Protocol for the creation and characterization of SARS-CoV-2 variant testing panels using remnant clinical samples for diagnostic assay testing.

The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Alpha variant in 2020 demonstrated the need for reanalysis of diagnostic tests to ensure detection of emerging variants. Here, we present a protocol for creating and characterizing SARS-CoV-2 variant testing panels using remnant clinical samples for diagnostic assay testing. We describe steps for characterizing SARS-CoV-2 remnant clinical samples and preparing them into pools and their use in preparing varying quantities of virus. We then detail procedures for verifying variant detection using the resulting sample panel. For complete details on the use and execution of this protocol, please refer to Rao et al.1,2.

Performance of the Xpert™ Mpox PCR assay with oropharyngeal, anorectal, and cutaneous lesion swab specimens.

Anorectal and oropharyngeal exposures are implicated in sexual transmission of mpox, but authorized assays in the United States are only validated with cutaneous lesion swabs. Diagnostic assays for anorectal and oropharyngeal swabs are needed to address potential future outbreaks. The Cepheid Xpert® Mpox is the first point-of-care assay to receive FDA emergency use authorization in the United States and would be a valuable tool for evaluating these sample types. Our exploratory study demonstrates 100 % positive agreement with our in-house PCR assay for natural positive anorectal and oropharyngeal specimens and 92 % sensitivity with low-positive spiked specimens. The Xpert® assay detected viral DNA in specimens not detected by our reference PCR assay from four participants with mpox DNA at other sites, suggesting it may be more sensitive at low viral loads. In conclusion, the validation of the Xpert® for oropharyngeal and anorectal sample types can be rapidly achieved if clinical need returns and prospective samples become available.

Multisite Mpox Infection and Viral Dynamics Among Persons With HIV in Metro Atlanta.

The 2022 mpox outbreak primarily involved sexual transmission among men who have sex with men and disproportionately affected persons with human immunodeficiency virus (HIV). We examined viral dynamics and clinical features in a cohort evaluated for mpox infection at a comprehensive HIV clinic in Atlanta, Georgia. Viral DNA was found in 8 oropharyngeal and 5 anorectal specimens among 10 mpox cases confirmed by lesion swab polymerase chain reaction. Within-participant anatomic site of lowest cycle threshold (Ct) value varied, and lower Ct values were found in oropharyngeal and anorectal swabs when corresponding symptoms were present. This provides insight into mpox infection across multiple anatomic sites among people with HIV.

Standardization and Comparison of Emergency Use Authorized COVID-19 Assays and Testing Laboratories.

Motivation: The motivation for this work was the need to establish a predefined cutoff based on genome copies per ml (GE/ml) rather than Ct, which can vary depending on the laboratory and assay used. A GE/ml-based threshold was necessary to define what constituted 'low positives" for samples that were included in data sets submitted to the FDA for emergency use approval for SARS-CoV-2 antigen tests. Summary: SARS-CoV-2, the causal agent of the global COVID-19 pandemic, made its appearance at the end of 2019 and is still circulating in the population. The pandemic led to an urgent need for fast, reliable, and widely available testing. After December 2020, the emergence of new variants of SARS-CoV-2 led to additional challenges since new and existing tests had to detect variants to the same extent as the original Wuhan strain. When an antigen-based test is submitted to the Food and Drug Administration (FDA) for Emergency Use Authorization (EUA) consideration it is benchmarked against PCR comparator assays, which yield cycle threshold (CT) data as an indirect indicator of viral load - the lower the CT, the higher the viral load of the sample and the higher the CT, the lower the viral load. The FDA mandates that 10-20% of clinical samples used to evaluate the antigen test have to be low positive. Low positive, as defined by the FDA, are clinical samples in which the CT of the SARS-CoV-2 target gene is within 3 CT of the mean CT value of the approved comparator test's Limit of Detection (LOD). While all comparator tests are PCR-based, the results from different PCR assays used are not uniform. Results vary depending on assay platform, target gene, LOD and laboratory methodology. The emergence and dominance of the Omicron variant further challenged this approach as the fraction of low positive clinical samples dramatically increased as compared to earlier SARS-CoV-2 variants. This led to 20-40% of clinical samples having high CT values and therefore assays vying for an EUA were failing to achieve the 80% Percent Positive Agreement (PPA) threshold required. Here we describe the methods and statistical analyses used to establish a predefined cutoff, based on genome copies per ml (GE/ml) to classify samples as low positive (less than the cutoff GE/ml) or high positive (greater than the cutoff GE/mL). CT 30 for the E gene target using Cobas® SARS-CoV-2-FluA/B platform performed at TriCore Reference Laboratories, and this low positive cutoff value was used for two EUA authorizations. Using droplet digital PCR and methods described here, a value 49,447.72 was determined as the GE/ml equivalent for the low positive cutoff. The CT cutoff corresponding to 49447.72 GE/ml was determined across other platforms and laboratories. The methodology and statistical analysis described here can now be used for standardization of all comparators used for FDA submissions with a goal towards establishing uniform criteria for EUA authorization.

Sensitivity of rapid antigen tests against SARS-CoV-2 Omicron and Delta variants.

Rapid antigen tests (RATs) have become an invaluable tool for combating the COVID-19 pandemic. However, concerns have been raised regarding the ability of existing RATs to effectively detect emerging SARS-CoV-2 variants. We compared the performance of 10 commercially available, emergency use authorized RATs against the Delta and Omicron SARS-CoV-2 variants using both individual patient and serially diluted pooled clinical samples. The RATs exhibited lower sensitivity for Omicron samples when using PCR cycle threshold (CT) value (a rough proxy for RNA concentration) as the comparator. Interestingly, however, they exhibited similar sensitivity for Omicron and Delta samples when using quantitative antigen concentration as the comparator. We further found that the Omicron samples had lower ratios of antigen to RNA, which offers a potential explanation for the apparent lower sensitivity of RATs for that variant when using C T value as a reference. Our findings underscore the complexity in assessing RAT performance against emerging variants and highlight the need for ongoing evaluation in the face of changing population immunity and virus evolution.

Sensitivity of Rapid Antigen Tests Against SARS-CoV-2 Omicron and Delta Variants.

Rapid Antigen Tests (RAT) have become an invaluable tool for combating the COVID-19 pandemic. However, concerns have been raised regarding the ability of existing RATs to effectively detect emerging SARS-CoV-2 variants. We compared the performance of eight commercially available, emergency use authorized RATs against the Delta and Omicron SARS-CoV-2 variants using individual patient and serially diluted pooled clinical samples. The RATs exhibited lower sensitivity for Omicron samples when using PCR Cycle threshold (C T ) value (a proxy for RNA concentration) as the comparator. Interestingly, however, they exhibited similar sensitivity for Omicron and Delta samples when using quantitative antigen concentration as the comparator. We further found that the Omicron samples had lower ratios of antigen to RNA, which offers a potential explanation for the apparent lower sensitivity of RATs for that variant when using C T value as a reference. Our findings underscore the complexity in assessing RAT performance against emerging variants and highlight the need for ongoing evaluation in the face of changing population immunity and virus 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.