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Abstract
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 (Ct) 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. Ct 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 (Ct values) were assessed. A total of 66 laboratories participated, contributing results from 101 distinct test systems and reporting Ct values for a total of 92 different protocols. In all three positive samples, the means of the Ct 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. In an attempt to use PCR diagnostics beyond the identification of infected individuals, laboratories are frequently requested to report Ct values along with a qualitative result. This study highlights the limitations of interpreting Ct values from the various SARS-CoV genome detection protocols and suggests that standardization is necessary in the reporting of Ct values with respect to the target gene.
Highlights
Virus genome-specific real-time reverse transcription PCR (RT-qPCR) is the gold standard for laboratory diagnosis of infections with SARS-CoV-2
In order to guarantee stability of SARS-CoV-2 genomic Ready /COVID- Nucleic acid (RNA), all samples were stored at −80 °C and one panel was tested before shipment as follows: after thawing, samples were kept at ambient temperature for two days before further storage at 4 °C for four days
Sixty-six laboratories participated with a total of 101 individual test systems
Summary
Virus genome-specific real-time reverse transcription PCR (RT-qPCR) is the gold standard for laboratory diagnosis of infections with SARS-CoV-2. The guidelines for the discharge management of patients were based on time-lines of infection (a minimum of 10 days since the development of first symptoms and an asymptomatic period of at least 48 h), and on momentary Ct values (time consideration and/or a Ct value >30) [4, 5]. These potentially significant decisions – both from a medical and an epidemiological point of view – require a high degree of reliability from the laboratory results on which they are based.
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