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Epidemiology and fungal species distribution of superficial mycoses in Northeast Brazil
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ORIGINAL ARTICLE/ARTICLE ORIGINAL
Epidemiology and fungal species distribution of superficial mycoses in Northeast Brazil ´Epide ´ miologie et distribution des espe ` ces fongiques des mycoses ´ sil superficielles dans le Nord-est du Bre W.P. Silva-Rocha, M.F. de Azevedo, G.M. Chaves * ´ rio de Micologia Me ´ dica e Molecular, Departamento de Ana ´ lises Clı ´ gicas, ´nicas e Toxicolo Laborato Universidade Federal do Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil Received 23 May 2016; received in revised form 16 August 2016; accepted 21 August 2016
KEYWORDS Dermatomycoses; Epidemiology; Fungal distribution; Mycological examination; Northeast Brazil
Summary Objective. — Dermatomycoses are superficial fungal infections which affect the skin, hair and nails of humans and animals. Male and female patients of all ages are affected by this condition. The main etiological agents of dermatomycoses are the dermatophytes fungi of the genera Trichophyton, Microsporum and Epidermophyton, while the main yeasts belong to the genera Candida, Malassezia and Trichosporon. The variation in the distribution of dermatomycoses worldwide justify the conduction of epidemiological studies in order to contribute for the better understanding of patterns of mycological cutaneous infections. This study was conducted from April 2013 to December 2014. Material and methods. — A total of 205 patients were evaluated, while 235 clinical specimens were obtained. From our positive cases of mycological examination, 73 (64.6%) patients were female, while 40 (35.4%) were male. Scales from the skin and nails were collected and observed at optical microscopy after potassium hydroxide clarification. Cultures were performed on Sabouraud Dextrose Agar added chloramphenicol. Identification was performed by classic methodology. Results. — We found that the glabrous skin was the largest source of dermatomycoses (30.11%), followed by toenails (27.4%) and fingernails (17.7%). Regarding onychomycosis, the most affected population was over 50 years old. Trichophyton rubrum was the dermatophyte fungal species more commonly found. Most of the patients with pityriasis versicolor were adults and female. Another important fact observed is that Candida parapsilosis was the most prevalent species. Finally, a high incidence of T. tonsurans in cases of superficial mycoses was observed.
* Corresponding author. Laboratório de Micologia Médica e Molecular, Departamento de Análises Clínicas e Toxicológicas, Universidade Federal do Rio Grande do Norte, Centro de Ciências da Saúde, Rua Gal. Gustavo Cordeiro de Faria S/N, Petrópolis, 59012-570 Natal-RN, Brazil. E-mail address: [email protected] (G.M. Chaves). http://dx.doi.org/10.1016/j.mycmed.2016.08.009 1156-5233/# 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Silva-Rocha WP, et al. Epidemiology and fungal species distribution of superficial mycoses in Northeast Brazil. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2016.08.009
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W.P. Silva-Rocha et al. Conclusion. — Our results clearly demonstrate peculiarities in terms of etiological agents of dermatophytoses distribution in a specific region of Brazil. # 2016 Elsevier Masson SAS. All rights reserved.
MOTS CLÉS Dermatophytoses ; Épidémiologie ; Distribution fongique ; Examen mycologique ; Nord-est du Brésil
Re ´sume ´ Objectif. — Les mycoses cutanées sont des infections fongiques superficielles qui affectent la peau, les cheveux et les ongles chez l’homme et les griffes des animaux. Les patients masculins et féminins de tout âge sont touchés par cette maladie. Les principaux agents étiologiques de la dermatomycose sont les dermatophytes des genres Trichophyton, Microsporum et Epidermophyton, alors que les principales levures appartiennent aux genres Candida, Malasezzia et Trichosporon. Les variations de la distribution mondiale des dermatomycoses justifient la conduction d’études épidémiologiques afin de contribuer à une meilleure compréhension des tendances des infections cutanées mycologiques. Cette étude a été réalisée entre avril 2013 et décembre 2014. Mate´riel et me´thodes. — Un total de 221 patients ont été évalués, 255 échantillons cliniques ont été obtenus. Dans les cas d’examen mycologique positif, 87 (65,4 %) patients étaient des femmes, alors 46 (34,6 %) étaient des hommes. Les produits de grattage de la peau et des ongles ont été recueillis et observés au microscope optique après clarification par l’hydroxyde de potassium. Les cultures ont été effectuées sur gélose Sabouraud additionné de chloramphénicol. Ainsi, l’identification a été réalisée par une méthodologie classique. Re ´sultats. — Nous avons constaté que la peau glabre était la principale source de mycoses superficielles, suivie par les ongles. En ce qui concerne l’onychomycose, la population de plus de 50 ans a été la plus touchée. Trichophyton rubrum a été l’espèce des dermatophytes plus fréquemment trouvée. La plupart des patients souffrant de pityriasis versicolor ont été des adultes et des femmes. Un autre fait important observé c’est que Candida parapsilosis était l’espèce de levure la plus répandue. Enfin, une forte incidence de T. tonsurans a été observée. Conclusion. — Nos résultats démontre clairement les particularités en termes d’agents étiologiques et de la distribution des dermatophytoses dans une région spécifique du Brésil. # 2016 Elsevier Masson SAS. Tous droits réservés.
Introduction Dermatomycosis are superficial fungal infections which affect the skin, hair and nails of humans and animals. They affect both male and female patients of all ages and have a worldwide distribution. However, hot and humid climates such as in tropical and subtropical areas contribute to the high prevalence of number of cases in countries from Latin America, Africa and Asia [2,11,39]. Superficial fungal infections are related to several predisposing factors, including: climate conditions, people migrations, sports activities, prolonged contact with water, lifestyle, immunological status, drug therapy and age of patients [11]. In addition, life conditions and the environment where people are inserted influence epidemiological characteristics of human infection [11]. The main etiological agents of dermatomycosis are the dermatophytes fungi of the genera Trichophyton, Microsporum and Epidermophyton. The non-dermatophyte agents include other filamentous fungi such as Fusarium and Aspergillus and mainly yeasts belonging to the genera Candida, Malasezzia and Trichosporon [9]. Dermatophytes fungi are filamentous, hyaline, septate and present arthroconidia in their micromorphology. The high incidence of these fungi on epidemiological data of dermatomycosis is influenced by the high production of keratinasis, an important enzyme used for acquisition
of nutrients from the environment, but also contributes for fungal invasion of the stratum corneum of epidermis and to degrade keratin of the skin, hair and nails [9]. Therefore, these fungi are known as keratinolytic [6]. Yeasts belonging to the Candida genus are also involved in superficial infections and the main species isolated from dermatomycosis are the Candida parapsilosis species complex, Candida albicans and Candida tropicalis. The lesion occurs mainly in intertriginous areas of the body, such as interdigital, inguinal, intergluteal and inframammary regions as well as nails and periungueal regions [37]. The genus Malassezia comprises lipophilic yeast-like fungi that are part of the normal skin microbiota in humans and warmblooded animals [15]. The variation in the distribution of dermatomycoses according to the local geography, inspires the conduction of epidemiological studies in order to contribute for the better understanding of patterns of mycological dermatomycoses and etiologic distribution of the most common fungal agents which cause infection in the skin and its annexes. Of note, each species presents peculiarities in terms of body sites and age of patients which they cause disease. Therefore, this study aimed to conduct an epidemiological survey of dermatomycoses derived from patients in a private laboratory located in Northeast Brazil. This data will contribute to increase the discussion on the epidemiology of dermatomycosis in a region of Brazil where this clinical scenario is poorly investigated.
