Development of a novel PCR assay for the identification of the black yeast, Exophiala (Wangiella) dermatitidis from adult patients with cystic fibrosis (CF).

Cystic fibrosis (CF) patients may suffer increased morbidity and mortality through colonisation, allergy and invasive infection from fungi. The black yeast, Exophiala dermatitidis (synonym Wangiella dermatitidis) has been found with increasing frequency in sputum specimens of CF patients, with reported isolation rates ranging from 1.1 to 15.7%. At present, no diagnostic PCR exists to aid with the clinical laboratory detection and identification of this organism. A novel species-specific PCR-based assay was developed for the detection of E. dermatitidis, based on employment of rDNA operons and interspacer (ITS) regions between these rDNA operons. Two novel primers, (designated ExdF & ExdR) were designed in silico with the aid of computer-aided alignment software and with the alignment of multiple species of Exophiala, as well as with other commonly described yeasts and filamentous fungi within CF sputum, including Candida, Aspergillus and Scedosporium. An amplicon of approximately 455 bp was generated, spanning the partial ITS1 region - the complete 5.8S rDNA region - partial ITS2 region, employing ExdF (forward primer [16-mer], 5'-CCG CCT ATT CAG GTC C-3' and ExdR (reverse primer [16-mer], 5'-TCT CTC CCA CTC CCG C-3', was employed and optimised on extracted genomic DNA from a well characterised culture of E. dermatitidis, as well as with high quality genomic DNA template from a further 16 unrelated fungi, including Candida albicans, C. dubliniensis, C. parapsilosis, C. glabrata, Scedosporium apiospermum, Penicillium sp., Aspergillus fumigatus, Aspergillus versicolor, Pichia guilliermondii, Rhodotorula sp., Trichosporon sp., Aureobasidium pullulans, Fusarium sp., Mucor hiemalis, Bionectria ochroleuca, Gibberella pulicaris. Results demonstrated that only DNA from E. dermatitidis gave an amplification product of the expected size, whilst none of the other fungi were amplifiable. Subsequent employment of this primer pair detected this yeast from mycological cultures from 2/50 (4%) adult CF patients. These two patients were the only patients who were previously shown to have a cultural history of E. dermatitidis from their sputum. E. dermatitidis is a slow-growing fungus, which usually takes up to two weeks to culture in the microbiology laboratory and therefore is slow to detect conventionally, with the risk of bacterial overgrowth from common co-habiting pan- and multiresistant bacterial pathogens from sputum, namely Pseudomonas aeruginosa and Burkholderia cepacia complex organisms, hence this species-specific PCR assay may help detect this organism from CF sputum more specifically and rapidly. Overall, employment of this novel assay may help in the understanding of the occurrence, aetiology and epidemiology of E. dermatitidis, as an emerging fungal agent in patients with CF.