PCR Assays for SARS-CoV-2: Testing the Tests

Many PCR-based SARS-CoV-2 tests have been granted CDC emergency use authorization to meet the enormous diagnostic need associated with the COVID-19 pandemic. The assays utilize different primer/probe sets and various swabs and transport media. Do all tests perform equally? And do transport media and conditions influence the results?

Lieberman and colleagues compared their laboratory-developed, CDC-based test (gold standard) with commercial SARS-CoV-2 molecular tests from Cepheid (Xpert Xpress), DiaSorin (Simplexa), Hologic (Panther Fusion), and Roche (Cobas 6800) in 169 nasopharyngeal swabs. All assays were 100% specific in a common panel of 26 clinical specimens (12 positive, 1 inconclusive, and 13 negative) stored at 4°C for <72 hours. An additional 115 clinical specimens were tested against the modified CDC assay. Among samples testing positive with the CDC assay, 13 of 13 were detected with Xpert Xpress, 11 of 11 with Simplexa, 24 of 26 with Panther Fusion, and 19 of 20 with Cobas 6800. Cycle threshold (Ct) value is inversely related to viral concentration; all specimens with Ct values <35 on the CDC assay were detected with all other assays.

Zhen and colleagues evaluated SARS-CoV-2 assays from DiaSorin (Simplexa), GenMark (ePlex), and Hologic (Panther Fusion) as well as the modified CDC protocol. Using a synthetic SARS-CoV-2 RNA standard, Simplexa showed the lowest limit of detection (39 copies/mL) followed by Panther Fusion (83 copies/mL), the modified CDC assay (779 copies/mL), and ePlex (1000 copies/mL). Of 104 clinical samples, the modified CDC assay, Simplexa, and Panther Fusion each detected 51 of 51 and ePlex detected 49 of 51. Specificity was 100% for ePlex and Simplexa, 98% for the CDC assay, and 96% for Panther Fusion.

Rogers and colleagues tested detection of SARS-CoV-2 RNA in various commercial transport media as well as 0.9% saline immediately after sampling and serially under different storage conditions (room temperature, refrigerated, frozen) for up to 14 days. Media and storage conditions minimally influenced test results, with Ct differences <3 at various time points. A high titer clinical specimen diluted 1:1000 in 0.9% saline showed minimal variation in mean Ct values (<1 Ct) over 14 days if kept frozen. At room temperature and refrigerated storage conditions, a linear 2-Ct increase was noticed over 14 days (which would not have altered the test result).