Variability in the Cycle Threshold Values of SARS-CoV-2 Real-Time Reverse Transcription Polymerase Chain Reaction Test: A Review of the Nationwide Proficiency Testing Survey in Korea

Abstract

Background: A nationwide proficiency test result for the severe acute respiratory syndrome coronavirus 2 real-time reverse transcription polymerase chain reaction (rRT-PCR) was analyzed to investigate the intra- and interlaboratory variations in the cycle threshold (Ct) values. Methods: Four different types of contrived samples, including five positive samples with three different concentrations and two negative samples, were sent to participating laboratories in March/October 2022. This study examined the variations in the extraction kits and PCR reagents used in the laboratories. Results: Overall, 278 and 301 laboratories responded to each challenge. In the first challenge, five laboratories (1.8%) provided incorrect results—three false positives and two false negatives from one positive and one weak-positive sample. In the second challenge, only two laboratories (0.7%) provided incorrect results—one false positive and one false negative from one positive sample. The intralaboratory variations of the Ct values in the first and second challenges were deemed unacceptable (Ct >1.66) in 7 (2.5%) and 14 (4.7%) laboratories, respectively; one laboratory showed unacceptable intralaboratory variations for both positive and weak-positive samples in the second challenge. The interlaboratory variability of the Ct values for two commonly used rRT-PCR reagents was remarkable, with a difference of up to eight cycles observed for the same specimen. Conclusions: The overall performance and intralaboratory variability of the test were acceptable in most participating laboratories. However, caution should be exercised when interpreting the results from other institutions due to the remarkable interlaboratory variability of the Ct values.