Amplitude multiplexed wastewater surveillance for campus health: tracking SARS-CoV-2, influenza A, and norovirus

The University of Southern California implemented a wastewater-based surveillance program (USC-WS) to proactively prevent viral outbreaks and maintain a safe campus environment for students. The main objective of this project was to develop a novel amplitude multiplex assay to quantify SARS-CoV-2, influenza A, norovirus, and PMMoV. The study aimed to 1) describe the temporal trends of the target viruses from the start of Fall 2022 to the end of the Spring 2023 academic semester; 2) establish correlations between USC-WS and USC Student Health clinical data; and 3) assess the impact of varying wastewater lead-time and PMMoV normalizations on correlations with clinical data. Two-step RT-ddPCR assays quantified the viral targets in an amplitude multiplex to overcome the limitation of two fluorescent channels on the QX200 Droplet Reader. Statistical analysis demonstrated statistically significant ( p -value < 0.05 and Pearson r ≥ 0.50) correlations between SARS-CoV-2 and influenza A wastewater data and reported clinical case data, but not for norovirus. Further, normalization by PMMoV improved correlations for SARS-CoV-2 and norovirus, but not for influenza A. We also found that SARS-CoV-2 wastewater data provides an earlier lead-time to the onset of clinical cases than influenza A. This study validated the amplitude multiplex of SARS-CoV-2, influenza A, norovirus, and PMMoV for wastewater surveillance and the lead-time building-scale wastewater can provide ahead of clinical cases. This study implements wastewater surveillance on a university campus to monitor highly infectious communal diseases utilizing ddPCR and overcoming the limitation of two fluorescent channels of a ddPCR reader by employing an amplitude multiplex.