Analysis of external quality assessment samples revealed crucial performance differences between commercial RT-PCR assays for SARS-CoV-2 detection when taking extraction methods and real-time-PCR instruments into account

Abstract
In limelight of the ongoing pandemic SARS-CoV-2 testing is critical for the diagnosis of infected patients, contact-tracing and mitigating the transmission. Diagnostic laboratories are expected to provide appropriate testing with maximum accuracy. Real-time reverse transcriptase PCR (RT-PCR) is the diagnostic standard. However, only a handful of studies have reviewed their performance in clinical settings. The aim of this study was to compare the performance of the overall analytical matrix including the extraction kit (BD MAX, Promega, Qiagen), the PCR instrument (Agilent Mx3005 P, BD MAX, Qiagen Rotor-Gene, Roche Cobas z 480) and the RT-PCR assay (Altona Diagnostics, CerTest Biotec, R-Biopharm AG) using predefined samples from proficiency testing organizers. The greatest difference of the cycle threshold values between the matrices was nine cycles. One borderline sample could not be detected by three out of twelve analytical matrices and yielded a false negative result. We therefore conclude that diagnostic laboratories should take the complete analytical matrix in addition to the performance values published by the manufacturer for a respective RT-PCR kit into account. With limited resources laboratories have to validate a wide range of kits to determine appropriate analytical matrices for detecting SARS-CoV-2 reliably. The interpretation of clinical results has to be adapted accordingly.
Keywords: COVID-19; Cycle threshold; In vitro diagnostics; RT-PCR; SARS-CoV-2.