Limit of Detection for Rapid Antigen Testing of the SARS-CoV-2 Omicron and Delta Variants of Concern Using Live-Virus Culture

To bolster coronavirus disease 2019 (COVID-19) pandemic mitigation efforts, the U.S. Food and Drug Administration (FDA) issued an emergency use authorization (EUA) for easy-to-use rapid antigen (Ag) tests for the diagnosis and surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (1, 2). Unlike sensitive molecular tests that detect multiple SARS-CoV-2 genes, antigen tests target a singular yet genetically conserved nucleocapsid viral protein (3–6). As the pandemic continues, some hypothesized that the performance of available antigen tests may differ between SARS-CoV-2 variants. As of February 2022, the most recent SARS-CoV-2 strains declared variants of concern (VoC) by the U.S. Centers for Disease Control and Prevention are Omicron (strain B.1.1.529) and Delta (strain B.1.617.2) (7). Beyond striking amino acid mutations in the spike protein, Omicron also harbors P13L, D31–33, R203K, and G204R nucleocapsid mutations, while Delta strains carry D63G, R203M, and D377Y nucleocapsid mutations (8, 9). However, the limits of detection (LoDs) of many FDA EUA antigen tests were established with gamma-irradiated or heat-inactivated preparations of the USA WA1/2020 (WA1) strain (10) lacking these nucleocapsid mutations. This includes at-home lateral flow tests like the BinaxNOW COVID-19 Ag card (Abbott Diagnostics Scarborough, Inc., Scarborough, ME), the CareStart COVID-19 antigen home test (Access Bio, Inc., Somerset, NJ), and the GenBody Covid-19 Ag test (GenBody, Inc., Chungcheongnam-do, Republic of Korea) and also the LumiraDx SARSCoV-2 Ag test (LumiraDx UK Ltd., Alloa, Great Britain), a microfluidic immunofluorescence assay for clinical laboratory testing (11–14). In the present study, we used cultured plaque-titered live Omicron, Delta, and WA1 viruses to assess differences in the LoDs with the BinaxNOW, CareStart, GenBody, and LumiraDx tests. The titers of the Omicron lh01 (NCBI accession number OL719310), Delta (BEI Resources catalog number NR-55671, isolate hCoV-19/USA/MD-HP05285/2021; Johns Hopkins University), and WA1 (10) viruses were determined by a plaque assay (10) and further calibrated with the Abbott RealTime SARS-CoV-2 assay (Abbott Molecular, Inc., Des Plaines, IL) (15). The genomes of the Omicron, Delta, and WA1 viral stocks used in our analysis were also sequenced using the NEBNext ARTIC SARS-CoV-2 companion kit (New England BioLabs, Ipswich, MA) and MinION (Oxford Nanopore Technologies, Oxford, UK) technology (16–20), confirming the lack of mutation acquisition during propagation. For LoD evaluation, 10-fold serial dilutions in phosphate-buffered saline (PBS) ranging from 2.5 104 to 2.5 PFU/mL were applied to swabs in 50-mL volumes and tested EditorMelissa B. Miller, UNC School of Medicine Copyright © 2022 American Society for Microbiology. All Rights Reserved. Address correspondence to Phyllis J. Kanki, pkanki@hsph.harvard.edu, or James E. Kirby, jekirby@bidmc.harvard.edu.