Please cite this article in press as: Silva-Rocha WP, et al. Epidemiology and fungal species distribution of superficial mycoses in Northeast Brazil. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2016.08.009
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Materials and methods Collection of clinical specimens Strains were collected from April 2013 to December 2014 at a private clinical laboratory analysis from the city of Natal, Rio Grande do Norte State, Northeast Brazil and sent for further identification at the Laboratory of Medical and Molecular Mycology, Department of Clinical and Toxicological Analysis, Federal University of Rio Grande do Norte. The patients were referred by dermatologists from different clinical ambulatories of the state. During the period of the study, 205 patients of both genders and different ages ranging from 3 to 83 years old were evaluated, according to the presence of lesions suggestive of superficial mycosis in different areas of the skin, scalp, hair and nails. Samples were collected according to the anatomical site and the aspect of lesion and clinical indication. Circular lesions of glabrous skin with dry appearance and well-defined edges and peeling areas were collected by scraping the edges of the lesion with the aid of a sterile scalpel. Scales were stored in sterile Petri dishes. Lesions from intertriginous areas were collected with a sterile swab moistened with sterile saline solution at 0.9%. Scales from the scalp and nails were collected by scraping with a sterilized scalpel. When white piedra was suspected, a lock of hair was collected with scissors. Lesions with hypopigmented or hyperpigmented maculae suggestive of pityriasis versicolor were collected using the Method of Porto [22].
Sample processing and culture Samples collected from different clinical specimens were analyzed according with the nature of the lesion. Hair, skin and nail scales were treated with 20% Potassium hydroxide (KOH) during 30 min. Samples collected with a sterile swab were observed with optical microscopy without staining and clarification (CX21, Olympus, Japan). All the clinical samples were analyzed with optical microscopy at 400 of magnification and the presence of fungal structures such as blastoconidia, pseudohyphae, true hyphae and conidia were observed. Clinical samples were seeded on the surface of Sabouraud Dextrose Agar (Dextrose 40 g, Peptone 10 g, Agar 15 g, Distilled water 1000 mL) added chloramphenicol (50 mg/mL), at seven equidistant points and incubated at 30 8C and culture growth was analyzed daily. Cultures without growth during a period of up to four weeks were considered negative.
Mycological diagnose criteria Besides the fact that all the patients showed lesions suggestive of superficial mycoses as forwarded by dermatologists, for molds, yeasts and yeast-like fungi that can be found as commensals of several anatomic sites, mycological examination was only considered positive when besides positive culture, fungal structures were found at the direct examination, including some degree of evagination or pseudohyphae for the Candida species that may undergo morphogenesis. Other criteria adopted were the positive mycological examination (direct examination and culture) within two
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different occasions (different days of collections) and if colonies growth was found for the seven equidistant points seeded on the surface of the Petri dishes.
Identification of isolates grown in culture The clinical isolates were identified based in their phenotypic, biochemistry and nutritional characteristics. The identification of yeasts was performed by classical methodology [21]. The presumptive identification of the main Candida species was performed by analyzing the color of colonies grown on Chromagar Candida1 (CHROMagar Microbiology, Paris, France). The micromorphological aspects were observed by culturing pure colonies on the surface of cornmeal agar added Tween 80. Carbon auxanogram and zymogram tests were performed to evaluate biochemical characteristics of yeasts [41]. The filamentous fungi were identified by analyzing the macromorphological aspects of colonies (appearance, texture, development time, and coloring of verse and reverse). The micromorphological aspects were observed by microculturing the colonies in potato dextrose agar. The typical structures of genus and species such as microconidia, macroconidia, chlamydoconidia, and coiled spirals were observed by staining a colony fragment with lactophenol cotton blue, visualized by 400 of magnification with optical microscopy [17].
Results In the present study a total of 205 patients were evaluated, while 235 clinical specimens were obtained. A total of 225 samples were analyzed by direct microscopic examination (DME) and culture. Of note, 113 clinical specimens were positive, while 122 were negative for mycological examination. We obtained 42.7% (96 cases) correspondence between direct examination and culture (Table 1). From our positive cases of mycological examination, 73 (64.6%) patients were female, while 40 (35.4%) were male. The main body sites infected with superficial mycosis were the glabrous skin (including the cases of pityriasis versicolor), followed by the nails and scalp (Table 2). Of note, Table 3 describes the distribution of dermatomycosis Table 1 Correspondence between direct examination and culture of mycological examination performed in Natal City, Rio Grande do Norte State, Brazil from April 2013 to December 2014. Correspondance entre l’examen direct et culture mycolo´ alise ´ s `a la ville de Natal, Rio Grande do Norte, Bre ´ sil gique re ´ cembre 2014. entre avril 2013 et de Direct microscopic examination (DME)
Culture (CULT)
Number of cases
Percentage (%)
DME + DME DME Total
CULT + CULT + CULT
96 07 122 225 a
42.7 3.1 54.2 100
a The total number of 225 excludes 10 samples collected by method of Porto for pityriais versicolor diagnosis, whose cultures were not performed.
Please cite this article in press as: Silva-Rocha WP, et al. Epidemiology and fungal species distribution of superficial mycoses in Northeast Brazil. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2016.08.009
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W.P. Silva-Rocha et al. Table 2 Percentage of positive mycological examination according to the different body sites of patients affected with superficial mycoses separated by age range. Samples were collected in Natal City, Rio Grande do Norte State, Brazil from April 2013 to December 2014. Pourcentages des ´echantillons avec examen positif par rap´ par les mycoses superficielles port au site corporel affecte ˆ ge. ´Echantillons collecte ´ s `a la ville de ´ pare ´ s par tranche d’a se ´ sil entre avril 2013 et Natal, Rio Grande do Norte, Bre ´ cembre 2014. de Body site
N (%)
Age range (years)
Fingernail Toenail Glabrous skin Feet Scalp Inguinocrural Total
20 (17.7) 31 (27.4) 33 (29.2) 5 (4.4) 13 (11.5) 11 (9.7) 113
28—83 26—83 6—73 17—57 03—26 22—60 03—83
among different body sites according to the age class. We could observe that for the infants (up to 10 years old), most of positive cases were from the scalp, whereas in adolescents and young adults (from 11 to 40 years old) the glabrous skin was the predominant site. In middle age adults and the elderly (40 years old and above), most of the positive mycological examinations were obtained from the nails. Ten samples with hypopigmented scaly areas were collected by the Method of Porto [22] to analyze the presence of Malassezia spp. In six occasions, mycological examinations were positive, as we could observe the presence of blastoconidia and short and tortuous hyphae with an aspect previously described as ‘‘spaghetti and meatballs’’ at the direct examination [19]. All the six positive cases were obtained from the glabrous skin, on the upper region of the body. Five of the six positive cases occurred in female patients and the mean age of all the subjects was 36 years old (6, 19, 21, 34, 67 and 69 years old). Besides, only a single case was found for a 6-year-old female infant (Table 3). When analyzing the distribution of cases of superficial mycosis in this study, we observe a difference for the prevalence of infections caused by either yeasts or filamentous
fungi. From the 113 positive cases, yeast infections were observed in 45 of cases (39.8%) while filamentous fungi were observed in 68 of cases (60.2%). The superficial mycoses due filamentous fungi were predominantly caused by dermatophytes (59 positive samples; 86.8%; Table 4). Regarding to the etiological agents found, we identified ten genera of clinically important fungi causing superficial infections, as follows: Candida, Malassezia, Trichosporon, Geotrichum, Trichophyton, Microsporum, Epidermophyton, Aspergillus and Fusarium. When we analyzed species distribution within the different positive clinical samples, we observed that T. rubrum was the most prevalent species in both the glabrous skin and inguinocrural region, whereas T. tonsurans and M. canis were specifically found on the scalp. Interestingly, C. parapsilosis was the main species found in the nails. Taken all the body sites evaluated together, the most prevalent and representative species were C. parapsilosis, T. rubrum and T. tonsurans (Table 4). When we specifically investigated the different clinical presentation of dermatophytosis, Tinea corporis was more prevalent, followed by Tinea capitis, Tinea cruris and Tinea ungueum, respectively (Table 5).
