Genotypic heterogeneity and molecular basis of 5-flucytosine resistance among Candida dubliniensis isolates recovered from clinical specimens in Kuwait.

There is a paucity of information about genotypic heterogeneity among Candida dubliniensis isolates recovered from different geographic regions. This study explored genotypic heterogeneity among 103 C. dubliniensis strains obtained over a six-year period from clinical specimens in Kuwait. Genotype assignment was based on amplification with genotype-specific primers and sequencing of rDNA. Susceptibility to 5-flucytosine was determined by means of the Etest. DNA sequencing of cytosine deaminase was performed to determine the molecular basis of resistance to 5-flucytosine. DNA sequencing of rDNA identified seven different genotypes, i.e., 68 (66%) isolates were found to belong to genotype 1, 25 to genotype 4, six to genotype 5 and one each to genotypes 6-9. Strains of genotype 2 or genotype 3 were not detected. All isolates of genotype 4 but none of other genotypes were resistant to 5-flucytosine and the resistant strains all contained S29L mutation. Isolates of all other genotypes contained wild-type codon 29 in cytosine deaminase. A simple, PCR-RFLP-based method has been developed to facilitate rapid detection of S29L mutation in cytosine deaminase. A noteworthy observation of our study is the identification of five new genotypes of C. dubliniensis isolates, recovered from oral/respiratory specimens from patients of Middle Eastern origin. Furthermore, all 5-flucytosine resistant C. dubliniensis isolates in Kuwait belonged to genotype 4 only.