Development of a PCR-based line probe assay for identification of fungal pathogens

We report on a reverse-hybridization line probe assay (LiPA) which when com bined with PCR amplification detects and identifies clinically significant fungal pathogens including Candida, Aspergillus, and Cryptococcus species. DNA probes have been designed from the internal transcribed-spacer (ITS) re gions of Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida krusei, Candida dubliniensis, Cryptococcus neoformans, Aspergillus fumigatus, Aspergillus versicolor, Aspergillus nidulans and Asp ergillus flavus. The probes were incorporated into a LiPA for detection of biotinylated ITS PCR products, and the specificity of the probes was evalua ted. We established LiPA detection limits for ITS 1 and for full ITS amplic ons for genomic DNA from C. albicans, A. fumigatus, and C, neoformans. Furt her evaluation of the LiPA was carried out on clinical fungal isolates, One hundred twenty-seven isolates consisting of dimorphic yeasts and dermatoph ytic and filamentous fungi were tested by the LiPA, which correctly identif ied 77 dimorphic yeasts and 23 of the filamentous isolates; the remaining 2 7 isolates represented species of Fungi for which probes were not included in the LiPA. The fungal-PCR-LiPA technology was applied to blood samples in oculated with Candida cells which were pretreated by minibead beating to me chanically disrupt the cells, with the DNA extracted by either a previously described guanidium thiocyanate-silica method or the commercially availabl e QIAmp tissue kit, PCR amplification of the extracted DNA and subsequent D NA probe hybridization in the LiPA assay yielded detection limits of 2 to 1 0 cells/ml, An internal standard control was included in the PCR amplificat ion to monitor for PCR inhibition. This fungal PCR-LiPA assay is robust and sensitive and can easily be integrated into a clinical-testing laboratory with the potential for same-day diagnosis of fungal infection.