Update on dermatophytosis in Mashhad, Northeastern Iran, emergence of infection with Trichophyton persicum.

Background: Dermatophytosis is a common global superficial mycosis caused by a group of keratinophilic moulds known as dermatophytes that invade the skin and keratinized tissues such as hair and nails of humans and animals. This study takes identification of a collection of clinical dermatophyte isolates by using partial sequencing of translation elongation factor-1α (Tef-1α) gene aiming both to update the epidemiological status of dermatophytosis in Mashhad, Northeastern Iran and to corroborate the efficacy of Tef-1α for species-level identification of dermatophytes. Method: The demographic data related to 87 culture-positive dermatophytes isolated from patients clinically suspected to have dermatophytosis were collected. The dermatophyte isolates were subjected to a partial polymerase chain reaction (PCR)-sequencing of Tef-1α gene by using specific pan-dermatophyte primers. The data were analysed by SeqMan software, the sequences were compared and aligned with the GenBank database and the isolates were identified.
Results: Identification based on Tef-1α partial sequence was successful for all isolates. The identified dermatophyte isolates in decreasing order were as Trichophyton interdigitale 19 (22%), T. tonsurans 19 (22%), T. mentagrophytes 13 (15%), T. persicum 10 (11.5%), Epidermophyton floccosum 9 (10.3%), Microsporum canis 7 (8%), T. rubrum 5 (5.7%), T. violaceum 2 (2.2%), Nannizzia fulva 2 (2.2%) and N. persicolor 1 (1.1%). The isolates have been associated with clinical forms of tinea corporis (n = 38; 43.7%), tinea faciei (n = 13; 15%), tinea cruris (n = 12; 13.9%), tinea manuum (n = 7; 8%), tinea unguium (n = 7; 8%), tinea capitis (n = 7; 8%) and tinea pedis (n = 3; 3.4%).
Conclusion: Dermatophytosis has yet remained a public health problem in Northeastern Iran, and infection with new and less frequent species, e.g., T. persicum, N. fulva and N. persicolor have emerged. The Tef-1α gene partial sequencing reconfirmed the resolution power of this locus for the determination of species boundaries in dermatophytes. Copyright:

Introduction

The dermatophytosis is the term used to explain infections of the skin, hair and nails caused by the globally distributed keratinophilic fungi known as dermatophytes. The prevalence of dermatophytosis is different in geographical regions, and depends on factors such as lifestyle, type of population, health, migration of people and climatic conditions.[12] On the other hand, diagnosis of dermatophyte infection is essential for appropriate antifungal therapy that can save time and cost.[3] The causative agents of dermatophytosis (dermatophytes) can transfer from human resources to humans and soil, and vice versa. Dermatophytes are a group of more than 50 species in various genera of Trichophyton, Microsporum, Epidermophyton, Arthroderma, Lophophyton, Nannizzia and Paraphyton.[4] The spectrum of dermatophytes has experienced a great evolution, and the distribution of species varies significantly by region.[2] For instance, social activities, population density, the growth of immigrant populations, socioeconomic alterations and some sports activities can influence the dermatophytes distribution.[1] Reliable and accurate species identification is helpful to upgrade the epidemiological status of dermatophytosis, especially in cases with therapy failure.[5] Moreover, true identification of species with potential treatment resistance can reduce the cost of therapy.[6] Recently, different genetic loci have been introduced for identification, phylogenetic analysis and taxonomy of dermatophytes.[478] Although sequencing of internal transcribed spacer-ribosomal DNA (ITS-rDNA) regions is currently the golden standard used for identification of dermatophytes, however translation elongation factor-1α (Tef-1α) gene has recently been shown as an alternative and stable genetic marker for discrimination of dermatophyte species.[910] The present study was conducted for sequencing of the Tef-1α gene in a collection of clinical dermatophyte isolates in Mashhad, Northeastern Iran aiming both to update the epidemiological status of dermatophytosis in this part of Iran and to corroborate the efficacy of Tef-1α for species-level identification of dermatophytes.

Methods

This research was approved by the Ethics Committee of Mashhad University of Medical Sciences, Mashhad, Iran (Ethical code: IR.MUMS.fm.REC.1395.320).

Fungal strains

In total, 87 clinical dermatophyte isolates collected from various specimens, such as skin, hair and nail of patients referred to a medical mycology laboratory in Mashhad, Iran were included in the study. All clinical specimens were examined using 15–20% KOH (potassium hydroxide) and cultured on Sabouraud dextrose agar with chloramphenicol and cycloheximide (SCC) medium (Merck, Germany) and incubated at 27°C for 21–28 days, which resulted in the achievement of dermatophyte colonies.

