Abstract
Background: Onychomycosis represents the most frequently encountered nail disease, which is difficult to eradicate with drug treatment. Aim: This study was undertaken to document the clinicomycological pattern of onychomycosis in Iran. Results of mycological tests of nail scrapings collected over a 4-year period were analyzed. Methods: Both direct microscopy and cultures of the nail material were performed to identify the causative agents. Results: The microscopic and/or cultural detection of fungi was positive in 40.2% of samples. The most common clinical type noted was distolateral subungual onychomycosis in 48.4% of cases. Etiological fungal agents were 50% dermatophytes, 46.8% yeasts, and 3.2% saprophytic moulds. The most frequently detected dermatophyte species were Trichophyton rubrum（48.4%）and T. mentagrophytes（41.9%） . Among yeasts, Candida albicans（58.6）was most common, followed by C. parapsilosis（17.2%） , C. glabrata（10.3%） , C. krusei and C. tropicalis（each 6.9%） . Aspergillus niger and A. flavus were the most frequent saprophytic moulds. Females were affected more frequently than males, and in both sexes those most infected were between 40-49 years of age. Fingernails were affected more frequently than toenails. Conclusions: The clinico-epidemiological data collected can serve as reference for future research and may be useful in the development of preventive and educational strategies.
Introduction
Among nail diseases, onychomycosis is still considered as a major public health problem in many parts of the world. These diseases are relatively common with a universal prevalence and relapsing condition mostly in adult populations, which have increased during the past few decades. Large-scale epidemiological studies performed worldwide have demonstrated different epidemiological results. However, onychomycosis represents up to 50% of all nail disorders and 30% of all superficial skin fungal infections diagnosed 1） . Clinically, onychomycosis is classified into various types: distolateral subungual onychomycosis（DLSO） , superficial white onychomycosis（SWO） , proximal subungual onychomycosis（PSO） , candidal onychomycosis（CO）and total dystrophic onychomycosis （TDO） 2,3） . These infections are produced by yeasts, dermatophytes, or saprophytic moulds. The nails infected with yeasts are usually hard and thick, brown colored, and grooved, whereas the nails infected with dermatophytes are fragile. A chronic infection with yeasts can lead to complete destruction of the nail tissues. Candidal onychomycosis is seen in people whose hands or feet are put in water frequently or for long periods in relation with their job, and/or in patients with diabetes, neoplasias, or those under treatment with cytotoxic or immunosuppressive drugs. The impairment of the immune system of the normal host by drugs provides suitable conditions for colonization of Candida species. Onychomycosis with dermatophytes is often produced by scratching an infected area of the body, clipping the nail with a contaminated clipper, rasping nails with a contaminated file, or extension of an infection from infected skin to the toenails. Onychomycosis with saprophytic moulds are frequently produced by a trauma to the nail and connection of this injured nail with a polluted source 4） . The most common opportunistic moulds are Scopulariopsis, Aspergillus, and Fusarium
5）
. Candida albicans is the most frequent genus of candidas among the yeasts causing onychomycosis, but C. parapsilosis, C. tropicalis, C. glabrata, and other candidas can also involve nails 6） .
To the best of our knowledge, this is the first report on different aspects of onychomycosis in Iran and with respect to the importance of the subject; this study was carried out over a period of four years in order to determine the epidemiology, causative agents and different types of onychomycosis among people resident in Qazvin. There is a need to educate the general population concerning the danger of acquiring infections from infected persons and animals.
Materials and methods
The study was conducted from June 2004 to June 2007 in Qazvin province, situated northwest of the capital, Tehran carried out using an observational method and samples of nail scrapings were taken by scalpel from the fingernail or toenail of patients referred to the Medical Mycology Department of the Medical School of Qazvin.
