Dermatophyte identification usually depends on conventional methods based on detection of phenotypic characteristics, such as direct microscopy and in-vitro culture 1. Morphological and physiological characteristics can frequently vary; in fact the phenotypic features can be easily influenced by external factors such as temperature and the medium used 2.
In the last few years, molecular approaches have been proven to be useful for solving taxonomic problems regarding dermatophytes. Genotypic differences are considered more stable and more precise than phenotypic characteristics 3. Molecular methods, such as PCR, random amplification of polymorphic DNA, arbitrarily primed PCR, PCR fingerprinting, and restriction fragment length polymorphism (RFLP), have brought greater efficiency in distinguishing between species and strains of dermatophytes 1,4–7. On the other hand, PCR immunosorbant assay (PCR-ELISA), line block PCR (PCR-RLP), and multiplex real-time PCR are propagated for the detection of dermatophytes in clinical materials 8–10. The present work was done to study the phenotypic and molecular methods for identification of dermatophytes.
Materials and methods
Fifty-five skin scrapings and/or hair were collected from patients suffering from dermatophytosis who were attending the Dermatology Outpatient Clinic of Misr University for Science and Technology (MUST) Hospital during the period from May 2010 to September 2011. The study protocol was approved by the Research Ethics Committee of MUST. The cases included were 25 tinea capitis, 15 tinea corporis, 10 tinea cruris, and 5 tinea pedis.
The following media were used: dermasel agar (Oxoid; OXOID Ltd., Basingstoke, Hampshire UK), dermatophyte test medium (DTM; HiMedia, Mumbai, Maharashtra, India), InTray DM (Biomed Diagnostic, White City, Oregan, USA), Derm-Duet (Hardy Diagnostics, Santa Maria, California, USA) (containing a biplate with one section of DTM and the other section of rapid sporulation medium), bromocresol purple (BCP) 11, rice lactritmel agar (RLA) 12, milk honey bromothymol blue (MHB) medium 13, and rice grain (RG) medium 14.
Isolation of dermatophytes
After cleaning the lesions with 70% ethyl alcohol, skin scrapings and/or hair were collected from each case in a sterile Petri dish (6 mm). Direct microscopic examination was performed using KOH (20%). Specimens of skin scrapings and hair were inoculated in dermasel agar, DTM, Derm-Duet, and InTray DM. All inoculated media were incubated at 30°C for 2 weeks.
Identification of isolated dermatophytes
Phenotypic identification was made as follows 15,16:
- Through macromorphological examination, on the basis of the reported rate of growth, consistency, surface color, reverse color, and change in DTM color.
- Through micromorphological examination: using InTray DM, the cartilage was examined through a clear viewing window under a microscope for hyphae and conidia; in the case of other media, first, subculturing on dermasel agar was performed, and then specimens from the growth were placed on a slide with drops of lactophenol cotton blue (HiMedia), overlaid with a coverslip, and examined under a microscope for modification of hyphae, macroconidia, and microconidia.
- Through cultivation on differential media: subculture of the isolated dermatophytes was performed on RG, BCP, RLA, and MHB.
Dermatophytes: Nineteen isolates formerly identified by phenotypic methods were subjected to molecular identification.
DNA extraction: This was performed using a Qiagen extraction kit (Qiagen, Hilden, North Rhine-Westphalia, Germany) according to the manufacturer’s instructions.
Detection of DNA by PCR
Using general primers (internal transcribed spacer – ITS1 and ITS4) 17
Amplification reactions were carried out with 100 μl total reaction volume containing 50 μl DreamTag Green Master Mix 2× (Thermo Scientific Fermentas, Waltham, Massachusetts, USA) and 30 pmol of each of primers, ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) 17 prepared by Sigma (Hamburg, Germany), and 10 μl DNA. The PCR cycling conditions were 35 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 2 min, followed by an extension step of 72°C for 10 min. PCR was carried out using a thermal cycler (S1000; Bio-Rad, Hercules City, California, USA). The resulting PCR products were digested with the restriction endonuclease enzyme MvaI (Takara, Ostu, Shiga, Japan), which recognizes the sequence 5′-CC(T/A)GG-3′. The resulting products were separated in 2% agarose gel stained with ethidium bromide, 1× tris–borate–EDTA, and DNA ladder 100 bp plus DNA, Gene Ruler (Thermo Scientific Fermentas). Images were captured using ultraviolet transillumination and a digital camera (Canon, Ota, Tokyo, Japan).
Using repetitive primer (GACA)41 (prepared by Sigma)
Amplification reactions were carried out with total reaction volume of 50 μl containing 25 μl reaction buffer and 30 pmol of the primer. PCR was carried out for 39 cycles of denaturation at 93°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min, followed by a final extension step at 72°C for 7 min. The resulting PCR products were separated in 1% agarose gel stained with ethidium bromide in 0.5× tris–borate–EDTA buffer and stained with ethidium bromide; images were then obtained as described above.
