Experience with the MicroSeq D2 Large-Subunit Ribosomal DNA Sequencing Kit for Identification of Commonly Encountered, Clinically Important Yeast Species

Abstract

Experience with a MicroSeq D2 large-subunit (LSU) ribosomal DNA (rDNA) sequencing kit for identification of yeast species commonly encountered in the mycology laboratory at Mayo Clinic is described here. A total of 131 isolates of yeasts recovered from clinical specimens were included in the study. Phenotypic methods used for initial identification included germ tube formation, urease production, microscopic morphological features on cornmeal agar, and an API 20C AUX system; all isolates were sequenced using a MicroSeq D2 LSU rDNA sequencing kit. Nucleic acid sequencing identified 93.9% of the isolates to the correct genus and species. A total of 100 of the isolates (representing 19 species of Candida) were sequenced, and 98% gave results concordant with identifications made by the API 20C AUX system; distance scores ranged from 0 to 1.88%, with an average value of 0.23%. Candida dubliniensis was not included in the MicroSeq database and was identified as Candida albicans. A total of 32 isolates representing 9 other genera (including Cryptococcus, Filobasidium, Kloeckera, Malassezia, Pichia, Sporidiobolus, Rhodotorula, Zygosaccharomyces, and Trichosporon) were included, and 81.3% showed concordant results when phenotypic and sequencing results were compared. Most discrepancies were attributed to the lack of inclusion of the species in the MicroSeq or API 20C AUX database. The MicroSeq D2 LSU rDNA sequencing kit appears to be accurate and useful for the identification of yeasts that might be seen in a clinical laboratory.

Improved Identification of Yeast Species Directly from Positive Blood Culture Media by Combining Sepsityper Specimen Processing and Microflex Analysis with the Matrix-Assisted Laser Desorption Ionization Biotyper System

“…The specimen with discrepant identification results (specimen 43-154) between the conventional methods and the MALDI Biotyper system was further analyzed by 28S rRNA gene sequencing. The nucleotide sequence of a partial 603-bp sequence of the 28S rRNA gene (GenBank accession number HQ214057) was identical to that of a clinical C. tropicalis isolate identified and reported previously (11).…”

“…A loopful of the purified yeast isolate was put into 1 ml of distilled water and heated at 95°C for 10 min, the suspension was centrifuged, and 1 l of supernatant was used for PCR amplification. PCR amplification was carried out as previously described (11,29). Nucleotide sequences were determined bidirectionally and were analyzed with BLAST (Basic Local Alignment Search Tool) on the NCBI website.…”

Lodderomyces elongisporus Masquerading as Candida parapsilosis as a Cause of Bloodstream Infections

“…Molecular identification of fungal isolates is becoming an important diagnostic tool (5,12,13). There have been a number of recent cases where two species are so similar phenotypically that only molecular techniques can distinguish them (1,4,11,19).…”

Performance and Cost Analysis of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Routine Identification of Yeast

“…All other yeasts were identified using the API 20C AUX yeast identification system (bioMérieux, Hazelwood, MO) and microscopic morphology on cornmeal agar with Tween 80 (17). Additionally, 116 strains (IMA subcultures at 30°C) from an archived collection of less common yeasts that were previously identified using API 20C and D2 sequencing were identified using MALDI-TOF MS (13).…”