Assessment of RNA quality from airplane wastewater

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 effective wastewater-based epidemiology, yet systematic evaluation of AWW processing methods remains limited. This study (i) assessed the impact of disinfectant on SARS-CoV-2 RNA recovery and (ii) developed an optimized workflow to maximize RNA yield and quality for PCR-based quantification and sequencing. Even low disinfectant concentrations caused substantial RNA loss (e.g. >90% after 2 h with 0.25% disinfectant), underscoring the need for cautious interpretation of results. A comparative evaluation of enrichment methods across all AWW fractions identified the supernatant as most effective, with sample dilution further improving recovery and sequencing quality. Inhibitor trap treatment additionally increased viral recovery for some protocols but didn´t improve sequencing outcomes, while DNase treatment reduced performance in our hands. Optimizing sequencing protocols enhanced genome coverage and variant detection, supporting robust monitoring of viral diversity. A case study applying our optimized AWW workflow showed 72% SARS-CoV-2 positivity and successfully identified circulating variants among the passengers. Overall, the established workflow enables sensitive, reproducible detection of SARS-CoV-2 in AWW, strengthening pathogen surveillance and early-warning capacities for cross-border public health threats.