Lateral flow antigen tests can sensitively detect live cultured virus of the SARS-CoV-2 B1.1.7 lineage
Authors: Konstantina Kontogianni, Ana I. Cubas-Atienzar, Dominic Wooding, Kate Buist, Caitlin R. Thompson, Christopher T. Williams, Lisa Baldwin, Camille Escadafal, Jilian A. Sacks, Emily R. Adams, Thomas Edwards
Journal: Journal of Infection (2021)

We read with interest recent evaluations of SARS-CoV-2 antigen rapid diagnostic tests (Ag-RDTs), showing high sensitivity for detecting cases with higher viral loads.1Alemanya A. Baróc B. Ouchia D. Analytical and clinical performance of the panbio COVID-19 antigen-detecting rapid diagnostic test.J. Infect. 2021; 82: 186-230Abstract Full Text Full Text PDF Scopus (55) Google Scholar The clinical sensitivity of these tests relies on their limit of detection (LOD), and there are concerns that new SARS-CoV-2 variants may affect test performance. New variants of SARS-CoV-2 have arisen worldwide, including the lineage B.1.1.7 (Variant of Concern (VOC) strain 202,012/01), which has become the dominant circulating SARS-CoV-2 strain in the U.K., causing >95% of new infections as of April 2021.2Davies N.G. Abbott S. Barnard R.C. et al.Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England.Science. 2021; 372Crossref Scopus (1520) Google Scholar Lineage B.1.1.7 has now been detected in over 114 countries in Europe, America, Australia, Asia and Africa. B.1.1.7 has 17 mutations compared with the original circulating virus, including 8 in the spike protein (S), and 2 in the nucleoprotein (N).3A. Rambaut, N. Loman, O. Pybus, W. Barclay, et al. Preliminary genomic characterisation of an emergent SARS-CoV-2 lineage in the UK defined by a novel set of spike mutations.. 2020. https://virological.org/t/preliminary-genomic-characterisation-of-an-emergent-sars-cov-2-lineage-in-the-uk-defined-by-a-novel-set-of-spike-mutations/563Google Scholar Most Ag-RDTs target N protein with some targeting the S protein. Therefore, these mutations may affect antibody binding in the Ag-RDT, and consequently affect the assay sensitivity. The impact of these mutations on molecular diagnostics has been demonstrated by the failure of S gene detection probes in some nucleic acid tests.4Kidd M. et al.S-variant SARS-CoV-2 lineage B1.1.7 is associated with significantly higher viral loads in samples tested by ThermoFisher TaqPath RT-qPCR.J Infect Dis. 2021; Crossref Scopus (152) Google Scholar It is essential that Ag-RDTs are re-evaluated using new variants to determine if there is any change in test sensitivity due to mutations in the target antigen. The aim of this study was to determine the LOD of seventeen commercially available RDTs using the B.1.1.7 lineage and compare results obtained with the original dominant strain (B.1). A clinically isolated SARS-CoV-2 strain from the B.1.1.7 lineage (Genbank accession number: MW980115), was used for the study. Frozen aliquots of the third passage of the virus were quantified via plaque assay as previously described.5Edwards T. A Kay G. Aljayyoussi G. SARS-CoV-2 transmission risk from sports equipment (STRIKE).MedRxiv. 2021; PubMed Google Scholar For the determination of LODs, a fresh aliquot was serially diluted from 1.0 × 106 plaque forming units (pfu)/ml to 1.0 × 102 pfu/ml. Each dilution was tested in triplicate. Two-fold dilutions were made below the ten-fold LOD dilution to confirm the lowest LOD. Culture media was used as negative control. Viral RNA was extracted from each dilution using QIAmp Viral RNA mini kit (Qiagen, Germany) according to the manufacturer's instructions, and quantified using TaqPath COVID-19 CE-IVD RT-PCR (ThermoFisher). Genome copy number/ml (gcn/ml) were calculated as previously described.6Cubas-Atienzar A.I. et al.Limit of detection in different matrices of nineteen commercially available rapid antigen tests for the detection of SARS-CoV-2.MedRxiv. 2021; Google Scholar We evaluated 17 commercially available Ag-RDT tests (Table 1) following the instructions for use (IFU). The LOD was defined as the lowest dilution at which all three replicates were positive. Results were interpreted by two operators, each blinded to the result of the other. If a discrepant result was obtained, a third operator read any discrepant tests as a tie-breaker.Table 1Characteristics of the Ag-RDT tested.In this StudyTest/CompanyTargetLOD (pfu/ml)LOD (gcn/ml)ActiveXpressActivXpress+ COVID-19 Ag Complete Kit/ Edinburgh Genetics Ltd, UKNucleocapsid5.0 × 1021.0 × 104BiocreditBiocredit COVID-19 Ag, Rapidgen Inc., Rep.KoreaNucleocapsid5.0 × 1044.7 × 106EsplineESPLINE® SARS-CoV-2/ Fujirebio