Detection and identification of Candida species in experimentally infected tissue and human blood by rRNA-specific 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.

Improved DNA-FISH for cytometric detection ofCandidaspp

“…Therefore, we used a simple hypotonic ⁄ detergent (H ⁄ D) lysis step (0AE1% Triton X-100 in distilled water, as described in Materials and methods) to quickly remove intact red blood cells from the system prior to fixation, hybridization and analysis. Similar approaches taking advantage of the differential lability of red blood cells and yeasts have been reported previously (Jones 1990;Lischewski et al 1997;Kempf et al 2005). Figure 3, panel B, provides an overlay-based comparison of C. albicans populations recovered from blood using the H ⁄ D lysis approach and hybridized with two different probe treatments.…”

“…2). As noted in Results, a simple hypotonic ⁄ detergent lysis step similar to pre-analytical approaches used by other groups (Jones 1990;Lischewski et al 1997;Kempf et al 2005) was used to remove potentially interfering red blood cells from the sample prior to fixation and hybridization. Removal of red blood cells was an essential step, as their clumping interfered with yeast detection via flow cytometry (data not shown).…”

“…them critical in a clinical setting (Campanha et al, 2005). Fluorescence in situ hybridization (FISH) combined with epifluorescence microscopy or flow cytometry has been introduced as a rapid alternative to traditional cultural techniques (Lischewski et al 1997;Kempf et al 2000Kempf et al , 2005. However, previously reported FISH methods using DNA-based probes for detection of C. albicans from blood have been limited by relatively lengthy or dim hybridizations, and the specificities of some probes have not been thoroughly reported.…”

Mycotic keratitis: an overview of diagnosis and therapy

“…119 This technique has been reported for the identification of Candida spp. 131,132 and Aspergillus spp. 133,134 Although the entire procedure is rapid and easy to perform, the sensitivity of the assay is often lower than those of other molecular biological assays, especially those including nucleic acid amplification.…”

Rapid differentiation of Candida albicans from non-C. albicans directly in a variety of clinical specimens using fluorescent in situ hybridisation

“…4,7-9 However, only a few studies have investigated FISH assays for other clinical samples such as infected tissue and human blood. 11 This study focused on optimising the hybridisation parameters for different types of clinical samples, facilitating the detection and identification of Candida species in different samples. In this study, we showed that only minor adjustments in the fixation and hybridisation time are necessary to make the protocol applicable for different samples.…”