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 is necessary in the reporting of C_t values with respect to the target gene.