Infections caused by yeasts have increased in previous decades due primarily to the increasing population of immunocompromised patients. In addition, infections caused by less common species such as Pichia, Rhodotorula, Trichosporon, and Saccharomyces spp. have been widely reported. This study extensively evaluated the feasibility of sequence analysis of the rRNA gene internal transcribed spacer (ITS) regions for the identification of yeasts of clinical relevance. Both the ITS1 and ITS2 regions of 373 strains (86 species), including 299 reference strains and 74 clinical isolates, were amplified by PCR and sequenced. The sequences were compared to reference data available at the GenBank database by using BLAST (basic local alignment search tool) to determine if species identification was possible by ITS sequencing. Since the GenBank database currently lacks ITS sequence entries for some yeasts, the ITS sequences of type (or reference) strains of 15 species were submitted to GenBank to facilitate identification of these species. Strains producing discrepant identifications between the conventional methods and ITS sequence analysis were further analyzed by sequencing of the D1-D2 domain of the large-subunit rRNA gene for species clarification. The rates of correct identification by ITS1 and ITS2 sequence analysis were 96.8% (361/373) and 99.7% (372/373), respectively. Of the 373 strains tested, only 1 strain (Rhodotorula glutinis BCRC 20576) could not be identified by ITS2 sequence analysis. In conclusion, identification of medically important yeasts by ITS sequencing, especially using the ITS2 region, is reliable and can be used as an accurate alternative to conventional identification methods.

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Yeasts are emerging as important etiological agents of nosocomial bloodstream infections. A multiplex PCR method was developed to rapidly identify clinically important yeasts that cause fungemia. The method amplified the internal transcribed spacer 1 (ITS1) region between the 18S and 5.8S rRNA genes and a specific DNA fragment within the ITS2 region of Candida albicans. With this method, C. albicans produced two amplicons, whereas other species produced only one. Through sequence analysis, the precise lengths of the PCR products were found to be as follows: C. glabrata (482 or 483 bp), C. guilliermondii (248 bp), C. parapsilosis (229 bp), C. albicans (218 or 219 and 110 bp), C. tropicalis (218 bp), Cryptococcus neoformans (201 bp), and C. krusei (182 bp). The PCR products could be effectively separated by disk polyacrylamide gel electrophoresis. The method was used to test 249 positive blood cultures (255 isolates), from which the following species (strain number) were isolated: C. albicans (128), C. tropicalis (51), C. glabrata (28), C. parapsilosis (23), C. neoformans (9), C. krusei (5), C. guilliermondii (3), and other, minor species (8). The test sensitivity of the method was 96.9% (247 of 255 isolates). The eight minor species were either misidentified (one strain) or not identified (seven strains). From the time at which a positive bottle was found, the multiplex PCR could be completed within 8 h; the present method is simpler than any previously reported molecular method for the identification of blood yeasts.

The incidence of yeast infections has increased in the recent decades, with Candida albicans still being the most common cause of infections. However, infections caused by less common yeasts have been widely reported in recent years. Based on the internal transcribed spacer 1 (ITS 1) and ITS 2 sequences of the rRNA genes, an oligonucleotide array was developed to identify 77 species of clinically relevant yeasts belonging to 16 genera. The ITS regions were amplified by PCR with a pair of fungus-specific primers, followed by hybridization of the digoxigenin-labeled PCR product to a panel of oligonucleotide probes immobilized on a nylon membrane for species identification. A collection of 452 yeast strains (419 target and 33 nontarget strains) was tested, and a sensitivity of 100% and a specificity of 97% were obtained by the array. The detection limit of the array was 10 pg of yeast genomic DNA per assay. In conclusion, yeast identification by the present method is highly reliable and can be used as an alternative to the conventional identification methods. The whole procedure can be finished within 24 h, starting from isolated colonies.