Effective Inhibitor Removal from Wastewater Samples Increases Sensitivity of RT-dPCR and Sequencing Analyses and Enhances the Stability of Wastewater-Based Surveillance

Wastewater-based surveillance (WBS) is a proven tool for monitoring population-level infection events. Wastewater contains high concentrations of inhibitors, which contaminate total nucleic acids (TNA) extracted from these samples. We found that TNA extracts from raw influent of Berlin wastewater treatment plants contained highly variable amounts of inhibitors that impaired molecular analyses like dPCR and next-generation sequencing (NGS). By using dilutions, we were able to detect inhibitory effects. To enhance WBS sensitivity and stability, we applied a combination of PCR inhibitor removal and TNA dilution (PIR+D). This approach led to a 26-fold increase in measured SARS-CoV-2 concentrations, practically reducing the detection limit. Additionally, we observed a substantial increase in stability of the time series. We define suitable stability as a mean absolute error (MAE) below 0.1 log10 copies/l and a geometric mean relative absolute error (GMRAE) below 26%. Using PIR+D, the MAE could be reduced from 0.219 to 0.097 and the GMRAE from 65.5% to 26.0% and even further in real-world WBS. Furthermore, PIR+D improved SARS-CoV-2 genome alignment and coverage in amplicon-based NGS for low to medium concentrations. In conclusion, we strongly recommend both the monitoring and removal of inhibitors from samples for WBS. Overall design: Communal wastewater of three wastewater treatment plants (EU UWW Codes: Ruhleben DETP_BE01, Schönerlinde DETP_BE02, Waßmannsdorf DETP_BE03) in Berlin and the airport BER was collected from raw influent. Samples were collected during low SARS-CoV-2 levels (February/March 2024) and high levels (September 2024). Total TNA was extracted and then, with or without inhibitor removal, subjected to high-throughput SARS-CoV-2 sequencing.