DNA (deoxyribonucleic acid) extraction

The DNA of dermatophyte colonies was extracted and purified using a DNA extraction kit (Dena zist Asia, Mashhad, Iran). The quality and quantity of extracted DNA were checked by nanodrop (Thermo Scientific, Wilmington, DE, USA), and stored at −20°C until used for polymerase chain reaction (PCR) amplification.

DNA amplification and sequencing

Tef-1α amplicons were generated with primers EF-derm F (5×-CACATTAACTTGGTGGTTATGG-3×) and EF-derm R (5×- CATCCTTGGAGATACCAGC-3×).[11] The PCR was performed in a 25 μl volume reaction mixture with 7 μl red master mix (Ampliqon, Denmark), 1 μl of each primer (25 pmol), 1 μl DNA and 15 μl distilled water. Amplification was performed in an ABI PRISM 2720 thermocycler (Applied Biosystems, Foster City, USA) as follows: 95°C for 4 min, followed by 35 cycles consisting of 95°C for 45 s, 58°C for 30 s and 72°C for 1 min, with a delay at 72°C for 10 min. 3 μl of the PCR products were electrophoresed onto 2% agarose gel in TAE (Tris base, acetic acid and EDTA) buffer and observed and photographed under ultraviolet (UV) irradiation. The amplicons were subjected to direct sequencing, with PCR regimen as follows: 95°C for 1 min followed by 30 cycles consisting of 95°C for 10 s, 50°C for 5 s and 60°C for 2 min. Sequencing was done using ABI PRISM BigDyeTM terminator cycle sequencing kit (Applied Biosystems) and amplicons were analysed on an ABI PRISM 3730XL Sequencer. The data were analysed by SeqMan software (DNASTAR, Wisconsin, USA) and sequences were compared and aligned with those of sequences maintained at GenBank (http://www.ncbi. nlm.nih.gov/BLAST/).

Results

The Tef-1α gene partial sequence was successfully amplified in all 87 dermatophyte isolates with bands ranging around 700–770 base pair (bp) in size. All isolates could be identified to 10 species, i.e., T. tonsurans, T. interdigitale, T. mentagrophytes, T. persicum, E. floccosum, M. canis, T. rubrum, T. violaceum, Nannizzia fulva and N. persicolor in decreasing order of frequency [Table 1]. The patients’ ages ranged from 2 to 78 with a mean age of 21.7 years and the 11–20 years as the most frequent age group, while the gender distribution comprised 70% males and 30% females [Table 2]. In terms of clinical manifestations, almost all forms of dermatophytosis were observed except tinea barbae, and the most common form was tinea corporis 38 (43.7%) followed by tinea faciei, tinea cruris, tinea manuum, tinea unguium, tinea capitis and tinea pedis [Table 3]. The relation between different tinea infections and the aetiologic species is shown in Table 1. The Tef-1α intra-species variation was as 0–1% and new sequences generated in this study were deposited in the NCBI GenBank database with the accession numbers MG356854 to MG356942.

Table 1

Frequency of different clinical manifestations of dermatophytosis in association with dermatophyte species in Mashhad, Iran.

Clinical manifestations	Dermatophyte species										Total
	T. interdigitale	T. tonsurans	T. mentagrophytes	T. persicum	E. floccosum	M. canis	T. rubrum	T. violaceum	N. fulva	N. persicolor
Tinea corporis	6	9	7	7	4	3	0	1	0	1	38
Tinea faciei	1	8	0	2	1	0	0	1	0	0	13
Tinea cruris	1	1	2	0	3	0	5	0	0	0	12
Tinea unguium	6	0	1	0	0	0	0	0	0	0	7
Tinea manuum	1	0	1	0	1	2	0	0	2	0	7
Tinea capitis	2	1	1	1	0	2	0	0	0	0	7
Tinea pedis	2	0	1	0	0	0	0	0	0	0	3
Total	19	19	13	10	9	7	5	2	2	1	87

Table 2

Details of clinical manifestations and outcomes of different age groups and gender among patients affected by dermatophytosis in Mashhad, Iran

Clinical manifestations	Age groups							Gender		Total
	0-10	11-20	21-30	31-40	41-50	50-60	>60	Male	Female
Tinea corporis	7	11	6	10	2	2	0	27	11	38 (43.7%)
Tinea faciei	2	6	3	2	0	0	0	11	2	13 (14.9%)
Tinea cruris	0	2	5	1	2	0	2	10	2	12 (13.9%)
Tinea unguium	0	0	1	1	2	3	0	5	2	7 (8%)
Tinea manuum	0	3	1	2	1	0	0	3	4	7 (8%)
Tinea capitis	6	0	0	0	1	0	0	2	5	7 (8%)
Tinea pedis	0	0	1	1	0	0	1	3	0	3 (3.3%)
Total	15	22	17	17	8	5	3	61	26	87 (100)

