key: cord-0692881-yf667e29 authors: Stanley, Sydney; Hamel, Donald J.; Wolf, Ian D.; Riedel, Stefan; Dutta, Sanjucta; Contreras, Elisa; Callahan, Cody J.; Cheng, Annie; Arnaout, Ramy; Kirby, James E.; Kanki, Phyllis J. title: Limit of Detection for Rapid Antigen Testing of the SARS-CoV-2 Omicron and Delta Variants of Concern Using Live-Virus Culture date: 2022-04-20 journal: J Clin Microbiol DOI: 10.1128/jcm.00140-22 sha: dee9e82ae3c3d6d4c5020d53c3bd6d5372a55c34 doc_id: 692881 cord_uid: yf667e29 nan The authors declare a conflict of interest. LumiraDx provided instrumentation and diagnostic kits; Abbott provided diagnostic Ag kits; and Ginkgo Bioworks provided CareStart and Genbody antigen testing kits for the study. The BIDMC authors (J.E.K., A.C.) received support from Abbott Molecular unrelated to this study under a COVID-19 Diagnostics Evaluation Agreement. Abbott, LumiraDx, and Ginkgo Bioworks had no role in study design, manuscript preparation or decision to publish. All authors, no other conflicts of interest. in triplicate according to the manufacturers' instructions (11) (12) (13) (14) . SteriPack sterile polyester spun nasal swabs (catalog number 60566REVA; LumiraDx UK Ltd., Alloa, Great Britain) and iClean foam swabs (catalog number CY-FS742; Supera, Houston, TX) were used with the LumiraDx test. After identifying the lowest 10-fold dilution with three replicate positive tests, we iteratively tested 3-fold dilutions around this concentration until identifying the lowest dilution (the LoD) in which at least 19 of 20 replicates ($95%) were positive. We found that Omicron had a 95% LoD threshold similar to or lower than that of WA1 for all four tests ( Fig. 1 ). In contrast, the LoDs were 40-to 140-fold higher for Delta than for WA1 for every test examined except for CareStart (Fig. 1) . The equal detection of all three variants by CareStart and the only relatively modest increase in the ratio of PFU to genome copies per milliliter for Delta (Fig. 2) suggest that this represents a true reduction in analytical sensitivity for Delta rather than an artifact of enhanced plaquing efficiencies for Delta relative to antigen levels and associated levels of genome copies. We previously found that the CareStart and LumiraDx antigen tests were excellent in the detection of presumptively WA1-infected individuals (15) . We expect, however, that the observed magnitude of the loss in Delta sensitivity could result in a .20% loss in the detection of potentially infectious individuals based on our previous examination of the effect of LoD on clinical sensitivity (21) . Nevertheless, the most infectious individuals should still be detected. Of note, our use of live virus, analyte volume, and swab type may explain the slight discrepancy with the manufacturers' determined LoDs. Our results for variant detection were also not completely consistent with similar reports, but these studies either fell short of the FDA's EUA requirement of 20 LoD replicates, examined tests unavailable in the United States, and/or tested gamma-irradiated or heat-killed virus, inactivation processes which may artifactually affect test performance (22) (23) (24) . In summary, we demonstrate that the rapid antigen tests evaluated detect Omicron effectively. However, our unexpected findings of decreased detection of Delta virus suggest that antigen test performance needs to (11) (12) (13) (14) , converted from 50% tissue culture infective doses (TCID 50 ) per milliliter to PFU per milliliter by multiplying the TCID 50 per milliliter by 0.7, a standard conversion based on the Poisson distribution, for BinaxNOW (dotted line) (1.4 Â 10 2 TCID 50 /mL; 9.8 Â 10 1 PFU/mL), CareStart (dashed and dotted line) (8.0 Â 10 2 TCID 50 /mL; 5.6 Â 10 2 PFU/mL), GenBody (dashed line) (1.1 Â 10 2 TCID 50 /mL; 7.8 Â 10 1 PFU/mL), and LumiraDx (solid line) (3.2 Â 10 1 TCID 50 /mL; 2.2 Â 10 1 PFU/mL). be reevaluated for emerging variants to ensure that they still meet the intended public health testing goals of the pandemic. US Food and Drug Administration. 2022. In vitro diagnostics EUAs-antigen diagnostic tests for SARS-CoV-2. US Food and Drug Administration Overview of testing for SARS-CoV-2, the virus that causes COVID-19 Diagnostic accuracy of a SARS-CoV-2 rapid antigen test in real-life clinical settings A real-world comparison of SARS-CoV-2 rapid antigen testing versus PCR testing in Florida CDC COVID-19 Surge Laboratory Group. 2021. Performance of an antigen-based test for asymptomatic and symptomatic SARS-CoV-2 testing at two university campuses-Wisconsin A sequence homology and bioinformatic approach can predict candidate targets for immune responses to SARS-CoV-2 SARS-CoV-2 variant classifications and definitions Genomic epidemiology of novel coronavirus-global subsampling High diversity in Delta variant across countries revealed by genome-wide analysis of SARS-CoV-2 beyond the spike protein Severe acute respiratory syndrome coronavirus 2 from patient with coronavirus disease, United States BinaxNOW COVID-19 Ag cardinstructions for use CareStart COVID-19 antigen test-instructions for use GenBody COVID-19 AG-instructions for use SARS-CoV-2 antigen (Ag) product insert. Lumi-raDx UK Ltd SARS-CoV-2 antigen tests predict infectivity based on viral culture: comparison of antigen, PCR viral load, and viral culture testing on a large sample cohort ARTIC. 2022. The ARTIC field bioinformatics pipeline Minimap2: pairwise alignment for nucleotide sequences ARTIC nanopore protocol for nCoV2019 novel coronavirus Nextclade: clade assignment, mutation calling and quality control for viral genomes A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology The limit of detection matters: the case for benchmarking severe acute respiratory syndrome coronavirus 2 testing FIG 2 Correlation of PFU per milliliter and viral load in genome copies per milliliter. Stocks of each strain were serially diluted 10-fold in PBS and analyzed by PFU (10) and calibrated reverse transcription-quantitative PCR (RT-qPCR) assays (15) Detection of the omicron variant virus with the Abbott BinaxNow SARS-CoV-2 rapid antigen assay Sensitivity of SARS-CoV-2 antigen-detecting rapid tests for Omicron variant Assessment of the analytical sensitivity of ten lateral flow devices against the SARS-CoV-2 omicron variant We thank Ginkgo Bioworks for providing CareStart and GenBody SARS-CoV-2 antigen test kits and also Abbott and LumiraDx for providing their respective SARS-CoV-2 antigen test kits used in this study.