Seborrheic dermatitis: predisposing factors and ITS2 secondary structure for Malassezia phylogenic analysis

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1 Medical Mycology November 2013, 51, Seborrheic dermatitis: predisposing factors and ITS2 secondary structure for Malassezia phylogenic analysis YULIEN AMADO *, ANELVI PATI Ñ O-UZC Á TEGUI *, MARIA C. CEPERO DE GARC Í A *, JAVIER TABIMA *, ADRIANA MOTTA, MARTHA C Á RDENAS *, ADRIANA BERNAL *, SILVIA RESTREPO * & ADRIANA CELIS * * Laboratorio de Micolog í a y Fitopatolog í a, Departamento de Ciencias Biol ó gicas Universidad de Los Andes, Bogot á, Universidad El Bosque, Bogot á, and Hospital Sim ó n Bol í var, Bogot á, Colombia Seborrheic dermatitis (SD) is a chronic, widespread skin condition, which is considered a multifactorial disease influenced, in part, by Malassezia spp. opportunistic activities, as well as various endogenous and exogenous factors. Malassezia species are lipophilic, lipid-dependent yeasts that are members of the normal mycobiota of the human skin. Their isolation from SD lesions varies around the world and the study of the relationship among factors such as gender, age, immunosuppressive condition of the patient and SD development, can lead to a better understanding of this disease. To elucidate the association of age and gender with the development of SD and to precisely determine the Malassezia species involved in the disease, samples were obtained from 134 individuals, including individuals without lesions, human immunodeficiency virus positive patients, individuals with seborrheic dermatitis, and HIV patients with seborrheic dermatitis. Malassezia spp. were identified by phenotypic and genotypic methods and a phylogenetic analysis was performed using Bayesian inference. This study revealed that age and gender are not predisposing factors for SD development, and that the most frequent species of Malassezia related to SD development among the Colombian population is M. restricta. We also report the isolation of M. yamatoensis for the first time in Colombia, and propose an ITS2 secondary structure from Malassezia taxa that can be used for precise identification and to establish more robust phylogenetic relationships. Keywords seborrheic dermatitis (SD), Malassezia, secondary structure ITS2, HIV Introduction Scaling, inflammation and redness are the main clinical characteristics of seborrheic dermatitis (SD), a chronic, widespread skin condition occurring mainly on body areas rich in sebaceous glands, such as the scalp, face and chest [1]. The disorder is present in 3 5% of immunocompetent adults, and is far more frequently observed (30 85%) in AIDS patients. Its appearance is considered an early marker of HIV infection and usually occurs when CD4 counts are less than 400 cells per millimeter [2 4]. Received 19 November 2012 ; Received in final revised form 14 May 2013; Accepted 24 June Correspondence: Adriana Celis, Laboratorio de Micolog í a y Fitopatolog í a, Departamento de Ciencias Biol ó gicas Universidad de Los Andes, Bogot á, Colombia. Tel: 57 (1) , ext. 3757; acelis@uniandes.edu.co The etiology of the disease is unknown, but it has been related to the opportunistic activity of Malassezia yeasts, which usually appear as commensals on human skin [5] Malassezia spp. are part of the normal microbiota of humans and other warm-blooded animals. The genus comprises 14 species of which nine may be isolated from human skin ( M. furfur, M. sympodialis, M. restricta, M. globosa, M. slooffi ae, M. obtusa, M. dermatis, M. yamatoensis and M. japonica ) [6 9]. The identification of Malassezia species was based for many years of the organisms phenotypic traits, but interpreting the results is difficult and contributed to variable results which eventually lead to the development of molecular identification methods. Thus, more studies of the taxonomy, epidemiology and genomics of Malassezia are being undertaken in order to discover new species and to make advances in terms of understanding the relationship 2013 ISHAM DOI: /

