Authorized SARS-CoV-2 molecular methods show wide variability in the limit of detection.

On February 29th, 2020, the U.S. Food and Drug Administration issued the first Emergency Use Authorization (EUA) for a SARS-CoV-2 assay outside of the U.S. Centers for Disease Control and Prevention. As of May 3rd, 2021, 289 total EUAs have been granted. Like influenza, there is no standard for defining limit of detection (LoD), but rather guidance that analytical sensitivity/LoD be established as the level that gives a 95% detection rate in at least 20 replicates. Here we compare the performance characteristics of SARS-CoV-2 tests receiving EUA by standardizing sensitivity to a common unit of measure and assess the variability in LoD between tests. Additionally, we looked at factors that may impact sensitivities due to lack of standardization of the test development process and compare results for a standardized reference panel for comparative analysis within a subset of EUA tests offered by the U.S. Food and Drug Administration.

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.

BACKGROUND: We evaluated the analytical sensitivity and specificity of 4 rapid antigen diagnostic tests (Ag RDTs) for severe acute respiratory syndrome coronavirus 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: Three hundred fifty (150 negative and 200 RT-qPCR positive) residual 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 to 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.

Targeted Detection of SARS-CoV-2 Nucleocapsid Sequence Variants by Mass Spectrometric Analysis of Tryptic Peptides.

COVID-19 vaccines are becoming more widely available, but accurate and rapid testing remains a crucial tool for slowing the spread of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus. Although the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) remains the most prevalent testing methodology, numerous tests have been developed that are predicated on detection of the SARS-CoV-2 nucleocapsid protein, including liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunoassay-based approaches. The continuing emergence of SARS-CoV-2 variants has complicated these approaches, as both qRT-PCR and antigen detection methods can be prone to missing viral variants. In this study, we describe several COVID-19 cases where we were unable to detect the expected peptide targets from clinical nasopharyngeal swabs. Whole genome sequencing revealed that single nucleotide polymorphisms in the gene encoding the viral nucleocapsid protein led to sequence variants that were not monitored in the targeted assay. Minor modifications to the LC-MS/MS method ensured detection of the variants of the target peptide. Additional nucleocapsid variants could be detected by performing the bottom-up proteomic analysis of whole viral genome-sequenced samples. This study demonstrates the importance of considering variants of SARS-CoV-2 in the assay design and highlights the flexibility of mass spectrometry-based approaches to detect variants as they evolve.

Evaluation of the genalyte maverick SARS-CoV-2 multi-antigen serology panel.

Serologic testing for SARS-CoV-2 can be used for evaluation of past infection in individual patients and for community seroprevalence studies. We evaluated the analytical and clinical performance of the Genalyte Maverick SARS-CoV-2 Multi-Antigen Serology Panel compared to the Roche Elecsys Anti-SARS-CoV-2 nucleocapsid (NC) qualitative immunoassay, using well characterized clinical serum samples. A total of 143 pre-pandemic sera and 48 sera collected from patients with a negative molecular SARS-CoV-2 result were used for specificity studies. For sensitivity analyses, 179 sera were used, obtained 3-7 days, 8-14 days, or ≥ 15 days after symptom onset from patients with confirmed SARS-CoV-2 infection. Specificity was determined to be 95.3% (182/191) for the Genalyte Maverick. Overall sensitivity of the Genalyte Maverick was similar to that observed for the Roche Elecsys NC test, 79.3% (142/179) vs. 76.5% (137/179), respectively. Genalyte Maverick trended, without statistical significance, towards higher sensitivity as compared to the Roche Elecsys NC test in the 3-7 days (11/25 vs. 9/25, respectively) and 8-14 days (21/28 vs. 19/28, respectively) post-symptom onset sample sets, but was identical in the ≥ 15 days post-symptom onset group (106/116 vs. 106/116, respectively). Therefore, the Genalyte Maverick serologic test had similar overall sensitivity to the Roche Elecsys NC assay, but may have slightly improved sensitivity for early seroconversion. The lower Genalyte Maverick specificity as compared to the Roche Elecsys NC assay as reported by other studies (>99%), may necessitate confirmatory testing of positive Genalyte Maverick results if implemented for clinical use.