Circulation of SARS-CoV-2 Omicron sub-lineages revealed by multiplex genotyping RT-qPCR assays for sewage surveillance.

Sewage surveillance has proven to be an essential complementary tool to clinical diagnosis in combating the COVID-19 pandemic by tracking the spread of the SARS-CoV-2 virus and evaluating infection levels in populations. With the striking spreading and continuous evolution of SARS-CoV-2 Omicron VOC that characterized with higher transmissibility and potential immune evasion, there is an urgent need for the rapid surveillance of this prevalent strain and its sub-lineages in sewage. In this study, based on three multiplex allele-specific (AS) RT-qPCR assays, we established a rapid and high-throughput detection workflow for the simultaneous discrimination of Omicron sub-lineages BA.2.2, BA.2.12.1, BA.4 and BA.5 (hereafter referred to as BA.4/BA.5) to track their community circulation in Hong Kong. All primer-probe sets in the multiplex assays could correctly discriminate and quantitate their target genotypes with high sensitivity and specificity, even when multiple variants co-existed in the sewage samples. Using the established multiplex assays, the trends of SARS-CoV-2 total viral load and variant dynamics in influent samples collected from 11 wastewater treatment plants (WWTPs) during June 2022 and September 2022, aligned with the clinical data, successfully unveiling the swift emergence and predominance of Omicron BA.4/BA.5 in Hong Kong. The study highlights the feasibility and applicability of multiplex RT-qPCR assays for monitoring epidemic trends and tracking variant displacement dynamics in sewage samples, providing a more rapid, high-throughput and cost-effective alternative to enhance the current sewage surveillance system.