Discussion In the present study, we observed that among positive cases of mycological examination there was a higher prevalence of female patients with dermatomycosis. It seems that differences in distribution of positive cases of superficial fungal infections between both genders are variable worldwide. For instance, in a retrospective study from 2004 to 2014 performed in China, male patients were predominant (55.3%) rather than female (44.7%) [4]. The same distribution per gender was observed in a French Teaching Hospital in Grenoble Area, where 52.7% of patients were male [11]. In a study performed in a research institute in the north region of Brazil, it was observed a higher prevalence of female positive cases in dermatomycosis (52%) [38]. As it can be observed from data obtained in the literature, gender prevalence in cases of fungal superficial infections may be equally balanced. A correlation of DME + CULT+ was observed in 42.7% occasions (considering that 54.2% of mycological examinations were negative for both DME and Culture). In fact, a DME
Table 3 Age class distribution of positive mycological examination according to the different body sites affected with superficial mycoses. Samples were collected in Natal City, Rio Grande do Norte State, Brazil from April 2013 to December 2014. ˆ ge des examens mycologiques positifs selon les diffe ´ rentes parties du corps affecte ´ es par mycoses Distribution des classes d’a ´ collecte ´ s `a la ville de Natal, Rio Grande do Norte, Bre ´ sil entre avril 2013 et de ´ cembre 2014. superficielles. Les ´echantillons ont ´ete Age class (years)
Glabrous skin
Fingernail
Toenail
Feet
Scalp
Inguinocrural
Total N (%)
0—10 11—20 21—30 31—40 41—50 51—60 61—70 71—80 81—90 Total
02 05 10 06 03 02 03 02 — 33
— — 03 07 02 05 02 — 1 20
— — 01 02 01 12 10 02 03 31
— — — 02 — 01 01 — 01 05
11 01 01 — — — — — — 13
— — 04 02 02 03 — — — 11
13 06 19 19 08 23 16 04 05 113
(11.5) (5.3) (16.8) (16.8) (7.1) (20.4) (14.2) (3.5) (4.4) (100)
Please cite this article in press as: Silva-Rocha WP, et al. Epidemiology and fungal species distribution of superficial mycoses in Northeast Brazil. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2016.08.009
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Table 4 Fungal species distribution of positive mycological examination according to the different body sites affected with superficial mycoses. Samples were collected in Natal City, Rio Grande do Norte State, Brazil from April 2013 to December 2014. ` ces fongiques des examens mycologiques positifs selon les diffe ´ rentes parties du corps affecte ´ es. Les Distribution des espe ´ collecte ´ s `a la ville de Natal, Rio Grande do Norte, Bre ´ sil entre avril 2013 et de ´ cembre 2014. ´echantillons ont ´ete
A. flavus Candida albicans C. krusei C. parapsilosis C. tropicalis Candida sp. Epidermophyton floccosum Fusarium sp. Geotrichum sp. Microsporum canis M. gypseum Malassezia sp. Trichophyton rubrum T. mentagrophytes T. tonsurans Trichophyton sp. Trichosporon sp. Total
Glabrous skin
Fingernail
Toenail
Feet
Scalp
Inguinocrural
Total N (%)
— — — — — — 01 — — 02 02 06 12 — 08 02 — 33
01 02 01 08 01 03 — 02 — — — — — — — 01 01 20
— 01 — 10 03 04 — 06 01 — 01 — 02 01 — 02 — 31
— — — — — — — — — — — — 03 — — 02 — 05
— — — — — — — — — 05 — — — — 06 02 — 13
— 01 02 — — — — — — 01 — — 04 01 01 — 01 11
01 04 03 18 04 07 01 08 01 08 03 06 21 02 15 09 02 113
(0.9) (3.5) (2.7) (15.9) (3.5) (6.2) (0.9) (7.1) (0.9) (7.1) (2.7) (5.3) (18.6) (1.8) (13.3) (8) (1.8) (100)
Table 5 Dermatophytosis distribution of positive mycological examination according to the different body sites affected with superficial mycoses. Samples were collected in Natal City, Rio Grande do Norte State, Brazil from April 2013 to December 2014. ´ rentes parties du corps touche ´ es. Les Distribution des dermatophytes des examens mycologiques positifs selon les diffe ´ collecte ´ s `a la ville de Natal, Rio Grande do Norte, Bre ´ sil. ´echantillons ont ´ete
Mean age (SD) Microsporum canis M. gypseum Trichophyton rubrum T. tonsurans T. mentagrophytes Trichophyton sp. Epidermophyton floccosum N (%)
Tinea unguium
Tinea capitis
Tinea pedis
Tinea corporis
Tinea cruris
Total N (%)
26—82 — 01 02 — 01 03 — 07
3—13 05 — — 06 — 02 — 13
34—82 — — 03 — — 02 — 05
4—82 02 02 12 8 — 02 01 27
22—60 01 — 04 01 01 — — 07
08 03 21 15 02 09 01 59
performed with high quality and accuracy contributes significantly to the clinical outcome of patient. Accuracy of mycological examination depends on several factors including correct sample collection followed by a detailed direct microscopic analysis. These factors associated with different techniques used for culturing clinical samples and a possible previous use of antifungal drugs by the patients, contribute for the different rates of correspondence among the mycological tests in the literature. For instance, Di Chiacchio et al. [9] observed a positive correlation (DME + CULT + ) in 34% of the sample analyzed. The same correlation was observed in 95.6% of cases by Faure-Cognet et al. [11] and only in 33.8% in a study performed by Sariguzel et al. [35]. In the present study, a higher number of positive mycological examinations were found from scales obtained from glabrous skin, followed by nails. In the study of Faure-Cognet et al. [11], the prevalence of lesions in glabrous skin was observed in 51% of cases and the age group of 15—50 years
(13.6) (5.1) (35.6) (25.4) (3.4) (15.3) (1.7) (100)
was the most prevalent (42.1%), followed by 50—70 years (26.1%). Glabrous skin lesions higher prevalence was also observed in 35% of cases in the study of Kaur et al. [20]. They also observed that this body site was more affected in patients within 21—30 years old patients (23.3%), followed by the age class of 31—40 (20.5%) and 41—50 (14.8%). The high exposure of glabrous skin to the environment, associated with the different daily habits, life style, the higher probability to trauma and the exposure to chemical products, increases the incidence of superficial fungal infections in this specific body site [23]. Therefore, our data corroborates with the literature that indicates that young patients are more susceptible to develop glabrous skin fungal infections, rather than lesions in other body sites. The nails were the second most positive clinical samples found in our study with 31 (27.4%) cases found for fingernails and 20 (17.7%) for the toenails. Onychomycosis is frequently associated with several predisposing factors, such as the
Please cite this article in press as: Silva-Rocha WP, et al. Epidemiology and fungal species distribution of superficial mycoses in Northeast Brazil. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2016.08.009
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prolonged use of shoes during the day, contact with animals, trauma in the region, excessive sweating feet and the habit of not using sandals in common use bathrooms [23]. In a fouryear study performed in Northern Greece with 503 clinical specimens collections from 438 patients from different anatomical sites, onychomycosis was observed in 56.31% of cases [27]. In our study, we found that patients in the age class of 51—60 years (17 cases) and 61—70 (13 cases) were more affected with onychomycosis than the younger. In fact, these findings are consistent with literature data. Dias et al. [10] observed that the mean age in a group of patients with onychomycosis in Portugal was 73.9 years old. In an epidemiological study of onychomycosis performed in Italy, patients above 60 years old were the most prevalent with nails infections (20.7%) followed by those with 46—60 years old (16.1%) [31]. Several studies, such as the ones performed in Africa [28], Asia [40] and South America [1] describe a high incidence of onychomycosis in patients over 50 years. Species distribution in cases of onychomycoses is variable within different studies. C. parapsilosis is described as the most prevalent yeast in epidemiological data from Mexico [24] and China [12]. Other groups relate C. albicans as the most isolated yeast in cases of onychomycosis [14,28,36]. In our study, we found C. parapsilosis as the most frequently isolated Candida species in fingernails onychomycosis (followed by C. C. albicans) and toenails (followed by C. tropicalis). A study performed by another group in northeast of Brazil found C. albicans as the most frequent species, followed