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Journal of Clinical Microbiology, June 2000, p. 2302-2310, Vol. 38, No. 6
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Identification of Medically Important Yeasts Using PCR-Based Detection of DNA Sequence Polymorphisms in the Internal Transcribed Spacer 2 Region of the rRNA Genes

Y. C. Chen,1 J. D. Eisner,1 M. M. Kattar,1 S. L. Rassoulian-Barrett,1 K. LaFe,1 S. L. Yarfitz,2 A. P. Limaye,1,3 and B. T. Cookson1,4,*

Departments of Laboratory Medicine,1 Infectious Diseases,3 and Microbiology,4 and Health Sciences Library and Department of Medical Education, Division of Bioinformatics,2 University of Washington, Seattle, Washington

Received 11 January 2000/Returned for modification 26 February 2000/Accepted 27 March 2000

Identification of medically relevant yeasts can be time-consuming and inaccurate with current methods. We evaluated PCR-based detection of sequence polymorphisms in the internal transcribed spacer 2 (ITS2) region of the rRNA genes as a means of fungal identification. Clinical isolates (401), reference strains (6), and type strains (27), representing 34 species of yeasts were examined. The length of PCR-amplified ITS2 region DNA was determined with single-base precision in less than 30 min by using automated capillary electrophoresis. Unique, species-specific PCR products ranging from 237 to 429 bp were obtained from 92% of the clinical isolates. The remaining 8%, divided into groups with ITS2 regions which differed by <= 2 bp in mean length, all contained species-specific DNA sequences easily distinguishable by restriction enzyme analysis. These data, and the specificity of length polymorphisms for identifying yeasts, were confirmed by DNA sequence analysis of the ITS2 region from 93 isolates. Phenotypic and ITS2-based identification was concordant for 427 of 434 yeast isolates examined using sequence identity of >= 99%. Seven clinical isolates contained ITS2 sequences that did not agree with their phenotypic identification, and ITS2-based phylogenetic analyses indicate the possibility of new or clinically unusual species in the Rhodotorula and Candida genera. This work establishes an initial database, validated with over 400 clinical isolates, of ITS2 length and sequence polymorphisms for 34 species of yeasts. We conclude that size and restriction analysis of PCR-amplified ITS2 region DNA is a rapid and reliable method to identify clinically significant yeasts, including potentially new or emerging pathogenic species.

* Corresponding author. Mailing address: Departments of Laboratory Medicine and Microbiology, University of Washington, 1959 NE Pacific St., NW 120, Box 357110, Seattle, WA 98195. Phone: (206) 598-6131. Fax: (206) 598-6189. E-mail: cookson{at}u.washington.edu.

Journal of Clinical Microbiology, June 2000, p. 2302-2310, Vol. 38, No. 6
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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