Presumptive positive with the Cepheid Xpert Xpress SARS-CoV-2 assay due to N mutations in the Delta variant.

The Cepheid Xpert® Xpress SARS-CoV-2 assay is 1 of the several real-time reverse transcription polymerase chain reaction (RT-PCR) assays that received Emergency Use Authorization from the United States Food and Drug Administration (FDA) for detection of SARS-CoV-2. Here we report 4 SARS-CoV-2 samples that were reported as presumptive positives on the Cepheid platform while reported as positives on alternative RT-PCR platforms. Whole genome sequencing indicated that the samples were Delta variants and had point mutations in the N gene which potentially interfered with SARS-CoV-2 detection. Two types of point mutations were found in these samples in the US CDC 2019-nCoV Real time PCR N2 Probe region: C29203T and C29200T. C29203T is a novel point mutation, and C29200T has not been previously reported in the Delta variants. This underlines the fact that mutations in the real-time RT-PCR assay target region could hinder accurate detection of SARS-CoV-2.

Understanding false positives and the detection of SARS-CoV-2 using the Cepheid Xpert Xpress SARS-CoV-2 and BD MAX SARS-CoV-2 assays.

Several real-time RT-PCR assays have received Emergency Use Authorization from the United States Food and Drug Administration. The BD MAX™ SARS-CoV-2 assay, run by the BD MAX™ system, is a qualitative test that detects the SARS-CoV-2 specific nucleocapsid phosphoprotein gene regions, N1 and N2. The human RNase P gene is used as the endogenous nucleic acid extraction control. The Cepheid Xpert® Xpress SARS-CoV-2 assay, run by the GeneXpert system, detects the pan-sarbecovirus E gene and the N2 region of the N gene. We evaluated the performance characteristics of the BD and Cepheid assays using matched patient samples. We also analyzed comparative Ct values for both assays using 183 positive samples tested at this facility. In addition, we mitigated reporting false positive results without relying on interpretive software. We found that both systems showed comparable sensitivity. We found an approximately 3.5% false positive rate from the BD MAX™ system results.