2 Malassezia species and aspects associated with seborrheic dermatitis 869 between endogenous factors (age, gender and immune status) and exogenous factors (geographical area and environmental conditions) in dermatological conditions such as pityriasis versicolor, psoriasis, folliculitis, atopic dermatitis and SD [5,10 16]. The isolation of the Malassezia species associated with SD varies around the world. For example, M. globosa and M. restricta have been the principal species isolated from samples in Colombia, while M. sympodialis is the predominate species in Mexico and M. furfur is the principal cause of SD in Iran and France [11,17 20]. Previous studies showed that SD occurs more frequently in men compared to women [5,12,21] and the frequency of Malassezia spp. is higher in men entering adolescence and in women over the age of 30. In addition, more Malassezia cells are present on human skin in summer than in winter, suggesting that changes in humidity and temperatures could be a predisposing factor to the development of SD [21,22]. The precise identification of Malassezia species is important to expand our knowledge of various aspects of SD. Genotypic studies revealed high levels of genetic diversity among and within the Malassezia species [16,23], and that the use of molecular markers such as 26S rdna, ITS and 5.8S allows species identification. Phylogenetic studies using these regions confirmed intra-species variation among some Malassezia species [24]. The use of secondary structures of rrna regions, in particular the ITS2 region, may help resolve intra-species variations [25,26] due to similarities in structure that cannot be inferred using the primary sequence alone. The ITS2 secondary structure increases the resolution of the phylogenetic reconstructions [27 29] and introduces compensatory base change (CBS) analysis which has been reported as a powerful tool in determining species boundaries [30,31]. SD is one of the most frequently found multifactorial skin disorders which is highly prevalent in HIV-positive patients. In view of this, the objectives of this study were: (a) to determine the frequency of Malassezia species associated with SD in individuals in Colombia; (b) to elucidate the relationships between age and gender and the development of this condition; and (c) to determine the phylogenetic relationships among Malassezia isolates in order to obtain a precise identification of the species. Materials and methods Study population A total of 134 men and women, ranging in age from 3 86 years participated in the study. Patients were selected from May 2010 through April 2011 during dermatologic consultations from one of the following groups: (i) those without lesions (NL); (ii) human immunodeficiency virus-positive individuals (HIV); (iii) those with seborrheic dermatitis (SD); and (iv) human immunodeficiency virus-positive patients with seborrheic dermatitis (SD/HIV). They were either outpatients or hospitalized HIV patients at the Simon Bolivar Hospital, Bogot á, Colombia. In each case, a dermatologist performed a thorough dermatological examination and patients were informed about the sampling procedure, which was then performed after they had all signed the informed consent form. The Ethics Committee of the Universidad de los Andes approved the protocol and the investigations were conducted according to the Declaration of Helsinki Principles. Sample collection and processing Samples were taken according to the study groups, in that for HIV patients and non-lesion individuals, the sampling was performed by Dacron swab of the scalp and the nasolabial region (Copan Diagnostics, Corona, CA, USA). Scales from the scalp of patients with seborrheic dermatitis were taken using the blunt end of a scalpel and collected in sterile Petri dishes to be processed as previously described [18]. Another 29 strains from individuals among the same population were obtained from a previous study (32). The sources of the strains are listed in Table 1. Phenotypic characterization We conducted macroscopic identification of the primary isolates to recognize and select different morphotypes that were compatible with Malassezia spp. These were cultured on modified Dixon agar and incubated over 4 5 days at 33 C [15]. To identify the Malassezia species we analyzed: (a) biochemical characteristics such as reactions for catalase, urease and β -glucosidase activities; (b) the assimilation capacity of cremophor EL, and Tween 20, 40, 60 and 80; (c) pigment production on tryptophan-based medium; and (d) the ability to grow on Sabouraud agar, Sabouraud plus 10% Tween 20, Sabouraud plus 0.1% Tween 80 and Dixon agar at 37 and 42 C [9,15,18,33,34]. Molecular characterization Pure culture were stored at 80 C overnight to be freezedried for 2 days and DNA was then extracted as previously described [35]. PCR was performed, in a final volume of 25 μ l [24], to amplify the region 5.8S rdna-its2 using the primers ITS3 and ITS4 [14,36]. Amplified products were visualized in 1% (w/v) agarose gel electrophoresis in TBE buffer, stained with ethidium bromide and revealed by UV. PCR products were sequenced using an ABI3730xl DNA analyzer (PE Applied Biosystems). Sequence assembly