Microscopic examination of nail clippings or subungual scrapings samples was performed following treatment with an aqueous solution of 20%（w/v）potassium hydroxide（KOH） . A portion of each sample was placed on a slide, a drop of KOH added and the slide was stored in a humid chamber for 1-2 h. This incubation period was necessary in order to make the test material transparent. The wet preparation was then examined under low（× 100）and high（× 400）magnification for the presence of mycelia or arthroconidia, yeast cells, blastoconidia, pseudohyphae and hyphae and even spores in some saprophytic moulds. These microscopic features indicate the presence of a fungal infection but do not reveal the species or genus of the pathogen. All samples from suspected cases were cultured irrespective of the negative or positive examination result. Each sample was cultured on two plates of Sabouraud's glucose agar（BioMeriéux, Marcy-1, Etoile, France） , with 0.5 mg/ml cycloheximide and 0.05 mg/ml chloramphenicol（SCC） , which was prepared according to the manufacturer's instructions. The cultures were kept at 28-30℃ for 4-6 weeks, and examined macroscopically once a week for evidence of growth. It was also required to do a repeat culture where at least five out of 20 inocula planted demonstrated that the mould was the sole etiologic agent. However, it was not always possible to carry out repeated sampling. After the growth of the dermatophytes was established, a subculture was made on Sabouraud's glucose agar and potato dextrose agar plates（Oxoid, Basingstoke, UK）for further identification. The dermatophytes were diagnosed on the basis of their gross and microscopic morphologies, especially according to their formation of macroconidia and microconidia in slide cultures. In order to differentiate between T. mentagrophytes and T. rubrum, additional special methods including a urea medium test, hair penetration, and production of pink color pigments on corn meal agar medium plus 1% glucose were used if needed. In order to determine T. mentagrophytes varieties, the isolates were differentiated according to macroscopic and microscopic characteristics and divided into T. mentagrophytes var. interdigitale and T. mentagrophytes var. mentagrophytes.
Diagnosis of the isolated yeasts was made on the basis of their microscopic morphology on cornmeal agar containing Tween 80 and production of their colors on CHROMagar Candida culture medium （Becton-Dickinson） . The saprophytic moulds were diagnosed by their morphological characteristics and confirmation of the diagnosis was made by incubation on Sabouraud's glucose agar with chloramphenicol （SC） culture medium. Information of age, gender, and injury location were collected and evaluated using descriptive statistics procedures.
Results
Samples were taken from 308 patients referred for suspicion of onychomycosis, of which 124 cases（40.2%） were positive by both direct microscopy and culture, of those, 62 cases（50%）were infected with dermatophytes, 58（46. 8%）with candidas, and 4（3. 2%） with saprophytic moulds. The most common dermatophyte isolated from nails was T. rubrum（48.4%） , the predominant yeast isolated was C. albicans（58.6%） and the sole mould isolated was the genus Aspergillus （Table 1） . As shown in Table 1 , the frequency of dermatophytes isolated was as follows: T. rubrum 24.2%, T. mentagrophytes var. interdigitale 16.9%, T. mentagrophytes var. granular or mentagrophytes 4.0%, T. verrucosum 1.6 % and Epidermophyton floccosum 3.2%. After C. albicans（27.5%）the most commonly isolated non-albicans species were C. parapsilosis （8.1%） , C. glabrata（4.9%） , C. krusei and C. tropicalis （each 3.2%） .
Eighty patients（64.5%）had fungal infection on their hands and 44（35.5%）on their feet. All patients showed only fingernail or toenail involvement. Of the 58 patients with onychomycosis of candidal origin, 42（72.4%）were females and 16（27.6%）males. Of the 62 patients with onychomycosis of dermatophytic origin, 26（41.9%）and 36（58.1%）were females and males, respectively.
