Identification of medically important yeasts using PCR-based detection of DNA sequence polymorphisms in the internal transcribed spacer 2 region of the rRNA genes

Identification of medically relevant yeasts can be time-consuming and inacc urate with current methods. We evaluated PCR-based detection of sequence po lymorphisms in the internal transcribed spacer 2 (ITS2) region of the rRNA genes as a means of fungal identification. Clinical isolates (401), referen ce strains (6), and type strains (27), representing 34 species of yeasts we re examined. The length of PCR-amplified ITS2: region DNA was determined wi th single-base precision in less than 30 min by using automated capillary e lectrophoresis, Unique, species-specific PCR products ranging from 237 to 4 29 bp were obtained from 92% of the clinical isolates. The remaining 8%, di vided into groups with ITS2 regions which differed by less than or equal to 2 bp in mean Length, all contained species-specific DNA sequences easily d istinguishable by restriction enzyme analysis, These data, and the specific ity of length polymorphisms for identifying yeasts, were confirmed by DNA s equence analysis of the ITS2 region from 93 isolates, Phenotypic and ITS2-b ased identification was concordant for 427 of 434 yeast isolates examined u sing sequence identity of greater than or equal to 99%. Seven clinical isol ates contained ITS2 sequences that did not agree with their phenotypic iden tification, and ITS2-based phytogenetic analyses indicate the possibility o f 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 fur 34 species of yeas ts. We conclude that size and restriction analysis of PCR-amplified ITS2 re gion DNA is a rapid and reliable method to identify clinically significant yeasts, including potentially new or emerging pathogenic species.