Identification of candida species using MP65 gene and evaluation of the candida albicans MP65 gene expression in BALB/C mice

Abstract

Background: Systemic candidiasis is a major public health concern. In particular, in immune compromised people, such as patients with neutropenia, patients with Acquired Immune Deficiency Syndrome (AIDS) and cancer who are undergoing antiballistic chemotherapy or bone marrow transplants, and people with diabetes. Since the clinical signs and symptoms are non specific, early diagnosis is often difficult. The 65-kDa mannoprotein (MP65) gene of Candida albicans is appropriate for detection and identification of systemic candidiasis. This gene encodes a putative b-glucanase mannoprotein of 65 kDa, which plays a major role in the host-fungus relationship, morphogenesis and pathogenicity. Objectives: The current study aimed to identify different species of Candida (C. albicans, C. glabrata and C. parapsilosis) using the Polymerase Chain Reaction (PCR) technique and also to evaluate C. albicans MP65 gene expression in BALB/C mice. Materials and Methods: All yeast isolates were identified on cornmeal agar supplemented with tween-80, germ tube formation in serum, and assimilation of carbon sources in the API 20 C AUX yeast identification system. Polymerase Chain Reaction was performed on all samples using species-specific primers for the MP65 65 kDa gene. After RNA extraction, cDNA synthesis was performed by the Maxima RT Pre Mix kit. Candida albicans MP65 gene expression was evaluated by quantitative Real-Time (q Real-Time) and Real-Time (RT) PCR techniques. The 2-ΔΔCT method was used to analyze relative changes in gene expression of MP65. For statistical analysis, nonparametric Wilcoxon test was applied using the SPSS version 16 software. Results: Using biochemical methods, one hundred, six and one isolates of clinical samples were determined as C. albicans, C. glabrata and C. parapsilosis, respectively. Species-specific primers for PCR experiments were applied to clinical specimens, and in all cases a single expected band for C. albicans, C. glabrata and C. parapsilosis was obtained (475, 361 and 124 base pairs, respectively). All species isolated by culture methods (100% positivity) were evaluated with PCR using species-specific primers to identify Candida species. Relative expression of Mp65 genes increased significantly after C. albicans injection into the mice (P < 0.05). Conclusions: The results of the current study showed that the PCR method is reproducible for rapid identification of Candida species with specific primers. Mp65 gene expression of Calbicans after injection into the mice was 2.3 folds higher than before injection, with this difference being significant. These results indicated that increase of Mp65 gene expression might be an early stage of infection; however definitive conclusions require further studies.