title: A real-time polymerase chain reaction assay for detection of Pneumocystis from bronchoalveolar lavage fluid.
abstract: Pneumocystis jiroveci is an important cause of pneumonia in immunocompromised individuals. This organism cannot be cultured, and therefore, diagnosis relies on microscopic identification of the organism using stains or antibodies. Although simple, these tests are insensitive and require expertise for accurate interpretation. We developed a real-time polymerase chain reaction (PCR) assay that provides sensitive and objective detection of Pneumocystis from bronchoalveolar lavage fluid. Primers and fluorescence resonance energy transfer probes were developed that target the cdc2 gene of P. jiroveci. Assay sensitivity is 6 copies of target per microliter of sample. No cross-reactivity occurs with other pathogens, and the PCR assay has a 21% increase in clinical sensitivity as compared with Calcofluor white staining. The real-time PCR assay provides a sensitive, rapid, and objective method for the detection of Pneumocystis from bronchoalveolar lavage fluid.
authors.affiliation: Division of Clinical Microbiology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
authors.forename: Rodney C
authors.initials: RC
authors.lastname: Arcenas
authors.forename: James R
authors.initials: JR
authors.lastname: Uhl
authors.forename: Seanne P
authors.initials: SP
authors.lastname: Buckwalter
authors.forename: Andrew H
authors.initials: AH
authors.lastname: Limper
authors.forename: Dana
authors.initials: D
authors.lastname: Crino
authors.forename: Glenn D
authors.initials: GD
authors.lastname: Roberts
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Wengenack
mesh_terms: D001557:Benzenesulfonates; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology*; D017931:DNA Primers; D004271:DNA, Fungal / Q000032:analysis; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D005656:Fungal Proteins / Q000235:genetics; D006801:Humans; D045363:Pneumocystis carinii / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods
chemical_list: D001557:Benzenesulfonates; D017931:DNA Primers; D004271:DNA, Fungal; D005456:Fluorescent Dyes; D005656:Fungal Proteins; C007061:C.I. Fluorescent Brightening Agent 28