Internal Transcribed Spacer Sequencing versus Biochemical Profiling for Identification of Medically Important Yeasts

Abstract

In this study, we established an in-house database of yeast internal transcribed spacer (ITS) sequences. This database includes medically important as well as colonizing yeasts that frequently occur in the diagnostic laboratory. In a prospective study, we compared molecular identification with phenotypic identification by using the ID32C system (bioMérieux) for yeast strains that could not be identified by a combination of CHROMagar Candida and morphology on rice agar. In total, 113 yeast strains were included in the study. By sequence analysis, 98% of all strains were identified correctly to the species level. With the ID32C, 87% of all strains were identified correctly to the species or genus level, 7% of the isolates could not be identified, and 6% of the isolates were misidentified, most of them as Candida rugosa or Candida utilis. For a diagnostic algorithm, we suggest a three-step procedure which integrates morphological criteria, biochemical investigation, and sequence analysis of the ITS region.Yeast infections are increasing due to the growing number of immunocompromised and severely ill patients (5, 9). In addition, widespread use of antibiotics and invasive procedures facilitate infections with yeasts (18). Although Candida albicans is still the most frequently encountered yeast species, others have gained increasing importance in the last few years (1). Some species, such as Candida krusei (resistance to fluconazole) or Trichosporon sp. (reduced susceptibility to amphotericin B), may show inherent resistance to antimycotics (13). Rapid and accurate identification is thus essential for proper treatment. Various identification methods have been proposed in the past, including morphology, physiological properties, nucleic acid amplification, restriction fragment length polymorphism analysis, and sequencing (2).For molecular identification, we have chosen sequence analysis since this procedure is simple and can be fully automated. In addition, interpretation of nucleic acid sequences is straightforward and does not depend on too much expertise compared to morphological analyses. As target, we have chosen the internal transcribed spacer (ITS) region, which is located between the highly conserved genes coding for 18S and 28S rRNA. The ITS encompasses the two noncoding regions ITS1 and ITS2, which are separated by the highly conserved 5.8S rRNA gene (20). The ITS1 and ITS2 regions are more variable than the adjacent rRNA gene sequences and thus promise a better separation of closely related species. As the inspection of yeast ITS sequences which are available in the public database GenBank (NCBI) suggested that some entries are incorrect and because certain medically relevant species are not included, we decided to establish an in-house database. Since the number of known yeast species is enormous, we restricted our database to species occurring in the medical diagnostic laboratory.In this study, we compared sequence-based identification with conventional identification. Based on these results, we establish...

Systematic Internal Transcribed Spacer Sequence Analysis for Identification of Clinical Mold Isolates in Diagnostic Mycology: a 5-Year Study

“…Identification by phenotypic traits is often challenging, since this requires growth on selected media at various temperatures and expertise in morphological characterization. For identification of clinically relevant fungi, molecular techniques based on sequencing of specific target regions are valuable alternatives to traditional phenotypic identification procedures, since sequence-based identification is independent of growth conditions and the formation of specific morphological structures (4,6,8).…”

Characterization of Saccharomyces cerevisiae Isolated from Flower and Algal Beach Casts in Ishikawa, Japan, Using the One-step Small Moromi Model

“…Identification and characteristics of selected strains The selected 24 strains were identified by the carbohydrate utilization assay kit API-ID32c (bioMéreux, Marcy-l'Etoile, France; apiweb: https://apiweb.biomerieux.com) and ITS gene sequencing (primers: ITS1Fb 5῍-GTAACAAG-GTTTCCGT-3῍; ITS1R 5῍-CGTTCTTCATCGA-TG-3) 4,17) . ITS gene sequencing was carried out by BEX Co. (Tokyo, Japan).…”

White piedra: molecular identification of Trichosporon inkin in members of the same family

“…The identification of this microorganism using ribosomal genes represents consistent evolutionary markers, including alternating conserved and variable regions, which may be useful for species identification and phylogenetic studies 13,14 . Universal primers, ITS4 and ITS5, are sufficiently discriminatory for the identification of a considerable number of species of the genus Trichosporon 15 .…”