Wastewater-based surveillance has been emerging as an efficient and advantageous tool to predict COVID-19 prevalence in the population, much earlier (7–28 days) than reported clinical cases, thus providing sufficient time to organize resources and optimize their use in managing COVID-19. Since the commencement of the COVID-19 pandemic, SARS-CoV-2 genetic lineages have emerged and are circulating all over the world. The assessment of SARS-CoV-2 variants of concern (VOCs) in wastewater has recently been proven to be successful. The present research demonstrates a case study utilizing an established approach to perform monitoring of SARS-CoV-2 variants from 11 distinct wastewater treatment plants across Jaipur (India) during the second peak period of COVID-19 (from 19 February 2021 to 8 June 2021). The sequences obtained were analyzed to detect lineage using the Pangolin tool and SNPs using the mpileup utility of Samtools, which reported high genome coverage. The mutation analyses successfully identified the penetration of the B.1. in the first two weeks of sampling (19–26 February), followed by the B.1.617.2 variant into Jaipur in the first week of March 2021. B.1.617.2 was initially discovered in India in October 2020; however, it was not reported until early April 2021.The present study identified the presence of B.1.617.2 in early March, which correlates well with the clinical patient’s data (290 cases were reported much later by the government on 10 May 2021). The average total genome coverage of the samples is 94.39% when mapped onto the severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1; a complete genome (NC_045512.2) sequence and SNP analysis showed that 37–51 SNPs were identified in each sample. The current study demonstrates that sewage surveillance for variant characterization is a reliable and practical method for tracking the diversity of SARS-CoV-2 strains in the community that is considerably faster than clinical genomic surveillance. As a result, this method can predict the advent of epidemiologically or clinically important mutations/variants, which can help with public health decision making.

The COVID-19 pandemic showcased the power of genomic sequencing to tackle the emergence and spread of infectious diseases. However, metagenomic sequencing of total microbial RNAs in wastewater has the potential to assess multiple infectious diseases simultaneously and has yet to be explored. A retrospective RNA-Seq epidemiological survey of 140 untreated composite wastewater samples was performed across urban (n = 112) and rural (n = 28) areas of Nagpur, Central India. Composite wastewater samples were prepared by pooling 422 individual grab samples collected prospectively from sewer lines of urban municipality zones and open drains of rural areas from 3rd February to 3rd April 2021, during the second COVID-19 wave in India. Samples were pre-processed and total RNA was extracted prior to genomic sequencing. This is the first study that has utilised culture and/or probe-independent unbiased RNA-Seq to examine Indian wastewater samples. Our findings reveal the detection of zoonotic viruses including chikungunya, Jingmen tick and rabies viruses, which have not previously been reported in wastewater. SARS-CoV-2 was detectable in 83 locations (59%), with stark abundance variations observed between sampling sites. Hepatitis C virus was the most frequently detected infectious virus, identified in 113 locations and co-occurring 77 times with SARS-CoV-2; and both were more abundantly detected in rural areas than urban zones. Concurrent identification of segmented virus genomic fragments of influenza A virus, norovirus, and rotavirus was observed. Geographical differences were also observed for astrovirus, saffold virus, husavirus, and aichi virus that were more prevalent in urban samples, while the zoonotic viruses chikungunya and rabies, were more abundant in rural environments. RNA-Seq can effectively detect multiple infectious diseases simultaneously, facilitating geographical and epidemiological surveys of endemic viruses that could help direct healthcare interventions against emergent and pre-existent infectious diseases as well as cost-effectively and qualitatively characterising the health status of the population over time. 10.13039/100014013 UK Research and Innovation (UKRI) 10.13039/100016270 Global Challenges Research Fund (GCRF) grant number H54810, as supported by Research England.

