SARS-CoV-2 molecular testing and whole genome sequencing following RNA recovery from used BinaxNOW COVID-19 antigen self tests.

Widespread use of over-the-counter rapid diagnostic tests for SARS-CoV-2 has led to a decrease in availability of clinical samples for viral genomic surveillance. As an alternative sample source, we evaluated RNA isolated from BinaxNOW swabs stored at ambient temperature for SARS-CoV-2 rRT-PCR and full viral genome sequencing. 81 of 103 samples (78.6%) yielded detectable RNA, and 46 of 57 samples (80.7 %) yielded complete genome sequences. Our results illustrate that SARS-CoV-2 RNA extracted from used Binax test swabs provides an important opportunity for improving SARS-CoV-2 genomic surveillance, evaluating transmission clusters, and monitoring within-patient evolution.

SARS-CoV-2 molecular testing and whole genome sequencing following RNA recovery from used BinaxNOW COVID-19 Antigen Self Tests

Widespread use of over-the-counter rapid diagnostic tests for SARS-CoV-2 has led to a decrease in availability of clinical samples for viral genomic surveillance. As an alternative sample source, we evaluated RNA isolated from BinaxNOW swabs stored at ambient temperature for SARS-CoV-2 rRT-PCR and full viral genome sequencing. 81 of 103 samples (78.6%) yielded detectable RNA, and 46 of 57 samples (80.7 %) yielded complete genome sequences. Our results illustrate that SARS-CoV-2 RNA extracted from used Binax test swabs provides an important opportunity for improving SARS-CoV-2 genomic surveillance, evaluating transmission clusters, and monitoring within-patient evolution.

Deep mutational scanning identifies SARS-CoV-2 Nucleocapsid escape mutations of currently available rapid antigen tests.

The effects of mutations in continuously emerging variants of SARS-CoV-2 are a major concern for the performance of rapid antigen tests. To evaluate the impact of mutations on 17 antibodies used in 11 commercially available antigen tests with emergency use authorization, we measured antibody binding for all possible Nucleocapsid point mutations using a mammalian surface-display platform and deep mutational scanning. The results provide a complete map of the antibodies' epitopes and their susceptibility to mutational escape. Our data predict no vulnerabilities for detection of mutations found in variants of concern. We confirm this using the commercial tests and sequence-confirmed COVID-19 patient samples. The antibody escape mutational profiles generated here serve as a valuable resource for predicting the performance of rapid antigen tests against past, current, as well as any possible future variants of SARS-CoV-2, establishing the direct clinical and public health utility of our system.

Assessment of the Abbott BinaxNOW SARS-CoV-2 rapid antigen test against viral variants of concern.

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 (B.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 RADx Tech Test Verification Core and the ACME POCT in the Evaluation of COVID-19 Testing Devices: A Model for Progress and Change.

Faced with the COVID-19 pandemic, the US system for developing and testing technologies was challenged in unparalleled ways. This article describes the multi-institutional, transdisciplinary team of the "RADxSM Tech Test Verification Core" and its role in expediting evaluations of COVID-19 testing devices. Expertise related to aspects of diagnostic testing was coordinated to evaluate testing devices with the goal of significantly expanding the ability to mass screen Americans to preserve lives and facilitate the safe return to work and school. Focal points included: laboratory and clinical device evaluation of the limit of viral detection, sensitivity, and specificity of devices in controlled and community settings; regulatory expertise to provide focused attention to barriers to device approval and distribution; usability testing from the perspective of patients and those using the tests to identify and overcome device limitations, and engineering assessment to evaluate robustness of design including human factors, manufacturability, and scalability.

RADx Variant Task Force Program for Assessing the Impact of Variants on SARS-CoV-2 Molecular and Antigen Tests.

Goal: Monitoring the genetic diversity and emerging mutations of SARS-CoV-2 is crucial for understanding the evolution of the virus and assuring the performance of diagnostic tests, vaccines, and therapies against COVID-19. SARS-CoV-2 is still adapting to humans and, as illustrated by B.1.1.7 (Alpha) and B.1.617.2 (Delta), lineage dynamics are fluid, and strain prevalence may change radically in a matter of months. The National Institutes of Health's Rapid Acceleration of Diagnostics (RADxSM) initiative created a Variant Task Force to assess the impact of emerging SARS-CoV-2 variants on in vitro diagnostic testing. Working in tandem with clinical laboratories, the FDA, and the CDC, the Variant Task Force uses both in silico modeling and in vitro testing to determine the effect of SARS-CoV-2 mutations on diagnostic molecular and antigen tests. Here, we offer an overview of the approach and activities of the RADx Variant Task Force to ensure test performance against emerging SARS-CoV-2 lineages.

Single-Amplicon Multiplex Real-Time Reverse Transcription-PCR with Tiled Probes To Detect SARS-CoV-2 spike Mutations Associated with Variants of Concern.

To provide an accessible and inexpensive method to surveil for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutations, we developed a multiplex real-time reverse transcription-PCR (rRT-PCR) assay, the Spike single-nucleotide polymorphism (SNP) assay, to detect specific mutations in the spike receptor binding domain. A single primer pair was designed to amplify a 348-bp region of spike, and probes were initially designed to detect K417, E484K, and N501Y. The assay was evaluated using characterized variant sample pools and residual nasopharyngeal samples. Variant calls were confirmed by SARS-CoV-2 genome sequencing in a subset of samples. Subsequently, a fourth probe was designed to detect L452R. The lower limit of 95% detection was 2.46 to 2.48 log10 genome equivalents (GE)/ml for the three initial targets (∼1 to 2 GE/reaction). Among 253 residual nasopharyngeal swabs with detectable SARS-CoV-2 RNA, the Spike SNP assay was positive in 238 (94.1%) samples. All 220 samples with threshold cycle (CT) values of <30 for the SARS-CoV-2 N2 target were detected, whereas 18/33 samples with N2 CT values of ≥30 were detected. Spike SNP results were confirmed by sequencing in 50/50 samples (100%). Addition of the 452R probe did not affect performance for the original targets. The Spike SNP assay accurately identifies SARS-CoV-2 mutations in the receptor binding domain, and it can be quickly modified to detect new mutations that emerge.

Multidisciplinary assessment of the Abbott BinaxNOW SARS-CoV-2 point-of-care antigen test in the context of emerging viral variants and self-administration.

While there has been significant progress in the development of rapid COVID-19 diagnostics, as the pandemic unfolds, new challenges have emerged, including whether these technologies can reliably detect the more infectious variants of concern and be viably deployed in non-clinical settings as "self-tests". Multidisciplinary evaluation of the Abbott BinaxNOW COVID-19 Ag Card (BinaxNOW, a widely used rapid antigen test, included limit of detection, variant detection, test performance across different age-groups, and usability with self/caregiver-administration. While BinaxNOW detected the highly infectious variants, B.1.1.7 (Alpha) first identified in the UK, B.1.351 (Beta) first identified in South Africa, P.1 (Gamma) first identified in Brazil, B.1.617.2 (Delta) first identified in India and B.1.2, a non-VOC, test sensitivity decreased with decreasing viral loads. Moreover, BinaxNOW sensitivity trended lower when devices were performed by patients/caregivers themselves compared to trained clinical staff, despite universally high usability assessments following self/caregiver-administration among different age groups. Overall, these data indicate that while BinaxNOW accurately detects the new viral variants, as rapid COVID-19 tests enter the home, their already lower sensitivities compared to RT-PCR may decrease even more due to user error.