Exploring beyond the limit: How comparative stochastic performance affects retesting outcomes in six commercial SARS CoV-2 nucleic acid amplification tests

Abstract

ObjectivesTo examine the comparative stochasticity profile of six commercial SARS-CoV-2 nucleic acid amplification tests (NAATs) and how this may affect retesting paradigms. MethodsCommercial quality control (QC) material was serially diluted in viral transport media to create a panel covering 10–10,000 copies/ml. The panel was tested across six commercial NAATs. A subset of high cycle threshold results was retested on a rapid PCR assay to simulate retesting protocols commonly used to discriminate false positives. ResultsPerformance beyond the LOD differed among assays, with three types of stochasticity profiles observed. The ability of the rapid PCR assay to reproduce a true weak positive specimen was restricted to its own stochastic performance at the corresponding viral concentration. ConclusionStochastic performance of various NAATs overlap across low viral concentrations and affect retesting outcomes. Relying on retesting alone to discriminate false positives risk missing true positives even when a more sensitive assay is deployed for confirmatory testing.