Aircraft Wastewater as a Lens On Global Pathogen Spread: Matrix-Induced Challenges and Workflow Optimization for SARS-CoV-2 RNA Detection and Sequencing

Abstract

Aircraft wastewater (AWW) surveillance emerged as a promising tool to monitor transboundary pathogen introduction during the COVID-19 pandemic. Reliable viral RNA detection and sequencing are essential for wastewater-based epidemiology, yet systematic evaluation of AWW processing methods remains limited. This study assessed the impact of disinfectant on SARS-CoV-2 RNA recovery and developed an optimized workflow to maximize RNA yield and quality for PCR-based quantification and sequencing. Even low concentrations of disinfectant (Aeroclean-T-600) caused substantial RNA loss in municipal wastewater (e.g., half-life of 1.1 h at 0.25%), underscoring the need for cautious data interpretation. However, high SARS-CoV-2 RNA positivity rates in this study (72%) and elsewhere suggest that sample matrix effects and different disinfectant chemistry might mitigate degradation in AWW. A comparative evaluation of enrichment methods across all AWW fractions identified the liquid fraction (supernatant) as the most effective, with sample dilution further enhancing recovery and sequencing quality. Inhibitor trap treatment additionally improved recovery for some protocols but not sequencing outcomes, while DNase treatment reduced the performance in our hands. Optimizing sequencing protocols enhanced genome coverage and variant detection, supporting robust monitoring of SARS-CoV-2 diversity. Overall, the established workflow enabled sensitive, reproducible SARS-CoV-2 surveillance in the AWW, strengthening early warning capacities for cross-border public health threats.