Comparison of the automated Phoenix with the Vitek 2 for the identification of Streptococcus pneumoniae.

Rapid and accurate identification of Streptococcus pneumoniae is a critical component in the optimal management of infected patients. The performance of the BD Phoenix Automated Microbiology System (BD Diagnostic Systems, Sparks, Md.) was evaluated for identification of S. pneumoniae (n = 311) and was compared to the Vitek 2 (bioMérieux, Marcy l'Etoile, France). Strains with discordant identification between methods were resolved with 16S rRNA gene sequencing as the gold standard. The Phoenix and the Vitek 2 correctly identified 96.8% (n = 301) and 95.2% (n = 296) of S. pneumoniae strains, respectively. Overall, there was no statistically significant difference in the performance of the 2 automated systems for the identification of S. pneumoniae in this study. The Vitek 2 mean time-to-results for all streptococcal identification was 1.5 h faster than that for the Phoenix. We conclude that the automated Phoenix and the Vitek 2 systems are comparable in their ability to identify S. pneumoniae and are preferable to the use of routine biochemical assays, which have delayed time-to-results and are not dependably accurate.

Comparison of BD phoenix to vitek 2, microscan MICroSTREP, and Etest for antimicrobial susceptibility testing of Streptococcus pneumoniae.

The performance of the BD Phoenix Automated Microbiology System (BD Diagnostic Systems) was compared to those of the Vitek 2 (bioMérieux), the MicroScan MICroSTREP plus (Siemens), and Etest (bioMérieux) for antibiotic susceptibility tests (AST) of 311 clinical isolates of Streptococcus pneumoniae. The overall essential agreement (EA) between each test system and the reference microdilution broth reference method for S. pneumoniae AST results was >95%. For Phoenix, the EAs of individual antimicrobial agents ranged from 90.4% (clindamycin) to 100% (vancomycin and gatifloxacin). The categorical agreements (CA) of Phoenix, Vitek 2, MicroScan, and Etest for penicillin were 95.5%, 94.2%, 98.7%, and 97.7%, respectively. The overall CA for Phoenix was 99.3% (1 very major error [VME] and 29 minor errors [mEs]), that for Vitek 2 was 98.8% (7 VMEs and 28 mEs), and those for MicroScan and Etest were 99.5% each (19 and 13 mEs, respectively). The average times to results for Phoenix, Vitek 2, and the manual methods were 12.1 h, 9.8 h, and 24 h, respectively. From these data, the Phoenix AST results demonstrated a high degree of agreement with all systems evaluated, although fewer VMEs were observed with the Phoenix than with the Vitek 2. Overall, both automated systems provided reliable AST results for the S. pneumoniae-antibiotic combinations in half the time required for the manual methods, rendering them more suitable for the demands of expedited reporting in the clinical setting.

Distinctive modulatory effects of five human auxiliary beta2 subunit splice variants on L-type calcium channel gating.

Sequence analysis of the human genome permitted cloning of five Ca(2+)-channel beta(2) splice variants (beta(2a)-beta(2e)) that differed only in their proximal amino-termini. The functional consequences of such beta(2)-subunit diversity were explored in recombinant L-type channels reconstituted in HEK 293 cells. Beta(2a) and beta(2e) targeted autonomously to the plasma membrane, whereas beta(2b)-beta(2d) localized to the cytosol when expressed in HEK 293 cells. The pattern of modulation of L-type channel voltage-dependent inactivation gating correlated with the subcellular localization of the component beta(2) variant-membrane-bound beta(2a) and beta(2e) subunits conferred slow(er) channel inactivation kinetics and displayed a smaller fraction of channels recovering from inactivation with fast kinetics, compared to beta(2b)-beta(2d) channels. The varying effects of beta(2) subunits on inactivation gating were accounted for by a quantitative model in which L-type channels reversibly distributed between fast and slow forms of voltage-dependent inactivation-membrane-bound beta(2) subunits substantially decreased the steady-state fraction of fast inactivating channels. Finally, the beta(2) variants also had distinctive effects on L-type channel steady-state activation gating, as revealed by differences in the waveforms of tail-activation (G-V) curves, and conferred differing degrees of prepulse facilitation to the channel. Our results predict important physiological consequences arising from subtle changes in Ca(2+)-channel beta(2)-subunit structure due to alternative splicing and emphasize the utility of splice variants in probing structure-function mechanisms.

Systematic identification of splice variants in human P/Q-type channel alpha1(2.1) subunits: implications for current density and Ca2+-dependent inactivation.

P/Q-type (Ca(v)2.1) calcium channels support a host of Ca2+-driven neuronal functions in the mammalian brain. Alternative splicing of the main alpha1A (alpha1(2.1)) subunit of these channels may thereby represent a rich strategy for tuning the functional profile of diverse neurobiological processes. Here, we applied a recently developed "transcript-scanning" method for systematic determination of splice variant transcripts of the human alpha1(2.1) gene. This screen identified seven loci of variation, which together have never been fully defined in humans. Genomic sequence analysis clarified the splicing mechanisms underlying the observed variation. Electrophysiological characterization and a novel analytical paradigm, termed strength-current analysis, revealed that one focus of variation, involving combinatorial inclusion and exclusion of exons 43 and 44, exerted a primary effect on current amplitude and a corollary effect on Ca2+-dependent channel inactivation. These findings significantly expand the anticipated scope of functional diversity produced by splice variation of P/Q-type channels.

Novel functional properties of Ca(2+) channel beta subunits revealed by their expression in adult rat heart cells.

Recombinant adenoviruses were used to overexpress green fluorescent protein (GFP)-fused auxiliary Ca(2+) channel beta subunits (beta(1)-beta(4)) in cultured adult rat heart cells, to explore new dimensions of beta subunit functions in vivo. Distinct beta-GFP subunits distributed differentially between the surface sarcolemma, transverse elements, and nucleus in single heart cells. All beta-GFP subunits increased the native cardiac whole-cell L-type Ca(2+) channel current density, but produced distinctive effects on channel inactivation kinetics. The degree of enhancement of whole-cell current density was non-uniform between beta subunits, with a rank order of potency beta(2a) approximately equal to beta(4) > beta(1b) > beta(3). For each beta subunit, the increase in L-type current density was accompanied by a correlative increase in the maximal gating charge (Q(max)) moved with depolarization. However, beta subunits produced characteristic effects on single L-type channel gating, resulting in divergent effects on channel open probability (P(o)). Quantitative analysis and modelling of single-channel data provided a kinetic signature for each channel type. Spurred on by ambiguities regarding the molecular identity of the actual endogenous cardiac L-type channel beta subunit, we cloned a new rat beta(2) splice variant, beta(2b), from heart using 5' rapid amplification of cDNA ends (RACE) PCR. By contrast with beta(2a), expression of beta(2b) in heart cells yielded channels with a microscopic gating signature virtually identical to that of native unmodified channels. Our results provide novel insights into beta subunit functions that are unattainable in traditional heterologous expression studies, and also provide new perspectives on the molecular identity of the beta subunit component of cardiac L-type Ca(2+) channels. Overall, the work establishes a powerful experimental paradigm to explore novel functions of ion channel subunits in their native environments.