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

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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 fromHistoplasma capsulatum,Blastomyces dermatitidis,Coccidioides immitis,Paracoccidioides brasiliensis,Penicillium marneffei,Sporothrix schenckii,Cryptococcus neoformans, fiveCandidaspecies, andPneumocystis 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 fromH.capsulatum,B.dermatitidis,C.immitis,P.brasiliensis, andP.marneffeibut not with DNA from nondimorphic fungi. Specific probes forH.capsulatum,B.dermatitidis,C.immitis,P.brasiliensis,P.marneffei,S.schenckii,C.neoformans, andP.cariniihybridized with homologous but not heterologous DNA. Minor cross-reactivity was observed for theB.dermititidisprobe used againstC.immitisDNA and for theH.capsulatumprobe used againstCandida albicansDNA. However, theC.immitisprobe did not cross-react withB.dermititidisDNA, nor did the dimorphic probe hybridize withC.albicansDNA. 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.