Variability of cycle threshold values in an external quality assessment scheme for detection of the SARS-CoV-2 virus genome by RT-PCR

Objectives: The qualitative results of SARS-CoV-2 specific real-time reverse transcription (RT) PCR are used for initial diagnosis and follow-up of Covid-19 patients and asymptomatic virus carriers. However, clinical decision-making and health management policies often are based additionally on cycle threshold (C-t) values (i.e., quantitative results) to guide patient care, segregation and discharge management of individuals testing positive. Therefore, an analysis of inter-protocol variability is needed to assess the comparability of the quantitative results. Methods: C-t values reported in a SARS-CoV-2 virus genome detection external quality assessment challenge were analyzed. Three positive and two negative samples were distributed to participating test laboratories. Qualitative results (positive/negative) and quantitative results (C-t values) were assessed. Results: A total of 66 laboratories participated, contributing results from 101 distinct test systems and reporting C-t values for a total of 92 different protocols. In all three positive samples, the means of the C-t values for the E-, N-, S-, RdRp-, and ORF1ab-genes varied by less than two cycles. However, 7.7% of reported results deviated by more than +/- 4.0 (maximum 18.0) cycles from the respective individual means. These larger deviations appear to be systematic errors. Conclusions: In an attempt to use PCR diagnostics beyond the identification of infected individuals, laboratories are frequently requested to report C-t values along with a qualitative result. This study highlights the limitations of interpreting C-t values from the various SARS-CoV genome detection protocols and suggests that standardization isnecessary in the reporting of C-t values with respect to the target gene.