The identification of Candida species isolated from clinical specimens of immunocompromised patients with PCR and determination of antifungal resistance genes with RFLP and sequencing analysis

Objectives: The aim of this study is to investigate PCRtechnique and antifungal resistance genes with RFLP andsequencing analysis in Candida species isolated fromclinical specimens of immune-compromised patients.Materials and methods: Clinical samples (96 bronchoalveolarlavages, 56 biopsy-abscess, 8 blood specimens,15 peritoneal fluid specimens, 15 pleural fluid, 5 cerebrospinalfluid and 5 pericard fluid specimens) from 200 immunosuppressedpatients were studied by conventionaland molecular methods. Antifungal susceptibility testingwas performed by the E-test method. Firstly, fungal DNAwas isolated from specimens, and then the resultantproducts are defined with multiplex PCR. Antifungal resistanceand resistance genes were established by E-testand RFLP analysis.Results: Thirty of 200 samples (15%) were culture positive[20 Candida albicans (67%), five Candida parapsilosis(17%), five Candida tropicalis (17%)], and 170 ofsamples were found culture negative (85%). PCR with theuniversal primers detected fungal DNA in all 30 culturepositive samples. One strain was determined as resistant;2 strains were dose dependent susceptible and 27 strainswere sensitive to fluconazole by E-test. The resistancegene (ERG11) was detected by BamHI and SalI enzymesrevealed fluconazole resistance in one of C.albicansstrains. The identification was successful in Candida dubliniensis(950 bp) and Candida krusei (360 bp) with multiplexPCR. D132E and E216D mutations were detected insequencing of ERG 11 gene of this isolate and comparedwith reference gene in GenBank by clustal analysis.Conclusion: The molecular test methods supplies correcttherapy rather early in immunosuppressive patientstherefore it is important for the survival.