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Journal of Clinical Microbiology, November 2000, p. 4186-4192, Vol. 38, No. 11
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.
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
*
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.
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