by C. tropicalis and C. parapsilosis [3]. Regarding to the filamentous fungi isolated from onychomycosis, T. rubrum was the most frequent etiological agent found in our study and this finding is in agreement with the literature data everywhere [13,19,28]. Species distribution of fungi isolated from dermatomycosis in this study was very variable, with a total of ten genera involved. Among the yeasts isolated, the most prevalent species was C. parapsilosis, followed by C. tropicalis and C. albicans. In a study performed by our group from September, 2010 to May, 2011 [37], C. parapsilosis was isolated from 21.9% of all the clinical specimens from different body sites, while C. albicans was the second species most isolated, with 17.1% of positive cases. However, another study performed by another group in France [12] observed that C. albicans was the most prevalent species, with 60.6%, while C. parapsilosis was isolated in 30.8% of cases. The high prevalence of C. parapsilosis in the studies involving the epidemiological aspects of dermatomycosis may be attributed to the fact that this and others Candida species belong to the normal human microbiota of the skin. It is important to emphasize that colonization precedes and is a considered risk factor for the establishment of fungal infections [30]. Malassezia spp. is an opportunistic yeast-like fungi associated with pityriasis versicolor. In the present study, women were more affected by this infirmity (5/6 cases). Although most cases in the literature report a male predominance in cases of pityriasis versicolor, other have reported a high prevalence of cases in women [25,32,34]. In our study, Malassezia spp. was observed in 4.5% of positive cases of dermatomycosis. In a 7-year survey of superficial and cutaneous mycoses in a public hospital in Natal city, Rio Grande do Norte state, Brazil, performed by Calado et al. [12], Malassezia spp. was identified in 9.5% of
cases. In the study performed by Di Chiacchio et al. [9], Southwest Brazil, pityriasis versicolor was diagnosed in 2.1% of all cases of dermatomycosis. In our study, the average age of patients was 36 years old, with all the cases observed on the upper region of the body. A possible explanation for this finding is the lipophilic nature of this yeast and the post-puberty hormonal stimulation inherent in this group, which is associated with an increased stimulation of the activity of the sebaceous glands in the region, promoting a favorable environment for the growth of Malassezia spp [9,15]. Our single case found in a female child occurred on the face as described everywhere [8,18,26]. In an European study analyzing the epidemiology of superficial mycosis, it was observed that T. rubrum was the most prevalent species isolated when yeasts and filamentous fungi are taken together (32% of cases), followed by C. parapsilosis (15.1%), and M. canis (13.4%) [27]. Another study on epidemiological data from dermatomycoses also performed in Rio Grande do Norte State, Northeastern Brazil found that T. rubrum was the most prevalent species (21.3%), followed by C. albicans (12.5%) and T. tonsurans (10.8%) [5]. This data reinforces that differences among species distributions may occur even in different studies performed in the same city in distinct periods of time, reflecting possible changes on the epidemiology of dermatomycosis. Dermatophytes were isolated in 59 out of 68 (86. 8%) cultures of filamentous fungi and T. rubrum (35. 6%) was the most frequent species followed by T. tonsurans (25.4%). In a review performed by Nweze et al. [29] on the incidence of dermatophytes in Africa, the authors describe T. rubrum as the most frequent agent of dermatophytosis in Morocco (83.6%), Egypt (79%), Tunisia (57.1%), and Algeria (20.9%). In a 16-year retrospective study performed in the southern Brazil, T. rubrum was also the most prevalent dermatophyte observed in 59.54% of cases, followed by T. interdigitale (33.97%), a species not isolated in our study [16]. Interestingly, in this study [16], T. tonsurans showed low frequency of isolation in the cases of dermatophytosis, with only 0.93% of cases, which differs from the data presented in the present study, where T. tonsurans was the second most frequently isolated species. A 10-year epidemiological study on dermatophytosis in Northeast Brazil describes T. rubrum as the most prevalent species, found in 26.7% of clinical specimens, followed by T. tonsurans with 26.2% of cases, which confirms a high incidence of T. tonsurans in Northeastern Brazil [7], a peculiarity on dermatophytes distribution in Brazil, a continental size country. This data is also reinforced in the study of Calado et al. [5], also performed in Natal city, Rio Grande do Norte state, Brazil. T. tonsurans was the most frequent dermatophyte found on lesions of the scalp (42.6%), followed by M. canis (23.4%), the same trend observed in the present study. The same prevalence was also described by Leite et al., in a military population in Central-West Region of Brazil [23]. In this study, T. tonsurans was found in 33.3% of dermatophytosis in scalp and M. canis isolation corresponded to 16.7% of all cases [23]. A higher prevalence of T. tonsurans against M. canis in cases of tinea capitis was also described by FaureCognet in a French study which reported 23.8% versus 9.5% of cases, for these species, respectively [11]. Tinea corporis was the main clinical form of dermatophytosis found in this study, followed by Tinea capitis. The
Please cite this article in press as: Silva-Rocha WP, et al. Epidemiology and fungal species distribution of superficial mycoses in Northeast Brazil. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2016.08.009
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Epidemiology and fungal species distribution of superficial mycoses frequency of clinical presentation of dermatophytosis may have some differences among different epidemiological studies, but these two clinical forms seem to be highly prevalent in several cases. A study of clinical and epidemiological profiling of dermatophytosis performed in the State of Para, in North Brazil, Tinea corporis was observed in 24.1% of cases, while Tinea capitis was diagnosed in 22.1% [33]. Another study evaluating the prevalence of dermatophytosis in a Scientific Institute also in North Brazil, observed that Tinea corporis and Tinea capitis were the most prevalent dermatophytosis clinical presentation, present in 31.8% of cases [38]. The high incidence of cases of Tinea corporis and T. capitis reflects the large area of body exposure to the environment, mainly by increasing the risk of contact with anthropophilic, zoophilic and geophilic fungi as the dermatophytes isolated in these studies [7,11,16,33,38]. In conclusion, although this study has some limitations such as the number of patients involved, we observed the high prevalence of superficial mycosis in female patients and that the analysis of clinical samples by direct microscopy examination is extremely important, given the fact that in some cases the recovery of fungi in the samples cannot occur. We also verified that the glabrous skin is the largest source of Dermatomycoses, followed by nails. Regarding onychomycosis, the population of over 50 years old is the most affected. Another important fact observed is that colonizing yeasts of skin are highly involved in cases of superficial infections, depending on favorable conditions for this occurrence, specifically C. parapsilosis. Finally, we can corroborate to increase the knowledge of dermatomycosis, through this study, by the fact that we have a different epidemiology of dermatophytosis found in studies performed in the South and Southeast regions of Brazil. In the Northeast region a high incidence of T. tonsurans was observed, clearly demonstrating peculiarities in terms of etiological agents of dermatophytoses distribution within a large size and population country.
Disclosure of interest
[4]
[5]
[6]
[7]
[8] [9]
[10]
[11]
[12]
[13]
[14] [15]
[16]
The authors declare that they have no competing interest. [17]
Acknowledgements [18]
We are very grateful to Brazilian Ministry of Education for financial fundings. We also would like to thank Professor Arnaldo Lopes Colombo for donation of reference strains used as controls for fungal identification and to Professor Marcelo Lancellotti for the critical review of the French translation.
