مرکز منطقه ای اطلاع رساني علوم و فناوري - 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

Abstract :

Objectives: The aim of this study is to investigate PCR technique and antifungal resistance genes with RFLP and sequencing analysis in Candida species isolated from clinical specimens of immune-compromised patients. Materials and methods: Clinical samples (96 bronchoalveolar lavages, 56 biopsy-abscess, 8 blood specimens, 15 peritoneal fluid specimens, 15 pleural fluid, 5 cerebrospinal fluid and 5 pericard fluid specimens) from 200 immunosuppressed patients were studied by conventional and molecular methods. Antifungal susceptibility testing was performed by the E-test method. Firstly, fungal DNA was isolated from specimens, and then the resultant products are defined with multiplex PCR. Antifungal re-sistance and resistance genes were established by E-test and 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 of samples were found culture negative (85%). PCR with the universal primers detected fungal DNA in all 30 culture positive samples. One strain was determined as resistant; 2 strains were dose dependent susceptible and 27 strains were sensitive to fluconazole by E-test. The resistance gene (ERG11) was detected by BamHI and SalI enzymes revealed fluconazole resistance in one of C.albicans strains. The identification was successful in Candida dub-liniensis (950 bp) and Candida krusei (360 bp) with multiplex PCR. D132E and E216D mutations were detected in sequencing of ERG 11 gene of this isolate and compared with reference gene in GenBank by clustal analysis. Conclusion: The molecular test methods supplies correct therapy rather early in immunosuppressive patients therefore it is important for the survival.