A Recurrent Mutation at Position 26340 of SARS-CoV-2 Is Associated with Failure of the E Gene Quantitative Reverse Transcription-PCR Utilized in a Commercial Dual-Target Diagnostic Assay

Abstract

Control of the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic requires accurate laboratory testing to identify infected individuals, while also clearing essential staff to continue work. At the current time a number of qRT-PCR assays have been developed to identify SARS-CoV-2, targeting multiple positions in the viral genome. While the mutation rate of SARS-CoV-2 is moderate, given the large number of transmission chains it is prudent to monitor circulating viruses for variants that might compromise these assays. Here we report the identification of a C-to-U transition at position 26,340 of the SARS-CoV-2 genome which is associated with failure of the cobas® SARS-CoV-2 E-gene qRT-PCR in eight patients. As the cobas® SARS-CoV-2 assay targets two positions in the genome, the individuals carrying this variant were still called as SARS-CoV-2 positive. Whole genome sequencing of SARS-CoV-2 showed all to carry closely related viruses. Examination of viral genomes deposited on GISAID showed this mutation has arisen independently at least four times. This work highlights the necessity of monitoring SARS-CoV-2 for the emergence of SNPs which might adversely affect RT-PCRs used in diagnostics. Additionally, it argues that two regions in SARS-CoV-2 should be targeted to avoid false negatives.

Clinical performance and sample freeze-thaw stability of the cobas®6800 SARS-CoV-2 assay for the detection of SARS-CoV-2 in oro-/nasopharyngeal swabs and lower respiratory specimens

“…On the other hand, two samples were single positive in the RdRP target. This could be related to a previously described C26340 T mutation in the SARS-CoV-2 E-gene, resulting in a mismatch with primers/probes and thus escape from detection in samples with lower viral loads [ 19 ]. This supports the dual-target detection strategy for fast-evolving viruses to limit this risk for false-negative results.…”

IFCC Interim Guidelines on Molecular Testing of SARS-CoV-2 Infection

“…local prevalence of SARS-CoV-2 infection, exposure history, and symptoms) and test sensitivity [28]. The potential for false negative results to occur is estimated to be higher in high prevalence settings [28], or due to inappropriate timing of sample collection, insufficient collection by healthcare personnel, low viral load, presence of PCR inhibitor, preanalytical or analytical issues [29] or viral mutations [30]. Rates of false negatives have been shown to vary with time since symptom onset [31], and ranged from 2 to 29% in one systematic review [32].…”

“…Assays for molecular diagnosis should employ a minimum of two gene targets to minimize the risk of false negatives [30,35].…”

Diagnosis of SARS-CoV-2 infection in the setting of the cytokine release syndrome

“…However, a more accurate assessment is a comparison of assays' performance using parallel testing of low positive clinical samples. Recently, falsely negative NAAT testing has been reported with mutated strains of SARS-CoV-2 highlighting the need for epidemiologic monitoring of circulating SARS-CoV-2 viruses [81,82].…”