Simple PCR-based DNA microarray system to identify human pathogenic fungi in skin

Abstract

Fungal diseases in immunocompromised hosts pose significant threats to their prognoses. An accurate diagnosis and identification of the fungal pathogens causing the infection are critical to determine the proper therapeutic interventions, but these are often not achieved, due to difficulties with isolation and morphological identification. In an effort to ultimately carry out the simultaneous detection of all human pathogenic microbes, we developed a simple system to identify 26 clinically important fungi by using a combination of PCR amplification and DNA microarray assay (designated PCR-DM), in which PCR-amplified DNA from the internal transcribed spacer region of the rRNA gene was hybridized to a DNA microarray fabricated with species-specific probes sets using the Bubble Jet technology. PCR-DM reliably identified all 26 reference strains; hence, we applied it to cases of onychomycosis, taking advantage of the accessibility of tissue from skin. PCR-DM detected fungal DNA and identified pathogens in 92% of 106 microscopy-confirmed onychomycosis specimens. In contrast, culture was successful for only 36 specimens (34%), 3 of which had results inconsistent with the results of PCR-DM, but sequence analysis of the isolates proved that the PCR-DM result was correct. Thus, PCR-DM provides a powerful method to identify pathogenic fungi with high sensitivity and speed directly from tissue specimens, and this concept could be applied to other fungal or nonfungal infectious human diseases in less accessible anatomical sites.

FIG. 1.

Identification of representative fungal strains by PCR-DM. Data were obtained by PCR-DM after hybridization with Cy3-labeled target DNA from C. albicans (JCM 1542), T. rubrum (ATCC 10218), and A. fumigatus (JCM 10253). (A) Actual scanned fluorescence image; (B) graphs representing fluorescence intensities as arbitrary units. (C and D) Layout of the microarray, on which nine blocks are set per glass slide, but for simplicity, only three blocks are shown. Each block is composed of a unique matrix of oligonucleotide probes designed to target 26 clinically important fungi. The target fungi and the corresponding probe sequences are listed in Table S1 in the supplemental material.

FIG. 2.

PCR-DM specifically identifies 26 targeted fungal species. Normalized signal intensities of all hybridization experiments are listed by probe position and fungal species. Signals were normalized by dividing each absolute value by the background values. Averages were then taken from duplicates or triplicates within a chip and then the average of chip replicates from the same experiments. Normalized signal intensities of 1-, 5-, 10-, 20-, 50-, and 100-fold are indicated in the different pattern images. The abbreviations for the probe names are listed in Fig. 1.