Identification of dermatophytes by arbitrarily primed PCR

The study was approved by the Ethics Committee of Ahvaz Jundishapur University of Medical Sciences.

A total of 52 isolates belonging to any of 3 genera, Microsporum, Trichophyton, and Epidermophyton containing 10 species were analyzed with AP-PCR.

The genomic DNA of 10 dermatophytes was amplified with random primers OPAA11, OPU15, and OPD18 separately. However, primer OPAA17 was used to amplify the genomic DNA of 9 dermatophytes (Table 1, Figures 1–4). No band was generated from the negative control without a DNA template. For consistency, the experiences were repeated and the same results were obtained.

Using primer OPAA11, the PCR exclusively amplified different products for dermatophytes between 300 bp to 1800 bp. The results indicated that all 10 dermatophyte species displayed a distinct DNA band pattern after amplification with random primer OPAA11 (Figure 1). This primer amplified the genomic DNA of E. floccosum and M. canis with a similar band pattern 350, 450, 650, and 750 bp. Therefore, E. floccosum and M. canis must be recognized with another random primer, for example, OPU15. Primer OPAA11 generated only 2 bands, 350 and 750 bp from T. mentagrophytes. AP-PCR with primer OPU15 amplified various products for dermatophytes between 250 bp to 1800 bp. All 10 species of dermatophytes could be identified with random primer OPU15 by their distinctive DNA band pattern (Figure 2).The fewest distinctive bands belonged to T. verrucosum, with 3 bands 250, 350, and 800 bp for primer OPU15.

Figure 1

Figure 2

As shown in Figure 1, the PCR with the primer OPAA17 generated different products, from 300 bp to 1400 bp, for 9 species of dermatophytes. No PCR product was obtained for T. schoenleinii with the primer OPAA17 (Figure 3). Nine species of dermatophytes could be distinguished with the random primer OPAA17 by different DNA band pattern. The fewest distinctive bands belonged to M. ferrugineum, with 3 bands 300, 500, and 700 bp, and T. verrucosum, with 300, 500, and 850 bp for primer OPAA17.

Figure 3

With the use of primer OPD18, AP-PCR amplified different PCR products with distinct DNA band patterns between 250 bp to 1600 bp for 10 dermatophyte species (Figure 4). Similar band patterns were obtained for M. gypseum and T. verrucosum with the OPD18 primer. Therefore, these 2 dermatophytes must be identified with another random primer, for example OPAA17. The fewest distinctive bands belonged to T. mentagrophytes with 4 bands 350, 400, 750, and 1000 bp for primer OPD18. Amplification success rate for primers OPAA11, OPU15, and OPD18 was 100%, and for primer OPAA17 was 90%.

Figure 4

Identification of dermatophytes is not straightforward because of the existence of various species. They grow slowly and the morphology of the isolates is different. The conventional diagnosis of dermatophytosis is founded on microscopic detection of fungal hyphae directly from lesion followed by culture. Although this technique is rapid and economical, but it is not species specific, and is relatively insensitive, presenting false-negative results of up to 15% [2]. Molecular methods are more helpful for the diagnosis of dermatophytes because these procedures are more sensitive and rapid. Furthermore, these methods rely on genetic structures, which are more constant than phenotypic features, and they are able to recognize atypical dermatophytes that cannot be distinguished with morphological approaches [14-16]. The molecular approaches can recognize the dermatophyte species or the strain levels [8, 17]. The use of molecular techniques has facilitated great progress in identification and phylogenetic analysis of dermatophytes [18, 19].

In this study, the AP-PCR with four random primers OPAA11, OPU15, OPAA17, and OPD18 was used for identification of ten species of dermatophytes. After examining 52 isolates from 10 species of dermatophytes, it was revealed that these primers are able to identify DNA from tested dermatophytes, generating bands between 250 bp to 1800 bp in PCR. However, OPAA17 could not identify the DNA of T. schoenleinii.

Three random primers, OPAA11, OPAA17, and OPU15 are not able to differentiate among three dermatophytes T. rubrum, T. soudanense, and T. gourvilii with AP-PCR, but random primer OPD18 generates slight differences among these species of dermatophytes [6, 20].

Neji et al. identified 3 dermatophytes T. mentagrophytes, T. rubrum, and M. ferrugineum with a 450 bp fragment obtained from CHS1 gene. Their method requires sequencing and only can identify 3 dermatophytes compared with 10 dermatophytes in our AP-PCR [21].

DNA based techniques rely on the recognition of genotypic variations in pathogenic microorganisms [22]. The genotypic features are less possibly affected by external influences, for example temperature differences and chemotherapy. The PCR methods such as arbitrarily primed PCR (AP-PCR) or random amplified polymorphic DNA (RAPD) [6, 23-25] have improved the sensitivity of the diagnostic procedures. A number of laboratories have successfully applied the PCR method to the diagnosis and phylogeny of fungal pathogen in the small (18S) [26] and large (25S) [27] ribosomal DNA regions, and the gene for chitin synthase 1 [28]. These approaches resulted in a more precise assessment of phylogenic associations among dermatophytes and other fungi [29], and improved the identification of several frequent dermatophytes [30]. However, because of the techniques commonly required to the additional treatments such as restriction enzyme digestion, sequencing or hybridization after amplification, they are not simply accepted for regular use in clinical laboratories. Furthermore, usually these methods are able to distinguishing only a few dermatophyte species and, therefore, they are more suitable for the genus-specific rather than species-specific recognition.

Our results showed that AP-PCR analysis of the internal transcribed spacer (ITS) region is a rapid, reliable and reproducible method, which allows the identification of major dermatophytes at a species level.

There are several reports from prevalence of dermatophytosis in Iran. An epidemiological study of dermatophytosis in Tehran, Iran, showed that the most frequent dermatophytes isolated were E. floccosum (32%), T. rubrum (26%), and T. mentagrophytes (19.9%). Identification of dermatophyte isolates was on the basis of morphological characteristics and conventional methods [31]. Mirzahoseini et al. among 160 skin scrapings examined in Teheran (Iran) identified 6 species of dermatophyte including T. mentagrophytes, T. rubrum, T. verrucosum, T. tonsurans, M. canis, and E. floccosum were based on morphology characteristics and molecular methods. Their study showed that specific PCR products from the ITS region and the use of restriction fragment length polymorphism (RFLP) contributed to the differentiation of isolated dermatophytes at the genus or species level [32].

An epidemiological aspect of dermatophytosis in Southwest Iran demonstrated that tinea cruris and tinea corporis were the most frequent clinical appearance in both men and women. E. floccosum was the most commonly isolated dermatophyte (39.25%), followed by T. verrucosum (27.33%), and T. rubrum (8.41%) [33].

By comparison with other studies of dermatophytes in Iran, our study demonstrated the rapid and reliable identification of common dermatophytes at a species level. Regarding the detail of the different fragments of DNA amplified from dermatophytes with one of the four random oligonucleotides, gene regions were distinguished, and these oligonucleotides could be evolutionarily variable. Nucleotide sequence analysis of amplified bands is required for clarification of the accurate structures and functions of the gene regions.

In conclusion, AP-PCR offers a convenient solution to the problems encountered in the recognition of morphological features of dermatophyte species in comparison to conventional morphological methods. This approach shows a technological progress in identification of dermatophytosis.