Avoiding False-Positive SARS-CoV-2 Rapid Antigen Test Results with Point-of-Care Molecular Testing on Residual Test Buffer

LeBlanc, Jason J.; McCracken, Gregory R.; Goodall, Barbara; Hatchette, Todd; Barrett, Lisa; Ross, John J.; Davidson, Ross; Patriquin, Glenn

doi:10.1128/spectrum.00639-22

Cited by 5 publications

(8 citation statements)

References 25 publications

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“…For most dilutions, G29195T resulted in an approximately 10-fold reduction in sensitivity; however, sporadic N2 gene detection failures were observed near the detection limit and at high-mutated target concentrations ( Fig. 2 ; Table S4 ), which is a well-recognized concept ( 34 – 37 ). However, inconsistencies at high viral concentrations are rarely described.…”

Section: Discussionmentioning

confidence: 95%

“…Using NGS on amplicons derived from the Cepheid Xpert ( 34 ), Roche Cobas 6800 ( 34 ), or Abbott ID NOW ( 35 ) SARS-CoV-2 assays, target sites were identified in high-quality data with sequence depth reads ranging from 6,665 to 99,381. Compared to manufacturer-described gene targets, the sizes of the NGS-defined target sites were 1.5- to 151-fold smaller, thereby reducing the genomic regions to monitor for mutations during genetic surveillance ( Table 1 ).…”

Section: Resultsmentioning

confidence: 99%

“…When compared to clinical specimens harboring the same NAAT target site mutations for the Xpert N2 gene target, synthetic DNAs showed near perfect concordance ( R 2 = 0.9936; Table S3). For methods without a quantifiable output signal like the ID NOW assay ( 35 ), a quantifiable calibrator can be fused to the target of interest in the synthetic DNA to allow sample normalization and mutation impact assessment through serial dilution and endpoint comparisons. For real-time RT-PCR technologies like Xpert and Cobas ( 34 ), access to Ct values greatly facilitated sample normalization and mutation impact assessments as these acted as surrogates for virus concentration and rapidly identified mutations resulting in target detection failure or reduced sensitivity.…”

Section: Discussionmentioning

confidence: 99%

“…Assessments of the target site for the isothermal NAAT (i.e., Abbott ID NOW) were important, as any mutation could potentially lead to diagnostic failure in this single-target assay ( 35 ). Interestingly, none of the 23 mutations observed in its 36 bp target site compromised detection.…”

Section: Discussionmentioning

confidence: 99%

“…This study aimed to: (i) identify the target site locations of three commonly used commercial NAATs ( 34 , 35 ); (ii) query these regions against SARS-CoV-2 genome databases to reveal any mutations; (iii) evaluate mutation impact; (iv) and communicate findings of any mutations compromising target detection. This proof-of-principle framework for commercial NAAT target site quality assurance could be a useful model for other NAATs, targets, and applications.…”

Section: Introductionmentioning

confidence: 99%

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Neglected SARS-CoV-2 variants and potential concerns for molecular diagnostics: a framework for nucleic acid amplification test target site quality assurance

McCracken,

Gaston,

Pettipas

et al. 2023

Microbiol Spectr

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During the COVID-19 pandemic, SARS-CoV-2 detection using nucleic acid amplification tests (NAATs) played a key role in clinical management and public health interventions. However, mutations could jeopardize NAAT-based detection if they occur in the NAAT target site, potentially resulting in false negative results. However, mutation monitoring is challenged as the exact location of commercial NAAT target sites is not divulged by manufacturers. This study sequenced commercial SARS-CoV-2 NAAT target sites to assess the impact of mutations occurring in these regions. The resulting sequences for the Xpert, Cobas, and ID NOW SARS-CoV-2 assays were queried against SARS-CoV-2 genome databases to identify mutations in circulating strains. Synthetic DNAs and clinical specimens harboring NAAT target site mutations were used to assess mutation impact. Of 17,600 NAAT target site mutation occurrences in a genome database, 269 compromised target detection. These represented 24 unique mutations that reduced NAAT target sensitivity and nine led to target detection failure. Only seven of these mutations were previously recognized. Overall, this reactive strategy along with passive surveillance identified 29 novel mutations that compromised detection with Xpert and Cobas targets. Knowledge of commercial NAAT target sites, paired with a strategy for mutation impact assessment and ongoing genetic surveillance, provided a robust framework for commercial NAAT target site quality assurance. The question remains of who should be responsible for NAAT target site quality assurance, but collaborative efforts between methods users, industry, and regulatory agencies would be ideal. Molecular tests like polymerase chain reaction were widely used during the COVID-19 pandemic but as the pandemic evolved, so did SARS-CoV-2. This virus acquired mutations, prompting concerns that mutations could compromise molecular test results and be falsely negative. While some manufacturers may have in-house programs for monitoring mutations that could impact their assay performance, it is important to promptly report mutations in circulating viral strains that could adversely impact a diagnostic test result. However, commercial test target sites are proprietary, making independent monitoring difficult. In this study, SARS-CoV-2 test target sites were sequenced to monitor and assess mutations impact, and 29 novel mutations impacting SARS-CoV-2 detection were identified. This framework for molecular test target site quality assurance could be adapted to any molecular test, ensuring accurate diagnostic test results and disease diagnoses.

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Section: Discussionmentioning

confidence: 95%