Clinical Evaluation of BD Veritor SARS-CoV-2 and Flu A+B Assay for Point-Of-Care System.

Differential diagnosis of COVID-19 and/or influenza (flu) at point of care is critical for efficient patient management and treatment of both these diseases. The study presented here characterizes the BD Veritor System for Rapid Detection of SARS-CoV-2 and Flu A+B ("Veritor SARS-CoV-2/Flu") triplex assay. The performance for SARS-CoV-2 detection was determined using 298 specimens from patients reporting COVID-19 symptoms within 7 days from symptom onset (DSO) in comparison with the Lyra SARS-CoV-2 RT-PCR (reverse transcriptase PCR) assay ("Lyra SARS-CoV-2") as the reference. The performance for flu A and flu B detection was determined using 75 influenza-positive and 40 influenza-negative retrospective specimens in comparison with the previously FDA-cleared BD Veritor System for Rapid Detection of Flu A+B assay ("Veritor Flu") as the reference. The Veritor SARS-CoV-2/Flu assay met the FDA EUA acceptance criteria (86.7%; 95% confidence interval [95% CI]: 75.8 to 93.1) for SARS-CoV-2 testing compared to Lyra SARS-CoV-2. The Veritor SARS-CoV-2/Flu assay also demonstrated 100% agreement with the Veritor Flu for Flu A+B assay. For flu A detection, the lower bound of the 95% CI was 91.2%; for flu B detection, the lower bound was 90.0%. The dual detection capability of Veritor SARS-CoV-2/Flu for the etiologic agents causing COVID-19 and flu will allow efficient differentiation between the two illnesses, inform disease management, and facilitate optimal treatment. IMPORTANCE COVID-19 and flu are two respiratory illnesses which share similar clinical symptoms. The BD Veritor SARS-CoV-2/Flu assay has high sensitivity and specificity for detecting the SARS-CoV-2 and influenza A/B, the two etiologic agents causing COVID-19 and flu, respectively. This dual detection capability is critical when overlap occurs between the COVID-19 pandemic and the flu season. This triplex assay will allow efficient differentiation between the two respiratory illnesses and support a point-of-care physician diagnosis to facilitate the proper treatment and disease management for patients exhibiting overlapping symptoms.

Clinical Evaluation of BD Veritor SARS-CoV-2 Point-of-Care Test Performance Compared to PCR-Based Testing and versus the Sofia 2 SARS Antigen Point-of-Care Test.

The clinical performance of the BD Veritor System for Rapid Detection of SARS-CoV-2 nucleocapsid antigen (Veritor), a chromatographic immunoassay used for SARS-CoV-2 point-of-care testing, was evaluated using nasal specimens from individuals with COVID-19 symptoms. Two studies were completed to determine clinical performance. In the first study, nasal specimens and either nasopharyngeal or oropharyngeal specimens from 251 participants with COVID-19 symptoms (≤7 days from symptom onset [DSO], ≥18 years of age) were utilized to compare Veritor with the Lyra SARS-CoV-2 PCR assay (Lyra). In the second study, nasal specimens from 361 participants with COVID-19 symptoms (≤5 DSO, ≥18 years of age) were utilized to compare performance of Veritor to that of the Sofia 2 SARS Antigen FIA test (Sofia 2). The positive, negative, and overall percent agreement (PPA, NPA, and OPA, respectively) were the primary outcomes. In study 1, the PPA for Veritor, compared to Lyra, ranged from 81.8 to 87.5% across the 0 to 1 and 0 to 6 DSO ranges. In study 2, Veritor had PPA, NPA, and OPA values of 97.4, 98.1, and 98.1%, respectively, with Sofia 2. Discordant analysis showed one Lyra positive missed by Veritor and five Lyra positives missed by Sofia 2; one Veritor positive result was negative by Lyra. Veritor met FDA emergency use authorization (EUA) acceptance criteria for SARS-CoV-2 antigen testing for the 0 to 5 and 0 to 6 DSO ranges (PPA values of 83.9% and 82.4%, respectively). Veritor and Sofia 2 showed a high degree of agreement for SARS-CoV-2 detection. The Veritor test allows for more rapid COVID-19 testing utilizing easy-to-collect nasal swabs but demonstrated <100% PPA compared to PCR.

