Analytical sensitivity and specificity of four point of care rapid antigen diagnostic tests for SARS-CoV-2 using real-time quantitative PCR, quantitative droplet digital PCR, and a mass spectrometric antigen assay as comparator methods

Karon, Brad S; Donato, Leslie; Bridgeman, Amber R; Blommel, Joseph H; Kipp, Benjamin; Maus, Anthony; Renuse, Santosh; Kemp, Jennifer; Madugundu, Anil K; Vanderboom, Patrick M; Chavan, Sandip; Dasari, Surendra; Singh, Ravinder J; Grebe, Stefan K G; Pandey, Akhilesh

BACKGROUND: We evaluated the analytical sensitivity and specificity of four rapid antigen diagnostic tests (Ag RDTs) for SARS-CoV-2, using reverse transcription quantitative PCR (RT-qPCR) as the reference method; and further characterizing samples using droplet digital quantitative PCR (ddPCR) and a mass spectrometric antigen test. METHODS: 350 (150 negative and 200 RT-qPCR positive) residual phosphate buffered saline (PBS) samples were tested for antigen using the BD Veritor lateral flow (LF), ACON LF, ACON fluorescence immunoassay (FIA), and LumiraDx FIA. ddPCR was performed on RT-qPCR positive samples to quantitate the viral load in copies/mL applied to each Ag RDT. Mass spectrometric antigen testing was performed on PBS samples to obtain a set of RT-qPCR positive, antigen positive samples for further analysis. RESULTS: All Ag RDTs had nearly 100% specificity compared to RT-qPCR. Overall analytical sensitivity varied from 66.5% to 88.3%. All methods detected antigen in samples with viral load >1,500,000 copies/mL RNA, and detected ≥75% of samples with viral load of 500,000 to 1,500,000 copies/mL. The BD Veritor LF detected only 25% of samples with viral load between 50,000-500,000 copies/mL, compared to 75% for the ACON LF device and >80% for LumiraDx and ACON FIA. The ACON FIA detected significantly more samples with viral load <50,000 copies/mL compared to the BD Veritor. Among samples with detectable antigen and viral load <50,000 copies/mL, sensitivity of the Ag RDT varied between 13.0% (BD Veritor) and 78.3% (ACON FIA). CONCLUSIONS: Ag RDTs differ significantly in analytical sensitivity, particularly at viral load <500,000 copies/mL.