Phenotypic and Molecular Characterization of Candida dubliniensis Isolates from Clinical Specimens in Kuwait

Objective: This study was carried out to characterize Candida dubliniensis using phenotypic and molecular methods and to determine the occurrence of C. dubliniensis in clinical specimens in Kuwait. Materials and Methods: A total of 880 clinical specimens for isolation of fungi were processed according to standard procedures. Of these, 390 germ-tube-positive clinical isolates of Candida species were examined for rough colonies with hyphal fringes and chlamydospore production on simplified sunflower seed agar for their presumptive phenotypicidentification as C. dubliniensis. The identification of C. dubliniensis isolates was further confirmed by the Vitek 2 yeast identification system, semi-nested (sn) PCR amplification of high-copy rDNA and direct DNA sequencing of the internally transcribed spacer 2 (ITS2) region. Results: Of the 390 isolates of Candida species investigated, 12 were identified as C. dubliniensis, giving an overall occurrence of 3%. All the C. dubliniensis isolates formed rough colonies with hyphal fringes and abundant chlamydospores on sunflower seed agar, did not assimilate trehalose, lactate and α-methyl-D-glucoside, and were isolated from human immunodeficiency virus (HIV)-negative patients. Four C. dubliniensis isolates utilized D-xylose. The species-specific primer derived from the ITS2 sequence of C. dubliniensis and used together with the panfungal reverse primer in the reamplification step of the snPCR specifically amplified rDNA from reference and clinical C. dubliniensis isolates and not from C. albicans or other Candida species. The identity of two representative isolates was confirmed by DNA sequencing of the ITS2 region. Conclusions:The identity of 12 C. dubliniensis isolates was first established by phenotypic characteristics and then by snPCR using species-specific primers derived from ITS2 sequences. The recovery of C. dubliniensis from HIV-negative patients from Kuwait reinforces the existing view that this novel yeast species has a worldwide distribution and its occurrence is not restricted to any particular immunocompromised population.