The prevalence of onychomycosis of dermatophytic origin in toenails and fingernails was 51.6% and 48.4% and of candidal origin was 13.8% and 86.2%, respectively. Aspergillus niger and A. flavus were the only saprophytic moulds observed in toenails of 4（3.2%） male subjects（Table 2） . In fungal infections of fingernails, after yeasts the most common isolated agent was dermatophytes and no saprophytic moulds were observed. Table 4 shows the various clinical types. DLSO was the most common clinical type, being encountered in 60 patients（48.4%） , followed by PSO with paronychia（33 patients; 26.6%） , PSO without paronychia（23 patients; 18.5%）and TDO（8 patients; 6.5%） . SWO was observed in none of the patients. Discoloration（yel-low-brown to brown-black）was observed in all of the patients and subungual hyperkeratosis leading to detachment of distal part of the plate from its bed with a furrowed base and ragged edge and onycholysis were the common features of dermatophyte infections that were seen in 83.3% of T. rubrum infections. In PSO with paronychia cases, the skin appeared edematous with erythema and sometimes painful and in 75.8% the nails were rough, convex and 24.2% had transverse grooves. 
2（1.6）
Females outnumbered males: 68（54.8%）of the 124 patients were females and 56（45.2%） were males. The female to male ratio being 1.22：1. The age range was 1-79 years（Tables 2 and 3） . In this study, females were affected more frequently than males and in both sexes, onychomycosis occurred most frequently in the age group of 40-49. 
Discussion
Because of difficulties in treatment of onychomycosis, determining the identity of the causative organism of the disease is essential in choosing the most appropriate therapy. In our study, 40.2% of the samples were positive in both culture and direct vision. The etiological fungal agents were 50% dermatophytes, 46.8% yeasts, and 3.2% saprophytic moulds. According to Mügge et al.
7）
, 54% of the nail samples were positive in both culture and by direct vision. The disease is known to occur at any age, but is more common between 40 and 60 years and is unusual prior to puberty 3） ; but in our study, however, 12.9% of the patients showed the disease before puberty. Overall, onychomycosis is more common in adults than children and its prevalence increases with age
8）
. Investigators assert that the slowly advancing age of the population is the single reason behind the rise in incidents of onychomycosis 8） . In the present study, the majority of subjects（65%）were under 49 years of age and the age group of 40-49 contained the highest prevalence of onychomycosis. This observation is in agreement with other studies . T. mentagrophytes is a common worldwide dermatophyte species composed of two chief varieties: T. mentagrophytes var. interdigitale（anthropophilic form）and T. mentagrophytes var. mentagrophytes（zoophilic form） . In this study we identified these two varieties of T. mentagrophytes, which was a positive point for our study. However, only a small number of the mentioned studies provided different varieties of T. mentagrophytes isolates. In this study, T. rubrum（24.2%）was the main etiologic agent followed by T. mentagrophytes var. interdigitale（16.9%）and the presence of the zoophilic variety of T. mentagrophytes（var. granulare or mentagrophytes）was infrequent and isolated only in 4.0% of the diseased nails（Table 1） . This finding is in concordance with a Turkish study on onychomycosis, in which T. rubrum（48.0%）and T. mentagrophytes var. interdigitale（26. 6%）were responsible for most of the nail infections , T. rubrum has been the principal or one of the main dermatophyte species responsible for tinea unguium, whit the exception of one Iranian study which found only two cases（7%）of T. rubrum infection
27）
. Moreover, our results are in accordance with a report in Brazil that 10. 4% of toenails were contaminated with T. rubrum（80%）and T. mentagrophytes（20%）
28）
. Similar results in other studies found that T. rubrum was the most common pathogen causing onychomycosis 15,29−33） , whereas Gupta et al. reported that T. mentagrophytes was the main etiological agent followed by T. rubrum in distal and lateral subungual and white superficial onychomycosis in toenails 34） .
The results of the present study showed that onychomycosis was more frequent in fingernails （64. 5%） , whereas Ilkit 25） reported that toenails were more involved in the Turkish people in his study. This difference might be due to the lifestyle differences between members of the two populations. The 真菌誌 第 51 巻 第１号 平成 22 年 26 . Furthermore, in fungal infections of toenails, after dermatophytes the most common isolated agent was yeasts and then saprophytic moulds.
