Performance Evaluation of the BD SARS-CoV-2 Reagents for the BD MAX System.

Nucleic acid amplification testing (NAAT) for SARS-CoV-2 is the standard approach for confirming COVID-19 cases. This study compared results between two emergency use authorization (EUA) NAATs, with two additional EUA NAATs utilized for discrepant testing. The limits of detection (LOD) for the BD SARS-CoV-2 reagents for the BD MAX system (MAX SARS-CoV-2 assay), the bioMérieux BioFire respiratory panel 2.1 (BioFire SARS-CoV-2 assay), the Roche cobas SARS-CoV-2 assay (cobas SARS-CoV-2 assay), and the Hologic Aptima SARS-CoV-2 assay Panther (Aptima SARS-CoV-2 assay) NAAT systems were determined using a total of 84 contrived nasopharyngeal specimens with 7 target levels for each comparator. The positive and negative percent agreement (PPA and NPA, respectively) of the MAX SARS-CoV-2 assay, compared to the Aptima SARS-CoV-2 assay, was evaluated in a postmarket clinical study utilizing 708 nasopharyngeal specimens collected from suspected COVID-19 cases. Discordant testing was achieved using the cobas and BioFire SARS-CoV-2 NAATs. In this study, the measured LOD for the MAX SARS-CoV-2 assay (251 copies/ml; 95% confidence interval [CI], 186 to 427) was comparable to the cobas SARS-CoV-2 assay (298 copies/ml; 95% CI, 225 to 509) and the BioFire SARS-CoV-2 assay (302 copies/ml; 95% CI, 219 to 565); the Aptima SARS-CoV-2 assay had an LOD of 612 copies/ml (95% CI, 474 to 918). The MAX SARS-CoV-2 assay had a PPA of 100% (95% CI, 97.3% to 100.0%) and an NPA of 96.7% (95% CI, 94.9% to 97.9%) compared to the Aptima SARS-CoV-2 assay. The clinical performance of the MAX SARS-CoV-2 assay agreed with another sensitive EUA assay.

Multicenter Clinical Validation of the Molecular BD Max Enteric Viral Panel for Detection of Enteric Pathogens.

The conventional methodology for gastrointestinal pathogen detection remains time-consuming, expensive, and of limited sensitivity. The objective of this study was to evaluate the performance of the BD Max enteric viral panel (Max EVP) assay for identification of viral pathogens in stool specimens from individuals with symptoms of acute gastroenteritis, enteritis, or colitis. Prospective and archival stool specimens from adult and pediatric patients with diarrhea were collected in Cary-Blair medium or unpreserved containers. The results for specimens tested by the Max EVP (on the BD Max platform) were compared to those obtained by the reference method (alternate PCR assays, followed by bidirectional sequencing). Positive percent agreement (PPA) and negative percent agreement (NPA) were calculated. A total of 2,239 specimens were collected, with 2,148 being included for analysis. In this population, 39.6% of specimens were from outpatients, 42.1% were from patients <21 years old, and 49.7% were from females. Prevalence rates for prospective specimens were 7.3%, 4.5%, 3.5%, 2.4%, and 1.2% for norovirus, sapovirus, astrovirus, rotavirus, and adenovirus, respectively. PPA was 92.8%, 84.9%, 93.0%, 100%, and 95.6%, for norovirus, sapovirus, astrovirus, rotavirus, and adenovirus, respectively. NPA was ≥99.4% for all targets. In conjunction with the clinical presentation, laboratory findings, and epidemiological information, the Max EVP assay is effective for the differential diagnosis of enteric disease caused by norovirus, sapovirus, astrovirus, rotavirus, and adenovirus. This assay can be used individually for patients at high risk for a viral enteropathogen (e.g., in outbreak settings) or as an adjunct to other enteric bacterial panels.

BD Vaginal Panel assay results on the high-throughput BD COR system compared to the BD MAX system.

