ONYCHOMYCOSIS: CLINICAL FINDINGS, ETIOLOGICAL AGENTS AND EVALUATION OF LABORATORY METHODS

- The aim of this study was to identify the etiological agents in patients with suspected onychomycosis, and to carry out comparative testing of individual or combinations of tests: direct microscopy with KOH and Blankophor (BP), culturing on Sabouraud’s dextrose agar (SDA), diluted Sabouraud’s dextrose agar (D-SDA) and dermatophyte test medium (DTM). From 70 nail samples (65 toenails, 5 fingernails), 46 (60.5%) had at least one of five positive tests. Isolation was possible in 41, while in 5 samples the presence of fungi was observed by KOH and/or BP. Dermatophytes were most frequently isolated (80.5%) where Trichophyton rubrum was predominant. Candida spp. was isolated in 9.8%, Aspergillus spp. 4.9%, Alternaria spp. 2.4% and Fusarium spp. 2.4%. Application of BP as an individual test was the most sensitive method. The combination of BP with DTM or D-SDA provides the best sensitivity and allows the identification of fungi to the species/genus level.


INTRODUCTION
Onychomycosis is a fungal infection of one or more of the nail units and accounts for up to 50% of all nails disorders.Among superficial mycoses, onychomycosis is most frequently diagnosed and treated and represents about 30% of all mycotic cutaneous infections (Nazar et al., 2012).Although onychomycosis is rarely life threatening, it affects approximately 5% of the population worldwide and therefore represents an important public health problem (Kaur et al., 2008).Some studies report even higher prevalence of onychomycosis of more than 10% of the general population due to differences in socioeconomic and cultural factors (Grover et al., 2012).Predisposing factors of this disease include the presence of positive history of onychomycosis, increasing age, trauma to the nail, poor hygiene, hyperhidrosis, diabetes, immunosuppression, contact with animals and soil, poor peripheral circulation, HIV infection, avid sports participation and many others (Takehara et al., 2011;Sabadin et al., 2011;Tchernev et al., 2013;Ozkan et al., 2013;Pierard, 2001).
Onychomycosis can be classified into four clinical types according to the pattern of infection (Gupta et al., 2000;Vennewald et al., 2008).Distal and lateral subungual onychomycosis, the most common type, affects the distal portion of the nail bed and the underside of the nail.In proximal subungual onychomycosis, the fungus enters through the cuticle to invade the proximal portion of the nail bed.In superficial white onychomycosis, direct fungal invasion of the nail plate surface produces a white crumbly appearance.Candidal onychomycosis commonly affects fingernails and is usually caused by Candida albicans.
Progression of any of the four types so that the entire nail unit becomes involved is known as total dystrophic onychomycosis (Gupta et al., 2000;Vennewald et al., 2008).Onychomycosis can be caused by dermatophytes, yeasts and nondermatophyte molds.Most cases of disease are caused by dermatophytes (approximately 90% in toenails and at least 50% in fingernails).The dominant dermatophyte species are T. rubrum and T. mentagrophytes (Wisselink et al., 2011).Laboratory confirmation of fungal infection is very important, since many disorders, such as psoriasis, nail trauma, nail tumors, lichen planus and other diseases can have similar clinical manifestations.Peripheral vascular disease, many systemic diseases and aging can cause changes in the nail that can also mimic onychomycosis (Lawry et al., 2000).Therefore, an accurate diagnosis of infection needs to be made before initiation of antifungal therapy because of the long duration of the treatment, its high cost and potential side effects of the drugs.In addition, some antifungals are more effective against certain organisms than others (Tchernev et al., 2013).Knowledge of the zoophilic, geophilic or anthropophilic origin of the dermatophyte may allow the setting-up of prophylactic measures (Robert, 2008).
The aim of this study was to identify the etiological agents in patients with suspected onychomycosis, and to carry out comparative testing of individual or combination of tests that were used in this study: direct microscopic preparation with potassium hydroxide, and Blankophor, culturing on Sabouraud's dextrose agar, diluted Sabouraud's dextrose agar and dermatophyte test medium.

Patients
The study population comprised 65 suspected cases of onychomycosis attending the dermatology outpatient department from November 2012 till February 2013.The criterion for enrolment was nail dystrophy highly suggestive for onychomycosis.We excluded patients who had used topical or systemic antifungal drugs within the previous 2 and 4 weeks, respective-ly, and patients who did not consent to have a sample clipped from their nail.All patients were referred for sampling to the Medical Mycology Reference Laboratory, Institute of Microbiology and Immunology, Faculty of Medicine, University of Belgrade.Seventy samples were taken as in five patients suggestive changes were found on the fingernails besides their toenails.All subjects were informed about the aims of the study and were eligible to participate if they provided informed consent.For each patient we recorded basic demographic information, site of infection (toenails or fingernails), clinical type of onychomycosis and presence of risk factors (diabetes, poor peripheral circulation, contact with animals and soil).

