Fast, simple and highly sensitive double‐rounded polymerase chain reaction assay to detect medically relevant fungi in dermatological specimens

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Abstract
Early detection of fungal infection is essential for beginning of prompt and specific therapy. In this study we describe a rapid and sensitive procedure to detect, by polymerase chain reaction (PCR) assays, a wide range of medically important opportunistic and pathogenic fungi in dermal specimens from dermatomycoses-affected patients. Three primer pairs, amplifying fragments of the highly conserved gene coding for small ribosomal RNA (18S rDNA) and the adjacent internal transcribed spacer (ITS) rDNA, previously published by others, were probed on DNA from pure cultures of medically relevant human and animal fungal species. In order to evaluate the specificity of the assay, amplifications of control DNAs from other eukaryotes and prokaryotes were also carried out, and they gave negative results. These primer sets, in single amplification or double-rounded PCR assays, allowed specific amplification when applied to a wide number of fungal DNA from human and animal tissue specimens, including dermatophytes (genera Trichophyton, Microsporum), several yeast species (Candida, Saccharomyces, Cryptococcus, Malassezia) and moulds (Aspergillus, Penicillium). The PCR assay was able to detect as little as 10 pg of fungal DNA, corresponding to approximately 25 fungal genomes per sample specimen. A small-scale DNA extraction method was also developed. This simple, time-saving and sensitive procedure was successfully applied to 40 human and veterinary specimens, and the diagnosis was confirmed with cultural techniques, being shown to work even in the presence of other lesions or contaminating organisms. This method allows early recognition of fungal pathogen cells in clinical samples as an alternative tool to conventional detection techniques.