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Print version ISSN 0074-0276On-line version ISSN 1678-8060
Mem. Inst. Oswaldo Cruz vol.103 no.8 Rio de Janeiro Dec. 2008
Alba Regina MagalhãesI, II, +; Silvia Susana Bona de MondinoII; Manuela da SilvaIII; Marilia Martins NishikawaIV
ILaboratório de Micologia, Instituto Biomédico, Universidade Federal Fluminense, Rua Marquês de Paraná 303, 24030-210, Niterói, RJ, Brasil
IIPrograma de Pós-Graduação em Patologia, Departamento de Patologia, Universidade Federal Fluminense, Rua Marquês de Paraná 303, 24030-210, Niterói, RJ, Brasil
IIIPrograma de Pós-Graduação em Vigilância Sanitária, Instituto Nacional de Controle de Qualidade-Fiocruz, Rio de Janeiro, RJ, Brasil
IVSetor de Fungos de Referência, Departamento de Microbiologia, Instituto Nacional de Controle de Qualidade-Fiocruz, Rio de Janeiro, RJ, Brasil
The Trichosporon genus is constituted by many species, of which Trichosporon ovoides and Trichosporon inkin are the causative agents of white piedra. They can cause nodules in genital hair or on the scalp. At present, Brazilian laboratory routines generally do not include the identification of the species of Trichosporon genus, which, although morphologically and physiologically distinct, present many similarities, making the identification difficult. The aim of this study was to identify the aetiological agents at the species level of white piedra from clinical specimens. Therefore, both the macro and micro morphology were studied, and physiological tests were performed. Trichosporon spp. was isolated from 10 clinical samples; T. ovoides was predominant, as it was found in seven samples, while T. inkin was identified just in two samples. One isolate could not be identified at the species level. T. inkin was identified for the first time as a white piedra agent in the hair shaft on child under the age of 10.
Key words: Trichosporon - white piedra - mycosis - phenotypic identification
Trichosporon Behrend is a genus that belongs to the Basidiomycota phylum, the Hymenomycetes class and the Trichosporonales order (Fell et al. 2000). It is characterised by blastoconidia and arthroconidia and has been traditionally considered as the agent of white piedra, a superficial mycosis that attacks genital hair or hair shafts, producing bright, soft and yellowish nodules (Lacaz et al. 1984). For many years, this genus was regarded as consisting of few species that were morphologically and biochemically variable and its main representative was Trichosporon beigelii (Küchenm & Rabenh) Vuill., a synonym of Trichosporon cutaneum (Kreger-van Rij 1984). However, Guého et al. (1992b), after a thorough revision of the genus based on morphological, biochemical and molecular characterisations, identified six human pathogenic species: Trichosporon ovoides Behrend, Trichosporon inkin (Oho ex Ota) do Carmo-Souza and van Uden, Trichosporon asteroides, Trichosporon cutaneum, Trichosporon asahii and Trichosporon mucoides. In another study published in the same year, Guého et al. (1992a) demonstrated that T. ovoides should replace T. beigelii, considered to be of doubtful identity, which should not be retained or be applied to T. cutaneum, a species already identified by de Beurmann, Gougerot and Vaucher from skin samples (de Beurmann & Gougerot 1909). T. ovoides and T. inkin that have been to man, are scalp and genital white piedra agents, respectively (Guého et al. 1992b, 1994).
White piedra, a mycosis that occurs in some animals, such as horses, monkeys and domestic animals, as well as humans (Kaplan 1959), is found within a wide geographical distribution, including tropical and temperate regions, occurring mainly in South America, Africa, Europe and Asia (Kamalam et al. 1981, Thérizol-Ferly et al. 1994, Ghorpade 2004, Schwartz 2004). There are also some cases in the USA (Elmer et al. 2002, Youker et al. 2003, Kiken et al. 2006) and it has been found to be endemic to Texas (Kalter et al. 1986). Although some authors recorded the disease in India, after three years of investigation, Kamalam et al. (1981) found just four cases of white piedra in that country.
