title: Development and evaluation of a real-time PCR assay for detection of Pneumocystis jirovecii on the fully automated BD MAX platform.
abstract: 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.
authors.affiliation: Department of Infectious Diseases, Medical Microbiology and Hygiene, University Hospital Heidelberg, Heidelberg, Germany. alexander.dalpke@med.uni-heidelberg.de
authors.forename: Alexander H
authors.initials: AH
authors.lastname: Dalpke
authors.forename: Marjeta
authors.initials: M
authors.lastname: Hofko
authors.forename: Stefan
authors.initials: S
authors.lastname: Zimmermann
mesh_terms: D057205:Automation, Laboratory / Q000379:methods*; D006801:Humans; D008828:Microbiological Techniques / Q000379:methods*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016720:Pneumocystis Infections / Q000175:diagnosis* / Q000382:microbiology; D045363:Pneumocystis carinii / Q000235:genetics / Q000302:isolation & purification*; D011446:Prospective Studies; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012189:Retrospective Studies; D012680:Sensitivity and Specificity