October compared to September 2020, followed by a sharp increment in November 2020. Correspondingly, the descending order of average effective gene concentration was: November (~10,729 copies/L) > September (~3047 copies/L) > October (~454 copies/L). Monthly variation of SARS-CoV-2 RNA in the wastewater samples may be ascribed to a decline of 20.48% in the total number of active cases in October 2020 and a rise of 1.82% in November 2020. Also, the monthly recovered new cases were 16.61, 20.03, and 15.58% in September, October, and November 2020, respectively. The percentage change in the gene concentration was observed in the lead of 1–2 weeks with respect to the provisional figures of confirmed cases. SWEEP data-based city zonation was matched with the heat map of the overall COVID-19 infected population in Ahmedabad city, and month-wise effective RNA concentration variations are shown on the map. The results expound on the potential of WBE surveillance of COVID-19 as a city zonation tool that can be meaningfully interpreted, predicted, and propagated for community preparedness through advanced identification of COVID-19 hotspots within a given city.