Simultaneous detection and characterization of common respiratory pathogens in wastewater through genomic sequencing.

Genomic surveillance of SARS-CoV-2 has given insight into the evolution and epidemiology of the virus and its variant lineages during the COVID-19 pandemic. Expanding this approach to include a range of respiratory pathogens can better inform public health preparedness for potential outbreaks and epidemics. Here, we simultaneously sequenced 38 pathogens including influenza viruses, coronaviruses and bocaviruses, to examine the abundance and seasonality of respiratory pathogens in urban wastewater. We deployed a targeted bait capture method and short-read sequencing (Illumina Respiratory Virus Oligos Panel; RVOP) on composite wastewater samples from 8 wastewater treatment plants (WWTPs) and one associated hospital site. By combining seasonal sampling with whole genome sequencing, we were able to concurrently detect and characterise a range of common respiratory pathogens, including SARS-CoV-2, adenovirus and parainfluenza virus. We demonstrated that 38 respiratory pathogens can be detected at low abundances year-round, that hospital pathogen diversity is higher in winter vs. summer sampling events, and that significantly more viruses are detected in raw influent compared to treated effluent samples. Finally, we compared detection sensitivity of RT-qPCR vs. next generation sequencing for SARS-CoV-2, enteroviruses, influenza A/B, and respiratory syncytial viruses. We conclude that both should be used in combination; RT-qPCR allowed accurate quantification, whilst genomic sequencing detected pathogens at lower abundance. We demonstrate the valuable role of wastewater genomic surveillance and its contribution to the field of wastewater-based epidemiology, gaining rapid understanding of the seasonal presence and persistence for common respiratory pathogens. By simultaneously monitoring seasonal trends and early warning signs of many viruses circulating in communities, public health agencies can implement targeted prevention and rapid response plans.

Near-source passive sampling for monitoring viral outbreaks within a university residential setting.

Wastewater-based epidemiology (WBE) has proven to be a powerful tool for the population-level monitoring of pathogens, particularly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For assessment, several wastewater sampling regimes and methods of viral concentration have been investigated, mainly targeting SARS-CoV-2. However, the use of passive samplers in near-source environments for a range of viruses in wastewater is still under-investigated. To address this, near-source passive samples were taken at four locations targeting student hall of residence. These were chosen as an exemplar due to their high population density and perceived risk of disease transmission. Viruses investigated were SARS-CoV-2 and its variants of concern (VOCs), influenza viruses, and enteroviruses. Sampling was conducted either in the morning, where passive samplers were in place overnight (17 h) and during the day, with exposure of 7 h. We demonstrated the usefulness of near-source passive sampling for the detection of VOCs using quantitative polymerase chain reaction (qPCR) and next-generation sequencing (NGS). Furthermore, several outbreaks of influenza A and sporadic outbreaks of enteroviruses (some associated with enterovirus D68 and coxsackieviruses) were identified among the resident student population, providing evidence of the usefulness of near-source, in-sewer sampling for monitoring the health of high population density communities.