Accurate identification of yeasts isolated from patient specimens is necessary for specific diagnoses and selection of appropriate antifungal therapy. The use of automated biochemical instrumentation such as bioMérieux Vitek Yeast Biochemical Card (YBC) along with growth morphology on cornmeal agar with Tween 80 (Dalmau plate) can provide rapid identification of commonly isolated yeasts. However, some species are not distinguished with confidence from each other and unusual or rare species are not identified or are misidentified as other species. We developed a yeast identification protocol that incorporates the use of sequence analyses for the identification of yeast species when instrumentation fails or provides an identification that is inconsistent with cornmeal agar morphology. Germ tube-negative yeast are tested with YBC and streaked onto cornmeal agar with Tween 80. Criteria for acceptance of results are YBC with a •90% probability limit and agreement with cornmeal agar growth morphology. Yeast that fail such criteria are identified by sequence analyses. Sequence polymorphisms in the internal transcribed spacer regions and 2 (ITS 1 and 2) of the rRNA operon provide accurate identification of yeast. We have shown that ITS sequences provide phylogenetically valid information, therefore, comparing the ITS sequences from an unknown yeast to a database of validated ITS sequences provides taxonomic information about the unknown. Conspecific yeasts display >99% identity at the ITS loci. Our ITS database consists of DNA sequences from 542 yeast isolates of 68 species verified by morphological analyses. ITS sequence analyses identified 40 species of yeast and yeast-like isolates that were not identified, or misidentified by YBC. We conclude that DNA sequence analyses provide accurate and complete identification of yeast and its incorporation into routine clinical testing provides a valuable tool in patient diagnosis and therapy.
Full conference title:
The 15 th Congress of the International Society for Human and Animal Mycology
- ISHAM 15th (2003)