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.

Keywords: Antigen; False negative; Nucleocapsid; Quidel; Sars-cov-2; Sofia 2; Variant.

Fig. 1

Confirmation of analytical sensitivity of the using clinical specimens. Specimens are depicted in descending order by copies/swab from left to right. Specimens depicted with purple circles were detected by the Quidel Sofia 2 while gold circles were undetected. The analytical sensitivity determined using dilutions of a pooled positive of 54,900 copies/swab is denoted by the dotted line. The arrow denotes the specimen that was negative on repeat testing and subjected to whole genome sequencing.

Fig. 2

Prevalence of D399N mutations in deposited GISAID consensus sequences. A) Distribution of deposited GISAID genomes with the D399N mutation across the globe. Each dot represents sequences in a GISAID-defined subregion, with area of the dot proportional to the number of sequences. Dots are colored by PANGO lineage. B) Distribution of deposited GISAID genomes with the D399N mutation over time. Countries and subregions are indicated on the left. Each dot represents a unique deposited sequence, colored by PANGO lineage.

Fig. 3

D399N in the context of other N gene mutations in deposited GISAID consensus sequences. Matrix shows co-occurrences of D399N with other N gene mutations, sorted in descending frequency from left to right, with frequencies plotted above as a bar plot. Frequency of each individual mutation is plotted to the left. Highlighted in orange are the genomes with D399N without any co-occurring N gene mutations. N gene mutations with <10 total co-occurrences are collapsed into an “Other N mutations” category.

Fig. 4

Western blot quantitation of ectopically expressed recombinant SARS-CoV-2 nucleocapsid protein in 293T cells. To evaluate N protein expression, total protein normalized lysates from 293T cells transfected with N Wuhan-Hu-1 (WT), N T205I, N D399N, N T205I/D399M, or empty vector and either 250 ng, 125 ng, or 62.5 ng of recombinant N were subjected to SDS-PAGE followed by ɑN western blot. The estimated nanograms of N loaded per lane for each variant is shown beneath the western blot determined from a standard curve generated by quantification of the commercial recombinant N standards.