LAUSR

Purpose Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a worldwide spreading disease with acute respiratory distress syndrome as one of the major complications. In the early disease stage, COVID-19 cannot be distinguished from influenza based on the clinical symptoms. During viraemia, direct pathogen detection by reverse transcription polymerase chain reaction (RT-PCR) is the diagnostic gold standard. This study evaluated a novel real-time RT-PCR test for fast detection and differentiation of RNA from SARS-CoV-2 and influenza virus types A and B. Methods & Materials The assays´ diagnostic performance was compared to CE-IVD/FDA-EUA-marked reference PCR tests. RNA was extracted from patient samples collected as nasopharyngeal or oropharyngeal swabs. Virus-specific RNA was amplified after reverse transcription using the EURORealTime SARS-CoV-2/Influenza A/B PCR test (EUROIMMUN) allowing simultaneous detection of two target sequences in the SARS-CoV-2 ORF1ab and N genes as well as one target sequence each for influenza virus A and B. Assays were carried out on the CFX96 cycler (Bio-Rad) and evaluated with the EURORealTime Analysis Software (EUROIMMUN). The 95% limit of detection (LoD) was determined by Probit analysis using a dilution series of quantified target RNA. To exclude cross-reactivity and interference, the assay was run against human genomic DNA/RNA, nucleic acids from different viral, bacterial and fungal pathogens, and potentially interfering substances. Results Compared to the reference PCR tests, the EURORealTime SARS-CoV-2/Influenza A/B showed positive agreements of 97.8%, 93.0% and 100% and negative agreements of 100%, 100% and 98.9% for SARS-CoV-2, influenza A and influenza B, respectively. The 95% LoD values were calculated to be 0.55cp/µl for SARS-CoV-2, 0.92cp/µl for influenza A H3N2, 0.67cp/µl for influenza A H1N1 and 1.21cp/µl for influenza B. No cross-reactivities with human or pathogen-specific nucleic acids or interferences were detected. Conclusion The novel test is able to detect SARS-CoV-2, influenza A and influenza B with high sensitivity and clearly discriminate between these viruses. It is therefore optimally suited for differential diagnostics for patients presenting with symptoms compatible with COVID-19 and influenza. Combined detection of the three pathogens in one multiparameter assay helps to save time and resources in the diagnostic workup.