title: Development and validation of a Pneumocystis jirovecii real-time polymerase chain reaction assay for diagnosis of Pneumocystis pneumonia.
abstract: BACKGROUND
  Pneumocystis jirovecii (PJ), a pathogenic fungus, causes severe interstitial Pneumocystis pneumonia (PCP) among immunocompromised patients. A laboratory-developed real-time polyermase chain reaction (PCR) assay was validated for PJ detection to improve diagnosis of PCP.
  
  
  METHODS
  Forty stored bronchoalveolar lavage (BAL) samples (20 known PJ positive [PJ+] and 20 known PJ negative [PJ-]) were initially tested using the molecular assay. Ninety-two sequentially collected BAL samples were then analyzed using an immunofluorescence assay (IFA) and secondarily tested using the PJ real-time PCR assay. Discrepant results were resolved by retesting BAL samples using another real-time PCR assay with a different target. PJ real-time PCR assay performance was compared with the existing gold standard (ie, IFA) and a modified gold standard, in which a true positive was defined as a sample that tested positive in two of three methods in a patient suspected to have PCP.
  
  
  RESULTS
  Ninety of 132 (68%) BAL fluid samples were collected from immunocompromised patients. Thirteen of 92 (14%) BALs collected were PJ+ when tested using IFA. A total of 40 BAL samples were PJ+ in the present study including: all IFA positive samples (n=13); all referred PJ+ BAL samples (n=20); and seven additional BAL samples that were IFA negative, but positive using the modified gold standard. Compared with IFA, the PJ real-time PCR had sensitivity, specificity, and positive and negative predictive values of 100%, 91%, 65% and 100%, respectively. Compared with the modified gold standard, PJ real-time PCR had a sensitivity, specificity, and positive and negative predictive values of 100%.
  
  
  CONCLUSIONS
  PJ real-time PCR improved detection of PJ in immunocompromised patients.
authors.affiliation: Division of Microbiology, Calgary Laboratory Services, Departments of Pathology & Laboratory Medicine, the University of British Columbia, Vancouver, British Columbia ; Division of Medical Microbiology and the University of British Columbia, Vancouver, British Columbia ; Medicine, Alberta Health Services and the University of Calgary, Calgary, Alberta, and Department of Pathology & Laboratory Medicine, the University of British Columbia, Vancouver, British Columbia.
authors.forename: Deirdre L
authors.initials: DL
authors.lastname: Church
authors.affiliation: Medicine, Alberta Health Services and the University of Calgary, Calgary, Alberta, and Department of Pathology & Laboratory Medicine, the University of British Columbia, Vancouver, British Columbia.
authors.forename: Anshula
authors.initials: A
authors.lastname: Ambasta
authors.affiliation: Division of Medical Microbiology, the University of British Columbia, Vancouver, British Columbia.
authors.forename: Amanda
authors.initials: A
authors.lastname: Wilmer
authors.affiliation: Division of Microbiology, Calgary Laboratory Services, Departments of Pathology & Laboratory Medicine, the University of British Columbia, Vancouver, British Columbia.
authors.forename: Holly
authors.initials: H
authors.lastname: Williscroft
authors.affiliation: Division of Medical Microbiology, the University of British Columbia, Vancouver, British Columbia ; St Paul's Hospital and the University of British Columbia, Vancouver, British Columbia.
authors.forename: Gordon
authors.initials: G
authors.lastname: Ritchie
authors.affiliation: Division of Microbiology, Calgary Laboratory Services, Departments of Pathology & Laboratory Medicine, the University of British Columbia, Vancouver, British Columbia ; Division of Medical Microbiology and the University of British Columbia, Vancouver, British Columbia ; Medicine, Alberta Health Services and the University of Calgary, Calgary, Alberta, and Department of Pathology & Laboratory Medicine, the University of British Columbia, Vancouver, British Columbia.
authors.forename: Dylan R
authors.initials: DR
authors.lastname: Pillai
authors.affiliation: Division of Medical Microbiology, the University of British Columbia, Vancouver, British Columbia ; St Paul's Hospital and the University of British Columbia, Vancouver, British Columbia.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Champagne
authors.affiliation: Division of Microbiology, Calgary Laboratory Services, Departments of Pathology & Laboratory Medicine, the University of British Columbia, Vancouver, British Columbia ; Division of Medical Microbiology and the University of British Columbia, Vancouver, British Columbia ; Medicine, Alberta Health Services and the University of Calgary, Calgary, Alberta, and Department of Pathology & Laboratory Medicine, the University of British Columbia, Vancouver, British Columbia.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Gregson
keywords: Diagnosis; Immunocompromised; P jirovecii; Pneumocystis pneumonia; Real-time PCR