Single-Step PCR Using (GACA) ₄ Primer: Utility for Rapid Identification of Dermatophyte Species and Strains

Abstract

Dermatophytes are fungi that belong to three genera: Epidermophyton, Microsporum, and Trichophyton. Identification of dermatophyte species is essential for appropriate diagnosis and treatment of dermatophytosis. Routine identification depends on macroscopic and microscopic morphology, which is time-consuming and does not identify dermatophyte strains. In this study, two PCR-based methods were compared for their abilities to identify 21 dermatophyte isolates obtained from Egyptian patients to the species and strain levels. The first method employed a two-step method: PCR amplification, using ITS1 and ITS4 as primers, followed by restriction enzyme digestion using the endonuclease MvaI. The second method employed a one-step approach employing the repetitive oligonucleotide (GACA) 4 as a primer. Dermatophyte strains were also identified using a conventional culture method. Our results showed that the conventional culture method identified four species: Microsporum canis, Trichophyton mentagrophytes, Trichophyton rubrum, and Trichophyton violaceum. Moreover, both PCR methods agreed with the diagnosis made using the conventional approach. Furthermore, ITS1/ITS4-based PCR provided no strain differentiation, while (GACA) 4 -based PCR identified different varieties among the T. mentagrophytes isolates. Taken together, our results suggest that (GACA) 4 -based PCR has utility as a simple and rapid method for identification of dermatophyte species as well as utility for differentiation of T. mentagrophytes variants.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 topo...

Correlation Between Phenotypic and Genotypic Identification of Trichophyton Violaceum

“…This primer set amplified the ITS I, 5.8S, and ITS II regions of the ribosomal DNA in all 28 phenotypically identified T. violaceum tested isolates, resulting in amplified products of approximately 690 bp which means successful amplification of the DNA. Similar results obtained by Shehata et al 25 . Concerning with DNA extraction for isolates of T. violaceum two methods are compared in this work: the classic method with phenol/chloroform and that with spin column.…”

“…Similar findings reported by Shehata et al 25 . In conclusion, although genotypic identification results of T. violaceum in comparison to phenotypic identification results were identical, we recommended the use of genotypic method for rapid and accurate result especially in isolates showing atypical morphological characters.…”

PCR-RFLP: an alternative to culture (gold standard) in diagnosis of dermatophytes in dogs and cats

“…MachouartDubach et al (2001) Outras variações da PCR vêm sendo utilizadas no diagnóstico da dermatofitose em humanos e animais com resultados significativos quanto à identificação de gêneros e espécies a partir de amostras cultivadas, o que indica a possibilidade da identificação desses agentes diretamente da amostra colhida do paciente sem a necessidade da realização de uma cultura, reduzindo em muito o tempo de espera pelo resultado e a perda do cultivo por contaminantes (Gräser et al, 1998, Liu et al, 2000, Faggi et al, 2001, Shehata et al, 2008.…”

“…Várias técnicas moleculares têm sido aplicadas para a identificação laboratorial de espécies de dermatófitos, sendo consideradas mais estáveis, rápidas e precisas do que aquelas que se baseiam nas características fenotípicas (Gutzmer et al, 2004, Shehata et al, 2008. Dentre elas está a PCR (Shehata et al, 2008).…”

Genetic relationships and isozyme profile of dermatophytes and Candida strains from Egypt and Libya

“…Conventional methods on the basis of phenotype variations and molecular methods on the basis of molecular differences were used for identification of dermatophytes species. Due to some limitations in traditional methods such as the high degree of phenoltypic similarity between these relative species identification and time-consumption, many isolates reveal unusual characteristics (Weiyzman et al, 1995;Graser et al, 2008;Shehata et al, 2008;De Baere et al, 2010). To overcome these problems, in recent years, molecular marker approaches relying on genetic makeup are regarded as useful in the exact and rapid recognition of dermatophytes Kanbe et al, 2003;Liu et al, 2000;Graser et al, 1998).…”