Molecular-based assays demonstrate excellent sensitivity for the detection of vaginitis causes. Here, the high-throughput BD Vaginal Panel for BD COR System (VP-COR) performance was compared to that of the predicate, BD MAX Vaginal Panel for BD MAX System (VP-MAX). Clinical or contrived samples were used to determine the agreement between VP-COR and VP-MAX. Acceptance criteria for VP-COR agreement were as follows: bacterial vaginosis (BV) required a positive percent agreement (PPA) point estimate of ≥95% and a negative percent agreement (NPA) point estimate of ≥98%; Candida group, Candida glabrata, Candida krusei, and Trichomonas vaginalis (TV) required a PPA and NPA point estimate of ≥95% [with lower bound of 95% confidence interval (95% CI) ≥90%]. PPA was 99.5% (95% CI: 97.5-100) and 97.9% (95% CI: 96.5-98.8) for BV contrived (n = 516) and BV clinical (n = 1,050) specimens, respectively. For the Candida group (clinical; n = 724), C. glabrata (contrived; n = 544), C. krusei (contrived; n = 522), and TV (clinical; n = 702), PPA was 99.4% (95% CI: 98.0-99.9), 100% (95% CI: 97.9-100), 100% (95% CI: 97.6-100), and 99.7% (95% CI: 98.3-100), respectively; the lowest lower bound CI value was 97.6%. NPA was >95% for BV contrived and BV clinical specimens. For the Candida group, C. glabrata, C. krusei, and TV, NPA was ≥98.9%; the lowest lower bound CI value was 97.3%. These results demonstrate the equivalent performance of the VP-COR assay when compared to VP-MAX.IMPORTANCEVaginitis is common among women of reproductive age, resulting in around 10 million office visits a year. Diagnosis is often difficult due to its multiple causes-including bacterial vaginosis, vulvovaginal candidiasis, and trichomoniasis-as well as variation in symptom presentation. Typically, cases are identified with a combination of symptomology, medical history, physical examination, and office- or laboratory-based testing. These traditional techniques involve subjective elements and demonstrate varying sensitivity and specificity. Inaccurate or delayed diagnosis leads to continued symptoms, repeat visits, inappropriate treatment, and unnecessary costs. Alternatively, the use of molecular-based assays increases sensitivity for the detection of vaginitis causes. With the validation of the vaginal panel molecular assay on COR (a high-throughput platform), a workflow can be streamlined in high-demand laboratories while providing high sensitivity for vaginitis detection.

Validation of a New High-Throughput BD COR System Using the BD CTGCTV2 Assay.

This study determined the precision and reproducibility of results for the BD CTGCTV2 (CTGCTV2) assay on the BD COR System (COR). The clinical performance of the CTGCTV2 assay conducted on COR was compared with its performance on the BD MAX System (MAX) for detecting Chlamydia trachomatis, Neisseria gonorrhoeae, or Trichomonas vaginalis. The multiday precision and multisite reproducibility studies were conducted using contrived panels of positive and negative urine and PreservCyt specimens. A total of 433 panel members, generated from remnant clinical specimens, were tested in the clinical comparison study. Each panel member was tested three times on MAX and three times on COR. The results in the same testing group were compared for agreement by target. The cycle threshold scores from MAX and COR were analyzed by paired t-test and Deming regression. The CTGCTV2 assay on COR showed high reproducibility in the multiday and multisite precision analysis. The point estimates of positive percent agreement and negative percent agreement in the clinical comparison study for all three targets were greater than 95%, with all corresponding lower bounds of two-sided 95% CIs greater than 90%. Cycle threshold score comparison showed no systematic difference between the two systems. The results of this study show equivalent performance of the CTGCTV2 assay on the MAX and COR systems.

Functional and physical competition between phospholamban and its mutants provides insight into the molecular mechanism of gene therapy for heart failure.

