Identification of medically important yeasts by sequence analysis of the internal transcribed spacer and D1/D2 region of the large ribosomal subunit

Background: The prevalence of opportunistic yeast infections has increased in recent decades as the resultof an increasing immunocompromised patient population.Aims: To evaluate ribosomal RNA (rRNA) gene sequence to identify medically important yeast species, toinvestigate the performance of both the rRNA gene internal transcribed spacer (ITS) and D1/D2 region in identifying clinically relevant yeasts, and to compare these results with those of a standard phenotypicmethod.Methods: Both regions from 50 yeast strains, comprising 45 clinical isolates and 5 reference strains, wereamplified using PCR and then sequenced. The sequences were compared to reference data available fromthe GenBank database of the National Center for Biotechnology Information using the BLASTn tool.Results: Using ID32C, 88% (44/50) of all strains were identified accurately at the species level, although6% were misidentified; two Candida eremophila isolates were identified as Candida glabrata and Can-dida tropicalis, and one Saprochaete clavata isolate was identified as Saprochaete capitata. Two of the fourisolates identified by phenotypic methods as Trichosporon asahii were defined so by analyzing the ITSregion, but the remaining two were not distinguishable from closely related species. Based on the D1/D2region, these four isolates had 100% sequence identity with T. asahii, Trichosporon japonicum, and Tri-chosporon asteroides. The isolate identified as Trichosporon inkin using ID32C could not be distinguished from Trichosporon ovoides by analyzing the ITS and D1/D2 regions. Conclusions: Identifying medically important yeasts by sequencing the ITS and D1/D2 region is a rapidand reliable alternative to conventional identification methods. For a diagnostic algorithm, we suggest atwo-step procedure integrating conventional methods (e.g. microscopic morphology on corn meal agarwith Tween®80 and API ID32C®) and sequence analysis of the ITS and D1/D2 region.