Detection of Noroviruses GII in wastewater samples of Bhopal, India, using Droplet Digital PCR

Noroviruses are a significant cause of global gastroenteritis outbreaks, underscoring the importance of effective surveillance. Wastewater-based epidemiology helps identify viral pathogens in communities. Recent advancements in wastewater-based molecular surveillance have shown that viruses can be detected in feces and urine early, making sewage monitoring an essential tool for tracking viral presence. We aimed to create and validate a new method for detecting and monitoring Noroviruses GII in Bhopals wastewater using Automated Droplet Digital PCR (ddPCR) technology. In this study, a ddPCR assay targeting the ORF1-2 region of Norovirus GII was developed, allowing viral nucleic acid quantification without a standard curve. A total of 27 samples from five Sewage Treatment Plants located in Bhopal city were collected during the summer season (April and May 2023) at fortnightly intervals and analyzed for the presence of Norovirus using the novel ddPCR assay. Among the samples tested, 33% tested positive for Norovirus, with the highest detection rate observed as 72.72%, followed by 25%. The concentrations of Noroviruses GII in positive samples ranged from 0.06 to 6.60 copies/{micro}l. These findings indicate a potentially higher patient population within Bansal Hospitals catchment area than the other STPs in the Bhopal region. The study underscores Noroviruss varying prevalence and distribution in wastewater across different STPs in Bhopal. Moreover, it demonstrates the utility of wastewater surveillance and digital PCR in accurately and specifically detecting Norovirus in wastewater. The practical application of this wastewater surveillance strategy could serve as an early warning system for communities, enabling timely preparedness for impending viral outbreaks, implementation of effective administrative containment measures, and intensified vaccination campaigns.