key: cord-1039720-hrfcrsj9
authors: Rao, Anuradha; Bassit, Leda; Lin, Jessica; Verma, Kiran; Bowers, Heather B.; Pachura, Kimberly; Greenleaf, Morgan; Sullivan, Julie; Lai, Eric; Creager, Richard S.; Pribyl, Thomas; Blackwood, John; Piantadosi, Anne L.; Schinazi, Raymond; Martin, Greg S.; Lam, Wilbur A.
title: Assessment of the Abbott BinaxNOW SARS-CoV-2 rapid antigen test against viral variants of concern
date: 2022-02-22
journal: iScience
DOI: 10.1016/j.isci.2022.103968
sha: 26955e27583e5614334442cf0e3f29cbcaaec436
doc_id: 1039720
cord_uid: hrfcrsj9

As the emergence of SARS-CoV-2 variants brings the global pandemic to new levels, the performance of current rapid antigen tests against variants of concern and interest (VOC/I) is of significant public health concern. Here we report assessment of the Abbot BinaxNOW COVID-19 Antigen Self-Test. Using genetically sequenced remnant clinical samples collected from individuals positive for SARS-CoV-2, we assessed the performance of BinaxNOW against the variants that currently pose public health threats. We measured the limit of detection of BinaxNOW against various VOC/I in a blinded manner. BinaxNOW successfully detected the Omicron (B.1.1.529), Mu (B.1.621), Delta (B.1.617.2), Lambda (C.37), Gamma (P.1), Alpha (B.1.1.7), Beta (1.351), Eta (B.1.525), and P.2 variants and at low viral concentrations. BinaxNOW also detected the Omicron variant in individual remnant clinical samples. Overall, these data indicate that this inexpensive and simple-to-use, FDA-authorized and broadly distributed rapid test can reliably detect Omicron, Delta, and other VOC/I.

The emergence of new SARS-CoV-2 variants has pushed the pandemic to new levels, raising concerns of increased infectivity, breakthrough cases among vaccinated individuals, and the viability of current test strategies. In 2021, new SARS-CoV-2 variants of concern and interest (VOC/I) such as the Delta (B.1.617.2), Lambda (C37), Mu (B.1.621), and Omicron (B.1.1.529) variants have caused multiple surges of COVID-19 cases worldwide and raised concerns about evasion of the currently available SARS-CoV-2 vaccines. (Herlihy, 2021; Lopez Bernal et al., 2021; Shah et al., 2021b; Tchesnokova et al., 2021) With the high prevalence of rapid diagnostic assays currently available to the public at point-ofcare (POC) and as at-home over-the-counter (OTC) kits, an obvious question is whether these tests can even reliably detect Delta, Omicron, and other variants of concern/interest (VOC/I). The timing of detection is particularly important for Omicron, as it has been shown to have a shorter incubation period than previous variants, and thus may become transmissible faster after infection. (Brandal et al., 2021; Burki, 2021; Jansen, 2021) If these home and communitybased tests can indeed detect VOC/I, they can be implemented as part of broad public health strategies to help curtail the rapid spread of VOC/I. On the other hand, if these rapid tests cannot reliably detect the most prevalent VOC/I, their overall clinical utility at the current point of the pandemic should be called into question.

To that end, here we report our objective assessment of Abbott's BinaxNOW COVID-19 Antigen Self-Test, which has among the highest usage, availability, distribution, and production rates of rapid tests and was the first lateral flow assay (LFA)-based rapid antigen test to receive U.S. FDA Emergency Use Authorization (EUA) for the home OTC setting. (Shah et al., 2021a; Prince-Guerra, 2021; Pollock et al., 2021; Hodges, 2021) BinaxNOW is a SARS-CoV-2 diagnostic assay that detects the viral nucleocapsid (N) protein in samples collected by anterior nasal swab and reports a qualitative positive, negative, or invalid result ("BINAXNOW COVID-19 AG CARD"). We previously assessed the usability of BinaxNOW as a self-administered test and conducted initial experiments assessing the test's performance in detecting wildtype and various variants. (Frediani et al., 2021) Here we report with a higher molecular resolution on the performance of BinaxNOW using a comprehensive panel of VOC/I that are currently of the highest public health significance.

In September 2021, the Mu (B.1.621) variant had been detected in every state in the US and was designated as a VOC by the World Health Organization (WHO), though it was later reclassified as a VOI (WHO, 2021) . Importantly, the Mu variant harbors a mutation, E484K, that likely enables the virus to blunt vaccine and infection-induced immunity. (Wadman, M., 2021) Given the potential at the time for this variant to significantly worsen the outlook of the global pandemic, we tested the performance of BinaxNOW in duplicate and a blinded manner using remnant clinical samples (RCS) (Figure 1 ).

Most recently, the Omicron variant (B.1.1.529) has emerged and been designated a VOC by the WHO due to a high number of mutations in the spike protein (WHO, 2021). The Omicron variant has 32 additional mutations in the spike protein, which may allow it to partially evade vaccineelicited immunity by escaping neutralizing antibodies from previous strains of SARS-CoV-2. (Cao et al., 2021; Cele et al., 2021; Liu et al., 2021) Due to its increased transmissibility, Omicron has quickly become the dominant variant in the United States and around the world (CDC, 2020).

For these reasons, we assessed the performance of BinaxNOW using pooled, heat-inactivated RCS positive for Omicron (B.1.1.529) variant.

Overall, as detailed in Table 1 (Table 1) .