From the referenced literature, the cases of genital white piedra were the most frequently reported in Brazil (Brito & Costa 1966, Londero et al. 1966, Carneiro et al. 1971, 1973, Fishman et al. 1980, Zaror et al. 1989, Almeida et al. 1990); however, more recent studies refer mainly to scalp white piedra (Nora et al. 2002, Pontes et al. 2002b, Diniz & Souza Filho 2005), with the exception of five cases of genital white piedra recorded in 2002 (Pontes et al. 2002a). In Brazil, the disease has been recorded in the states of Pará, Rio Grande do Sul, São Paulo, Rio de Janeiro, Paraíba and Espírito Santo (Brito & Costa 1966, Londero et al. 1966, Fischman et al. 1980, Zaror et al. 1989, Gondim-Gonçalves et al. 1991, Nora et al. 2002, Pontes et al. 2002a, b, Diniz & Souza Filho 2005).
The laboratory diagnosis of white piedra is based on direct morphological observations of the fungal elements present on the hair. These elements are round blastoconidia and arthroconidia agglomerated and bind together by a cement-like substance produced by the fungal hyphae and are found in the extrafollicular region of the hair in any hairy areas, including eyelas-hes, eyebrows, beard, moustache, scalp, axilla and genitals (Smith et al. 1973, Zaror et al. 1989, Almeida et al. 1990, Kiken et al. 2006). When the fungus invades the interior of the hair, it becomes fragile, splits and breaks easily (Kalter et al. 1986).
The mode of infection is not clear: some authors suggest that poor hygienic habits and sexual transmission, in the case of genital white piedra, in addition to heat and humidity, are predisposing factors (Fischman et al. 1980, Torssander et al. 1985, Kalter et al. 1986, Stenderup et al. 1986). Despite cases of white piedra in domestic animals, they are unlikely the source of transmission to human beings (Walzman & Leeming 1989).
There are few reports on white piedra from which the aetiological agent has been identified at the species level. Consequently, the knowledge on the species that attack humans is very limited, as well as the information regarding this mycosis epidemiology. Thus, the purpose of this investigation was to identify at the species level the aetiological agents of white piedra in hospital patients.
PATIENTS, MATERIALS AND METHODS
This is a retrospective study using cultures that were macroscopically identified as the genus Trichosporon and preserved in freezers at -20ºC on Skin Milk medium (Difco, USA). The research project was submitted to the Research Ethical Committee of Santa Casa de Misericórdia of Rio de Janeiro (protocol number 016/07).
The fungal isolates were obtained from hair samples from patients with clinically suspected white piedra in metropolitan Rio de Janeiro and were sent to the Mycology Laboratory of the Dermatological Service of Santa Casa de Misericórdia of Rio de Janeiro from January 2003-December 2006. The identification of the isolates was based on macro and microscopic characteristics of the yeasts cultivated at 25ºC from 2-10 days on YMA (yeast malt extract agar, Difco, USA) for macroscopy and MEA (malt extract agar, Merck, Germany) and CMA (corn meal agar, Difco, USA) for microscopy. The results of carbon and nitrogen assimilation tests conducted with cultures on YNB (yeast nitrogen base, Difco, USA) and on YCB (yeast carbon base, Difco, USA), respectively, were also used as characteristics for species-level identification, as were tests on Christensen's media (Difco, USA) and from growth on the vitamin free medium YVFB (yeast vitamin free base, Difco, USA), on YNB with 0.5% glucose containing 0.1% or 0.01% cycloheximide and on YMB (yeast malt extract broth, Difco, USA) at 37ºC (Barnett et al. 2000). All assays were conducted over four weeks, with the exception of the urea hydrolysis test, which was up to seven days and nitrogen assimilation tests, which were up to 14 days.
Reference microorganisms T. ovoides INCQS 40270 (ATCC 90040) and T. inkin INCQS 40266 (ATCC 18020), acquired from the American Type Culture Collection and maintained in the Culture Collection of Instituto Nacional de Controle de Qualidade (INCQS) were used as control for the identification of the species in the different tests within this investigation.
