title: Detection of Blastomyces dermatitidis and Histoplasma capsulatum from culture isolates and clinical specimens by use of real-time PCR.
abstract: Blastomyces dermatitidis and Histoplasma capsulatum are dimorphic fungi that often cause self-limited respiratory infections. However, they may also cause severe disseminated disease, depending on the level of the exposure to the organism and the host immune status. In addition, patients with infections caused by these fungi may have very similar clinical presentations. Although microbiologic culture is a standard method for detecting these pathogens, their recovery may require days to weeks, and the manipulation of cultures presents a significant safety hazard to laboratory personnel. Therefore, the goal of this study was to design a rapid, real-time PCR assay to detect and differentiate B. dermatitidis and H. capsulatum from culture isolates and directly from clinical specimens. Primers and fluorescence resonance energy transfer hybridization probes were designed to target the histidine kinase and glyceraldehyde-3-phosphate dehydrogenase genes of B. dermatitidis and H. capsulatum, respectively. The analytical sensitivity of the assay was determined to be 100 copies/μl for both fungi. From culture isolates, the assay demonstrated 100% specificity and 100% sensitivity for B. dermatitidis and 100% specificity and 94% sensitivity for H. capsulatum. Detection directly from 797 clinical specimens demonstrated specificities and sensitivities of 99% and 86% for B. dermatitidis and 100% and 73% for H. capsulatum compared with the results for culture. This real-time PCR assay provides a rapid method for the detection of B. dermatitidis and H. capsulatum from culture isolates and directly from clinical specimens.
authors.affiliation: Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 55905, USA.
authors.forename: N Esther
authors.initials: NE
authors.lastname: Babady
authors.forename: Seanne P
authors.initials: SP
authors.lastname: Buckwalter
authors.forename: Leslie
authors.initials: L
authors.lastname: Hall
authors.forename: Kara M
authors.initials: KM
authors.lastname: Le Febre
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Binnicker
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Wengenack
mesh_terms: D001758:Blastomyces / Q000302:isolation & purification*; D001759:Blastomycosis / Q000175:diagnosis* / Q000382:microbiology; D017931:DNA Primers; D031541:Fluorescence Resonance Energy Transfer; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D000071677:Histidine Kinase; D006658:Histoplasma / Q000302:isolation & purification*; D006660:Histoplasmosis / Q000175:diagnosis* / Q000382:microbiology; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D015345:Oligonucleotide Probes; D011494:Protein Kinases / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D015345:Oligonucleotide Probes; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D011494:Protein Kinases; D000071677:Histidine Kinase