Panfungal PCR and multiplex liquid hybridization for detection of fungi intissue specimens

A procedure based on panfungal PCR and multiplex liquid hybridization was d eveloped for the detection of fungi in tissue specimens. The PCR amplified the fungal internal transcribed spacer (ITS) region (1TS1-5.8S rRNA-ITS2). After capture with specific probes, eight common fungal pathogens (Aspergil lus flavus, Aspergillus fumigatus, Candida albicans, Candida krusei, Candid a glabrata, Candida parapsilosis, Candida tropicalis, and Cryptococcus neof ormans) were identified according to the size of the amplification product on an automated sequencer. The nonhybridized products were identified by se quencing. The performance of the procedure was examined with 12 deep-tissue specimens and 8 polypous tissue biopsies from the paranasal sinuses. A det ection level of 0.1 to 1 pg of purified DNA (2 to 20 CFU) was achieved. Of the 20 specimens, PCR was positive for 19 (95%), of which 10 (53%) were hyb ridization positive. In comparison, 12 (60%) of the specimens were positive by direct microscopy, but only 7 (35%) of the specimens showed fungal grow th. Sequencing of the nonhybridized amplification products identified an in fecting agent in six specimens, and three specimens yielded only sequences of unknown fungal origin. The procedure provides a rapid (within 2 days) de tection of common fungal pathogens in tissue specimens, and it is highly ve rsatile for the identification of other fungal pathogens.