Similar to some Iranian 17,26,27） and a Japanese 36） study, the frequency rate of tinea unguium was higher in males than females and was rarely seen in children. In fact, this type of onychomycosis is more frequently seen among adults 37） and in a study on 382 Iranian children aged ＜ 16 years suspected to have dermatophytic lesions, tinea unguium has not been found
38）
. This could be due to smaller nail plate, diversity in nail plate structure, less exposure than adults to trauma and environments containing fungi and rapid growth of nails in children, which results in the elimination of dermatophytes from their nails. In our study, tinea unguium occurred in age group ＞ 19, most of which（38.5%）was in the 50-59 age group. This finding is somehow in accordance with results described by Pieʼrard 8） , that 15-80% of elderly people in Europe seem to be affected. Candida albicans has the greatest pathogenic capacity and has been described as the prevailing species in producing onyxis primary or secondary to paronychia . Yeast onychomycosis accounts for nearly half of all nail disorders 31） and 46. 8% of the suspected subjects examined in this study for this form of the disease proved to be infected. Incidence of onychomycosis of candidal origin is higher in adults and 2-3 times more in females than in males
4）
. In our study, similar to the previous Iranian study 17） , candidal onychomycosis of fingernails is also more prevalent （about 3-1）in women than in men which could be due to constant immersion of their fingers in water and mechanical trauma. Although onychomycosis is a very common superficial fungal disease, it is considered rare in children. In children, candidal onychomycosis has been seen in fingernails only due to finger sucking habits 40） . In the present study, only one case of candidal onychomycosis in a child the age of one year was seen.
Out of 124 onychomycosis-infected patients, 58 cases （46.8%）were infected with candida spp. of which the most common yeast isolated was C. albicans with other candida species; C. parapsilosis, C. glabrata, C. krusei, and C. tropicalis following. There is a vast variation of the percentage isolation of yeasts reported in diseased nails in Iranian and other publications. According to Moghaddami and Shidfar
. In the present study, the incidence of involvement of nails with saprophytic moulds was much less than other agents and was seen in the great toenails of males over 60 years of age. Bonifaz et al. 44） in his study, refers to contact with soil, trauma of nails, and peripheral vascular disease of nails as predisposing factors for their involvement with moulds and introduces the great toenail as the most common place for mould involvement. 
17）
, 66.04% of patients with onychomycosis were infected with Candida spp., of which C. albicans and C. parapsilosis were the predominant species. The most common species of Candida to have caused onychomycosis in our patients were C. albicans（27.5%）and C. parapsilosis（8.1%） , as has also been reported by others 16,17,20,21） . In two studies, 7,29） C.
parapsilosis and in another study C. tropicalis surpassed C. albicans to become the most common species isolated 25） . The last group of fungi causing onychomycoses is opportunistic moulds. This is the most debated fungal group since the majority of species tend to be found as contaminants in the environment or the skin 31） . Ungual infections caused by moulds account for 1-6% of all nail infections in European countries and up to 50% in certain areas of Africa . In Iran, only limited data are available on fungal nail infections, particularly by opportunistic moulds, and according to some restricted conducted studies, saprophytic moulds are one of the least important etiological agents.
The most frequently isolated mould from infected nails is Scopulariopsis brevicaulis, a species that has been demonstrated to have an affinity for keratin and has been a etiological agent of onychomycosis in most studies 5,7,16,21,44） . After Scopulariopsis, the most frequent saprophytic agents were Aspergillus（A. fumigatus, A. versicolor, A. terreus, A. flavus） , followed by Scytalidium, Fusarium, Acremonium, Cladosporium, Alternaria and Curvularia
Conclusions
An exact diagnosis of onychomycosis is based on clinical findings, direct microscopic investigation and mycological culture. Because the pattern of onychomycoses in any region changes with time, periodic studies are necessary to achieve novel information about prevalence and the causative agents of the infection; such information is extremely useful in the treatment of onychomycosis. Furthermore, as pathogens involved in onychomycosis vary according to each geographical area, treatments should be based on studies carried out in the same region.

{'paper_id': '45966997', 'title': 'Onychomycosis in Iran: epidemiology, causative agents and clinical features.', 's2_url': 'https://api.semanticscholar.org/CorpusID:45966997', 'source': 'S2ORC'}