Table 3

Frequency of different clinical manifestations of dermatophytosis in association with Trichophyton, Epidermophyton, Microsporum and Nannizzia species in Mashhad, Iran

Clinical manifestations	Dermatophyte species
	T. interdigitale	T. tonsurans	T. mentagrophytes	T. persicum	E. floccosum	M. canis	T. rubrum	T. violaceum	N. fulva	N. persicolor
T. corporis	6	9	7	7	4	3	0	1	0	1
T. faciei	1	8	0	2	1	0	0	1	0	0
T. cruris	1	1	2	0	3	0	5	0	0	0
T. unguium	6	0	1	0	0	0	0	0	0	0
T. manuum	1	0	1	0	1	2	0	0	2	0
T. capitis	2	1	1	1	0	2	0	0	0	0
T. pedis	2	0	1	0	0	0	0	0	0	0
Total	19	19	13	10	9	7	5	2	2	1

Discussion

The data from this study were significant from two viewpoints: 1)firstly, the value of Tef-1α gene as a novel genetic marker for identification of dermatophytes was reaffirmed by a new big collection of clinical isolates from Northeastern Iran, which were identified at the species level. 2) Moreover, they accentuated again this fact that detection/identification of novel or neglected species such as T. persicum and N. fulva can be achieved only by sequence-based strategies.

Mashhad, as a touristic city and the second-most populous city in Iran, is located in northeastern Iran with a warm and dry climate and annually hosts many tourists from Iran and abroad. Therefore, intermittent updating of the data on the epidemiological aspects of dermatophytosis in this city is necessary for the efficient control and prevention of this cutaneous disease.[14] Identification of dermatophytes was previously based on traditional methods, but these methods had low sensitivity and specificity. Hence, molecular methods such as PCR, Random amplification of polymorphic DNA (RAPD)-PCR, nested PCR, PCR-restriction fragment length polymorphism (RFLP), real-time PCR and sequencing can identify dermatophytes at genus and species levels.[2] In spite of its undeniable value, it was shown that sequencing of ITS-rDNA could not differentiate some closely related species, while other genetic regions such as β-tubulin and Tef-1α have been shown to be more informative in this regard.[7811]

In the present study, 10 species were identified using amplification and sequencing of the partial gene of Tef-1α. The most common aetiologic species were T. interdigitale and T. tonsurans that were similar to Rezaei-Matehkolaei et al.[12] in Tehran, and differs with Chadeganipour et al.[13] in Isfahan, and Naseri et al.[14] in Mashhad in that T. verrocosum was reported as the dominant agent. The most common dermatophytes in Europe were reported as T. rubrum and M. canis,[15] while in the United States and Central America T. rubrum, T. tonsurans, T. mentagrophytes, M. canis and E. floccosum were noted as the dominant agents in different investigations.[116] In a recent study by Ebrahimi et al.[2] from Mashhad, T. interdigitale was the most common clinical isolate, but T. tonsurans accounted for only 5% of cases. The discrepancy between the findings of Drakensjo et al. and the current survey can be due to increased prevalence of migration and travel, changes in the living conditions and cultures and more important to use of sequence base method in the current study. Given that T. interdigitale and T. tonsurans are anthropophilic species, direct and indirect communication and contacts in public environments between humans play an important role in their spread. Gyms and public swimming pools seem to be sources of the distribution of the anthropophilic species of dermatophytes, especially in athletes.[17] However, in several areas of the entire world, there have been lots of changes in the spread of certain species, and even emerging patterns of dermatophytes.

