Descriptive study of onychomycosis in a hospital in São Paulo

Onychomychosis, a nail fungus infection is the most frequent nail ailment, constituting about half of all nail disorders. It can be caused by dermatophytes, non-dermatophytes, yeasts and Prothoteca spp. Methods include 5407 samples of patients with suspected onychomycosis, studied from January 2002 to December 2006, by direct mycological examination and fungi culture. The diagnosis of onychomycosis was confirmed in samples from 3822 direct mycological and/or culture positive. The diagnosis was established by culture for fungi. Among the 1.428 identified agents, the dermatophytes were responsible for 68.6% (N = 980) of cases, followed by yeasts with 27.6% (N = 394), non-dermatophytes fungi with 2.2% (N = 31), Prothoteca spp with 0.1% (N = 2), and associations with 1.5% (N = 22). Females were more affected, with 66% (N = 2527) of cases, and the most affected age group ranged from 31 to 60 years of age (median 47 years). Fungal microbiota is often changed in the world, both quantitatively and qualitatively, and is affected by several environmental factors. Thus, the periodic review of the composition of this microbiota is important to evaluate the epidemiology and thus proportion a better therapeutic response.


Introduction
Onychomycosis is a fungal nail infection which can be caused by dermatophytes, yeast, non-dermatophyte filamentous fungi (NDFF) and currently Prototheca spp, affecting approximately 8% of the world population (Effendy I et al., 2005;Zaitz C et al., 2006). This nail ailment represents 30% of the fungal surface and around 50% of nail diseases (Ghannoum MA et al., 2000). The main onychomycosis etiologic agents are dermatophytes, which are isolated in 75% of cases. Candida yeasts occur in around 18-20% of cases and NDFF reach percentages ranging from one to five percent of cases (Agarwalla A et al., 2006). problem in our midst. Thus, onychomycosis affects the quality of a patient's life and causes esthetic losses, reflecting directly on self-esteem, vanity, social discrimination and the patient's working potential (Lopes JO et al., 1999).
Although nail infections are common, nowadays they are associated with difficult treatment and are reported as having high rates of recurrence and therapeutic inefficacy. The knowledge of ecology, etiology and distribution of the biota of the main etiological agents allows for a better comprehension of natural history, evolution and may contribute in the future for new therapeutic modalities (Schroeff J et al., 1992).
In this retrospective study, an analysis of all positive mycological examinations from patients with clinical onychomycosis diagnosis from the São Paulo Hospital Dermatology Department's Mycology Laboratory was performed, covering a period from January 2002 to December 2006 The objectives were: 1) To analyze the sensitivity of a diagnostic laboratory for onychomycosis in the São Paulo Hospital Dermatology Department's Medical Mycology Laboratory. 2) To analyze the distribution of cases of onychomycosis in relation to demographic data: age and sex. 3) To analyze the distribution of onychomycosis cases according to the main etiological agents: dermatophytes, non dermatophyte filamentous fungi (NDFF), protists and yeasts.

Material and Methods
This paper includes a descriptive study conducted at the São Paulo Hospital Dermatology Department, by reviewing the results of mycological examinations performed from January 2002 to December 2006.
All patients seen at the dermatology ambulatory that presented, among the possible diagnoses, a clinical suspicion of onychomycosis, were submitted to mycological examination in order to confirm the onychomycosis diagnosis.
Reagents, culture media, equipment and consumable material are routinely used in the medical mycology laboratory of the Dermatology Department in Hospital in São Paulo. The study was approved by the Ethics and Research Committee of the São Paulo Hospital.

Laboratory procedures Direct microscopic examination
The material was obtained by scraping with a dental explorer, a dental curette nail and examined between a slide and coverslip after clarification with potassium hydroxide with 20% dimethyl sulphoxide added (Lacaz CS et al., 2002;Sidrim JJC and Rocha MFG, 2004).

Culture
Macroscopic aspects: The culture media for isolation and analysis of morphological species of Dermatophytes are Sabouraud's medium with chloramphenicol and Sabouraud medium with cycloheximide. Non-dermatophyte filamentous fungi, yeast and Prototheca spp are grown on Sabouraud cloranfenicol. In our laboratory, the material was inoculated into three tubes of the means mentioned: first, collection of clinical material and isolation of agents; second, collection of clinical material after the first seven days from the initial collection and isolation of the agent; the third collection of clinical material after seven days from the second collection and isolation of agents. The growth period to occur during the full maturity of fungal structures is: dermatophytes and NDFF observed around 15 days, Prototheca spp and yeast about seven days (Lacaz CS et al., 2002;Sidrim JJC and Rocha MFG, 2004).
Microscopic aspects: The technique of microculture on slides with a culture medium with agar-cornmeal for the identification and determination of genus and species of dermatophytes, yeasts and NDFF was used. In Prototheca spp, an inoculum of the culture was withdrawn to be identified and was placed on a slide with lacto-phenol blue dye cotton. It was possible to observe the shaped structures of morula. Species identification is performed through biochemical tests. (Lacaz CS et al., 2002;Sidrim JJC and Rocha MFG, 2004).

