The Candida species are the most common fungal pathogens of systemic candidiasis. The diagnosis of invasive candidiasis remains a laboratory and clinical challenge. Thus, development of diagnostic assays to detect systemic candidiasis and to identify Candida virulence factors and associated pathogenesis through immunohistochemistry using specific monoclonals and polyclonals will be useful. Inbred Balb/c mice were immunized with C. albicans antigens, and blood was checked for the presence of reactive antibodies using ELISA. Fusion was performed using the harvested spleen cells and NS1 myeloma cells, and the clones were screened for the presence of antibody producing hybrid cells by dot-blot. Western blot analysis showed that the L2D10 monoclonal antibody was reactive against the antigens with molecular weight of 20 kDa. Experimental systemic candidiasis in mice was induced through intravenous injection of C. albicans and all the vital organs were collected for immunohistochemistry study. The monoclonal antibody reacted to surface epitopes on the yeast cells, germ tubes, and hyphae, and to immune complexes. It was used with the polyclonal antibody in a sandwich ELISA for the detection of circulating antigens in experimental candiadiasis in mice. Antibody levels were also determined using the ELISA method, and the antibody levels of C. albicans infected mice were increased compared with uninfected animals. The monoclonal antibody was used in immunoperoxidase and immunofluorescence techniques for the detection of fungal infection in tissue sections and was found to be more sensitive than conventional periodic acid Schiff or silver staining techniques. This monoclonal antibody may serve as potential primary capture antibodies for the development of a rapid diagnostic test for human systemic fungal infection.
The incidence of candidemia and invasive candidiasis have increased markedly due to the increasing number of immunocompromised patients. There are five major medically important species of Candida with their frequency of isolation in the diminishing order namely Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata and Candida krusei. In addition, there are numerous other species of Candida which differ in their genetic makeup, virulence properties, drug susceptibilities and sugar assimilation capabilities. In this report, an unusual Candida species was isolated from the blood of two leukaemic patients. Conventional culture and biochemical tests identified the Candida species as C. parapsilosis. Using fungal-specific oligonucleotide primers ITS1 and ITS4, we managed to amplify the ribosomal RNA gene and its internal transcribed spacer region from the genomic DNA of these isolates. The PCR products were then purified and subjected to automated DNA sequencing using BLAST and CLUSTAL sequence analysis identified these isolates to be Candida orthopsilosis. Candida orthopsilosis is a new species recently identified in 2005, being morphologically indistinguishable from C. parapsilosis and was previously classified as a subspecies of C. parapsilosis. This report highlights the importance of complementing traditional culture and biochemical-based identification methods with DNA-based molecular assays such as PCR as the latter is more superior in terms of its discriminatory power and speed.
A total of 102 Candida species were isolated from blood cultures from January 1997 to October 1999. Using assimilation of carbohydrate test, 52 (51.0%) of the Candida sp. were identified as C. parapsilosis, 25.5% (26) were C. tropicalis. C. albicans made up 11.8% (12), 6.9% (7) were C. rugosa, 3.8% (4) C. glabrata and 1% (1) C. guilliermondii. No C. dubliniensis was found in the study. In vitro antifungal susceptibility tests showed that all Candida species were sensitive to nystatin, amphotericin B and ketoconazole. Although all isolates remained sensitive to fluconazole, intermediate susceptibility was found in 3 C. rugosa isolates. Antifungal agents with high frequency of resistance were econazole, clotrimazole, miconazole and 5-fluorocytosine. Candida species found to have resistance to these antifungal agents were non-C. albicans.