title: 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.
authors.affiliation: Department of Dermatology, Keio University School of Medicine, Tokyo, Japan.
authors.forename: Tomotaka
authors.initials: T
authors.lastname: Sato
authors.forename: Atsushi
authors.initials: A
authors.lastname: Takayanagi
authors.forename: Keisuke
authors.initials: K
authors.lastname: Nagao
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Tomatsu
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Fukui
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kawaguchi
authors.forename: Jun
authors.initials: J
authors.lastname: Kudoh
authors.forename: Masayuki
authors.initials: M
authors.lastname: Amagai
authors.forename: Nobuko
authors.initials: N
authors.lastname: Yamamoto
authors.forename: Nobuyoshi
authors.initials: N
authors.lastname: Shimizu
mesh_terms: D002175:Candida / Q000235:genetics / Q000302:isolation & purification*; D004271:DNA, Fungal / Q000032:analysis / Q000302:isolation & purification; D006801:Humans; D009262:Nails / Q000382:microbiology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D014009:Onychomycosis / Q000175:diagnosis / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011237:Predictive Value of Tests; D013045:Species Specificity; D014249:Trichophyton / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D004271:DNA, Fungal