Sampling
Nail samples were obtained by scrapings/clippings depending on clinical type of onychomycosis.First, the nail area was cleansed with alcohol.Then, for distal and lateral subungual onychomycosis, the abnormal nail was clipped proximally to the nail bed and the underside of the nail plate was scraped with a curette.For proximal subungual onychomycosis, samples were taken by scraping around the area of the lunula.For superficial white onychomycosis, the white spots on the nail were scraped, and for the candidal onychomycosis the material closest to the proximal and lateral edges was obtained.For total dystrophic onychomycosis, any abnormal area of the nail plate or bed was used as a specimen.Samples obtained by clipping were cut into smaller pieces before subsequent examination.All samples were collected in sterile containers and labeled for identification.

Laboratory procedures
Each sample was divided into two parts: the first part was used for direct microscopy and the rest for fungal culture.Two types of direct microscopic preparations (DMP) were made: first with 15% potassium hydroxide (KOH) observed under a light microscope and second with fluorescent dye Blankophor (BP) and observed under a fluorescent microscope, both under 10× and 40× magnification.
The second part of each specimen was cultured on the following three mycological media: Sabouraud's dextrose agar (SDA), diluted Sabouraud's dextrose agar (D-SDA) and dermatophyte test medium (DTM).Plates were then incubated for 3 weeks at 28°C and 37°C and checked periodically for growth.If there was growth, the pathogen was identified by colony morphology and microscopic characteristics (Hoog et al., 2000).Plates with no growth even after 3 weeks of incubation were considered negative.

Statistical analysis
To analyze the data, a criterion standard for the diagnosis of onychomycosis was defined as clinical morphologic findings suggestive of onychomycosis plus at least one positive test result.
The sensitivity of each test was calculated as the proportion of positive results obtained in a specific test in individuals with a confirmed diagnosis of ony-chomycosis and with any other positive test used in this study.The negative predictive value (NPV) of each test was calculated as the proportion of negative results with a specific test compared with any negative test result.
Table 2 shows the statistical analysis of individual diagnostic tests, the number and frequency of positive results, as well as their sensitivity and negative predictive value.In order to increase the sensitivity of tests they were combined (Table 2).

