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Journal of Clinical Microbiology, November 2000, p. 4186-4192, Vol. 38, No. 11
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Panfungal PCR and Multiplex Liquid Hybridization for Detection of Fungi in Tissue Specimens

Panu H. Hendolin,1,* Lars Paulin,1 Pirkko Koukila-Kähkölä,2 Veli-Jukka Anttila,3 Henrik Malmberg,4 Malcolm Richardson,2 and Jukka Ylikoski4

Institute of Biotechnology, University of Helsinki, 00014 University of Helsinki,1 and Mycology Unit, Laboratory Diagnostics,2 Division of Infectious Diseases,3 and Department of Otorhinolaryngology,4 Helsinki University Central Hospital, 00029 HUS, Helsinki, Finland

Received 29 February 2000/Returned for modification 29 April 2000/Accepted 31 August 2000

A procedure based on panfungal PCR and multiplex liquid hybridization was developed for the detection of fungi in tissue specimens. The PCR amplified the fungal internal transcribed spacer (ITS) region (ITS1-5.8S rRNA-ITS2). After capture with specific probes, eight common fungal pathogens (Aspergillus flavus, Aspergillus fumigatus, Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, Candida tropicalis, and Cryptococcus neoformans) were identified according to the size of the amplification product on an automated sequencer. The nonhybridized products were identified by sequencing. The performance of the procedure was examined with 12 deep-tissue specimens and 8 polypous tissue biopsies from the paranasal sinuses. A detection 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 hybridization positive. In comparison, 12 (60%) of the specimens were positive by direct microscopy, but only 7 (35%) of the specimens showed fungal growth. Sequencing of the nonhybridized amplification products identified an infecting agent in six specimens, and three specimens yielded only sequences of unknown fungal origin. The procedure provides a rapid (within 2 days) detection of common fungal pathogens in tissue specimens, and it is highly versatile for the identification of other fungal pathogens.

* Corresponding author. Mailing address: Institute of Biotechnology, University of Helsinki, P.O. Box 56 (Viikinkaari 9), 00014 University of Helsinki, Helsinki, Finland. Phone: 358-9-191 59 591. Fax: 358-9-191 58 952. E-mail: Panu.Hendolin{at}Helsinki.fi.

Journal of Clinical Microbiology, November 2000, p. 4186-4192, Vol. 38, No. 11
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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