Rapid Identification of Dimorphic and Yeast-Like Fungal Pathogens Using Specific DNA Probes

Abstract

ABSTRACT Specific oligonucleotide probes were developed to identify medically important fungi that display yeast-like morphology in vivo. Universal fungal primers ITS1 and ITS4, directed to the conserved regions of ribosomal DNA, were used to amplify DNA from Histoplasma capsulatum , Blastomyces dermatitidis , Coccidioides immitis , Paracoccidioides brasiliensis , Penicillium marneffei , Sporothrix schenckii , Cryptococcus neoformans , five Candida species, and Pneumocystis carinii . Specific oligonucleotide probes to identify these fungi, as well as a probe to detect all dimorphic, systemic pathogens, were developed. PCR amplicons were detected colorimetrically in an enzyme immunoassay format. The dimorphic probe hybridized with DNA from H . capsulatum , B . dermatitidis , C . immitis , P . brasiliensis , and P . marneffei but not with DNA from nondimorphic fungi. Specific probes for H . capsulatum , B . dermatitidis , C . immitis , P . brasiliensis , P . marneffei , S . schenckii , C . neoformans , and P . carinii hybridized with homologous but not heterologous DNA. Minor cross-reactivity was observed for the B . dermititidis probe used against C . immitis DNA and for the H . capsulatum probe used against Candida albicans DNA. However, the C . immitis probe did not cross-react with B . dermititidis DNA, nor did the dimorphic probe hybridize with C . albicans DNA. Therefore, these fungi could be differentiated by a process of elimination. In conclusion, probes developed to yeast-like pathogens were found to be highly specific and should prove to be useful in differentiating these organisms in the clinical setting.