Abstract
Background: Cycle threshold (Ct) values from SARS-CoV-2 reverse transcription quantitative PCR (RT-qPCR) tests are used to measure viral burden. Calibration to the First WHO International Standard for SARS-CoV-2 RNA may improve quantitative inter-assay agreement.
Methods: WHO standard was tested using four emergency use authorized RT-qPCRs to generate calibration curves and evaluate Ct value differences. Harmonization of two assays, Cepheid Xpert Xpress SARS-CoV-2 targeting E and nucleocapsid (N2) [Xpert (E) and Xpert (N2)] and a laboratory-developed test targeting E [LDT (E)], was assessed using 93 positive upper respiratory samples. Platform (target) pairs were compared via Bland-Altman analysis and Passing-Bablok regression.
Results: Ct values with the WHO standard were comparable across platforms and targets, except Xpert (N2) for which the mean difference was a median of 3.68 cycles (Interquartile Range, IQR = 3.23 to 3.76 cycles) greater than other platform (target) pairs. Using clinical samples, the mean difference of Xpert (N2) to LDT (E) was 3.64 cycles (95% Confidence Interval, CI =1.51 to 5.76). After calibration, the mean difference of Xpert (N2) to LDT (E) was 0.08 log 10 IU/mL (95% CI = -0.56 to 0.71) and the regression was y = 1.00x * 0.08 (95% CI slope = 0.93 to 1.07, 95% CI intercept = 0.28 to 0.42).
Conclusions: Calibration to the WHO standard resulted in the harmonization of two RT-qPCR tests, whereas analysis by Ct value alone may have led to erroneous quantitation. Harmonization to the WHO standard has the potential to improve the generalizability of clinical associations with SARS-CoV-2 RNA levels.
【초록키워드】 SARS-CoV-2, clinical samples, RT-qPCR, cycle threshold, nucleocapsid, Ct value, target, WHO, reverse transcription, SARS-CoV-2 RNA, targets, harmonization, Cepheid, threshold, Quantitative, quantitative PCR, platform, Emergency use, international standard, association, Analysis, Regression, Ct values, Standard, confidence, Generalizability, interquartile range, 95% CI, 95% confidence interval, cycle, calibration, mean difference, RT-qPCRs, viral burden, quantitation, positive, upper respiratory, IMPROVE, log, greater, evaluate, assays, generate, median, comparable, the WHO, interval, the mean, IQR, Range, was tested, 【제목키워드】 SARS-CoV-2, International, WHO, reverse transcription,