Identification of Medically Important Yeast Species by Sequence Analysis of the Internal Transcribed Spacer Regions

Abstract

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.

Evaluation of Fluorescent Capillary Electrophoresis for Rapid Identification of Candida Fungal Infections

“…Another approach is based on PCR with panfungal-or genusspecific primers targeting conserved rRNA regions, followed mostly by sequencing (23) or also by other techniques like restriction analysis (restriction fragment length polymorphism [RFLP]) (24), high-resolution melting (HRM) analysis (25,26,27), microarray-based detection (28,29), pyrosequencing (30), or determination of amplicon size by using capillary electrophoresis (31,32,33). Capillary electrophoresis has been proven to be superior to classical electrophoresis for separating DNA due to its higher efficiency, higher speed, better sensitivity, and better suitability for automation (34).…”

Synergistic Activity of Chloroquine with Fluconazole against Fluconazole-Resistant Isolates of Candida Species

“…All of our isolates, which were stored at the Peking University Research Center for Medical Mycology, were collected from patients with candidemia. Isolates were identified by conventional methods such as the use of CHROMagar Candida (CHROMagar, Paris, France), the API 20C system (bioMérieux, Marcy l'Etoile, France), and molecular identification as described in a previous study (22).Broth microdilution (BMD) susceptibility was determined as described in the CLSI M27-A3 protocol (23). Inoculum concentrations were adjusted to 1 ϫ 10 6 to 5 ϫ 10 6 CFU/ml by using a 0.5 McFarland standard at the 530-nm wavelength, ensuring that the final concentration in the 96-well plates was 0.5 ϫ 10 3 to 2.5 ϫ 10 3 CFU/ml.…”

Development of Two Molecular Approaches for Differentiation of Clinically Relevant Yeast Species Closely Related to Candida guilliermondii and Candida famata

“…Because these genetically related species are difficult to identify by conventional methods, molecular techniques are more suitable for their differentiation (24,30). ITS sequence analysis has been widely used as the gold standard for yeast species identification and for phylogenetic analysis (24,25,38). In this study, D. nepalensis exhibits little divergence from D. hansenii based on ITS sequences, like the C. guilliermondii complex.…”

“…Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a promising technique that works well to distinguish yeast species, although a few closely related species (such as C. fermentati) may still be misidentified (34)(35)(36). Because the internal transcribed spacer (ITS) regions (ITS1, 5.8S, and ITS2) and the D1 and D2 regions of the large ribosomal subunit were confirmed as the most useful targets for species-level identification of yeasts (37)(38)(39), these regions were sequenced for these closely related yeast species, for further RFLP analysis. In addition, PCR analyses based on intron size differences or intron loss were used to easily differentiate closely related yeast species in previous studies (9,16,40,41).…”