Statisticalanalysis
To analyze the results, the chi-square test in MINITAB 14 and tables in EXCEL 2007 were used for characterization of the Clinical sample material obtained from nails of patients with suspected onychomycosis. This was done to evaluate the association between the variables of age, gender, test result, the affected site and agent. In all tests, the 0.05 significance level was set.

Results
Characterization of the samples studied Direct microscopic examination and culture Between January 2002 and December 2006, 5407 mycological tests were carried out with clinical suspicion of onychomycosis in 3541 patients of any age, sex and race, which were analyzed by direct microscopic examination and/or culture. Of the total amount tested, 71% (3.822/5.407) were positive for laboratory diagnosis of onychomycosis. Of the remaining mycological examinations that were analyzed, 29% (1585/5407) were negative.
The distribution of mycological examinations analyzed ( Figure 1) was based on laboratory confirmed cases of onychomycosis using direct microscopic examination and culture (both are criteria to define positivity).

Onychomycosis age and gender
Regarding age, it was observed in the table below that among the 3822 samples with a diagnosis of onychomycosis, 60% (2.287/3.822) presented data on age, with a minimum age of three months (0.25 years) and the maximum age of 98 years. The mean age was 46.9 years, standard deviation ± 17.02; observing a coefficient of variation of 36.3%, concluding that the most representative of the age of this population would be the median: 47 years old.
The total distribution of patient samples analyzed in relation to age is shown in Figure 2. Most samples of patients (60%) are aged from 31 to 60 years.
In terms of the distribution in relation to sex of the 3822 mycological positive tests for laboratory diagnosis of onychomycosis, 2527 patients were female (66%) and 1295 were male (34%). Table 1 shows the distribution according to age and sex in cases of onychomycosis.
The chi-square statistical test, performed to evaluate the association between the positive result of the test vari-Descriptive study of onychomycosis 487  ables and age of the patients by sex, showed a significant difference (p < 0.0001) between the percentage of positive results observed in relation to patients age by sex, for the level of significance; that is, the highest incidence of positive results was in the range of 31 to 60 years for females.

Identification of the etiologic agents of onychomycosis
The percentage of culture isolation and identification of agents etiology was 66% (1.428/2.162) of the samples.
The chi-square statistical test, conducted to evaluate the association between the positive result variables of the examination and the type of etiologic agent, showed a difference (p < 0.0001) between the percentage of positive results found in the type of etiologic agent for the level of significance, that is, a higher positive incidence refers to dermatophytes.