[19] [20]
[21]
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[34] Santana JO, Azevedo FL, Campos Filho PC. Pityriasis versicolor: clinical-epidemiological characterization of patients in the urban area of Buerarema-BA, Brazil. An Bras Dermatol 2013;88:216—21. [35] Sariguzel FM, Koc AN, Yagmur G, Berk E. Interdigital foot infections: Corynebacterium minutissimum and agents of superficial mycoses. Braz J Microbiol 2014;45:781—4. [36] Seck MC, Ndiaye D, Diongue K, Ndiaye M, Badiane AS, Sow D, et al. Mycological profile of onychomycosis in Dakar (Senegal). J Mycol Med 2014;24:124—8. [37] Silva WP, Lemos VL, Milan EP, Chaves GM. Species distribution and phospholipase activity of fungi isolated from children with dermatomycosis from child day care units in Natal, Brazil. J Eur Acad Dermatol Venereol 2013;27:1319—21. [38] Silveira-Gomes F, de Oliveira EF, Nepomuceno LB, Pimentel RF, Marques-da-Silva SH, Mesquita-da-Costa M. Dermatophytosis diagnosed at the Evandro Chagas Institute, Para, Brazil. Braz J Microbiol 2013;44:443—6. [39] Simonnet C, Berger F, Gantier JC. Epidemiology of superficial fungal diseases in French Guiana: a three-year retrospective analysis. Med Mycol 2011;49:608—11. [40] Soltani M, Khosravi AR, Shokri H, Sharifzadeh A, Balal A. A study of onychomycosis in patients attending a dermatology center in Tehran, Iran. J Mycol Med 2015;25:e81—7. [41] Yarrow D. Methods for the isolation, maintenance and identification of yeasts. In: Kurtzman CFJ, editor. The yeasts, a taxonomic study. 4th ed., Amsterdam: Elsevier Science; 1998 . p. 77—100.
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MYCMED-645; No. of Pages 8 Journal de Mycologie Médicale (2016) xxx, xxx—xxx
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ORIGINAL ARTICLE/ARTICLE ORIGINAL
Epidemiology and fungal species distribution of superficial mycoses in Northeast Brazil ´Epide ´ miologie et distribution des espe ` ces fongiques des mycoses ´ sil superficielles dans le Nord-est du Bre W.P. Silva-Rocha, M.F. de Azevedo, G.M. Chaves * ´ rio de Micologia Me ´ dica e Molecular, Departamento de Ana ´ lises Clı ´ gicas, ´nicas e Toxicolo Laborato Universidade Federal do Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil Received 23 May 2016; received in revised form 16 August 2016; accepted 21 August 2016
KEYWORDS Dermatomycoses; Epidemiology; Fungal distribution; Mycological examination; Northeast Brazil
Summary Objective. — Dermatomycoses are superficial fungal infections which affect the skin, hair and nails of humans and animals. Male and female patients of all ages are affected by this condition. The main etiological agents of dermatomycoses are the dermatophytes fungi of the genera Trichophyton, Microsporum and Epidermophyton, while the main yeasts belong to the genera Candida, Malassezia and Trichosporon. The variation in the distribution of dermatomycoses worldwide justify the conduction of epidemiological studies in order to contribute for the better understanding of patterns of mycological cutaneous infections. This study was conducted from April 2013 to December 2014. Material and methods. — A total of 205 patients were evaluated, while 235 clinical specimens were obtained. From our positive cases of mycological examination, 73 (64.6%) patients were female, while 40 (35.4%) were male. Scales from the skin and nails were collected and observed at optical microscopy after potassium hydroxide clarification. Cultures were performed on Sabouraud Dextrose Agar added chloramphenicol. Identification was performed by classic methodology. Results. — We found that the glabrous skin was the largest source of dermatomycoses (30.11%), followed by toenails (27.4%) and fingernails (17.7%). Regarding onychomycosis, the most affected population was over 50 years old. Trichophyton rubrum was the dermatophyte fungal species more commonly found. Most of the patients with pityriasis versicolor were adults and female. Another important fact observed is that Candida parapsilosis was the most prevalent species. Finally, a high incidence of T. tonsurans in cases of superficial mycoses was observed.
* Corresponding author. Laboratório de Micologia Médica e Molecular, Departamento de Análises Clínicas e Toxicológicas, Universidade Federal do Rio Grande do Norte, Centro de Ciências da Saúde, Rua Gal. Gustavo Cordeiro de Faria S/N, Petrópolis, 59012-570 Natal-RN, Brazil. E-mail address: [email protected] (G.M. Chaves). http://dx.doi.org/10.1016/j.mycmed.2016.08.009 1156-5233/# 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Silva-Rocha WP, et al. Epidemiology and fungal species distribution of superficial mycoses in Northeast Brazil. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2016.08.009
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W.P. Silva-Rocha et al. Conclusion. — Our results clearly demonstrate peculiarities in terms of etiological agents of dermatophytoses distribution in a specific region of Brazil. # 2016 Elsevier Masson SAS. All rights reserved.
MOTS CLÉS Dermatophytoses ; Épidémiologie ; Distribution fongique ; Examen mycologique ; Nord-est du Brésil
Re ´sume ´ Objectif. — Les mycoses cutanées sont des infections fongiques superficielles qui affectent la peau, les cheveux et les ongles chez l’homme et les griffes des animaux. Les patients masculins et féminins de tout âge sont touchés par cette maladie. Les principaux agents étiologiques de la dermatomycose sont les dermatophytes des genres Trichophyton, Microsporum et Epidermophyton, alors que les principales levures appartiennent aux genres Candida, Malasezzia et Trichosporon. Les variations de la distribution mondiale des dermatomycoses justifient la conduction d’études épidémiologiques afin de contribuer à une meilleure compréhension des tendances des infections cutanées mycologiques. Cette étude a été réalisée entre avril 2013 et décembre 2014. Mate´riel et me´thodes. — Un total de 221 patients ont été évalués, 255 échantillons cliniques ont été obtenus. Dans les cas d’examen mycologique positif, 87 (65,4 %) patients étaient des femmes, alors 46 (34,6 %) étaient des hommes. Les produits de grattage de la peau et des ongles ont été recueillis et observés au microscope optique après clarification par l’hydroxyde de potassium. Les cultures ont été effectuées sur gélose Sabouraud additionné de chloramphénicol. Ainsi, l’identification a été réalisée par une méthodologie classique. Re ´sultats. — Nous avons constaté que la peau glabre était la principale source de mycoses superficielles, suivie par les ongles. En ce qui concerne l’onychomycose, la population de plus de 50 ans a été la plus touchée. Trichophyton rubrum a été l’espèce des dermatophytes plus fréquemment trouvée. La plupart des patients souffrant de pityriasis versicolor ont été des adultes et des femmes. Un autre fait important observé c’est que Candida parapsilosis était l’espèce de levure la plus répandue. Enfin, une forte incidence de T. tonsurans a été observée. Conclusion. — Nos résultats démontre clairement les particularités en termes d’agents étiologiques et de la distribution des dermatophytoses dans une région spécifique du Brésil. # 2016 Elsevier Masson SAS. Tous droits réservés.
Introduction Dermatomycosis are superficial fungal infections which affect the skin, hair and nails of humans and animals. They affect both male and female patients of all ages and have a worldwide distribution. However, hot and humid climates such as in tropical and subtropical areas contribute to the high prevalence of number of cases in countries from Latin America, Africa and Asia [2,11,39]. Superficial fungal infections are related to several predisposing factors, including: climate conditions, people migrations, sports activities, prolonged contact with water, lifestyle, immunological status, drug therapy and age of patients [11]. In addition, life conditions and the environment where people are inserted influence epidemiological characteristics of human infection [11]. The main etiological agents of dermatomycosis are the dermatophytes fungi of the genera Trichophyton, Microsporum and Epidermophyton. The non-dermatophyte agents include other filamentous fungi such as Fusarium and Aspergillus and mainly yeasts belonging to the genera Candida, Malasezzia and Trichosporon [9]. Dermatophytes fungi are filamentous, hyaline, septate and present arthroconidia in their micromorphology. The high incidence of these fungi on epidemiological data of dermatomycosis is influenced by the high production of keratinasis, an important enzyme used for acquisition
of nutrients from the environment, but also contributes for fungal invasion of the stratum corneum of epidermis and to degrade keratin of the skin, hair and nails [9]. Therefore, these fungi are known as keratinolytic [6]. Yeasts belonging to the Candida genus are also involved in superficial infections and the main species isolated from dermatomycosis are the Candida parapsilosis species complex, Candida albicans and Candida tropicalis. The lesion occurs mainly in intertriginous areas of the body, such as interdigital, inguinal, intergluteal and inframammary regions as well as nails and periungueal regions [37]. The genus Malassezia comprises lipophilic yeast-like fungi that are part of the normal skin microbiota in humans and warmblooded animals [15]. The variation in the distribution of dermatomycoses according to the local geography, inspires the conduction of epidemiological studies in order to contribute for the better understanding of patterns of mycological dermatomycoses and etiologic distribution of the most common fungal agents which cause infection in the skin and its annexes. Of note, each species presents peculiarities in terms of body sites and age of patients which they cause disease. Therefore, this study aimed to conduct an epidemiological survey of dermatomycoses derived from patients in a private laboratory located in Northeast Brazil. This data will contribute to increase the discussion on the epidemiology of dermatomycosis in a region of Brazil where this clinical scenario is poorly investigated.
