Correlation of SARS-CoV-2 nucleocapsid antigen and RNA concentrations in nasopharyngeal samples from children and adults using an ultrasensitive and quantitative antigen assay

Abstract Background Diagnosis of COVID-19 by PCR offers high sensitivity, but the utility of detecting samples with high cycle threshold (Ct) values remains controversial. Currently available rapid diagnostic tests (RDTs) for SARS-CoV-2 nucleocapsid antigens (Ag) have sensitivity well below PCR. The correlation of Ag and RNA quantities in clinical nasopharyngeal (NP) samples is unknown. Methods An ultrasensitive, quantitative electrochemiluminescence immunoassay for SARS-CoV-2 nucleocapsid (the MSD ® S-PLEX ® CoV-2 N assay) was used to measure Ag in clinical NP samples from adults and children previously tested by PCR. Results The S-PLEX Ag assay had a limit of detection (LOD) of 0.16 pg/mL and a cutoff of 0.32 pg/mL. Ag concentrations measured in clinical NP samples (collected in 3.0 mL media) ranged from less than 160 fg/mL to 2.7 ug/mL. Log-transformed Ag concentrations correlated tightly with Ct values. In 35 adult and 101 pediatric PCR-positive samples, sensitivity was 91% (95% CI, 77-98%) and 79% (70-87%), respectively. In samples with Ct ≤ 35, sensitivity was 100% (88-100%) and 96% (88-99%), respectively. In 50 adult and 40 pediatric PCR-negative specimens, specificity was 100% (93-100%) and 98% (87-100%), respectively. Conclusions Nucleocapsid concentrations in clinical NP samples span 8 orders of magnitude and correlate closely with RNA concentrations (Ct values). The S-PLEX Ag assay had 96-100% sensitivity in samples from children and adults with Ct values ≤ 35, and 98-100% specificity. These results clarify Ag concentration distributions in clinical samples, providing insight into the performance of Ag RDTs and offering a new approach to diagnosis of COVID-19. Key points SARS-CoV-2 nucleocapsid concentrations in clinical nasopharyngeal samples, measured with an ultrasensitive assay, spanned an 8-log range and correlated closely with PCR Ct values. The assay was 96-100% sensitive in pediatric/adult samples with Ct values ≤ 35, and 98-100% specific.