title: Detection of Histoplasma capsulatum from clinical specimens by cycling probe-based real-time PCR and nested real-time PCR.
abstract: We developed new cycling probe-based real-time PCR and nested real-time PCR assays for the detection of Histoplasma capsulatum that were designed to detect the gene encoding N-acetylated α-linked acidic dipeptidase (NAALADase), which we previously identified as an H. capsulatum antigen reacting with sera from patients with histoplasmosis. Both assays specifically detected the DNAs of all H. capsulatum strains but not those of other fungi or human DNA. The limited of detection (LOD) of the real-time PCR assay was 10 DNA copies when using 10-fold serial dilutions of the standard plasmid DNA and 50 DNA copies when using human serum spiked with standard plasmid DNA. The nested real-time PCR improved the LOD to 5 DNA copies when using human serum spiked with standard plasmid DNA, which represents a 10-fold higher than that observed with the real-time PCR assay. To assess the ability of the two assays to diagnose histoplasmosis, we analyzed a small number of clinical specimens collected from five patients with histoplasmosis, such as sera (n = 4), formalin-fixed paraffin-embedded (FFPE) tissue (n = 4), and bronchoalveolar lavage fluid (BALF) (n = 1). Although clinical sensitivity of the real-time PCR assay was insufficiently sensitive (33%), the nested real-time PCR assay increased the clinical sensitivity (77%), suggesting it has a potential to be a useful method for detecting H. capsulatum DNA in clinical specimens.
authors.affiliation: Medical Mycology Research Center, Chiba University, Chiba, Japan y.muraosa@faculty.chiba-u.jp.
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Muraosa
authors.affiliation: Research Unit for Risk Analysis, Diagnostic Center for Animal Health and Food Safety, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan.
authors.forename: Takahito
authors.initials: T
authors.lastname: Toyotome
authors.affiliation: Medical Mycology Research Center, Chiba University, Chiba, Japan.
authors.forename: Maki
authors.initials: M
authors.lastname: Yahiro
authors.affiliation: Medical Mycology Research Center, Chiba University, Chiba, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Watanabe
authors.affiliation: Laboratory of Immunology, Hospital das Clínicas, Faculdade de Medicina, University of São Paulo and Department of Infectious and Parasitic Diseases, Faculdade de Medicina, University of São Paulo, São Paulo, Brazil.
authors.forename: Maria Aparecida
authors.initials: MA
authors.lastname: Shikanai-Yasuda
authors.affiliation: Medical Mycology Research Center, Chiba University, Chiba, Japan.
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Kamei
mesh_terms: D004271:DNA, Fungal / Q000032:analysis / Q000235:genetics; D006658:Histoplasma / Q000235:genetics*; D006660:Histoplasmosis / Q000382:microbiology*; D006801:Humans; D057230:Limit of Detection; D058889:Molecular Typing / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004271:DNA, Fungal
keywords: Cycling probe technology; Histoplasma capsulatum; Nested real-time PCR; Real-time PCR