title: Quantitative TaqMan PCR for detection of Pneumocystis jiroveci.
abstract: We developed a quantitative real-time PCR assay for detection and quantification of Pneumocystis jiroveci in bronchoalveolar lavage (BAL) specimens based on primers and probe targeting the gene encoding beta-tubulin. The assay was able to detect 50 DNA copies per ml of a standard plasmid containing the target sequence. The intra- and interassay coefficients of variation were 0.46%-4.27% and 0.05-2.00% over 5 log(10) values. Fifty-seven controls of human, viruses, bacteria and fungi DNA samples were amplified and found negative. Fifty-three BAL samples sent to the laboratory for diagnosis of pneumocystosis were prospectively investigated by real-time PCR and direct microscopic examinations (DME) using Giemsa stain and direct immunofluorescence. All PCR negative samples were negative by microscopy. Among the 24 (45%) BAL found PCR positive, 8 were positive by microscopy (35%). The copy numbers of the target gene were between 4.4 x 10(3) and 2.8 x 10(6) per ml for the microscopically positive samples and between 8 and 9.2 x 10(3) per ml for the microscopically negative samples. In conclusion, we developed a rapid, sensitive and specific real time PCR for the diagnosis and quantification of Pneumocystis jiroveci in BAL samples.
authors.affiliation: Laboratory of Microbiology, Erasme Hospital-ULB, 808 Route de Lennik, 1070 Brussels, Belgium. frbranca@ulb.ac.be
authors.forename: Françoise
authors.initials: F
authors.lastname: Brancart
authors.forename: Hector
authors.initials: H
authors.lastname: Rodriguez-Villalobos
authors.forename: Pierre-Alain
authors.initials: PA
authors.lastname: Fonteyne
authors.forename: Daliana
authors.initials: D
authors.lastname: Peres-Bota
authors.forename: Corinne
authors.initials: C
authors.lastname: Liesnard
mesh_terms: D001483:Base Sequence; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology*; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D009172:Mycology / Q000379:methods* / Q000706:statistics & numerical data; D045363:Pneumocystis carinii / Q000235:genetics* / Q000302:isolation & purification*; D011020:Pneumonia, Pneumocystis / Q000175:diagnosis* / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D014404:Tubulin / Q000235:genetics
chemical_list: D004271:DNA, Fungal; D014404:Tubulin