We have used functional co-reconstitution of purified sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) with phospholamban (PLB), its inhibitor in the heart, to test the hypothesis that loss-of-function (LOF) PLB mutants (PLB(M)) can compete with wild-type PLB (PLB(W)) to relieve SERCA inhibition. Co-reconstitution at varying PLB-to-SERCA ratios was conducted using synthetic PLB(W), gain-of-function mutant I40A, or LOF mutants S16E (phosphorylation mimic) or L31A. Inhibitory potency was defined as the fractional increase in K(Ca), measured from the Ca(2+)-dependence of ATPase activity. At saturating PLB, the inhibitory potency of I40A was about three times that of PLB(W), while the potency of each of the LOF PLB(M) was about one third that of PLB(W). However, there was no significant variation in the apparent SERCA affinity for these four PLB variants. When SERCA was co-reconstituted with mixtures of PLB(W) and LOF PLB(M), inhibitory potency was reduced relative to that of PLB(W) alone. Furthermore, FRET between donor-labeled SERCA and acceptor-labeled PLB(W) was decreased by both (unlabeled) LOF PLB(M). These results show that LOF PLB(M) can compete both physically and functionally with PLB(W), provide a rational explanation for the partial success of S16E-based gene therapy in animal models of heart failure, and establish a powerful platform for designing and testing more effective PLB(M) targeted for gene therapy of heart failure in humans.

Clinical evaluation of the molecular-based BD SARS-CoV-2/Flu for the BD MAX™ system.

BACKGROUND: COVID-19 and influenza (flu) share similar clinical symptoms. Therefore, differential detection of these viruses during the respiratory virus season will be an important component for proper patient triage, management, and treatment. OBJECTIVES: Establish the diagnostic performance related to SARS-CoV-2 and Flu A/B detection for the BD SARS-CoV-2/Flu for BD MAX™ System ("MAX SARS-CoV-2/Flu") multiplex assay. MATERIALS AND METHODS: Two hundred and thirty-five (235) retrospective nasopharyngeal specimens were obtained from external vendors. The BD BioGx SARS-CoV-2 Reagents for BD MAX™ System ("BioGx SARS-CoV-2″) and the Cepheid Xpert® Xpress Flu/RSV ("Xpert Flu/RSV") were utilized as reference methods. RESULTS: By reference methods, 52 specimens were SARS-CoV-2-positive, 59 were Flu A-positive, and 60 were Flu B-positive. MAX SARS-CoV-2/Flu had positive percent agreement (PPA) and negative percent agreement (NPA) values for SARS-CoV-2 detection of 96.2% ([95%CI]:87.0-98.9) and 100% [95%CI:88.7-100], respectively; PPA values for Flu A and Flu B of 100% [95%CI:93.9-100] and 98.3% [95%CI:91.1-99.7], respectively, and NPA values for Flu A and Flu B of 98.9% [95%CI:94.0-99.8] and 100% [95%CI:95.9-100], respectively. CONCLUSIONS: The MAX SARS-CoV-2/Flu assay met FDA-EUA performance criteria for SARS-CoV-2 (≥95% for PPA and NPA) and FDA clearance criteria for Flu A/B (PPA ≥90%; lower bound of the 95%CI ≥80% and NPA ≥95%; lower bound of the 95%CI ≥90%).

Site-specific methionine oxidation initiates calmodulin degradation by the 20S proteasome.

The proteasome is a key intracellular protease that regulates processes, such as signal transduction and protein quality control, through the selective degradation of specific proteins. Signals that target a protein for degradation, collectively known as degrons, have been defined for many proteins involved in cell signaling. However, the molecular signals involved in recognition and degradation of proteins damaged by oxidation have not been completely defined. The current study used biochemical and spectroscopic measurements to define the properties in calmodulin that initiate degradation by the 20S proteasome. Our experimental approach involved the generation of multiple calmodulin mutants with specific Met replaced by Leu. This strategy of site-directed mutagenesis permitted site-selective oxidation of Met to Met sulfoxide. We found that the oxidation-induced loss of secondary structure, as measured by circular dichroism, correlated with the rate of degradation for wild-type and mutants containing Leu substitutions in the C-terminus. However, no degradation was observed for mutants with Met to Leu substitution in the N-terminus, suggesting that oxidation-induced structural unfolding in the N-terminal region is essential for degradation by the 20S proteasome. Experiments comparing the thermodynamic stability of CaM mutants helped to further localize the critical site of oxidation-induced focal disruption between residues 51 and 72 in the N-terminal region. This work brings new biochemical and structural clarity to the concept of the degron, the portion of a protein that determines its susceptibility to degradation by the proteasome.