The most important findings of the present study were new cases of isolation and identification of Trichophyton persicum. T. persicum is a new species in the T. benhamiae species complex that was recently discovered and described by Čmoková et al.[18] Taghipour et al.[19] have recently shown that some dermatophyte species/genotypes are associated with particular geographic areas and clinical manifestations. Until 2021, all 37 retrospectively identified strains were exclusively from seven different provinces in Iran and isolated from individuals with tinea corporis except one which was from an ectothrix tinea capitis case. In keeping with the two mentioned studies, here, we reported 10 new cases of T. persicum from Mashhad, Northeastern Iran, among which nine were isolated from skin infections and one from a scalp infection. In a similar study by Ebrahimi et al.[2] in Mashhad, using ITS gene location, 1.3% of dermatophyte isolates were reported as T. benhamiae while a reassessment of the isolate reveals its identity as T. persicum. Recently, Zareshahrabadi et al.[20] also reported six cases of infection by T. benhamiae solely based on ITS sequencing, while more concentration on ITS sequences by Čmoková et al.[18] showed that those isolates were T. persicum, in fact. It seems that most cases of isolates originated from Iran and those formerly reported as T. benhamiae senso stricto, were in fact T. persicum and the species is currently indigenous to Iran. Isolation of M.canis in this study is important as regards keeping of the pets, especially dogs, increased in Mashhad in recent years, and this finding is not in accordance with Naseri et al.[14] in Mashhad. In most cases, this species as a zoophilic dermatophyte is isolated from clinical form tinea capitis cases, especially in children.

Geophilic dermatophytes have historically constituted the lowest proportion of infectious agents in Iran among which N. gypsea (formerly M. gypseum) has been reported as the dominant species. In the present investigation, two N. fulva and one N. persicolor isolates were solely identified based on the sequencing method. In 2013 and 2016, the first proven cases of infection by N. fulva and N. incurvata in Iran were reported in studies by Nouripour-Sisakht et al.[21] and Rezaei-Matehkolaei et al.[22] They accentuated the fact that many clinical isolates formerly described as N. gypsea in Iran, solely based on the morphological criteria, were indeed other morphological closely related species such as N. fulva. Recently, a comprehensive nationwide sequence-based study by Taghipour et al.[23] showed that N. fulva is the main dermatophyte species resident in the soils of different parts of Iran.

In this study, the prevalence rate of dermatophytosis in men and women was 67.8% and 28.9%, respectively. Moreover, most cases of dermatophytosis were observed in the age group of 11–20 years. These findings are similar to most of the other studies in Iran.[2425] In other studies, dermatophytosis has been reported in all of the age groups, and its frequency is different in both sexes. These results may be dependent on various factors such as job, hygienic and climatic conditions.[2627] There are some contradictory reports about forms of dermatophytosis in different areas in Iran. In previous reports in Iran, the most commonly reported clinical forms were tinea pedis,[1228] tinea cruris[2] and tinea capitis.[29] These incompatibilities can be seen due to various factors such as age, sex, job, climate, lifestyle, hygiene level and cultural and socioeconomic condition.

The frequency of tinea corporis in our study was consistent with some previous studies performed in Iran such as Mashhad,[14] Khorramabad[30] and Tehran.[24] In this study, tinea faciei (15%) had a high frequency among other clinical forms, while it was reported rarely in most studies. Tinea cruris (13.9%) was in the third level of frequency among the clinical forms. This form of dermatophytosis was prevalent in adults because anatomically this location is suitable to get dermatophytosis and factors such as heat and moisture support the growth of dermatophytes to colonize. The main causative agents of this kind of tinea in our study were E. floccosum and T. rubrum that is similar to other studies in Iran and other parts of the world.[122425]

In the current study, the frequency of tinea unguium was about 8% and mainly was caused by T. interdigitale. This was in agreement with results from Zarrin et al.[29] in Ahvaz, Chadeganipour et al.[13] in Isfahan and Rezeai-Matehkolaei et al.[12] in Tehran. Humidity, hyper sweating and some superficial vessel disorders are the main risk factors to get tinea unguium, particularly foot nails. Tinea pedis with the frequency of 3.3% by T. interdigitale has high similarity with most of the other studies in Iran,[122831] but in other countries T. rubrum is the main causative agent of tinea pedis.[32]

Conclusion

Our study was one of the limited investigations that successfully applied sequencing of a locus other than ITS-rDNA (Tef-1α) for identification and epidemiological purposes. The Tef-1α gene partial sequencing reconfirmed the resolution power of this locus for the determination of species boundaries in dermatophytes. Dermatophytosis has yet remained as a public health problem in Northeastern Iran, and infection with new and less frequent species, e.g., T. persicum, N. fulva and N. persicolor have emerged.

Statement of ethics

This study obtained the ethical committee code no. of IR.MUMS.fm.REC.1395.320.

Author contributions

F. Afsharzadeh performed the drafting of the manuscript. H. Zarrinfar and AM. Fata analysed the data. MJ. Najafzadeh conceived the original idea, supervised the project and provided critical revision of the manuscript.

Financial support and sponsorship

This work was extracted from a master thesis by Fatemeh Afsharzadeh and was financially supported by the Deputy of Research of MUMS, Mashhad, Iran (Grant No. 941862).

Conflicts of interest

There are no conflicts of interest.