Mycological results - Ten clinical isolates of Trichosporon from different hair samples and reference strains were studied. Fig. 1 shows piedric nodule caused by a white piedra agent on a hair shaft. The causative agent could be any Trichosporon species, since the macroscopic aspect of piedric nodule is the same regardless the white piedra agent. All the isolates hydrolysed urea and did not ferment carbohydrates. The physiological and biochemical tests in addition to the macro and microscopic characteristics allowed the identification at the species level of nine isolates, among them seven T.ovoides and two T.inkin. One isolate remained unidentified at the species level (Table I). The physiological and biochemical tests conducted for this isolate indicated 50% of T. ovoides and 50% of T. asteroids characteristics; however, the morphology observations did not allow differentiation between these two species. Therefore, further characterisations will be needed in order to identify this species. The identified T. ovoides and T. inkin species, including the reference strains T. ovoides INCQS 40270 (ATCC 90040) and T. inkin INCQS 40266 (ATCC 18020), showed variable profiles toward the assimilation of carbon and nitrogen sources (Table II). However, the hit probability calculated using the Yeast Identification PC Program version 4 (Barnett et al. 2000) for T. ovoides was 95-100% and 98-100% for T. inkin, both satisfactory values. The morphological features of the identified species T. ovoides and T. inkin are described below.
Trichosporon ovoides - Colonies of the isolates, including T. ovoides INCQS 40270 (ATCC 90040), on YMA after 10 days at 25ºC attained a diameter of around 15 mm and were dry, farinose, irregularly folded at the centre with deep radial fissures in some isolates and with a flat marginal zone, at first appearing as cream coloured then becoming white (Fig. 2A). The microscopic observations of the cultures on MEA after two days incubation at 25ºC showed blastoconidia, many septate hyphae and arthroconidia. After 10 days, the cultures were nearly all filamentous with many well-developed arthroconidias that had rectangular or oval shapes (Fig. 2B).
Trichosporon inkin (Oho ex Ota) do Carmo-Souza and van Uden - On YMA after 10 days at 25ºC the colonies, including T. inkin INCQS 40266 (ATCC 18020), attained 9-12 mm diameter and were finely cerebriform, without a marginal zone, often cracked the agar, and with a creamy, slightly yellow colour, and older colonies sometimes darkened (Fig. 3A). Microscopically cylindrical or rectangular arthroconidia measuring approximately 2-5 x 3-16 µm were observed after two days of incubation at 25ºC on MEA. Blastoconidia and sarcinae were observed after 10 days (Fig. 3B). Appressoria were observed in cultures on CMA after 10 days (Fig. 3C).
Clinical observations - The distribution of white piedra agents in patients according to age, sex and source is shown in Table I. Among the 10 fungal isolates collected from hair, nine were from scalp white piedra (90%) in female patients, and one was from genital white piedra (10%) in a male patient. T. ovoides was isolated as the aetiological agent of white piedra in seven cases (70%), among them six in hair shaft (86%) and one in genital hairs (14%). T. inkin was isolated from hair shaft samples from two patients (20%) and there were no occurrences of white piedra on genital hair produced by this fungus. The only isolate not identified at the species level (10%) was isolated from hair shaft. T. ovoides was the most frequently isolated (60%), once from male patient and six times from female patients. The isolates were deposited in the Culture Collection of INCQS and their corresponding accession numbers are listed in Table I.
The attack of hairs by Trichosporon spp. is known worldwide as white piedra and is considered a sporadic infection that occurs in tropical and temperate climates (Fishman et al. 1980, Thérizol-Ferly et al. 1994, Elmer et al. 2002, Youker et al. 2003, Ghorpade 2004, Kiken et al. 2006), mostly in rainy periods (Kamalam et al. 1981).