The alpha3(betaMet222Ser/Tyr345Trp)3gamma subcomplex of the TF1-ATPase does not hydolyze ATP at a significant rate until the substrate binds to the catalytic site of the lowest affinity.

The alpha(3)(betaM(222)S/Y(345)W)(3)gamma double-mutant subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)), free of endogenous nucleotides, does not entrap inhibitory MgADP in a catalytic site during turnover. It hydrolyzes 100 nM-2 mM ATP with a K(m) of 31 microM and a k(cat) of 220 s(-)(1). Fluorescence titrations of the introduced tryptophans with MgADP or MgATP revealed that both Mg-nucleotide complexes bind to the catalytic site of the highest affinity with K(d)()1 values of less than 1 nM and bind to the site of intermediate affinity with a common K(d)2 value of about 12 nM. The K(d)3 values obtained for the catalytic site of the lowest affinity from titrations with MgADP and MgATP are 25 and 37 microM, respectively. The double mutant hydrolyzes 200 nM ATP with a first-order rate of 1.5 s(-)(1), which is 0.7% of k(cat). Hence, it does not hydrolyze ATP at a significant rate when the catalytic site of intermediate affinity is saturated and the catalytic site of the lowest affinity is minimally occupied. After the addition of stoichiometric MgATP to the alpha(3)(betaM(222)S/Y(345)W)(3)gamma subcomplex, one-third of the tryptophan fluorescence remains quenched after 10 min. The product [(3)H]ADP remains bound when the wild-type and double-mutant subcomplexes hydrolyze substoichiometric [(3)H]ATP. In contrast, (32)P(i) is not retained when the wild-type subcomplex hydrolyzes substoichiometric [gamma-(32)P]ATP. This precludes assessment of the equilibrium at the high-affinity catalytic site when the wild-type TF(1) subcomplex hydrolyzes substoichiometric ATP.

The beta G156C substitution in the F1-ATPase from the thermophilic Bacillus PS3 affects catalytic site cooperativity by destabilizing the closed conformation of the catalytic site.

Fluorescence titrations of the alpha(3)(betaG(156)C/Y(345)W)(3)gamma, alpha(3)(betaE(199)V/Y(345)W)(3)gamma, and alpha(3)(betaY(345)W)(3)gamma subcomplexes of TF(1) with nucleotides show that the betaG(156)C substitution substantially lowers the affinity of catalytic sites for ATP and ADP with or without Mg(2+), whereas the betaE(199)V substitution increases the affinity of catalytic sites for nucleotides. Whereas the alpha(3)(betaG(156)C)(3)gamma and alpha(3)(betaE(199)V)(3)gamma subcomplexes hydrolyze 2 mM ATP at 2% and 0.7%, respectively, of the rate exhibited by the wild-type enzyme, the alpha(3)(betaG(156)C/E(199)V)(3)gamma hydrolyzes 2 mM ATP at 9% the rate exhibited by the wild-type enzyme. The alpha(3)(betaG(156)C)(3)gamma, alpha(3)(betaG(156)C/E(199)V)(3)gamma, and alpha(3)(betaG(156)C/E(199)V/Y(345)W)(3)gamma subcomplexes resist entrapment of inhibitory MgADP in a catalytic site during turnover. Product [(3)H]ADP remains tightly bound to a single catalytic site when the wild-type, betaE(199)V, betaY(345)W, and betaE(199)V/Y(345)W subcomplexes hydrolyze substoichiometric [(3)H]ATP, whereas it is not retained by the betaG(156)C and betaG(156)C/Y(345)W subcomplexes. Less firmly bound, product [(3)H]ADP is retained when the betaG(156)C/E(199)V and betaG(156)C/E(199)V/Y(345)W mutants hydrolyze substoichiometric [(3)H]ATP. The Lineweaver-Burk plot obtained with the betaG(156)C mutant is curved downward in a manner indicating that its catalytic sites act independently during ATP hydrolysis. In contrast, the betaG(156)C/E(199)V and betaG(156)C/E(199)V/Y(345)W mutants hydrolyze ATP with linear Lineweaver-Burk plots, indicating cooperative trisite catalysis. It appears that the betaG(156)C substitution destabilizes the closed conformation of a catalytic site hydrolyzing MgATP in a manner that allows release of products in the absence of catalytic site cooperativity. Insertion of the betaE(199)V substitution into the betaG(156)C mutant restores cooperativity by restricting opening of the catalytic site hydrolyzing MgATP for product release until an open catalytic site binds MgATP.

