Molecular epidemiology and antifungal susceptibility of Candida strains isolated from oral cavity of HIV-positive patients at king Chulalongkorn memorial hospital

Abstract

To perform the strain variation study and minimum inhibitory concentration (MIC) of antifungal drugs among Candida spp. isolated from the oral cavity of human immunodeficiency virus (HIV)-positive patients with oral candidiasis, the yeast isolates from oral swab of 98 patients were recruited. Two single colonies from homogeneous phenotype on Sabouraud dextrose agar and all heterogeneous colonies were selected. All cultures were identified by morphological and biochemical examinations. The electrophoretic karyotyping were assessed by pulsed-field gel electrophoresis (PFGE). Two isolates of Candida albicans with identical karyotype from individual were further characterized by restriction fragment length polymorphism (RFLP) using Smai restriction enzyme with Southern hybridization using RPS 102 specific probe. The MIC of antifungal drugs: amphotericin B, fluconazole, itraconazole, ketoconazole and 5-flucytosine against the mixed population of species and strains in individual were determined by Etest. The result showed that totally 197 isolates from 98 HIV-infected patients was selected. Based on the phenotypic pattern, the cultures from 91 cases illustrated homogeneous morphology and 7 cases showed heterogeneous type. C. albicans (180 isolates from 90 cases) and C. glabrata (2 isolates from 1 case) were identified from individual in the homogeneous type. In contrast, the mixed population of C. albicans with 1-2 different species of Candida in oral cavity from individual were found in heterogeneous type (7 cases). Those species were C. glabrata (217 cases), C. tropicalis (217 cases), C. krusei (117 case), and C. rugosa (1/7 case). One out of the seven cases illustrated C. albicans in combination with C. glabrata and C. tropicalis. Due to electrophoretic karyotype study, 101 C. albicans karyotypes, 4 C. glabrata karyotypes, 3 C. tropicalis karyotypes and 1 karyotype of each C. krusei and C. rugosa were found. Fifty-eight patients (59%) and thirty­ three patients (34%) were harbored two different and two identical karyotypes, respectively, in individual. All the identical karyotypes were identified as C. albicans except one which was C. glabrata. These C. albicans isolates (64 isolates/32 cases) were verified for the strain identity by PFGE and Sma I RFLP with RPS hybridization. The result showed that the isolates from 30 cases demonstrated the identical of both karyotypic and RPS hybridization profiles whereas two cases was difference. All the mixed population isolates in both species level and chromosomal level were used to determine the MIC. It is of interesting that all the non-albicans demonstrated the less susceptible to at least one or more kinds of antifungal drugs. Most of C. albicans isolates were susceptible to all five antifungal agents, except four isolates were resistant to fluconazole and itraconazole. No difference in antifungal susceptibility profile between two isolates in each individual were exhibited. In conclusion, PFGE and RFLP with hybridization is the useful method to show genetic heterogeneity within oral isolates. The mixed population of both species level and chromosomal level were found. The trend of resistant strains of non-albicans species and no correlation between antifungal susceptibility profile and genotypic profile were revealed.