In the few reports of mycoses caused by the genus Trichosporon in Brazil over the last 30 years, genital and scalp white piedra were recorded mainly in the states of São Paulo, Rio de Janeiro, Paraíba and Espírito Santo (Fischman et al. 1980, Gondim-Gonçalves et al. 1991, Pontes et al. 2002a, b, Diniz et al. 2005). In the present study of the 10 Trichosporon species mostly isolated from scalp, T. ovoides, considered as the main causative agent of white piedra (Guého et al. 1994), was the predominant species.
Based on Guého et al. (1994), the white piedra was very common in the beginning of the 20th century in Europe and South America. Nevertheless, in the present days, due to improvements in personal hygiene, it has become rarer. In the present investigation, all the patients seemed to have good personal hygiene habits and were of medium socioeconomic status. It was, therefore, not possible to associate the disease with poor personal hygiene. On the other hand, in some cases those in charge of the children reported that they habitually tied back their hair while still wet just after bathing and it was also observed that even the young children used cream conditioners and other hair creams. Diniz and Souza Filho (2005) suggested that those situations could be factors that are favourable for keeping hair wet and consequently for developing mycosis.
White piedra was recorded in Rio de Janeiro by Gondim-Gonçalves et al. (1991) on hair shafts from three children in the age group of 0-10 years. In Paraíba (Pontes et al. 2002b) and in Espírito Santo (Diniz & Souza Filho 2005), higher numbers of white piedra cases were found in children's scalps. The present investigation corroborates these earlier studies since the majority of the isolates (70%) were obtained from scalp white piedra found in children under the age of 10. The species found most frequently on these children was T. ovoides, which is in agreement to the literature that reports this species as the main agent of scalp white piedra (Guého et al. 1994). However, discrepant results were found by Kiken et al. (2006) in USA, who identified T. cutaneum, T. asahii and T. mucoides as the aetiological agents of white piedra in children.
Trichosporon inkin species currently related to some cases of systemic processes (Ramos et al. 1998, Koyanagi et al. 2006) were found in this investigation, causing white piedra in the hair shaft in two patients, one of them a four-year-old child. These results are unexpected in light of those of Guého et al. (1992b, 1994), who demonstrated the strong preference of this species to the genital areas, causing white piedra in pubic hair. To our knowledge this, is the first time that T. inkin is identified as a white piedra agent in hair shafts in children under the age of 10. On the other hand, T. ovoides, traditionally isolated from the hair shaft (Guého et al. 1994, 1998), was identified as the causative agent in the only case of genital white piedra observed in this study.
T. inkin and T. ovoides are molecularly similar species (Guého et al. 1992b) and even the sequencing of ITS, a DNA region that is highly conserved and very often used for species identification, does not allow the reliable distinction between them, causing some authors to prefer the biochemical characterisation for identification (Koyanagi et al. 2006). In the present investigation, biochemical and physiological characterisation analysed using Yeast Identification PC Program, version 4 (Barnett et al. 2000), resulted in the species identification with a 95-100% hit probability. These results, together with the morphological characterisations, allowed reliable identifications.
The patients included on this research were mostly females and were under the age of 10 (Table I). These results are in accordance to previous investigations that reported the white piedra cases in hair shaft mainly on female patients in the age group of 0-10 years (Pontes et al. 2002b, Diniz & Souza Filho 2005, Kiken et al. 2006). This is the first report on T. inkin identified as a white piedra agent in the hair shaft of a child under the age of 10. The species identification of the genus Trichosporon as white piedra agents will contribute to improve the knowledge on this mycosis and on these species.
To Dr. Regina Cazs Schetmann, dermatologist and head of the Mycology Laboratory at Santa Casa de Misericórdia do Rio de Janeiro, for giving the access to the patients records and to the photographs of the attacked hair, to Elzarlinda Eurich Reis, technician of this same Laboratory, for collecting, preserving and providing the fungal cultures used in the present study, to Miguel M. Fialho and Carlos Roberto S. do Nascimento, from Setor de Fungos de Referência from Departamento de Microbiologia, INCQS/FIOCRUZ, for their collaboration on the photographic documentation.
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Received 3 July 2008
Accepted 31 October 2008