The (alpha F(357)C)(3)(beta R(372)C)(3)gamma subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 has altered ATPase activity after cross-linking alpha and beta subunits at noncatalytic site interfaces.

In crystal structures of bovine MF(1), the side chains of alpha F(357) and beta R(372) are near the adenines of nucleotides bound to noncatalytic sites. To determine if during catalysis these side chains must pass through the different arrangements in which they are present in crystal structures, the catalytic properties of the (alpha F(357)C)(3)(beta R(372)C)(3)gamma subcomplex of the TF(1)-ATPase were characterized before and after cross-linking the introduced cysteines with CuCl(2). The unmodified mutant enzyme hydrolyzes MgATP at 50% the rate exhibited by wild type. Detailed comparison of the catalytic properties of the double mutant enzyme before and after cross-linking with those of the wild-type subcomplex revealed the following. Before cross-linking, the (alpha F(357)C)(3)(beta R(372)C)(3)gamma subcomplex has less tendency than wild type to release inhibitory MgADP entrapped in a catalytic site during turnover when MgATP binds to noncatalytic sites. Following cross-linking, ATPase activity is reduced 5-fold, and inhibitory MgADP entrapped in a catalytic site during turnover does not release under conditions wherein binding of ATP to noncatalytic sites of the wild-type enzyme promotes release of MgADP from the affected catalytic site. When assayed in the presence of lauryldimethylamine oxide, which prevents turnover-dependent entrapment of inhibitory MgADP in a catalytic site, ATPase activity of the cross-linked form is 47% that of the unmodified mutant enzyme. These results suggest that, during catalysis, the side chains of alpha F(357) and beta R(372) do not pass through the extremely different relative positions in which they exist at the three noncatalytic site interfaces in crystal structures.

The fluorescence spectrum of the introduced tryptophans in the alpha 3(beta F155W)3gamma subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 cannot be used to distinguish between the number of nucleoside di- and triphosphates bound to catalytic sites.

It has been reported that shifts in the fluorescence emission spectrum of the introduced tryptophans in the betaF155W mutant of Escherichia coli F(1) (bovine heart mitochondria F(1) residue number) can quantitatively distinguish between the number of catalytic sites occupied with ADP and ATP during steady-state ATP hydrolysis (Weber, J., Bowman, C., and Senior, A. E. (1996) J. Biol. Chem. 271, 18711--18718). In contrast, addition of MgADP, Mg-5'-adenylyl beta,gamma-imidophosphate (MgAMP-PNP), and MgATP in 1:1 ratios to the alpha(3)(betaF155W)(3)gamma subcomplex of thermophilic Bacillus PS3 F(1) (TF(1)) induced nearly identical blue shifts in the fluorescence emission maximum that was accompanied by quenching. Addition of 2 mm MgADP induced a slightly greater blue shift and a slight increase in intensity over those observed with 1:1 MgADP. However, addition of 2 mm MgAMP-PNP or MgATP induced a much greater blue shift and substantially enhanced fluorescence intensity over those observed in the presence of stoichiometric MgADP or MgAMP-PNP. It is clear from these results that the fluorescence spectrum of the introduced tryptophans in the betaF155W mutant of TF(1) does not respond in regular increments at any wavelength as catalytic sites are filled with nucleotides. The fluorescence spectrum observed after entrapping MgADP-fluoroaluminate complexes in two catalytic sites of the betaF155W subcomplex indicates that the fluorescence emission spectrum of the enzyme is maximally perturbed when nucleotides are bound to two catalytic sites. This finding is consistent with accumulating evidence suggesting that only two beta subunits in the alpha(3)beta(3)gamma subcomplex of TF(1) can simultaneously exist in the completely closed conformation.