Single-Step PCR Using (GACA)4 Primer: Utility for Rapid Identification of Dermatophyte Species and Strains
Authors: Atef S. Shehata, Pranab K. Mukherjee, Hassan N. Aboulatta, Atef I. El Akhras, Said H. Abbadi, and Mahmoud A. Ghannoum
Publication Year: 2008
Dermatophytes comprise a group of related fungi that belong to three genera, Epidermophyton, Microsporum, and Trichophyton, each of which includes several recognized species. These fungi are keratinophylic, as they infect the superficial keratinized tissues (skin, hair, and nails) of humans and animals (16), and can cause cutaneous mycoses which are called dermatophytoses, tinea, or ringworm infections. Health care costs associated with management of these mycoses are high (2, 14). Moreover, dermatophytoses are widespread and increasing in prevalence on a global scale and the recent increase in their incidence has been attributed to the increase of immunocompromised states, such as those associated with AIDS, diabetes mellitus, organ transplantation, and the use of corticosteroids and antineoplastic agents (3, 5, 11, 17).
The identification of dermatophyte species is essential for appropriate diagnosis and treatment of dermatophytosis. As the dermatophytes were reported to cause outbreaks of infection, especially in closed communities (15), their identification to the species as well as strain levels has a great epidemiological value in the investigations of such outbreaks with regard to identifying the sources of infections and establishing plans to manage and control them.
Routine laboratory procedures for the identification of dermatophytes rely on culturing of these fungi on appropriate growth media, followed by examination of the gross morphological characters of their colonies (e.g., rate of growth, colony topography, and pigmentation of the surface and reverse sides) as well as microscopic morphology (e.g., shape and size of macroconidia, microconidia, and hyphae). Further identification characteristics include nutritional requirements (such as vitamin and amino acid utilization), temperature tolerance, urease production, in vitro hair perforation, etc. (16). Although culture-based identification is specific and sensitive, it is time-consuming since some species need up to 2 to 3 weeks before diagnostic characteristics are fully developed in culture media. Additionally, many dermatophyte strains often develop atypical characteristics.
Many molecular approaches have been applied for identification of different dermatophyte species and strains. Such approaches are considered more stable and precise than those using phenotypic characteristics (7). One such approach employs PCR technology, which is simple, rapid, and able to generate species-specific DNA polymorphisms with many dermatophyte species on the basis of characteristic band patterns detected by agarose gel electrophoresis (4, 8).
Jackson et al. (8) used the internal transcribed spacer (ITS) region of ribosomal DNA as a target for PCR amplification using the ITS1 and ITS4 primers, followed by MvaI restriction enzyme digestion, for identification of 17 dermatophyte species. This method produced unique fragment patterns for most dermatophytic species studied but could not distinguish between closely related species, such as Trichophyton rubrum and Trichophyton soudanense or Trichophyton quinckeanum and Trichophyton schoenleinii. Faggi et al. (4) used a one-step PCR-based approach employing the simple repetitive oligonucleotide (GACA)4 as a single primer for identification of species of dermatophytes. Their data showed that this simple primer was able to amplify all the studied dermatophytes with production of species-specific PCR profiles. However, no head-to-head study comparing these two PCR-based approaches has been undertaken.
The present study aimed at comparing these two molecular PCR-based methods for identification of 21 dermatophyte strains isolated from patients in Egypt. The purpose of this study was to identify which of these methods is easier to perform and can differentiate between dermatophyte species as well as strains.
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