Fluorescent in Situ Hybridization

Abstract

Two 18S rRNA-targeted oligonucleotide probes specific for Candida albicans and Candida parapsilosis were used to detect and identify by fluorescent in situ hybridization these medically important Candida species in deep organs of mice after experimental systemic infection. The C. albicans-specific probe detected fungal cells in kidney, spleen, and brain sections of a mouse infected with C. albicans but not in a mouse infected with the closely related species C. parapsilosis. Conversely, the C. parapsilosis-specific probe detected fungal cells in the deep organs of a mouse infected with C. parapsilosis but not in the deep organs of a C. albicans-infected mouse. In addition, the C. albicans-specific probe was used to detect this species in human blood spiked with yeast cells by a lysis-filtration assay and subsequent fluorescent in situ hybridization. By this assay, as few as three yeast cells per 0.5 ml of blood were consistently detected. Our results demonstrate that fluorescent in situ hybridization with species-specific rRNA-targeted oligonucleotide probes provides a novel, culture-independent method for the sensitive detection and identification of Candida species in clinically relevant material.The opportunistic fungal pathogen Candida albicans and other species of the genus Candida are able to cause superficial and often fatal disseminated infections in immunocompromised patients. A clear-cut diagnosis is often difficult to obtain, and deep-seated candidiasis is in most cases only recognized postmortem. Disseminated candidiasis is usually diagnosed by blood culture, although blood cultures from patients with histologically proven disseminated candidiasis are often negative. In addition, even with an optimal blood culture system such as the lysis-centrifugation method, it takes a minimum of 1 to 2 days for detection and even longer for species identification (8). To reduce mortality in patients suffering from invasive candidiasis, early initiation of proper antifungal drug therapy is critical (2, 4). Because Candida species differ in their susceptibilities to widely used systemic antifungal agents, such as azoles (20), identification of the infecting species is important for ensuring effective therapeutic strategies. Direct histologic detection of the fungus in biopsy material from deep tissue offers the relatively best diagnostic assurance (11). The infecting species, however, cannot be identified by microscopic examination alone. Among other nonculture methods, like the less reliable detection of antigens or specific antibodies (19), PCR-based approaches for the detection of Candida infections have also been developed. Identification of the Candida species, however, relies on the application of one of a number of techniques which in most cases require additional steps after PCR (6, 9, 14-16, 21, 24, 26). The diagnostic value of these methods, however, remains to be established (19).Fluorescent in situ hybridization of whole cells with rRNAtargeted oligonucleotide probes has increasingly become a valuable t...