Molecular detection of respiratory viruses from breath samples: Evaluation of nucleic acid extraction systems and PCR panels

Background Reliable and scalable molecular testing is essential for risk assessment and monitoring of respiratory viruses with epidemic or pandemic potential. The AveloMask Kit (Avelo) is a non-invasive sampling device that collects exhaled breath aerosols (XBA) using a high-performance filter, which can be released into a stabilizing buffer for nucleic acid preservation. Detection of viral nucleic acids in breath samples remains challenging, as viral loads are typically lower than in conventional upper-respiratory swabs. Therefore, optimizing extraction and polymerase chain reaction (PCR) workflows is critical to maximizing analytical sensitivity. Methods In this proof-of-concept study, we compared the nucleic acid extraction efficiency of ELITe InGenius® (ELITech) and QIAsymphony (Qiagen) automated platforms in combination with an in-house real-time quantitative polymerase chain reaction (RT-qPCR) panel and a commercial PCR panel (RespiFinder® 2Smart, Pathofinder). Using synthetic ribonucleic acid (RNA) standards, we determined the limit of detection (LOD) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, influenza B, and respiratory syncytial virus (RSV) as a performance indicator for both extraction platforms and PCR panels. As proof-of-concept, we analyzed seven XBA samples from infected individuals, eight from healthy controls, and one from a person recovering from a cold. Results Our results demonstrate that ELITe InGenius and QIAsymphony provide comparable extraction efficiencies without negatively impacting downstream RT-qPCR. The combination of QIAsymphony and RespiFinder 2Smart achieved LODs below 100 viral copies/mL and detected all pathogens in clinical XBA samples (7/7) that were also detected in matched swabs. Conclusions These initial findings suggest the compatibility of high-throughput extraction systems and PCR panels with XBA samples collected using AveloMask and warrant further investigation of XBA sampling as a non-invasive method for respiratory virus detection.