Wastewater Genomic Surveillance Captures SARS-CoV-2 Early Detection, Cryptic Transmission, and Variant Dynamics

Wastewater-based epidemiology (WBE) addresses key biases and limitations in clinical surveillance by providing widespread, population-level coverage, capturing subclinical infections, and mitigating gaps and low rates in clinical testing. Despite its value during the COVID-19 pandemic, few phylogenetic studies of wastewater sequences have been conducted and inferences have been limited to comparative genomic diversity. Phylogenetic methods offer deeper insights into viral ancestry, transmission patterns, and epidemic dynamics, but their effectiveness using wastewater data remains unexplored. This is the first study to apply Bayesian phylodynamic approaches to wastewater sequences. We conducted a 2.5-year long longitudinal study (June 2020 - December 2022) using wastewater samples collected from three catchment sites in Clarke County, Georgia. Clarke County is home to Athens, Georgia, a college town with population changes driven by seasonal population shifts of [~]30,000 students. Leveraging an optimized SARS-CoV-2 enrichment and sequencing platform, we validated wastewater genomes for tracking SARS-CoV-2 variant dynamics (Alpha, Delta, and Omicron). As proof of concept for combining wastewater and clinical sequences to reconstruct spatial diffusion patterns, we reconstructed epidemic waves in Clarke County in the context of national and international transmission. Phylogenetic analyses of wastewater sequences estimated the time of the most recent common ancestors (TMRCAs) for Alpha and Delta in Clarke County in alignment with clinical genomes, inferred earlier emergence of Omicron sublineages compared to clinical surveillance, captured all variants from clinical sequencing, detected cryptic Beta transmission not captured by clinical sequences, and identified variants months before detection in clinical sequences. Lastly, we highlighted the potential importance of student movement events in clade expansion, increased transmission, and case surges. This study demonstrates the utility of wastewater surveillance in reconstructing the emergence and spread of SARS-CoV-2 variants. By integrating wastewater and clinical sequences, we establish a phylogenetic framework to identify cryptic transmission, reconstruct viral demographic history, and contextualize local viral circulation within broader transmission patterns. These findings underscore the importance of wastewater surveillance in pandemic preparedness and public health response.