Combination of Phenotypic Tests as a Screening Approach for the Differentiation of Cryptic Species Candida albicans and Candida dubliniensis

Abstract

Background: Candida albicans, an important pathogen for humans and animals, shares phenotypic features with Candida dubliniensis, leading to misidentification. Thus, the goal of this study was to apply a combination of phenotypic tests for the differentiation of these cryptic species. Methods and findings: Thirty-seven azole-resistant C. albicans from animals and 03 C. dubliniensis from humans were included in this study. Purity of strains was evaluated on CHROMagar Candida™, on which both species present green colonies. Then, phenotypic characterization was performed based on the growth pattern on sunflower seed agar, where C. albicans presents smooth colonies and C. dubliniensis presents rough colonies, and esterase production on Tween 80™ agar, which is positive for C. albicans, forming an opacification zone, and negative for C. dubliniensis. Molecular differentiation was performed with primers CALF/CALR for the amplification of ITS1/ITS2 regions of rDNA, yielding an amplicon of 100 bp for C. albicans, but not for C. dubliniensis. Of the 37 C. albicans, 35 showed green colonies on CHROMagar Candida Medium™, 36 presented smooth colonies on sunflower seed agar, while 34 showed an opacification zone on Tween 80™ agar. PCR yielded 100-bp-amplicons for all 37 C. albicans strains, confirming their identification. Control strains of C. dubliniensis showed the expected phenotypic features and amplicons were not obtained for the specific PCR reaction. Conclusions: The phenotypic methods used were not absolutely effective, however, the combined observation of smooth colonies on sunflower seed agar with an opacification zone on Tween 80™ agar leads to a reliable presumptive phenotypic identification of C. albicans.