The present study investigated the detection of severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2) genomes at each treatment stage of 14 aerobic wastewater treatment plants (WWTPs) serving the major municipalities in two states of Rajasthan and Uttarakhand in Northern India. The untreated, primary, secondary and tertiary treated wastewater samples were collected over a time frame ranging from under-lockdown to post-lockdown conditions. The results showed that SARS-CoV-2 RNA was detected in 13 out of 40 wastewater samples in Jaipur district, Rajasthan and in 5 out of 14 wastewater samples in the Haridwar District, Uttarakhand with the E gene predominantly observed as compared to the N and RdRp target genes in later time-points of sampling. The Ct values of genes present in wastewater samples were correlated with the incidence of patient and community cases of COVID-19. This study further indicates that the viral RNA could be detected after the primary treatment but was not present in secondary or tertiary treated samples. This study implies that aerobic biological wastewater treatment systems such as moving bed biofilm reactor (MBBR) technology and sequencing batch reactor (SBR) are effective in virus removal from the wastewater. This work might present a new indication that there is little to no risk in relation to SARS-CoV-2 while reusing the treated wastewater for non-potable applications. In contrast, untreated wastewater might present a potential route of viral transmission through WWTPs to sanitation workers and the public. However, there is a need to investigate the survival and infection rates of SARS-CoV-2 in wastewater.

The infection with SARS-CoV-2 is reported to be accompanied by the shedding of the virus in stool samples of infected patients. Earlier reports have suggested that COVID-19 agents can be present in the fecal and sewage samples and thus it can be a good indication of the pandemic extent in a community. However, no such studies have been reported in the Indian context so far. Since, several factors like local population physiology, the climatic conditions, sewage composition, and processing of samples could possibly affect the detection of the viral genome, it becomes absolutely necessary to check for the presence of the SARS-CoV-2 in the wastewater samples from wastewater treatment plants (WWTPs) serving different localities of Jaipur city, which has been under red zone (pandemic hotspots) since early April 2020. Samples from different local municipal WWTPs and hospital wastewater samples were collected and wastewater based epidemiology (WBE) studies for the presence of SARS-CoV-2 were carried out using the RT-PCR technique to confirm the presence of different COVID-19 target genes namely S gene, E gene, ORF1ab gene, RdRp gene and N gene in the viral load of wastewater samples. In the present study, the untreated wastewater samples from the municipal WWTPs and hospital wastewater samples showed the presence of SARS-CoV-2 viral genome, which was correlated with the increased number of COVID-19 positive patients from the concerned areas, as per reported in the publically available health data. This is the first study that investigated the presence of SARS-CoV-2 viral genome in wastewater, at higher ambient temperature (above 40°C), further validating WBE as a potential tool in predicting and mitigating outbreaks. Highlights: The study reports detection of SARS-CoV-2 in sewage in India. The presence of SARS-CoV-2 was confirmed by RT-PCR. The presence of viral genome was detected at high ambient temperatures of 40-45° C. Corroborates trends in the WWTPs showing viral genome with public health data. Treated effluent from WWTPs appears safe for reuse with low public health concern. Graphical

We investigated the presence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) RNA at different treatment stages of 15 wastewater treatment plants (WWTPs) in two North Indian states of Rajasthan and Uttarakhand. Untreated (influent), biologically treated, and disinfected wastewater samples were collected from May to August 2020. The qualitative analysis of the wastewater for the presence of SARS-CoV-2 RNA was done using different pre-processing methods. SARS-CoV-2 RNA was detected in 11 out of 39 wastewater samples in Jaipur district and 5 out of 17 wastewater samples in Haridwar District using Reverse-Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR) for qualitative detection. None of the 56 samples tested for post-secondary or tertiary treatment were found positive for SARS-CoV-2 RNA. The findings indicate that there are no SARS-CoV-2 related risks involved with using the treated effluent for non-potable applications. In contrast, untreated wastewater may be a potential route of viral transmission to the WWTP and sanitation workers. Future studies are imperative to understand the survival rates of these viruses in wastewater. Graphical Highlights: Mild to moderate genome load observed in the municipal wastewater samples. Increased patient numbers post-lockdown correspond to a decrease in the C _T value of genes. Presence of SARS-CoV-2 genome load was observed in untreated wastewater. E gene was present in abundance in wastewaters as compared to the N gene and RdRp gene. SARS-CoV-2 genome load was absent in secondary and tertiary treated effluent.