Effect of nucleic acid concentration method on Severe Acute Respiratory Syndrome Coronavirus 2 detection sensitivity in aircraft wastewater

<b>Introduction.</b> Global interconnectedness and rapid urbanization intensify the spread of infectious diseases, underscoring the critical need for effective and scalable surveillance. Wastewater-based epidemiology (WBE) has proven to be a practical and cost-effective approach for monitoring community-level pathogen prevalence, such as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).<b>Hypothesis/Gap Statement.</b> Despite WBE's success in municipal settings, its application to aircraft wastewater remains underexplored. This matrix presents unique analytical challenges due to its high particulate matter, concentrated inhibitory components and variable composition, creating a significant gap in surveillance capabilities.<b>Aim.</b> This study aimed to evaluate and directly compare the performance of two distinct virus concentration techniques - PEG precipitation and Nanotrap^® Microbiome Particles (NMPs) - for the detection and molecular characterization of SARS-CoV-2 in aircraft wastewater collected from Hamad International Airport.<b>Methodology.</b> Aircraft wastewater samples underwent thermal inactivation and concentration using both the PEG and NMP methods. The resulting extracted RNA was analysed by Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) targeting SARS-CoV-2 genes. All positive samples were subsequently analysed using next-generation sequencing to identify circulating viral variants.<b>Results.</b> The NMPs method detected SARS-CoV-2 RNA in 66.7% of samples, significantly exceeding the 20.8% detection rate achieved with PEG. NMPs also consistently yielded lower cycle threshold (Ct) values, indicating superior viral RNA recovery efficiency. Molecular analysis of positive samples successfully revealed circulating Omicron sublineages (XBB and XBB.1.16), demonstrating the efficacy of aircraft WBE for genomic surveillance.<b>Conclusion.</b> Although PEG precipitation is a cost-effective alternative, its high false-negative rate (72.2%) severely compromises its reliability for surveillance in this matrix. In stark contrast, NMPs proved to be highly sensitive and efficient, making it demonstrably better suited for rapid, large-scale screening. Future strategies could focus on standardized, automated protocols based on high-efficiency methods to enhance early warning and genomic surveillance of emerging pathogens in aviation settings.