Diagnostics Inc., JapanNucleocapsid2.5 × 1025.1 × 103ExcaliburRapid SARS-CoV-2 Antigen test card/ Excalibur Healthcare Services, UKNucleocapsid5.0 × 1021.0 × 104GenediaGENEDIA W COVID-19 Ag/ Green Cross Medical Sciences; Rep. KoreaNucleocapsid5.0 × 1021.0 × 104iChromaiChroma COVID-19 Ag Test/ Boditech Medical Inc.,Kep KoreaNucleocapsid2.5 × 1025.1 × 104InnovaInnova SARS-CoV-2 Antigen Rapid/ Innova Medical Group Ltd.,UKNucleocapsid2.5 × 1025.1 × 104JoysbioSARS-CoV-2 Antigen Rapid Test Kit./ Joysbio Biotechnology Ltd. ChinaNucleocapsid1.0 × 1026.3 × 103MologicMologic COVID-19 Ag Test device/ Mologic Ltd.UKNucleocapsid2.5 × 1025.1 × 104NowCheckNowCheck COVID-19 Ag test/ Bionote Inc./Mologic Ltd., Rep. KoreaNucleocapsid5.0 × 1021.0 × 104PanBioPanbio™ COVID-19 Ag Rapid Test/ Abbott Rapid Diagnostics. Rep. KoreaNucleocapsid5.0 × 1013.2 × 103RespiStripRespi-Strip COVID-19 Ag/ Coris Bioconcept, BelgiumNucleocapsid1.0 × 1039.8 × 104RocheSARS-CoV-2 Rapid Antigen Test/ SD Biosensor Inc., Rep. Korea / Roche Diagnostics, SwitzerlandNucleocapsid2.5 × 1025.1 × 104Standard-FStandard F COVID-19 Ag FIA., SD Biosensor Inc., Rep. KoreaNucleocapsid1.0 × 1026.3 × 102Standard-QStandard Q COVID-19, SD Biosensor Inc., Rep., KoreaNucleocapsid2.5 × 1025.1 × 103Sure-StatusSure-Status COVID-19 Antigen Card Test, Premier Medical Corporation, IndiaNucleocapsid5.0 × 1013.2 × 103WondfoWondfo 2019-nCoV Antigen Test/ Guangzhou Wondfo Biotech Co., ChinaNucleocapsid1.0 × 1032.1 × 104 Open table in a new tab We found 14/17 Ag-RDTs had an analytical LOD of ≤ 5.0 × 102 pfu/ml (ActiveXpress, Espline, Genedia, iChroma, Innova, Mologic, NowCheck, PanBio, Excalibur, Joysbio, Roche, Standard-F, Standard-Q and Sure-Status) fulfilling the British Department of Health and Social Care (DHSC) acceptable criteria. Additionally, 16/17 (including Wondfo and RespiStrip) had an LOD of ≤ 1.0 × 106 gcn/ml, fulfilling the recommendations in the WHO Target Product Profile for SARS-CoV-2 Ag-RDT (Table 1). With an early 2020 SARS-CoV-2 strain from the B.1 lineage (GISAID accession ID EPI_ISL_464,183), 13/17 Ag-RDTs had an analytical LOD of ≤ 5.0 × 102 pfu/ml. A total of 4/17 Ag-RDTs had a lower LOD when tested with the B.1.1.7 variant, 9/17 were higher, and 4/17 were the same. However, the performance of the Ag-RDTs was not significantly different (Mann Whitney test, P 0.937) between the two strains (Fig. 1) and only 1/17 (RespiStript) no longer met the DHSC acceptance criteria. Our results build on previous studies showing equivalent performance with the B.1.1.7 variant for a small panel of Ag-RDTs.7Pickering S. Batra R. Snell L.B et al.Comparative performance of SARS CoV-2 lateral flow antigen tests demonstrates their utility for high sensitivity detection of infectious virus in clinical specimens.MedRxiv. 2021; PubMed Google Scholar The B.1.1.7 variant has two mutations in the nucleoprotein, which may be insufficient to impact antibody binding for detection of the antigen. All Ag-RDTs evaluated in this study targeted the N protein. Ag-RDTs targeting the S protein may be expected to have more difficulties in detecting B.1.1.7 due to the larger number of mutations, however, further evaluations are required to investigate this. Other VOCs have different N gene mutations with the potential to affect Ag-RDT performance. For example, the P.1 lineage that has emerged in Manaus, Brazil, carries a single N gene mutation (P80R) 8Sabino E.C. Buss L.F. Carvalho M.P.S. et al.Resurgence of COVID-19 in Manaus, Brazil, despite high seroprevalence.Lancet. 2021; 397: 452-455Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar and B.1.243.1 carries mutation S194L, and further evaluations with these lineages are needed. New lineages and mutations in SARS-CoV-2 are constantly appearing and re-evaluation of SARS-COV-2 diagnostic tests including Ag-RDTs will be required once certain strains become more prevalent. This study demonstrates equivalent performance of 18 Ag-RDTs with SARS-CoV-2 B1.1.7 variant 6Cubas-Atienzar A.I. et al.Limit of detection in different matrices of nineteen commercially available rapid antigen tests for the detection of SARS-CoV-2.MedRxiv. 2021; Google Scholar as with the original dominant strain from the beginning of the pandemic, and that 16 tests meet WHO limits for analytical sensitivity. This supports the continued usage of these tests in areas where B.1.1.7 is dominant. The authors have no conflicts of interest to declare. Sources of funding: The study was supported by the Foundation for Innovative New Diagnostics (FIND). The funders of the study had no role in data collection and data analysis.