bWe developed a multiplex SYBR green real-time PCR for the BD Max instrument (BD Diagnostics, Sparks, MD) to detect a panel of carbapenemases. The assay was evaluated with 152 consecutive isolates sent to the German National Reference Laboratory, and 65/65 of the carbapenemase-positive and 87/87 of the carbapenemase-negative strains were identified correctly. R apid detection and differentiation of carbapenemases are important for epidemiological investigations and infection control measures, thereby contributing to the prevention of global spread and optimization of antimicrobial therapy (1). Phenotypebased techniques can be used to identify carbapenemases (2-7), yet the specificity and sensitivity of those procedures vary. Other assays use matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) (8), enzymatic measurements like the Carba NP test (9), and spectrophotometry (10). However, phenotypic and functional assays are often followed by molecular detection of the encoding carbapenemase genes. Molecular analysis is laborious, but fully automated PCR platforms might circumvent this restriction. Multiplexing allows detection in an economically reasonable manner (11).In our study, SYBR green real-time PCR on the BD Max system (BD Diagnostics, Sparks, MD) combined with melt curve analysis was developed to detect frequent carbapenemases in cultured isolates. Primers were mixed in two multiplex PCRs (master mix 1 [MM1] and MM2) that were selected on differences in the melting points of the amplicons. Panel 1 (MM1) amplified IMP-1, IMP-2, GES, KPC, VIM-2, and 16S rRNA as an internal amplification control. Panel 2 (MM2) detected OXA-23-like, VIM-1, OXA-48-like, and NDM (see Table S1 in the supplemental material). One to 2 colonies of a suspect isolate were added to 0.5 ml of 10 mM Tris-1 mM EDTA (pH 8.0) containing 0.1-mm glass beads (BioSpec, Bartlesville, OK). The sample was vortexed and incubated at 95°C for 10 min, and the cleared supernatant was directly used for the PCR. Each single reaction consisted of forward and reverse primers for each of the carbapenemases (final concentration, 0.25 M), a primer for 16S rRNA (only MM1, 0.05 M), 6.25 l of 2ϫ ABsolute SYBR green mix No Rox (Thermo Scientific, Schwerte, Germany), and PCR-grade water up to 10 l. Then 2.5 l of the strain lysate was combined with 10 l of the respective master mix and pipetted manually into a microfluidic BD Max PCR cartridge. The PCR was run in the PCR-only mode at 95°C for 15 min for 30 PCR cycles (98°C, 30 s; 56°C, 20 s; 72°C, 51.1 s) followed by melt curve analysis from 60°C to 100°C in 0.2°C steps.Using plasmids encoding the various carbapenemases, we observed clearly distinct melting temperatures ( Fig. 1A and B). Only IMP-1 and IMP-2 could not be differentiated. Melting temperatures were consistent and highly reproducible (Fig. 1C). The strains harboring a carbapenemase gene could be identified by comparison to the reference temperatures obtained with plasmids (an example is shown in Fig. 1D). Even the co...

We evaluated the fully automated molecular BD MAX Cdiff assay (BD Diagnostics) and the Xpert C. difficile test (Cepheid) for rapid detection of Clostridium difficile infection. Culture was done on chromogenic agar followed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry identification and toxin detection. Repeat testing was required for 1.8% and 6.0% of the BD MAX and Xpert tests, respectively. Sensitivities, specificities, positive predictive values (PPV), and negative predictive values (NPV) were 90.5%, 97.9%, 89.3%, and 98.1%, respectively, for BD MAX and 97.3%, 97.9%, 90.0%, and 99.5%, respectively, for Xpert. Clostridium difficile is the most important cause of hospitalacquired diarrhea. To implement timely infection control measures and appropriate treatment, rapid identification of toxigenic C. difficile is necessary (1). However, laboratory diagnostics remain challenging, as rapid test procedures relying on enzyme immunoassays (EIAs) show limited sensitivity (2), whereas the more-sensitive toxigenic culture and cytotoxicity assays are demanding and time-consuming. Two-step algorithms consisting of sensitive detection of glutamate dehydrogenase followed by a confirmatory toxin test have been proposed (2-4), yet the actual sensitivity has been questioned (5). Nucleic acid amplification techniques have been developed to combine low turnaround times with high sensitivity (6), but these assays require specific infrastructure. More recently, fully automated PCR assays that combine nucleic acid extraction, amplification, and detection have been developed. The Xpert C. difficile assay (Cepheid, Sunnyvale, CA) has high sensitivity and specificity and allows for accurate and rapid detection of Clostridium difficile infection (CDI). Recently, the BD MAX platform (BD Diagnostics, Franklin Lakes, NJ) has been made available; this allows for processing up to 24 samples in a fully automated PCR system. Clinical evaluation of the BD MAX Cdiff assay, based on detection of tcdB, has been published only for comparison with the BD GeneOhm assay (7).This study was conducted between April and July 2012 at the 2,000-bed tertiary care University Hospital Heidelberg. From 333 individual patients, 448 stool specimens, mostly (94.9%) soft or liquid, were examined. Samples were analyzed upon delivery or after overnight storage at 4°C. Specimens were analyzed by the BD MAX Cdiff assay and the Xpert C. difficile test. The standard technique used by the microbiology laboratory as the routine assay was the toxin A/B EIA (miniVIDAS; bioMérieux, Marcy l'Etoile, France). For the BD MAX Cdiff assay, stool samples were vortexed for 15 s and a 10-l loop was used to inoculate the sample tube. The Xpert C. difficile test and the miniVIDAS assay were done strictly in accordance with the manufacturers' protocols. As the reference method (8), stool samples were streaked onto chromID C. difficile agar plates (bioMérieux), incubated anaerobically at 35°C, and read after 24 and 48 h (9, 10). Suspicious col...

Pneumocystis jirovecii is an opportunistic pathogen in immunocompromised and AIDS patients. Detection by quantitative PCR is faster and more sensitive than microscopic diagnosis yet requires specific infrastructure. We adapted a real-time PCR amplifying the major surface glycoprotein (MSG) target from Pneumocystis jirovecii for use on the new BD MAX platform. The assay allowed fully automated DNA extraction and multiplex real-time PCR. The BD MAX assay was evaluated against manual DNA extraction and conventional real-time PCR. The BD MAX was used in the research mode running a multiplex PCR (MSG, internal control, and sample process control). The assay had a detection limit of 10 copies of an MSG-encoding plasmid per PCR that equated to 500 copies/ml in respiratory specimens. We observed accurate quantification of MSG targets over a 7-to 8-log range. Prealiquoting and sealing of the complete PCR reagents in conical tubes allowed easy and convenient handling of the BD MAX PCR. In a retrospective analysis of 54 positive samples, the BD MAX assay showed good quantitative correlation with the reference PCR method (R 2 ‫؍‬ 0.82). Cross-contamination was not observed. Prospectively, 278 respiratory samples were analyzed by both molecular assays. The positivity rate overall was 18.3%. The BD MAX assay identified 46 positive samples, compared to 40 by the reference PCR. The BD MAX assay required liquefaction of highly viscous samples with dithiothreitol as the only manual step, thus offering advantages for timely availability of molecular-based detection assays.