On the intractability of estrogen-related receptor alpha as a target for activation by small molecules.

The estrogen-related receptor alpha (ERRalpha) is a potential target for activation in the treatment of metabolic disease. To date, no small-molecule agonists of ERRalpha have been identified despite several high-throughput screening campaigns. We describe the synthesis and profiling of a small array of compounds designed on the basis of a previously reported agonist-bound crystal structure of the closely related receptor ERRgamma. The results suggest that ERRalpha may be intractable as a direct target for pharmacologic activation.

High-throughput FRET assay yields allosteric SERCA activators.

Using fluorescence resonance energy transfer (FRET), we performed a high-throughput screen (HTS) in a reconstituted membrane system, seeking compounds that reverse inhibition of sarcoplasmic reticulum Ca-ATPase (SERCA) by its cardiac regulator, phospholamban (PLB). Such compounds have long been sought to correct aberrant Ca(2+) regulation in heart failure. Donor-SERCA was reconstituted in phospholipid membranes with or without acceptor-PLB, and FRET was measured in a steady-state fluorescence microplate reader. A 20 000-compound library was tested in duplicate. Compounds that decreased FRET by more than three standard deviations were considered hits. From 43 hits (0.2%), 31 (72%) were found to be false-positives upon more thorough FRET testing. The remaining 12 hits were tested in assays of Ca-ATPase activity, and six of these activated SERCA significantly, by as much as 60%, and several also enhanced cardiomyocyte contractility. These compounds directly activated SERCA from heart and other tissues. These results validate our FRET approach and set the stage for medicinal chemistry and preclinical testing. We were concerned about the high rate of false-positives, resulting from the low precision of steady-state fluorescence. Preliminary studies with a novel fluorescence lifetime plate reader show 20-fold higher precision. This instrument can dramatically increase the quality of future HTS.

Lipid-mediated folding/unfolding of phospholamban as a regulatory mechanism for the sarcoplasmic reticulum Ca2+-ATPase.

The integral membrane protein complex between phospholamban (PLN) and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) regulates cardiac contractility. In the unphosphorylated form, PLN binds SERCA and inhibits Ca(2+) flux. Upon phosphorylation of PLN at Ser16, the inhibitory effect is reversed. Although structural details on both proteins are emerging from X-ray crystallography, cryo-electron microscopy, and NMR studies, the molecular mechanisms of their interactions and regulatory process are still lacking. It has been speculated that SERCA regulation depends on PLN structural transitions (order to disorder, i.e., folding/unfolding). Here, we investigated PLN conformational changes upon chemical unfolding by a combination of electron paramagnetic resonance and NMR spectroscopies, revealing that the conformational transitions involve mostly the cytoplasmic regions, with two concomitant phenomena: (1) membrane binding and folding of the amphipathic domain Ia and (2) folding/unfolding of the juxtamembrane domain Ib of PLN. Analysis of phosphorylated and unphosphorylated PLN with two phosphomimetic mutants of PLN (S16E and S16D) shows that the population of an unfolded state in domains Ia and Ib (T' state) is linearly correlated to the extent of SERCA inhibition measured by activity assays. Inhibition of SERCA is carried out by the folded ground state (T state) of the protein (PLN), while the relief of inhibition involves promotion of PLN to excited conformational states (Ser16 phosphorylated PLN). We propose that PLN population shifts (folding/unfolding) are a key regulatory mechanism for SERCA.