From crisis to clarity: tackling the sampling strategy challenge in airport wastewater surveillance during COVID-19

Wastewater surveillance at airports has gained interest as a tool for early pathogen detection, but current evidence remains largely feasibility-focused and provides limited guidance on where sampling efforts should be prioritised. In this study, we go beyond feasibility assessments and present a comparative framework for evaluating wastewater-based genomic surveillance at airports. From January to April 2023, wastewater samples were collected at Frankfurt and Berlin airports from single aircraft, lavatory trucks, a sewer combining aircraft and airfield wastewater, and an airport terminal. Viral RNA was concentrated and extracted, followed by short-read amplicon sequencing, digital PCR quantification, and variant annotation with SnpEff. Sequencing success and genome coverage varied by sample type. Aircraft wastewater showed variable coverage (median 20.2-52.1%) due to low SARS-CoV-2 concentrations, while coverage from airport terminal wastewater was consistently high (median: 99.3%). Terminal wastewater exhibited the highest mutation diversity, with 143-286 mutations per sample (grab vs composite). Notably, >95% of clinically known mutations were detected in the wastewater at least one week before clinical reporting, and over 9350 previously unreported mutations were identified-highlighting the early-warning potential of airport wastewater surveillance. These results demonstrate that differences in wastewater matrix and sampling location strongly influence genomic information yield. Wastewater surveillance can complement clinical surveillance and should be considered as part of broader global health monitoring. The framework established here offers replicable guidance for other transportation hubs and public health authorities seeking to strengthen early detection capabilities and pandemic preparedness.