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A SARS-CoV-2 Nucleocapsid Variant that Affects Antigen Test Performance.

ABSTRACT: More than one year into a global pandemic, SARS-CoV-2 is now defined by a variety of rapidly evolving variant lineages. Several FDA authorized molecular diagnostic tests have been impacted by viral variation, while no reports of viral variation affecting antigen test performance have occurred to date. While determining the analytical sensitivity of the Quidel Sofia SARS Antigen FIA test (Sofia 2), we uncovered a high viral load specimen that repeatedly tested negative by this antigen test. Whole genome sequencing of the specimen uncovered two mutations, T205I and D399N, present in the nucleocapsid protein of the isolate. All six SARS-CoV-2 positive clinical specimens available in our laboratory with a D399N nucleocapsid mutation and C_T < 31 were not detected by the Sofia 2 but detected by the Abbott BinaxNOW COVID-19 Ag Card, while clinical specimens with the T205I mutation were detected by both assays. Testing of recombinant SARS-CoV-2 nucleocapsid with these variants demonstrated an approximate 1000-fold loss in sensitivity for the Quidel Sofia SARS Antigen FIA test associated with the D399N mutation, while the BinaxNOW and Quidel Quickvue SARS Antigen tests were unaffected by the mutation. The D399N nucleocapsid mutation has been relatively uncommon to date, appearing in only 0.02% of genomes worldwide at time of writing. Our results demonstrate how routine pathogen genomics can be integrated into the clinical microbiology laboratory to investigate diagnostic edge cases, as well as the importance of profiling antigenic diversity outside of the spike protein for SARS-CoV-2 diagnostics.

SUBMITTER: Bourassa L

PROVIDER: S-EPMC8219478 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:In response to the COVID-19 pandemic, lateral flow assays (LFAs) for the detection of SARS-CoV-2 antigen have been proposed as a complementary option to the more costly and time consuming reverse-transcriptase polymerase chain reaction (RT-PCR). We assessed five commercially available SARS-CoV-2 antigen detecting LFAs (ASSUT EUROPE (Rome, Italy), Besthree (Taizhou, China), Encode (Zhuhai, China), Fortress (Antrim UK), and Hughes Medical (Buckinghamshire, UK), using samples collected from hospitalised individuals with COVID-19 and compared these results against established RT-PCR assays with the aim of estimating test performance characteristics. We performed a diagnostic accuracy study of the five LFAs on 110 inpatients with confirmed COVID-19 and 75 COVID-19 negative control participants. Assay evaluation was performed using a modified version of each manufacturer's protocol allowing for parallel testing of a single sample on multiple assays. Additional variables were studied including infection acquisition, oxygenation requirements at time of swabbing, and patient outcomes. The 110 patients were 48% (53) female, with mean age 67 years (range 26-100 years), and 77% (85) cases were community onset SARS-CoV-2. Across the five assays, sensitivity ranged from 64 (95% CI 53-73) to 76% (95% CI 65-85); Fortress performed best with sensitivity of 76% (95% CI 65-85). Specificity was high across all assays with 4/5 LFAs achieving 100%. LFA sensitivity was not dependant on RT-PCR cycle thresholds. SARS-CoV-2 antigen detecting LFAs may complement RT-PCR testing to facilitate early diagnosis and provide community testing strategies for identification of patients with COVID-19, however we find suboptimal test performance characteristics across a range of commercially available manufacturers, below WHO and MHRA pre-set sensitivity performance thresholds. With such variation in sensitivity between LFAs and PCR testing and between assay brands, we advise caution in the deployment of LFAs outside of environments with clinical oversight.

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A SARS-CoV-2 Nucleocapsid Variant that Affects Antigen Test Performance.

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