Please cite this article in press as: Silva-Rocha WP, et al. Epidemiology and fungal species distribution of superficial mycoses in Northeast Brazil. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2016.08.009
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Materials and methods Collection of clinical specimens Strains were collected from April 2013 to December 2014 at a private clinical laboratory analysis from the city of Natal, Rio Grande do Norte State, Northeast Brazil and sent for further identification at the Laboratory of Medical and Molecular Mycology, Department of Clinical and Toxicological Analysis, Federal University of Rio Grande do Norte. The patients were referred by dermatologists from different clinical ambulatories of the state. During the period of the study, 205 patients of both genders and different ages ranging from 3 to 83 years old were evaluated, according to the presence of lesions suggestive of superficial mycosis in different areas of the skin, scalp, hair and nails. Samples were collected according to the anatomical site and the aspect of lesion and clinical indication. Circular lesions of glabrous skin with dry appearance and well-defined edges and peeling areas were collected by scraping the edges of the lesion with the aid of a sterile scalpel. Scales were stored in sterile Petri dishes. Lesions from intertriginous areas were collected with a sterile swab moistened with sterile saline solution at 0.9%. Scales from the scalp and nails were collected by scraping with a sterilized scalpel. When white piedra was suspected, a lock of hair was collected with scissors. Lesions with hypopigmented or hyperpigmented maculae suggestive of pityriasis versicolor were collected using the Method of Porto [22].
Sample processing and culture Samples collected from different clinical specimens were analyzed according with the nature of the lesion. Hair, skin and nail scales were treated with 20% Potassium hydroxide (KOH) during 30 min. Samples collected with a sterile swab were observed with optical microscopy without staining and clarification (CX21, Olympus, Japan). All the clinical samples were analyzed with optical microscopy at 400 of magnification and the presence of fungal structures such as blastoconidia, pseudohyphae, true hyphae and conidia were observed. Clinical samples were seeded on the surface of Sabouraud Dextrose Agar (Dextrose 40 g, Peptone 10 g, Agar 15 g, Distilled water 1000 mL) added chloramphenicol (50 mg/mL), at seven equidistant points and incubated at 30 8C and culture growth was analyzed daily. Cultures without growth during a period of up to four weeks were considered negative.
Mycological diagnose criteria Besides the fact that all the patients showed lesions suggestive of superficial mycoses as forwarded by dermatologists, for molds, yeasts and yeast-like fungi that can be found as commensals of several anatomic sites, mycological examination was only considered positive when besides positive culture, fungal structures were found at the direct examination, including some degree of evagination or pseudohyphae for the Candida species that may undergo morphogenesis. Other criteria adopted were the positive mycological examination (direct examination and culture) within two
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different occasions (different days of collections) and if colonies growth was found for the seven equidistant points seeded on the surface of the Petri dishes.
Identification of isolates grown in culture The clinical isolates were identified based in their phenotypic, biochemistry and nutritional characteristics. The identification of yeasts was performed by classical methodology [21]. The presumptive identification of the main Candida species was performed by analyzing the color of colonies grown on Chromagar Candida1 (CHROMagar Microbiology, Paris, France). The micromorphological aspects were observed by culturing pure colonies on the surface of cornmeal agar added Tween 80. Carbon auxanogram and zymogram tests were performed to evaluate biochemical characteristics of yeasts [41]. The filamentous fungi were identified by analyzing the macromorphological aspects of colonies (appearance, texture, development time, and coloring of verse and reverse). The micromorphological aspects were observed by microculturing the colonies in potato dextrose agar. The typical structures of genus and species such as microconidia, macroconidia, chlamydoconidia, and coiled spirals were observed by staining a colony fragment with lactophenol cotton blue, visualized by 400 of magnification with optical microscopy [17].
Results In the present study a total of 205 patients were evaluated, while 235 clinical specimens were obtained. A total of 225 samples were analyzed by direct microscopic examination (DME) and culture. Of note, 113 clinical specimens were positive, while 122 were negative for mycological examination. We obtained 42.7% (96 cases) correspondence between direct examination and culture (Table 1). From our positive cases of mycological examination, 73 (64.6%) patients were female, while 40 (35.4%) were male. The main body sites infected with superficial mycosis were the glabrous skin (including the cases of pityriasis versicolor), followed by the nails and scalp (Table 2). Of note, Table 3 describes the distribution of dermatomycosis Table 1 Correspondence between direct examination and culture of mycological examination performed in Natal City, Rio Grande do Norte State, Brazil from April 2013 to December 2014. Correspondance entre l’examen direct et culture mycolo´ alise ´ s `a la ville de Natal, Rio Grande do Norte, Bre ´ sil gique re ´ cembre 2014. entre avril 2013 et de Direct microscopic examination (DME)
Culture (CULT)
Number of cases
Percentage (%)
DME + DME DME Total
CULT + CULT + CULT
96 07 122 225 a
42.7 3.1 54.2 100
a The total number of 225 excludes 10 samples collected by method of Porto for pityriais versicolor diagnosis, whose cultures were not performed.
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W.P. Silva-Rocha et al. Table 2 Percentage of positive mycological examination according to the different body sites of patients affected with superficial mycoses separated by age range. Samples were collected in Natal City, Rio Grande do Norte State, Brazil from April 2013 to December 2014. Pourcentages des ´echantillons avec examen positif par rap´ par les mycoses superficielles port au site corporel affecte ˆ ge. ´Echantillons collecte ´ s `a la ville de ´ pare ´ s par tranche d’a se ´ sil entre avril 2013 et Natal, Rio Grande do Norte, Bre ´ cembre 2014. de Body site
N (%)
Age range (years)
Fingernail Toenail Glabrous skin Feet Scalp Inguinocrural Total
20 (17.7) 31 (27.4) 33 (29.2) 5 (4.4) 13 (11.5) 11 (9.7) 113
28—83 26—83 6—73 17—57 03—26 22—60 03—83
among different body sites according to the age class. We could observe that for the infants (up to 10 years old), most of positive cases were from the scalp, whereas in adolescents and young adults (from 11 to 40 years old) the glabrous skin was the predominant site. In middle age adults and the elderly (40 years old and above), most of the positive mycological examinations were obtained from the nails. Ten samples with hypopigmented scaly areas were collected by the Method of Porto [22] to analyze the presence of Malassezia spp. In six occasions, mycological examinations were positive, as we could observe the presence of blastoconidia and short and tortuous hyphae with an aspect previously described as ‘‘spaghetti and meatballs’’ at the direct examination [19]. All the six positive cases were obtained from the glabrous skin, on the upper region of the body. Five of the six positive cases occurred in female patients and the mean age of all the subjects was 36 years old (6, 19, 21, 34, 67 and 69 years old). Besides, only a single case was found for a 6-year-old female infant (Table 3). When analyzing the distribution of cases of superficial mycosis in this study, we observe a difference for the prevalence of infections caused by either yeasts or filamentous
fungi. From the 113 positive cases, yeast infections were observed in 45 of cases (39.8%) while filamentous fungi were observed in 68 of cases (60.2%). The superficial mycoses due filamentous fungi were predominantly caused by dermatophytes (59 positive samples; 86.8%; Table 4). Regarding to the etiological agents found, we identified ten genera of clinically important fungi causing superficial infections, as follows: Candida, Malassezia, Trichosporon, Geotrichum, Trichophyton, Microsporum, Epidermophyton, Aspergillus and Fusarium. When we analyzed species distribution within the different positive clinical samples, we observed that T. rubrum was the most prevalent species in both the glabrous skin and inguinocrural region, whereas T. tonsurans and M. canis were specifically found on the scalp. Interestingly, C. parapsilosis was the main species found in the nails. Taken all the body sites evaluated together, the most prevalent and representative species were C. parapsilosis, T. rubrum and T. tonsurans (Table 4). When we specifically investigated the different clinical presentation of dermatophytosis, Tinea corporis was more prevalent, followed by Tinea capitis, Tinea cruris and Tinea ungueum, respectively (Table 5).
