title: Development and clinical application of a panfungal PCR assay to detect and identify fungal DNA in tissue specimens.
abstract: Given the rise in the incidence of invasive fungal infections (IFIs) and the expanding spectrum of fungal pathogens, early and accurate identification of the causative pathogen is essential. We developed a panfungal PCR assay that targets the internal transcribed spacer 1 (ITS1) region of the ribosomal DNA gene cluster to detect fungal DNA in fresh and formalin-fixed, paraffin-embedded (PE) tissue specimens from patients with culture-proven (n=38) or solely histologically proven (n=24) IFIs. PCR products were sequenced and compared with sequences in the GenBank database to identify the causal pathogen. The molecular identification was correlated with results from histological examination and culture. The assay successfully detected and identified the fungal pathogen in 93.6% and 64.3% of culture-proven and solely histologically proven cases of IFI, respectively. A diverse range of fungal genera were identified, including species of Candida, Cryptococcus, Trichosporon, Aspergillus, Fusarium, Scedosporium, Exophiala, Exserohilum, Apophysomyces, Actinomucor, and Rhizopus. For five specimens, molecular analysis identified a pathogen closely related to that identified by culture. All PCR-negative specimens (n=10) were PE tissues in which fungal hyphae were visualized. The results support the use of the panfungal PCR assay in combination with conventional laboratory tests for accurate identification of fungi in tissue specimens.
authors.affiliation: Faculty of Medicine, Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, University of Sidney, Australia.
authors.forename: Anna
authors.initials: A
authors.lastname: Lau
authors.forename: Sharon
authors.initials: S
authors.lastname: Chen
authors.forename: Tania
authors.initials: T
authors.lastname: Sorrell
authors.forename: Dee
authors.initials: D
authors.lastname: Carter
authors.forename: Richard
authors.initials: R
authors.lastname: Malik
authors.forename: Patricia
authors.initials: P
authors.lastname: Martin
authors.forename: Catriona
authors.initials: C
authors.lastname: Halliday
mesh_terms: D004271:DNA, Fungal / Q000032:analysis*; D021903:DNA, Ribosomal Spacer / Q000032:analysis*; D005557:Formaldehyde / Q000494:pharmacology; D005658:Fungi / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016533:Mycological Typing Techniques; D009181:Mycoses / Q000175:diagnosis* / Q000382:microbiology; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D016707:Tissue Fixation
chemical_list: D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer; D005557:Formaldehyde