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On November 26, 2021, the B.1.1.529 COVID-19 variant was classified as the Omicron variant of concern (VOC). Reports of higher transmissibility and potential immune evasion triggered flight bans and heightened health control measures across the world to stem its distribution. Wastewater-based surveillance has demonstrated to be a useful complement for clinical community-based tracking of SARS-CoV-2 variants. Using design principles of our previous assays that detect SARS-CoV-2 variants (Alpha and Delta), we developed an allele-specific RT-qPCR assay which simultaneously targets the stretch of mutations from Q493R to Q498R for quantitative detection of the Omicron variant in wastewater. We report their validation against 10-month longitudinal samples from the influent of a wastewater treatment plant in Italy. SARS-CoV-2 RNA concentrations and variant frequencies in wastewater determined using these variant assays agree with clinical cases, revealing rapid displacement of the Delta variant by the Omicron variant within three weeks. These variant trends, when mapped against vaccination rates, support clinical studies that found the rapid emergence of SARS-CoV-2 Omicron variant being associated with an infection advantage over Delta in vaccinated persons. These data reinforce the versatility, utility and accuracy of these open-sourced methods using allele-specific RT-qPCR for tracking the dynamics of variant displacement in communities through wastewater for informed public health responses. Ministry of Education (MOE) National Research Foundation (NRF) Published version This research is supported by the National Research Foundation, Prime Minister’s Office, Singapore, under its Campus for Research Excellence and Technological Enterprise (CREATE) program funding to the Singapore-MIT Alliance for Research and Technology (SMART) Antimicrobial Resistance Interdisciplinary Research Group (AMR IRG) and the Intra-CREATE Thematic Grant (Cities) grant NRF2019-THE001- 0003a to JT and EJA and funding from the Singapore Ministry of Education and National Research Foundation through an RCE award to Singapore Centre for Environmental Life Sciences Engineering (SCELSE). FW is supported by the Faculty Startup funding from the Center of Infectious Diseases at UTHealth and the UT system Rising STARs award.

Wastewater-based surveillance (WBS) has been widely used as a public health tool to monitor the emergence and spread of SARS-CoV-2 infections in populations during the COVID-19 pandemic. Coincident with the global vaccination efforts, the world is also enduring new waves of SARS-CoV-2 variants. Reinfections and vaccine breakthroughs suggest an endemic future where SARS-CoV-2 continues to persist in the general population. In this treatise, we aim to explore the future roles of wastewater surveillance. Practically, WBS serves as a relatively affordable and non-invasive tool for mass surveillance of SARS-CoV-2 infection while minimizing privacy concerns, attributes that make it extremely suited for its long-term usage. In an endemic future, the utility of WBS will include 1) monitoring the trend of viral loads of targets in wastewater for quantitative estimate of changes in disease incidence; 2) sampling upstream for pinpointing infections in neighborhoods and at the building level; 3) integrating wastewater and clinical surveillance for cost-efficient population surveillance; and 4) genome sequencing wastewater samples to track circulating and emerging variants in the population. We further discuss the challenges and future developments of WBS to reduce inconsistencies in wastewater data worldwide, improve its epidemiological inference, and advance viral tracking and discovery as a preparation for the next viral pandemic. Ministry of Education (MOE) National Research Foundation (NRF) This work was supported by the MIT Center for Microbiome Informatics and Therapeutics, funding from the Massachusetts Consortium on Pathogen Readiness (MassCPR) and China Evergrande Group (EJA), the National Research Foundation, Prime Minister’s Office, Singapore, under its Campus for Research Excellence and Technological Enterprise (CREATE) program funding to the Singapore-MIT Alliance for Research and Technology (SMART) Antimicrobial Resistance Interdisciplinary Research Group (AMR IRG), the Intra-CREATE Thematic Grant (Cities) grant NRF2019-THE001–0003a to JT and EJA and funding from the Singapore Ministry of Education and National Research Foundation through an RCE award to Singapore centre for Environmental Life Sciences Engineering (SCELSE) to SW and JT. FW is supported by the Faculty Startup funding from the Center of Infectious Diseases at UTHealth, the UT system Rising STARs award, and the Texas Epidemic Public Health Institute (TEPHI), which is housed within and supported administratively by The University of Texas Health Science Center at Houston (UTHealth).