The purified PCR products were sent to Macrogen Lab (Seoul, South Korea). The nucleic acid sequences obtained from forward and reverse primer cycle sequencing were analyzed by DNA baser software (http://www.dnabaser.com/index.html) to trim the sequences and align them with previously published sequence data in GenBank.
From 55 samples of skin scrapings and hair, positive microscopic results were obtained for 92.72% of samples on KOH, whereas dermatophytes were isolated from 48 (87.27%) samples on dermasel agar and DTM, 38 (69.09%) on InTray DM, and 32 (58.18%) isolates on Derm-Deut.
Phenotypic identification of isolated dermatophytes
Macromorphological and micromorphological identification
Forty-eight dermatophyte isolates were identified (as shown in Table 1 and Figs 1–3) and classified into Microsporum canis (n=15), Trichophyton violaceum (n=12), Trichophyton rubrum (n=12), Trichophyton mentagrophytes (n=4), and Epidermophyton floccosum (n=5). M. canis and T. violaceum were the causative agents of tinea capitis, and T. rubrum, M. canis, and T. violaeum were the causative of tinea corporis. Tinea cruris was caused by E. floccosum and T. rubrum, and tinea pedis was caused by T. mentagrophytes.
Identification by cultivation on differential media
Cultivation of dermatophyte isolates on RG, RLA, BCP, and MHB media confirmed the macromorphological and micromorphological identification of dermatophytes. Increased pigmentation, changed medium color, hydrolysis of casein (Fig. 4), and increased conidiation were the factors used for this type of identification.
Molecular identification of dermatophytes
In the present study three molecular methods were used for identification of dermatophytes: PCR for amplification of ITS1 and ITS4 by common primer, followed by RFLP using MvaI; PCR using single repetitive oligonucleotides [(GACA)4]; and DNA sequencing, which was done only for four representative isolates.
Identification of dermatophyte isolates by common primer and RFLP
PCR with the ITS1/ITS4 primer set for 19 dermatophyte isolates resulted in amplified products of ∼690 bp specific for T. rubrum, T. mentagrophytes, E. floccosum, and T. violaceum (Figs 5 and 6) and of 740 bp for M. canis (Fig. 7).
MvaI digestion of these amplified products from each of the dermatophyte isolates revealed unique restriction patterns, with no interspecies variation. M. canis isolates showed three band patterns, ranging from 100 to 500 bp in size, with a marked size difference between the largest and middle bands (Fig. 8).
On the other hand, both T. violaceum and T. rubrum isolates resulted in four bands, with sizes ranging between 50 and 400 bp (Fig. 9).
Identification of dermatophyte by (GACA)4-based PCR
Nineteen isolates were amplified with (GACA)4. The results showed that the numbers of PCR bands ranged from 9 to 13 (size range: 200–1300 bp).
(GACA)4-Based PCR of M. canis strains revealed the most complex profiles, with up to 11 bands, ranging from 170 to 1200 bp in size. There was no interspecies variation among M. canis isolates, all of which had the same band pattern (Fig. 10).
(GACA)4-Based PCR for T. rubrum showed 5–12 bands ranging from 200 to 2000. The result for E. floccosum showed bands ranging from 50 to 1500 bp. T. mentagrophytes showed different bands ranging from 50 to 800 bp (Fig. 11). T. violaceum gave nearly the same pattern, with PCR bands ranging from 400 to 2599 bp.
The query sequences were compared with those in GenBank database by Blast analysis. Whereas three of the representative isolates (M. canis, T. violaceum, and T. rubrum) were found to be identical in 97, 99, and 99% of cases to the same species in GenBank, the fourth isolate formerly identified as T. rubrum was found to be identical (99%) to Trichophyton raubitschekii, differing in only two bases, in nucleotides 150 and 160 (Table 2 and Fig. 12).
Conventional methods for dermatophyte identification are based on the detection of fungal elements by direct microscopy of clinical specimens, combined with culture. The aim of the present study was to compare between conventional and molecular methods for identification of dermatophyte isolates obtained from human superficial skin infection.
Fifty-five human samples were subjected to mycological examination. Whereas direct microscopic examination (KOH) was positive in 92.72% of cases, culture was positive in 87.27%. These results are in agreement with those of others 13,18 who attributed the higher positivity of KOH to the fact that the cases were under treatment, the culture was contaminated by rapidly growing fungi giving no chance for slowly growing dermatophytes to appear, and the use of unsuitable media for certain dermatophytes, which need higher nutritional requirements. In general, KOH and culture complete each other, and, although direct microscopic examination showed higher sensitivity, culture showed higher specificity, as reported by Levitt et al.19.