Discussion
Onychomycosis epidemiology has multifactorial influence and its prevalence is directly related to age and other population factors, such as lifestyle and association with other diseases. Furthermore, the distribution of pathogens, agents of onychomycosis is not uniform, depending on several factors such as geography, climate of the region, 488 Ribeiro et al.  and population migration (Faergemann J, Baran R, 2003;Brito A, 1992 (Arrese JE et al., 2005). A study conducted in India shows that of the 302 patients, 42.4% (128/302) had the diagnosis confirmed by laboratory examination (Sarma S et al., 2008).
Of the 3822 clinical samples with direct examination and / or positive cultures analyzed in our study, the direct microscopic examination was positive in 99.3% (3.796/3.822) of them, while the culture was positive in 66% (1.428/2.162) of the total samples. Studies in the literature reported 82.45% (94/114) positivity for the direct microscopic examination, and 17.5% (20/114) for culture (Brito A, 1992). The author explains the difference was due to the difficulties that the mycology laboratory went through some time, resulting in damage to research. In 1997, a positivity of 82% (1.707/2.065) for direct microscopic examination and 52% (1.069/2.065) for culture was observed (Elewski BE et al., 1997).
Culture was performed in 56.6% (2.162/3.822) of all samples studied; it was positive in 66% (1.428/2.162). In the studied laboratory, due to the large number of surveys collected in the daily routine, all positive culture direct mycological examination are not made. The cultivation limitations of all samples are the cost of the test and the period necessary for fungus growth.
Comparing our results with the literature, it can be stated that the studied laboratory is efficiently achieving results similar to the Mycological reference laboratories. Besides the difficulty related to culture in all samples studied, it was observed that the positivity obtained by direct examination is significantly higher than that one obtained by culture. This fact can be explained by the uneven distribution of fungi in lesions, the difficulty to collect material properly (especially in the subungueal region) and the ease by which the contamination by airborne fungi and bacterial microbiota, thus hindering the identification of the true etiologic agent, due to limitations inherent in the examination, such as the intense keratinization of nails, which makes microscopic observation of microorganisms becomes difficult; for fungal viability, which can result in false negative cultures and the use of antifungal medications by the patient prior to collection (Carvalho MTF, 1990;Martelozzo IC et al., 2005).
The age group with confirmed onychomycosis ranged from three months to 98 years of age, with a median age of 47 years, the age group 31-60 years, considered the economically active population, was involved in 60% of our sample. Our results are consistent with those of Martins et al., in 2007, who observed in a study with 184 patients, the mean age of onset was 36 to 64 years (62%). Already, in a study with 302 patients, it was found that onychomycosis occurred more in the age group between 21 and 30 years (36%) (Sarma S, 2008).
Descriptive study of onychomycosis 489 Of the 3822 positive mycological examinations for laboratory diagnosis of onychomycosis, 2527 were from female patients (66%) and 1295 male (34%).
According to statistical analysis by chi-square test, there is a significant difference between positive tests in both sexes. These data are consistent with the literature, as some authors have observed that the frequency of onychomycosis ranging from 67% to 74% in females (Souza EAF et al., 2007;Effendy I et al., 2005;Alvarez MI et al., 2005;Koussidou T et al., 2002;Vélez A et al., 1997;Kemna ME and Elewski BE, 1996;Sais G et al., 1995;Schroeff J et al., 1992). Other authors found exactly the opposite, with about 64% of cases occurring in males (Sarma S et al., 2008;Ghannoum MA et al., 2000, Sigurgeirsson B andSteingrimsson O, 2000;Perea S et al., 2000). The reason for these discrepancies may lie in the composition of the population studied, since the fungal infection depends on cultural habits and ecology, as previously described.
Similarly the sample shows the predominance of female patients. In this sampling, the most affected age group was between 31 and 60 years, where greater and more significant (p < 0.0001) incidence of positive results was seen in the range of 31 to 60 years for females. In the literature, studies like those of Ghannoum et al. in 2000 reported a higher incidence of onychomycosis in the active age group with a mean age of 57 years mainly in males (58%) (1063/1832). Patients with onychomycosis were in the range of 40-50 years old (67.4%) of which 71% were female (179/252) (Martins EA et al., 2007;Martelozzo IC et al., 2005).
Some authors explain the increased prevalence of onychomycosis with aging due to some factors, such as peripheral circulation slower, inactivity, inability to cut and care for nails, presence of comorbidities (diabetes, repeated nail trauma, longer exposure to pathogenic fungi, lower immunity). Moreover, the reason why the prevalence of onychomycosis was lower in children, can be justified by quicker nail growth, less exposure to the etiologic agents, a lower prevalence of tinea pedis and to a lesser extent nail invasion (Tosti A et al., 2005;Elewski BE, 1998).
On reviewing the literature, it was found that the main agents of onychomycosis are: T. rubrum, T. mentagrophytes and T. tonsurans (Ghannoum MA et al., 2000;Rodriguez JMT and Jodra OL, 2000;Brito A, 1992). T. raubistscheckii is considered by many mycologists a variant of the T. rubrum and it is rarely isolated in the nail (Brasch J, 2007, Papini M et al., 2004. Furthermore, non-dermatophyte filamentous fungi (NDFF) are cited in literature as etiologic agents of onychomycosis. In this study, Fusarium spp was the most isolated NDFF with 74.2% (23/31). In following came the Scytalidium hyalinum with 19.3% (6/31), Acremonium spp and Aspergillus spp, both with 3.2% (1/31).
It was found that 13.6% of onychomycosis were caused by NDFF. The main etiologic agent identified by the authors was Fusarium spp, 21.2% (28/132) (Brasch J, 2007).
For yeast isolates, Candida spp was found in 98.5% (388/394) (Souza EAF et al., 2007;Martelozzo IC et al., 2005). It was observed that the yeast Candida was most frequently isolated from cases of onychomycosis. The Trichosporum beigelli was isolated in 1.2% (5/394) of our cases as the etiological agent of onychomycosis by yeast and some studies also have similar results (Brasch, 2007;Papini M et al., 2004). Associations and fungi such as onychomycosis agents were also found.
Dermatophyte and yeast occurred in 1.1% of cases (16/1.428) NDFF with yeast at 0.4% of cases (5/1.428) and yeasts from other genera (not Candida) in 0.1% of cases (1/1.428). There are few studies which mention mixed onychomycosis. Studies reported association of onychomycosis caused by Candida spp and other fungi in immunosuppressed patients and reported possible associations of fungi in onychomycosis in a study of 2766 patients (Koussidou T et al., 2002). Onychomycosis "like" caused by Prototheca spp was a peculiarity observed in our study. In respect to this peculiarity, two patients were observed (patients with the same two nails affected), which correspond to 0.1% of the causative agents of onychomycosis. This case was published, the first case in Brazil of onychomycosis caused by Prototheca spp and 3rd case in the world (Zaitz C et al., 2006).

Conclusions
From January 2002 to December 2006, 3822 samples were analyzed with clinically suspected onychomycosis 490 Ribeiro et al.
which were confirmed in the laboratory and allowed for the following conclusions: 1 -The direct microscopic examination method is sensitive, rapid and inexpensive for general diagnosis of onychomycosis. In spite of its culture high specificity, it is difficult to perform, more costly and dependent on several factors such as: collection, culture medium and the skills of the professional who performs it.
2 -The most affected age group in the population studied was 31 to 60 years of age and predominantly female (66%).