Development of a whole workflow for multiplexed diagnosis of viral respiratory infections

Endemic cold waves caused by SARS-CoV-2, Influenza and RSV challenge global health systems by co-circulation in the population. This requires appropriate pre-analytical workflows that obtain valid multi-virus diagnostic test results and ensure the reliability of downstream molecular analyses. Various pre-analytical parameters like specimen stabilization, virus infectivity inactivation and nasal sprays as interfering substances were examined. The BD transport swab system including the UVT medium was able to stabilize the nucleic acid copy numbers of the tested viruses at 37 °C for up to 96 h, even when supplemented with the virus inactivating reagent transport medium additive-P (TMA-P). Viruses infectivity were inactivated by TMA-P, TMA-D and benzalkoniumchloride (BAC). However, only BAC is usable in combination with the QIAprep& high-throughput system. Furthermore, TMA-P and BAC are compatible with sequencing. Additionally, external substances like nasal sprays used prior to sample collection may interfere with test results. The BD system supplemented with inactivating substances reduce the biosafety requirements for transport and analysis while ensuring valid test results including sequencing. The use of the direct PCR QIAprep& system in combination with the multi-target detection assays minimizes the specimens processing time. Overall, a rapid and reliable detection workflow of respiratory viruses was developed for clinically relevant pre-analytical scenarios.