Rapid Discrimination among Dermatophytes, Scytalidium spp., and Other Fungi with a PCR-Restriction Fragment Length Polymorphism Ribotyping Method

Abstract

Dermatomycoses are very common infections caused mainly by dermatophytes. Scytalidiosis is a differential mycological diagnosis, especially in tropical and subtropical areas. Since a culture-based diagnosis takes 2 to 3 weeks, we set up a PCR-restriction fragment length polymorphism (RFLP) method for rapid discrimination of these fungi in clinical samples. The hypervariable V4 domain of the small ribosomal subunit 18S gene was chosen as the target for PCR. The corresponding sequences from 19 fungal species (9 dermatophytes, 2 Scytalidium species, 6 other filamentous fungi, and 2 yeasts) were obtained from databases or were determined in the laboratory. Sequences were aligned to design primers for dermatophyte-specific PCR and to identify digestion sites for RFLP analysis. The reliability of PCR-RFLP for the diagnosis of dermatomycosis was assessed on fungal cultures and on specimens from patients with suspected dermatomycosis. Two sets of primers preferentially amplified fungal DNA from dermatophytes (DH1L and DH1R) or from Scytalidium spp. (DH2L and DH1R) relative to DNA from bacteria, yeasts, some other filamentous fungi, and humans. Digestion of PCR products with EaeI or BamHI discriminated between dermatophytes and Scytalidium species, as shown with cultures of 31 different fungal species. When clinical samples were tested by PCR-RFLP, blindly to mycological findings, the results of the two methods agreed for 74 of 75 samples. Dermatophytes and Scytalidium spp. can thus be readily discriminated by PCR-RFLP within 24 h. This method can be applied to clinical samples and is suited to rapid etiologic diagnosis and treatment selection for patients with dermatomycosis.Dermatophytes, which belong to the genera Trichophyton, Microsporum, and Epidermophyton, are extremely widespread fungi that infect human skin, hair, and nails. They are responsible for most superficial fungal infections, causing 94.7% of cases of tinea pedis and 81.9% of cases of onychomycosis in the United States (13). Scytalidium hyalinum and Scytalidium dimidiatum are molds responsible for skin lesions and onychomycoses, which mimic those due to Trichophyton rubrum. These infections are frequent in tropical and subtropical areas. For example, S. dimidiatum accounts for 39% of dermatomycoses in Thai soldiers, whereas dermatophytes account for only 5% (6). In Gabon, S. dimidiatum was responsible for 34.2% of such cases, either alone or jointly with a dermatophyte or Candida albicans (14).Laboratory diagnosis of dermatomycosis is based on the demonstration of hyphae by direct microscopic examination of clinical samples, followed by species identification by culture. Microscopic examination is rapid, but it can be difficult to differentiate hyphae from dermatophytes or molds. Culture requires at least 2 to 3 weeks to obtain typical macroscopic and microscopic features for specific dermatophyte identification.In rare cases, identification is hindered by the absence of specific macroscopic and microscopic characteristics; subculture on specific media ...

Identification of Infectious Agents in Onychomycoses by PCR-Terminal Restriction Fragment Length Polymorphism

“…In the present study, a similar approach was adopted, and TRFLP analysis was used to identify infectious fungi based on differences in their 28S rDNA amplicons. Other DNA sequences, such as that of the chitin synthase 1 gene or small ribosomal subunit 18S rRNA, were successfully used for fungal species delineation and identification (7,27,28). The polymorphism of the internal transcribed spacers (ITS) of ribosomal DNA regions (ITS1 and ITS2) flanking the DNA sequence composing the 5.8S rDNA is the most discriminating tool for distinguishing different fungi (1).…”

Biochemical and Molecular Analysis of Laccase Enzyme in Saprobic Fungus; Sordaria fimicola

“…The quality of extracted DNA was checked by 1% agarose gel electrophoresis and quantification was carried out by taking absorbance at 260 nm using UV spectrophotometer (UV 1800 SHIMADZU). S. fimicola strains found efficient in laccase enzyme production were subjected to ribotyping by amplification of 431 bases long hypervariable (V4) region of 18S rRNA gene as described earlier (Machouart-Dubach et al, 2001).…”

Differentiation of clinically relevant Mucorales Rhizopus microsporus and R. arrhizus by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)

“…posaconazole MIC range) (Chowdhary et al, 2014). Machouart-Dubach et al (2001) introduced RFLP as a rapid method for the separation of the main species of Mucorales, but this technique requires availability of a set of specific primers. No serological indicator for mucormycosis is available at this moment (Griffin & Hanson, 2014).…”