Identification of CandidaSpecies by Seminested PCR in Candidemia

In recent decades, the incidence of fungal infections has increased. Invasive Candida nfection is an increasing cause of morbidity and mortality in the immunocompromised patients, for example, neuropenic patient with hematological malignancies, recipients of allogeneic transplants and HIV-infected individuals. There is an increasing incidence of bloodstream infections caused by Candida species. In this study, we applied Seminested PCR for detection and identification of Candida species in blood specimens.
A total of 2516 blood culture samples in Baghiyetollah hospital were investigated for the presence of yeast cells duringthe year 2009. Fourteen blood samples which were found to be infected with Candidaspecies, as proved by culture, were analysed. All yeasts cells were identified by germ tube test, chlamydospor production on cornmeal agar and using CHROMagar Candida medium. DNA was extracted by DNG-plus kit from whole blood. The universal outer primers (CTSF & CTSR) amplified the Internal Transcribed Spacer2 (ITS2). The species-specific primers (CADET, CPDET, CTDET, CGDET and CDDET), complementary to unique sequences within the ITS2 of each test species, amplified speciesspecific DNA in the reamplification step of snPCR. PCR products were identified by electrophoresis. Among the 14 (0.5%) blood samples, 16 Candidaspecies were detected and identified by using snPCR. Using snPCR for Candidaspecies identification indicated that 6 isolates were C. albicans, 4 isolates of C. parapsilosis, 4 isolates of C. glabrata, and 2 isolates of C. tropicalis. Two of the patients had dual infection with C. tropicalisand either C. albicansor C. glabrata. C. dubliniensiswasn’t detected in blood samples. The Seminested PCR that was applied and evaluated in this study is a rapid, specific and sensitive method for the diagnosis of candidemia or hematogenous candidiasis caused by common pathogenic Candidaspecies.