Dermatophytosis is a superficial infection caused by filamentous fungi belonging to the following three genera:
Colonization by dermatophytes is assisted by the release of various proteolytic and other enzymes produced by the fungi, which can cause inflammatory responses [2].
Almost 30 species of dermatophytes are considered involved in the etiology of dermatophytosis. Of dermatophyte species,
Laboratory diagnosis of dermatophyte species is helpful to treat the infection. Routinely, dermatophytes are detected by microscopic examination and by culture, but conventional routine procedures are not specific or sensitive, and the methods are time-consuming.
Several molecular methods have been developed for the rapid identification of various species of dermatophytes [5]. PCR is a very sensitive and precise technique for the diagnosis of microorganisms including fungi. Arbitrarily primed PCR (AP-PCR) is a rapid and sensitive procedure for the diagnosis of fungal species [6, 7]. Ribosomal and mitochondrial DNA of dermatophytes have shown variation among these agents [8-10].
The identification of dermatophytes has been improved as a result of the aforementioned studies to have more acceptable accuracy and speed. However, the techniques used have required extra manipulation such as restriction enzyme digestion and sequencing phylogenic relationships between different species. Recent research has focused on developing novel molecular techniques for the rapid detection of dermatophytes from clinical samples at the genus and species level [6, 11, 12]. The identification of dermatophytes with randomly amplified fragments enhanced laboratory discrimination of dermatophytes from other microorganisms [12, 13]. AP-PCR was used in the present study with four random primers for identification of 10 dermatophyte species recovered from patients in Iran. The aim of this study was to identify various dermatophyte species as rapidly and precisely as possible.
The study was approved by the Ethics Committee of Ahvaz Jundishapur University of Medical Sciences.
Fifty-two clinical dermatophyte isolates of 10 species were recovered from samples obtained from the Department of Medical Mycology and different parts of Iran. All species, except
PCR product patterns (bp) from dermatophyte species
Dermatophyte species | Number tested | OPAA11 | OPU15 | OPAA17 | OPD18 |
---|---|---|---|---|---|
8 | 350-450-650-750 | 250-400-600-650-750-1000 | 300-500-750-850 | 250-350-400-600-750-900-1100-1500 | |
4 | 350-450-650-750 | 250-350-450-600 | 200-300-450-700-900-1400 | 250-350-400-450-700-750-800-1000-1500 | |
8 | 300-450-600-700-800-1000-1200-1800 | 300-400-550-650-700-800-1000-1200-1500 | 300-450-500-600-800-900 | 350-450-600-700-800-900-1100-1600 | |
2 | 350-450-700 | 250-350-400-600-700-900-1300-1800 | 300-500-700 | 300-400-500-700-800-1000-1400 | |
10 | 350-750 | 250-400-450-600-1000 | 300-450-500-700-900 | 350-400-750-1000 | |
5 | 350-450-650 | 250-400-550-750-800-1000 | 300-500-700-800-1200-1400 | 350-400-650-800-900-1300-1500 | |
9 | 300-400-600 | 250-350- 800 | 300-500-850 | 350-450-600-700-800-900-1100-1600 | |
3 | 350-450-600-750-850 | 250-300-400-500-600-800-900-1400 | 300-400-500-700-900-1200 | 300-600-800-1000-1400-1500 | |
2 | 350-450-650 | 250-350-400-500-600-750-1000-1200 | 300-500-700-900-1000 | 250-300-500-700-800-1000-1500 | |
1 | 350-450-550-650 | 250-350-400-500-550-650-800-900-1000 | **************** | 250-350-400-450-500-600-700-800-1000-1300-1500 | |
Total | 52 |
The genomic DNA from all 52 dermatophytes was extracted and applied as templates for PCR amplifications. Dermatophyte isolates were grown in selective agar. A small portion of fresh culture was transferred into a 1.5 microtube containing 400 μL of lysis buffer (100 mM Tris-HCl, pH 8.0, 30 mM EDTA, pH 8.0, SDS 5% w/v) using a sterile tooth pick. The microtubes were boiled for 20 min, and then 150 μL of 3 M potassium acetate was added. The suspension was kept at -20°C for 10 min, and centrifuged at 12000 rpm at 4°C for 10 min. After transferring the supernatant to a new 1.5 mL microtube, 250 μL phenol-chloroform- isoamyl alcohol (25:24:1, v/v) was added. The tube was briefly vortexed and centrifuged at 12000 rpm for 10 min. The supernatant was transferred to a new 1.5 mL microtube and 250 μL chloroform-isoamyl alcohol was added. Then, it was kept at -20°C for 10 min and centrifuged at 12000 rpm for 10 min. After transfer of the supernatant to a new 1.5 mL tube, and the addition of an equal volume of iced-cold 2-propanol, the microtube was kept at -20°C for 10 min, then centrifuged at 12000 rpm for 10 min. The supernatant was discarded and the pellet was washed with 300 μL 70% ethanol. After that, the supernatant was discarded by centrifuging at 12000 rpm for 10 minutes. The DNA pellet was air dried and resuspended in 50 μL of distilled H2O.
AP-PCR was conducted for all isolates with each random primer separately. Amplification reactions were performed with volumes of 50 μL containing reaction buffer, 2.2 mM MgCl2, 200 μM each dNTP (dATP, dCTP, dGTP, and dTTP), 1.5 U of Taq DNA polymerase (Roche, Germany), 50 ng template DNA, 15 pmol of each random primer [6] OPAA11 (5′-ACCCGACCTG-3′), OPU15 (5′-ACGGGCCAGT-3′), OPAA17 (5′-GAGCCCGACT-3′), and OPD18 (5′-GAGAGCCAAC-3′). The PCR cycling conditions were 3 cycles of 94°C for 60 s, 36°C for 45 s, and 72°C for 90 s, followed by 32 cycles 94°C for 30 s, 36°C for 45 s, and 72°C for 90 s. The PCR products were electrophoresed in 1.5% agarose gel in 1× TAE buffer (40mM Tris, 20mM acetic acid, and 1mM EDTA) and stained with ethidium bromide.
A total of 52 isolates belonging to any of 3 genera,
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 (
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 (
As shown in
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 (
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
Three random primers, OPAA11, OPAA17, and OPU15 are not able to differentiate among three dermatophytes
Neji et al. identified 3 dermatophytes
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
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.
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.