DISCUSSION
Onychomycosis is widespread and in the general population about 3% and up to 5-15% of older people are affected (Pierard, 2001).In our study, the mean age of the patients was about 56 years.Many reports indicate that the prevalence of onychomycosis increases with age, with the highest prevalence among individuals above 60 years-of-age (Saunte et al., 2008).In our study, onychomycosis increased with age.The highest prevalence was in the age groups 61-70 and 71-80 years with an incidence of 26.1% and 28.3%, respectively.Elderly patients are more likely than the general population to have superficial infections due to structural changes, altered immunological response and different environmen-tal influences.Mechanical and functional changes, as well as the reduced immunological capacity of aging skin predispose the elderly to a variety of skin diseases, including onychomycosis (Pierard, 2001).
Our results showed a predominance of males compared to females (1.3:1) in onychomycosis cases and that toenails were more frequently affected than fingernails.In our study, big-toe onychomycosis was reported most frequently; this is in agreement with other studies, probably because of its bigger size, which predisposes it to increased trauma.A similar predominance of males in onychomycosis cases was reported in studies from the USA, Europe and Canada (Gupta et al., 2000).This is probably related to the frequent traumas and active involvement in sports in males with a higher risk of exposure, in particular to dermatophytes.By contrast, data from East Asia, Tunis, Northern Greece and Indonesia showed a predominance of females and that fingernails were affected more often than toenails.They found that the main predisposing factor is hand-wet work in female populations (Bramono, 2005).
Onychomycosis of fingernails was less common than toenail disease.The right thumb was the commonest fingernail involved.In our study, we had three cases of candidal onychomycosis and all grew Candida albicans on culture.We did not have any case of superficial white or proximal subungual onychomycosis.Other studies also showed a low frequency of these two clinical types as they mainly oc- cur in immunosuppressed patients, often associated with AIDS (Degreef, 2008).
In most studies, the most common cause of onychomycosis is dermatophytes.The most frequent are T. rubrum 60%, T. mentagrophytes var.interdigitale 20%, and E. floccosum 10% (Grover et al., 2012;Degreef, 2008;Kaur et al., 2008;Welsh et al., 2010).In our study, the most common fungal isolates were also dermatophytes, where the predominant species was T. rubrum (61%), but in contrast to other studies, we found greater frequency of E. floccosum (9.8%) than T. mentagrophytes (4.9%).Onychomycosis of toenails was present in 43 cases.As regards toenails, the most frequent clinical manifestation was distal and lateral subungual onychomycosis (28/43) followed by total dystrophic onychomycosis (15/43).Many reports from the literature have shown that the distal and lateral subungual and total dystrophic onychomycosis account for more than 90% of all cases of  As a standard for the diagnosis of onychomycosis, the majority of studies reported an association between clinical features and at least one positive laboratory test.Traditional methods used for diagnosis have been KOH preparation and fungal culture on SDA (Weinberg et al, 2003).These tests have poor sensitivity when they are used individually, so tests are often combined for better results (Lawry et al., 2000).
DMP with KOH is rapid, simple and the most inexpensive technique, which requires minimum infrastructure but a great amount of expertise to interpret the smears.Nail samples are placed on the slide in one drop of KOH that digests keratin and reveals fungal elements and is easily visible under a light microscope at 40× magnification.However, the KOH preparation can only detect the presence of fungal elements, while fungal identification is not possible (Manjunath et al., 2008).Various studies have shown varying sensitivity, some authors reported sensitivity of 50-60%, even up to 80%, whereas our study showed sensitivity of 45.7% (Grover C et al., 2012).In order to increase the sensitivity we did another test with fluorescent dye BP that binds to chitin and cellulose in the fungal cell wall and fluoresces when exposed to UV light.Studies have shown that the detection of fungal elements is faster and more accurate when, in addition to KOH, BP is used.In our study, the combination of BP with KOH increased the sensitivity up to 87%.Studies have also shown that BP has a higher sensitivity compared to KOH of up to 100% (Vennewald et al., 2008;Degreef, 2008).Our study also showed that the sensitivity of BP was the highest when used as an individual test (82.6%).The BP test is easy to perform and provides quick results, but its main drawback is that does not allow fungal identification, which is essential for proper treatment.
Culture is generally considered the gold standard since it is the only test in routine use that can identify the species of fungus causing onychomyco-sis.Reported sensitivity of fungal culture varies from 25% to 80% with approximately 30% false-negative results (Hamer et al., 2006).False-negative results in culture may arise if the nail sample contains only nonviable organisms, when an insufficient sample is collected, when the nail sample is clipped distal to the fungal growth, or if the nail sample is not crushed before subjecting to the test (Weinberg et a, 2003).A modification of the standard method of culture by pre-treatment of nail samples with trypsin enables improved sensitivity (Xess et al., 2007).
The recommendation for fungal culture is to use SDA and at least one additional medium (Grover C et al., 2012).In our study, nail samples were cultured on SDA, D-SDA and DTM.SDA is a basic mycological medium and reported sensitivity is from 50% to 70% (Tchernev et al., 2013).DTM is an alternative culture method that can be used to confirm diagnosis of onychomycosis.The growth of dermatophytes is indicated by a change in color of the media from yellow to red in response to alkaline metabolites that result from the growth of dermatophytes.Although change in color appeared in 3-7 days, it is not possible to identify specific organisms and identification by microscopic characteristics is possible after 14-21 days (Rich et al., 2003).D-SDA has a lower glucose concentration than standard SDA, which enables better fungal sporulation and facilitates identification (Takashio, 1972).Our study showed the lowest sensitivity in SDA (65.2%), and the best in D-SDA (78.3%) when used individually.By combining all three media, sensitivity increased to 87%, which reduced the number of false-negative results.Since these methods are time-consuming the main shortcoming is the delay in diagnosis of onychomycosis, which can lead to total nail dystrophy and nails may not regain their normal architecture in spite of adequate treatment.
In our study, the best sensitivity and NPV was shown by the combination of BP with DTM, followed by BP with D-SDA.The specificity and positive predictive value of each test could not be calculated because our criterion standard included all applied tests.The clinical relevance of specificity is unclear, since these tests would not be used to screen clinically normal nails for onychomycosis.The focus of such testing is to minimize the frequency of falsenegative test results in achieving the diagnosis.
The most common cause of onychomycosis in our patient population was similar to countries in Europe, is T. rubrum.Our study has shown that the application of BP as an individual test is the most sensitive method for the diagnosis of onychomycosis, but does not allow the identification of species/ genus.The combination of BP with DTM or D-SDA provides good sensitivity and allows the identification of fungi to the species/genus level, but takes time.Recommendations are to use a combination of laboratory tests for the diagnosis of onychomycosis.Further research on a larger number of samples and the introduction of new methods such as PCR are necessary in order to obtain accurate and timely diagnosis of onychomycosis.

Fig. 1 .
Fig. 1.Distribution of positive patients according to fungal organism and clinical type of onychomycosis

Table 1 .
Contact with animals and soil in positive patients by age

Table 2 .
Comparison of individual tests and combination of tests in laboratory diagnosis of onychomycosis