Combined RT-qPCR and Pyrosequencing of a SARS-CoV-2 Spike Glycoprotein Polybasic Cleavage Motif Uncovers Rare Pediatric COVID-19 Spectrum Diseases of Unusual Presentation

Abstract Background Surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is essential for the global containment measures with regard to the ongoing pandemic. Diagnostic gold standard is currently reverse transcription of the (+)RNA genome and subgenomic RNAs and subsequent quantitative polymerase chain reaction (RT-qPCR) from nasopharyngeal swabs or bronchoalveolar lavages. In order to further improve the diagnostic accuracy, particularly for the reliable discrimination between negative and false-negative specimens, we propose the combination of the RT-qPCR workflow with subsequent pyrosequencing of a S-gene amplicon. This extension might add important value mainly in cases with low SARS-CoV-2 load, where RT-qPCR alone can deliver conflicting results.s Results We successfully established a combined RT-qPCR and S-gene pyrosequencing method which can be optionally exploited after routine diagnostics or for epidemiologic studies. This allows a more reliable interpretation of conflicting RT-qPCR results in specimens with relatively low viral loads and close to the detection limits of qPCR (CT values >30). After laboratory implementation and characterization of a best practice protocol we tested the combined method in a large pediatric cohort from two German medical centers (n=769). Pyrosequencing after RT-qPCR enabled us to uncover 6 previously unrecognized cases of pediatric SARS-CoV-2 associated diseases, partially exhibiting unusual and heterogeneous presentation. Moreover, it is notable that in the course of RT-qPCR/pyrosequencing method establishment we did not observe any case of false-positive diagnosis when confirmed SARS-CoV-2-positive specimens were used from foregoing routine testing. Conclusions The proposed protocol allows a specific and sensitive detection of SARS-CoV-2 close to the detection limits of RT-qPCR. Combined RT-qPCR/pyrosequencing does not negatively affect preceding RT-qPCR pipeline in SARS-CoV-2 diagnostics and can be optionally applied in routine to inspect conflicting RT-qPCR results.