Discussion In the present study, we observed that among positive cases of mycological examination there was a higher prevalence of female patients with dermatomycosis. It seems that differences in distribution of positive cases of superficial fungal infections between both genders are variable worldwide. For instance, in a retrospective study from 2004 to 2014 performed in China, male patients were predominant (55.3%) rather than female (44.7%) [4]. The same distribution per gender was observed in a French Teaching Hospital in Grenoble Area, where 52.7% of patients were male [11]. In a study performed in a research institute in the north region of Brazil, it was observed a higher prevalence of female positive cases in dermatomycosis (52%) [38]. As it can be observed from data obtained in the literature, gender prevalence in cases of fungal superficial infections may be equally balanced. A correlation of DME + CULT+ was observed in 42.7% occasions (considering that 54.2% of mycological examinations were negative for both DME and Culture). In fact, a DME
Table 3 Age class distribution of positive mycological examination according to the different body sites affected with superficial mycoses. Samples were collected in Natal City, Rio Grande do Norte State, Brazil from April 2013 to December 2014. ˆ ge des examens mycologiques positifs selon les diffe ´ rentes parties du corps affecte ´ es par mycoses Distribution des classes d’a ´ collecte ´ s `a la ville de Natal, Rio Grande do Norte, Bre ´ sil entre avril 2013 et de ´ cembre 2014. superficielles. Les ´echantillons ont ´ete Age class (years)
Glabrous skin
Fingernail
Toenail
Feet
Scalp
Inguinocrural
Total N (%)
0—10 11—20 21—30 31—40 41—50 51—60 61—70 71—80 81—90 Total
02 05 10 06 03 02 03 02 — 33
— — 03 07 02 05 02 — 1 20
— — 01 02 01 12 10 02 03 31
— — — 02 — 01 01 — 01 05
11 01 01 — — — — — — 13
— — 04 02 02 03 — — — 11
13 06 19 19 08 23 16 04 05 113
(11.5) (5.3) (16.8) (16.8) (7.1) (20.4) (14.2) (3.5) (4.4) (100)
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Table 4 Fungal species distribution of positive mycological examination according to the different body sites affected with superficial mycoses. Samples were collected in Natal City, Rio Grande do Norte State, Brazil from April 2013 to December 2014. ` ces fongiques des examens mycologiques positifs selon les diffe ´ rentes parties du corps affecte ´ es. Les Distribution des espe ´ collecte ´ s `a la ville de Natal, Rio Grande do Norte, Bre ´ sil entre avril 2013 et de ´ cembre 2014. ´echantillons ont ´ete
A. flavus Candida albicans C. krusei C. parapsilosis C. tropicalis Candida sp. Epidermophyton floccosum Fusarium sp. Geotrichum sp. Microsporum canis M. gypseum Malassezia sp. Trichophyton rubrum T. mentagrophytes T. tonsurans Trichophyton sp. Trichosporon sp. Total
Glabrous skin
Fingernail
Toenail
Feet
Scalp
Inguinocrural
Total N (%)
— — — — — — 01 — — 02 02 06 12 — 08 02 — 33
01 02 01 08 01 03 — 02 — — — — — — — 01 01 20
— 01 — 10 03 04 — 06 01 — 01 — 02 01 — 02 — 31
— — — — — — — — — — — — 03 — — 02 — 05
— — — — — — — — — 05 — — — — 06 02 — 13
— 01 02 — — — — — — 01 — — 04 01 01 — 01 11
01 04 03 18 04 07 01 08 01 08 03 06 21 02 15 09 02 113
(0.9) (3.5) (2.7) (15.9) (3.5) (6.2) (0.9) (7.1) (0.9) (7.1) (2.7) (5.3) (18.6) (1.8) (13.3) (8) (1.8) (100)
Table 5 Dermatophytosis distribution of positive mycological examination according to the different body sites affected with superficial mycoses. Samples were collected in Natal City, Rio Grande do Norte State, Brazil from April 2013 to December 2014. ´ rentes parties du corps touche ´ es. Les Distribution des dermatophytes des examens mycologiques positifs selon les diffe ´ collecte ´ s `a la ville de Natal, Rio Grande do Norte, Bre ´ sil. ´echantillons ont ´ete
Mean age (SD) Microsporum canis M. gypseum Trichophyton rubrum T. tonsurans T. mentagrophytes Trichophyton sp. Epidermophyton floccosum N (%)
Tinea unguium
Tinea capitis
Tinea pedis
Tinea corporis
Tinea cruris
Total N (%)
26—82 — 01 02 — 01 03 — 07
3—13 05 — — 06 — 02 — 13
34—82 — — 03 — — 02 — 05
4—82 02 02 12 8 — 02 01 27
22—60 01 — 04 01 01 — — 07
08 03 21 15 02 09 01 59
performed with high quality and accuracy contributes significantly to the clinical outcome of patient. Accuracy of mycological examination depends on several factors including correct sample collection followed by a detailed direct microscopic analysis. These factors associated with different techniques used for culturing clinical samples and a possible previous use of antifungal drugs by the patients, contribute for the different rates of correspondence among the mycological tests in the literature. For instance, Di Chiacchio et al. [9] observed a positive correlation (DME + CULT + ) in 34% of the sample analyzed. The same correlation was observed in 95.6% of cases by Faure-Cognet et al. [11] and only in 33.8% in a study performed by Sariguzel et al. [35]. In the present study, a higher number of positive mycological examinations were found from scales obtained from glabrous skin, followed by nails. In the study of Faure-Cognet et al. [11], the prevalence of lesions in glabrous skin was observed in 51% of cases and the age group of 15—50 years
(13.6) (5.1) (35.6) (25.4) (3.4) (15.3) (1.7) (100)
was the most prevalent (42.1%), followed by 50—70 years (26.1%). Glabrous skin lesions higher prevalence was also observed in 35% of cases in the study of Kaur et al. [20]. They also observed that this body site was more affected in patients within 21—30 years old patients (23.3%), followed by the age class of 31—40 (20.5%) and 41—50 (14.8%). The high exposure of glabrous skin to the environment, associated with the different daily habits, life style, the higher probability to trauma and the exposure to chemical products, increases the incidence of superficial fungal infections in this specific body site [23]. Therefore, our data corroborates with the literature that indicates that young patients are more susceptible to develop glabrous skin fungal infections, rather than lesions in other body sites. The nails were the second most positive clinical samples found in our study with 31 (27.4%) cases found for fingernails and 20 (17.7%) for the toenails. Onychomycosis is frequently associated with several predisposing factors, such as the
Please cite this article in press as: Silva-Rocha WP, et al. Epidemiology and fungal species distribution of superficial mycoses in Northeast Brazil. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2016.08.009
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prolonged use of shoes during the day, contact with animals, trauma in the region, excessive sweating feet and the habit of not using sandals in common use bathrooms [23]. In a fouryear study performed in Northern Greece with 503 clinical specimens collections from 438 patients from different anatomical sites, onychomycosis was observed in 56.31% of cases [27]. In our study, we found that patients in the age class of 51—60 years (17 cases) and 61—70 (13 cases) were more affected with onychomycosis than the younger. In fact, these findings are consistent with literature data. Dias et al. [10] observed that the mean age in a group of patients with onychomycosis in Portugal was 73.9 years old. In an epidemiological study of onychomycosis performed in Italy, patients above 60 years old were the most prevalent with nails infections (20.7%) followed by those with 46—60 years old (16.1%) [31]. Several studies, such as the ones performed in Africa [28], Asia [40] and South America [1] describe a high incidence of onychomycosis in patients over 50 years. Species distribution in cases of onychomycoses is variable within different studies. C. parapsilosis is described as the most prevalent yeast in epidemiological data from Mexico [24] and China [12]. Other groups relate C. albicans as the most isolated yeast in cases of onychomycosis [14,28,36]. In our study, we found C. parapsilosis as the most frequently isolated Candida species in fingernails onychomycosis (followed by C. C. albicans) and toenails (followed by C. tropicalis). A study performed by another group in northeast of Brazil found C. albicans as the most frequent species, followed by C. tropicalis and C. parapsilosis [3]. Regarding to the filamentous fungi isolated from onychomycosis, T. rubrum was the most frequent etiological agent found in our study and this finding is in agreement with the literature data everywhere [13,19,28]. Species distribution of fungi isolated from dermatomycosis in this study was very variable, with a total of ten genera involved. Among the yeasts isolated, the most prevalent species was C. parapsilosis, followed by C. tropicalis and C. albicans. In a study performed by our group from September, 2010 to May, 2011 [37], C. parapsilosis was isolated from 21.9% of all the clinical specimens from different body sites, while C. albicans was the second species most isolated, with 17.1% of positive cases. However, another study performed by another group in France [12] observed that C. albicans was the most prevalent species, with 60.6%, while C. parapsilosis was isolated in 30.8% of cases. The high prevalence of C. parapsilosis in the studies involving the epidemiological aspects of dermatomycosis may be attributed to the fact that this and others Candida species belong to the normal human microbiota of the skin. It is important to emphasize that colonization precedes and is a considered risk factor for the establishment of fungal infections [30]. Malassezia spp. is an opportunistic yeast-like fungi associated with pityriasis versicolor. In the present study, women were more affected by this infirmity (5/6 cases). Although most cases in the literature report a male predominance in cases of pityriasis versicolor, other have reported a high prevalence of cases in women [25,32,34]. In our study, Malassezia spp. was observed in 4.5% of positive cases of dermatomycosis. In a 7-year survey of superficial and cutaneous mycoses in a public hospital in Natal city, Rio Grande do Norte state, Brazil, performed by Calado et al. [12], Malassezia spp. was identified in 9.5% of
cases. In the study performed by Di Chiacchio et al. [9], Southwest Brazil, pityriasis versicolor was diagnosed in 2.1% of all cases of dermatomycosis. In our study, the average age of patients was 36 years old, with all the cases observed on the upper region of the body. A possible explanation for this finding is the lipophilic nature of this yeast and the post-puberty hormonal stimulation inherent in this group, which is associated with an increased stimulation of the activity of the sebaceous glands in the region, promoting a favorable environment for the growth of Malassezia spp [9,15]. Our single case found in a female child occurred on the face as described everywhere [8,18,26]. In an European study analyzing the epidemiology of superficial mycosis, it was observed that T. rubrum was the most prevalent species isolated when yeasts and filamentous fungi are taken together (32% of cases), followed by C. parapsilosis (15.1%), and M. canis (13.4%) [27]. Another study on epidemiological data from dermatomycoses also performed in Rio Grande do Norte State, Northeastern Brazil found that T. rubrum was the most prevalent species (21.3%), followed by C. albicans (12.5%) and T. tonsurans (10.8%) [5]. This data reinforces that differences among species distributions may occur even in different studies performed in the same city in distinct periods of time, reflecting possible changes on the epidemiology of dermatomycosis. Dermatophytes were isolated in 59 out of 68 (86. 8%) cultures of filamentous fungi and T. rubrum (35. 6%) was the most frequent species followed by T. tonsurans (25.4%). In a review performed by Nweze et al. [29] on the incidence of dermatophytes in Africa, the authors describe T. rubrum as the most frequent agent of dermatophytosis in Morocco (83.6%), Egypt (79%), Tunisia (57.1%), and Algeria (20.9%). In a 16-year retrospective study performed in the southern Brazil, T. rubrum was also the most prevalent dermatophyte observed in 59.54% of cases, followed by T. interdigitale (33.97%), a species not isolated in our study [16]. Interestingly, in this study [16], T. tonsurans showed low frequency of isolation in the cases of dermatophytosis, with only 0.93% of cases, which differs from the data presented in the present study, where T. tonsurans was the second most frequently isolated species. A 10-year epidemiological study on dermatophytosis in Northeast Brazil describes T. rubrum as the most prevalent species, found in 26.7% of clinical specimens, followed by T. tonsurans with 26.2% of cases, which confirms a high incidence of T. tonsurans in Northeastern Brazil [7], a peculiarity on dermatophytes distribution in Brazil, a continental size country. This data is also reinforced in the study of Calado et al. [5], also performed in Natal city, Rio Grande do Norte state, Brazil. T. tonsurans was the most frequent dermatophyte found on lesions of the scalp (42.6%), followed by M. canis (23.4%), the same trend observed in the present study. The same prevalence was also described by Leite et al., in a military population in Central-West Region of Brazil [23]. In this study, T. tonsurans was found in 33.3% of dermatophytosis in scalp and M. canis isolation corresponded to 16.7% of all cases [23]. A higher prevalence of T. tonsurans against M. canis in cases of tinea capitis was also described by FaureCognet in a French study which reported 23.8% versus 9.5% of cases, for these species, respectively [11]. Tinea corporis was the main clinical form of dermatophytosis found in this study, followed by Tinea capitis. The
Please cite this article in press as: Silva-Rocha WP, et al. Epidemiology and fungal species distribution of superficial mycoses in Northeast Brazil. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2016.08.009
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Epidemiology and fungal species distribution of superficial mycoses frequency of clinical presentation of dermatophytosis may have some differences among different epidemiological studies, but these two clinical forms seem to be highly prevalent in several cases. A study of clinical and epidemiological profiling of dermatophytosis performed in the State of Para, in North Brazil, Tinea corporis was observed in 24.1% of cases, while Tinea capitis was diagnosed in 22.1% [33]. Another study evaluating the prevalence of dermatophytosis in a Scientific Institute also in North Brazil, observed that Tinea corporis and Tinea capitis were the most prevalent dermatophytosis clinical presentation, present in 31.8% of cases [38]. The high incidence of cases of Tinea corporis and T. capitis reflects the large area of body exposure to the environment, mainly by increasing the risk of contact with anthropophilic, zoophilic and geophilic fungi as the dermatophytes isolated in these studies [7,11,16,33,38]. In conclusion, although this study has some limitations such as the number of patients involved, we observed the high prevalence of superficial mycosis in female patients and that the analysis of clinical samples by direct microscopy examination is extremely important, given the fact that in some cases the recovery of fungi in the samples cannot occur. We also verified that the glabrous skin is the largest source of Dermatomycoses, followed by nails. Regarding onychomycosis, the population of over 50 years old is the most affected. Another important fact observed is that colonizing yeasts of skin are highly involved in cases of superficial infections, depending on favorable conditions for this occurrence, specifically C. parapsilosis. Finally, we can corroborate to increase the knowledge of dermatomycosis, through this study, by the fact that we have a different epidemiology of dermatophytosis found in studies performed in the South and Southeast regions of Brazil. In the Northeast region a high incidence of T. tonsurans was observed, clearly demonstrating peculiarities in terms of etiological agents of dermatophytoses distribution within a large size and population country.
Disclosure of interest
[4]
[5]
[6]
[7]
[8] [9]
[10]
[11]
[12]
[13]
[14] [15]
[16]
The authors declare that they have no competing interest. [17]
Acknowledgements [18]
We are very grateful to Brazilian Ministry of Education for financial fundings. We also would like to thank Professor Arnaldo Lopes Colombo for donation of reference strains used as controls for fungal identification and to Professor Marcelo Lancellotti for the critical review of the French translation.
[19] [20]
[21]
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Please cite this article in press as: Silva-Rocha WP, et al. Epidemiology and fungal species distribution of superficial mycoses in Northeast Brazil. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2016.08.009