Comparison between the four media – dermasel agar, DTM, InTray DM, and Derm-Deut – showed that dermasel and DTM succeeded in the isolation of 48 dermatophyte isolates from a total of 55 specimens, whereas InTray DM and Derm-Deut helped isolate 38 and 32 dermatophytes, respectively. The reason why the two readily prepared media failed to identify all dermatophyte isolates may be that the medium was subjected to dryness before the growth of slow-growing dermatophytes as T. violaceum and the plates of Derm-Deut were rapidly subjected to contamination by nondermatophyte molds. These coincide with the observations of Robert and Pihet 20, who found that the usefulness in office culture systems is still a matter of debate.
Phenotypic identification of dermatophyte species relies on the macromorphology of colonies (rate of growth, texture, and color of the surface and the reverse side), and micromorphology (presence and characters of macroconidia and microconidia as well as modification of hyphae – e.g. spirals, nodular organs, favic chandeliers, and pectinate bodies). If identification is not reached, biochemical tests such as urease, nutritional requirements, and in-vitro hair penetration will help in its identification 21.
Besides RG medium, which is used for differentiation of M. canis from Microsporum audouinii14, and BCP medium 15 for differentiation of T. mentagrophytes from T. rubrum, other differential media were also propagated 12,13,22,23 for differentiation between dermatophytes with ambiguous morphological and physiological characters.
In the present work the identification of 48 isolates obtained from human cases by macromorphology, micromorphology, and culture on four differential media (RG, LA, BCP, and MHB) revealed 15 isolates of M. canis, 12 of T. violaceum, 12 of T. rubrum, 5 of E. floccosum, and 4 of T. mentagrophytes. These results, although in accordance with other studies 13,24 in which M. canis was the predominant dermatophyte isolated in the last few years in Egypt followed by T. violaceum and T. rubrum, differs from others 25,26, which isolated T. violaceum as the predominant dermatophyte followed by M. canis in tinea capitis and tinea corporis. This variation in results may be due to the differences in study location.
Nucleic acid-based techniques rely on genotypic differences, and are more precise than those based on phenotypic features 27. Recently, a number of methods have been reported for molecular identification of dermatophytes. In the present study three methods were used: first, PCR for amplification of ITS1 and ITS4, followed by RFLP using MvaI for 19 isolates of dermatophyte formerly identified by phenotypic methods; second, application of PCR using single repetitive oligonucleotides [(GACA)4] for the same 19 dermatophyte isolates identified by phenotypic and RFLP methods; and lastly DNA sequencing, which was done only for four representative isolates.
The amplified products using universal primers ITS1 and ITS4 from T. violaceum, T. rubrum, T. mentagrophytes, and E. floccosum were found at 690 bp, whereas M. canis was at 740 bp. These results are identical to those of other studies 17.
MvaI digestion of amplified products in the first step revealed a unique restriction pattern. Analysis of the number and size of patterns identified the nineteen dermatophyte isolates examined as species typically detected by the phenotypic method. The results of the current work by RFLP coincide with those of Jackson et al.28, who found that PCR-RFLP of the ITS region is an easy method for identifying dermatophyte isolates.
In the current study all isolates identified by (GACA)4-based PCR was identical to those recognized by the phenotypic and PCR-RFLP methods. The present study is in agreement with that by Roque et al.29 and Liu et al.30, who found that repetitive primer (GACA)4 was able to differentiate all dermatophytes into species. A comparison of the two methods revealed that RFLP is complex, needing much effort and time, whereas the (GACA)4 method is simple and rapid.
On the other hand, four dermatophyte isolates [M. canis (n=1), T. violaceum (n=1), and T. rubrum (n=2)] formerly identified by RFLP and repetitive primer (GACA)4 were sent for sequencing. The data were analyzed by DNA software and compared with those in GenBank. Whereas M. canis, T. violaceum, and one isolate of T. rubrum were found to be identical, the other isolate of T. rubrum obtained from a case of tinea corporis was found to be identical (99%) for the sequence of T. raubitschekii, a dermatophyte considered atypical and confused with T. rubrum and rarely isolated from people who live or travel to the Mediterranean area 31.
Although sequencing provides a very accurate and useful method for the identification of dermatophytes, it is highly expensive to be used in routine identification. Therefore, it is recommended for identifying atypical isolates of dermatophytes 32,33.
In conclusion, although the current study revealed the capacity of phenotypic (morphological and differential media) and molecular (RFLP and repetitive primer) methods to classify dermatophytes into species, molecular methods are rapid and represent technological advancements in laboratory diagnosis. DNA sequencing proved to be more accurate and detected variants and subtypes, although it is expensive and face some problems in application in our country. Therefore, we recommended its use only for identification of dermatophytes in atypical isolates.
Conflicts of interest
There are no conflicts of interest.
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Keywords:© 2017 Egyptian Women's Dermatologic Society
dermatophytes; molecular identification; phenotypic identification