Development of a DNA microarray for detection and identification of fungal pathogens involved in invasive mycoses

Abstract

Invasive fungal infections have emerged as a major cause of morbidity and mortality in immunocompromised patients. Conventional identification of pathogenic fungi in clinical microbiology laboratories is time-consuming and, therefore, often imperfect for the early initiation of an adequate antifungal therapy. We developed a diagnostic microarray for the rapid and simultaneous identification of the 12 most common pathogenic Candida and Aspergillus species. Oligonucleotide probes were designed by exploiting the sequence variations of the internal transcribed spacer (ITS) regions of the rRNA gene cassette to identify Candida albicans, Candida dubliniensis, Candida krusei, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida guilliermondii, Candida lusitaniae, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus. By using universal fungal primers (ITS 1 and ITS 4) directed toward conserved regions of the 18S and 28S rRNA genes, respectively, the fungal ITS target regions could be simultaneously amplified and fluorescently labeled. To establish the system, 12 pre-characterized fungal strains were analyzed; and the method was validated by using 21 clinical isolates as blinded samples. As the microarray was able to detect and clearly identify the fungal pathogens within 4 h after DNA extraction, this system offers an interesting potential for clinical microbiology laboratories.

FIG. 1.

(a) Fluorescence image after hybridization with 0.5 pmol of C. albicans isolate VA 115839-03 target DNA for 1 h at 53°C. The signal intensity is encoded in the 65,636 gray scales of the 16-bit TIF image. (b) Layout of the capture probes on the array. All species- and genus-specific probes and the process controls were spotted in triplicate. The other control probes are positioned at the corners of each subarray. Each subarray contains a set of process controls. pos. hyb., positive hybridization, neg. hyb., negative hybridization. (c) The enlarged results for the C. albicans probes describe the arrangement of different probes for the same species or genus within the indicated fields of the array.

FIG. 2.

Relative intensities after hybridization with labeled target DNA from the 12 target species. Cutoff 1, I = 300; cutoff 2, RI = 10. The mean RIs and their standard deviations are calculated for the triplicate spots on one slide (n = 3).

FIG. 3.

Behaviors of I and RI of the species-specific probe Afum1 sense, the genus-specific probe Asp1 antisense, the process control PC3 antisense (a and c), and the A. niger-specific probes Anig1 antisense and 2 antisense (b and d) after hybridization with 0.5 pmol labeled A. fumigatus DSM 819 target DNA for 2 h at different hybridization temperatures. The columns depict the mean net signal intensities or the mean relative signal intensities and their standard deviations over three slides of the same experiment (n = 9).

FIG. 4.

Relative intensities after hybridization with labeled target DNA from blinded samples of clinical isolates. Cutoffs 1 and 2 are as described in the legend to Fig. 2. The columns depict mean relative signal intensities and their standard deviations over three slides of the same experiment (n = 9).

FIG. 4.

Relative intensities after hybridization with labeled target DNA from blinded samples of clinical isolates. Cutoffs 1 and 2 are as described in the legend to Fig. 2. The columns depict mean relative signal intensities and their standard deviations over three slides of the same experiment (n = 9).