Although the sensitivity of BinaxNOW appears to be slightly decreased against the Omicron (B.1.1.529) variant compared to previous VOC/I, the results still justify the continued use of this readily available, inexpensive and simple-to-use rapid test kit as part of the community-and/or home-based testing strategies to combat the ongoing public health crisis. Ultimately, laboratory experiments cannot fully recapitulate the real-world application of a test kit, and the utility of the BinaxNOW will depend on a review its clinical performance, which we are currently conducting.

We acknowledge the limitations of the current study in that we used RCS that may have some degradation and may not accurately reflect real-world testing conditions. In addition, the BinaxNOW kit lots used in this study varied from experiment to experiment and that could have generated slight differences in the test performance. Additional studies into the quantitative differences in the N antigen levels of Omicron variant patient samples will help to clarify the implications of the decreased performance. Future studies are also underway to compare BinaxNOW to other available rapid antigen tests.

Requests for further information and resources should be directed to the lead contact Wilbur Lam (wilbur.lam@emory.edu), or the co-corresponding author Greg Martin (greg.martin@emory.edu).

This study did not generate new materials.

The genetic sequence data was analyzed the Nextclade (https://clades.nextstrain.org/) version 1.7.1 or higher. SARS-CoV-2 strains to be tested were identified using the Rosalind (https://radx.rosalind.bio) bioinformatics platform, version 3.33.1.0 or higher. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request

The study abides by the ethical guidelines of research at Emory University. The deidentified remnant clinical samples in this study were provided without any patient information. Sequencing of the SARS-CoV-2 to confirm the variant was done by the party from which we obtained the remnant clinical samples.

Heat-inactivated RCS (provided by Helix OpCo, LLC) from individuals positive for SARS-CoV-2 (N=8) known to be infected with the Mu variant were selected based on genomic sequence quality (as determined by NextClade) and cycle threshold (Ct) value of N protein. We compared these results to samples obtained from individuals (N=8) infected with the B.1.2 strain of SARS-CoV-2, which is not considered to be a VOC/I by the WHO, and therefore served as our comparator. RT-qPCR for the SARS-CoV-2 N2 gene using CDC primers/probe set was performed on each RCS and N2 Ct values were used as estimates of viral load.

In addition, using genetically sequenced RCS collected from individuals positive for SARS-CoV-2 across the country, we created RCS "pools" of each of the VOC/I (Figure 1 ). The individual VOC/I pools were verified by repeating genetic sequencing to ensure quality control. Panels of the VOC/I pools of varying viral loads were then created by serial dilution using SARS-CoV-2 negative pooled human donor nasal wash (Lee Biosolutions, Catalog No. 991-26-P-1). Dilutions of every pool were then analyzed by RT-qPCR for the SARS-CoV-2 N2 gene using CDC primers/probe set (Figure 1 ). The performance of BinaxNOW was then assessed in a blinded manner in triplicate.

The pools for heat-inactivated Omicron variant were created using the same method as previously stated (Figure 1 ) and diluted to 10 dilutions that ranged from a Ct value of 21.2 to a Ct value of 31.7. The limit of detection was determined by testing each sample dilution 5 times with BinaxNOW. For testing, we used the direct spike method, where 20l of sample was spiked onto the swab provided with the test and subsequent steps were according to the BinaxNOW instructions for use (IFU). This limit of detection was further confirmed by testing the highest detectable dilution as well as the two neighboring dilutions a further 20 times.

We then assessed BinaxNOW using non heat inactivated RCS obtained from the University of Washington that were confirmed to be Omicron (B.1.1.529). These samples were pooled and diluted to a range of Ct values between 19.3 and 28.8. The dilutions were tested according to the IFU of BinaxNOW rapid antigen test to determine the limit of detection. Finally, we obtained 12 RCS sequence confirmed to be Omicron from LabCorp and measured N2 Ct in triplicate by RT-qPCR (Table 2 ). These samples were tested in duplicate using BinaxNOW, following the IFU.

The mean and standard deviation of the Ct values in Table 1 and Table 2 were calculated using Microsoft Excel. Table   REAGENT 

Abbot BinaxNOW COVID-19 Antigen Self-Test against the current viral variants of concern (VOC) and variants of interest (VOI). Pooled remnant clinical samples were diluted in SARS-CoV-2 negative nasal wash, and viral load was measured by RT-qPCR, following which the performance of BinaxNOW is assessed in a blinded manner and triplicate. (N2: nucleocapsid). J o u r n a l P r e -p r o o f sequenced remnant clinical samples (N=12) containing the N:D343G mutation were measured in triplicate using RT-qPCR, as a proxy for viral load. The samples were then tested in duplicate using the BinaxNOW COVID-19 Antigen Self-Test. The sample with the highest detectable Ct is highlighted in green.

BINAXNOW COVID-19 AG CARD (PN 195-000) -INSTRUCTIONS FOR USE

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Performance and Implementation Evaluation of the Abbott BinaxNOW Rapid Antigen Test in a High-Throughput Drive-Through Community Testing Site in Massachusetts

Evaluation of Abbott BinaxNOW Rapid Antigen Test for SARS-CoV-2 Infection at Two Community-Based Testing Sites -Pima County

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Classification of Omicron (B.1.1.529): SARS-CoV-2 Variant of Concern

Highlights of BinaxNOW VOC/I paper • Pooled remnant clinical samples with variants of concern are tested using BinaxNOW • BinaxNOW detects live and heat-inactivated virus in pooled and individual samples • BinaxNOW sensitivity slightly lower against Omicron variant in laboratory tests • BinaxNOW remains a useful public health tool to combat the COVID-19 pandemic J o u r n a l P r e -p r o o f Key Resources Table   REAGENT