3 870 Amado et al. Table 1 Origin of Malassezia isolates included in this study. Malassezia species Strain Origin GenBank Accession Number 5.8S rdna-its2 Malassezia furfur Malassezia globosa Malassezia restricta Malassezia sympodialis NL76- NL80- NL42 Healthy individual JQ JQ JQ SD41- SD45- SD84 Seborrheic dermatitis patient JQ JQ JQ SD/HIV14 - SD/HIV16 - SD/ HIV19B - SD/HIV20 - SD/HIV24A- SD/HIV51- SD/HIV63 Seborrheic dermatitis and HQ HQ HQ HQ JQ JQ JQ HIV23 - HIV30- HIV52- HIV59- HIV60- HIV62- HIV64- HIV65- HIV72- HIV13 - HIV50A- HIV86- HIV90- HIV95 HQ JQ JQ JQ JQ JQ JQ JQ JQ HQ JQ JQ JQ JQ NL10A- NL12 - NL32- NL39- NL69 Healthy individual JQ HQ JQ JQ JQ SD15 - SD31- SD48- SD44- SD36 Seborrheic dermatitis patient HQ JQ JQ JQ JQ HIV26B- HIV89- HIV91- HIV22 - HIV71- HIV96- HIV97 NL70- NL82- NL34- NL38- NL83- NL21 - NL79 SD3- SD43- SD46- SD47- SD53- SD54- SD55- SD4 - SD6 - SD49 SD/HIV29- SD/HIV56- SD/HIV58- SD/ HIV2 - SD/HIV88 HIV26A - HIV50B- HIV67- HIV87- HIV92 NL1 - NL9 - NL11 - NL33- NL35- NL37- NL68- NL75- NL77- NL78- NL81 Seborrheic dermatitis and Healthy individual Seborrheic dermatitis patient Seborrheic dermatitis and Healthy individual SD/HIV27 - SD/HIV19A- SD/ HIV24B - SD/HIV25 - SD/HIV61- HQ JQ HQ HQ JQ JQ JQ JQ HQ JQ JQ JQ JQ JQ JQ JQ JQ HQ JQ JQ JQ JQ JQ JQ JQ JQ HQ HQ JQ JQ JQ JQ HQ JQ HQ JQ JQ JQ JQ HQ HQ HQ JQ JQ JQ JQ JQ JQ JQ JQ SD5B - SD7 - SD40- Seborrheic dermatitis patient HQ HQ JQ J SD/HIV8 - SD/HIV17 - SD/HIV24C - SD/HIV28 Seborrheic dermatitis and HQ HQ HQ JQ HIV57- HIV73- HIV93- HIV94 HIV positive patient JQ JQ JQ JQ Malassezia slooffi ae SD 5A Seborrheic dermatitis patient HQ Malassezia yamatoensis NL 74 Healthy individual JQ Malassezia furfur CBS 1878 EU Malassezia globosa CBS 7986 AY Malassezia restricta CBS 7877 AY Malassezia sympodialis CBS 7222 AF Malassezia yamatoensis CBS 9726 AB Malassezia slooffi ae CBS 7971 AY Strains obtained from a previous study [32].

4 Malassezia species and aspects associated with seborrheic dermatitis 871 and editing were performed manually using Geneious software ( The nucleotide seq uences obtained in this study were submitted to GenBank and the GenBank accession numbers for the 5.8S rdna-its2 region sequences are listed in Table 1. We performed two phylogenetic analyses, the first of which was based on 5.8S and ITS2 rrna sequences. This data was aligned using the MUSCLE algorithm [37]. A Bayesian inference analysis was performed for the phylogenetic reconstruction using MrBayes [38] with 1 million MCMC generations, 4 chains (1 cold chain and 3 heated chains), two runs, model specification per matrix (GTR I G) and burn in of 25% of the total run. An ITS2 secondary structure was predicted using the mfold web server ( with the one chosen was one with a 4-helicoidal ring and the best value of free energy. Subsequently, structures were aligned and visualized using 4SALE (39), and the phylogenetic analyses were performed by Bayesian inference using the parameters described above. Statistical analyses Logistic and multinomial regression analyses were used for endogenous factors (age and gender) and species distribution analyses, respectively. Statistical analyses were performed with SPSS software (SPSS Inc., Chicago IL, USA). P -values 0.05 were considered significant. Results Of the 134 individual samples, 95 (54 men and 41 women) were positive for Malassezia spp., giving an isolation rate of 71%. The distribution by age and gender in each of the four populations studied is shown in Table 2. Age and gender are not predisposing factors for SD development The age and gender of individuals with SD were studied in order to elucidate the epidemiology of the condition. Of the total sample, we found that SD occurred more frequently in men (28%) than in women (16%), and more frequently in SD and SD/HIV patients aged 30 50, followed by those over 50 but least frequently in patients under 30 years of age. While the incidence of SD tended to decrease with age, we found that SD/HIV patients tended to develop this disease between the ages of (Fig. 1). However, it we noted in this investigation that age or gender were not significant factors in the development of SD ( P and 0.448, respectively). Malassezia restricta is the main species isolated from SD individuals Malassezia restricta (38%) was the most commonly isolated species among the SD individuals of all four populations studied. In NL and HIV individuals, M. furfur (47%) and M. sympodialis (43%) were predominant, respectively. The frequency of the isolation of Malassezia species from SD, NL, SD/HIV and HIV individuals is shown in Figure 2. To the best of our knowledge, our study is the first to report the recovery of M. yamatoensis from a non-lesion individual in Colombia. An ITS2 secondary structure complements the molecular and physiological species identifi cation A total of 101 Malassezia isolates were obtained (some samples contained two or three isolates). Typical morphological and physiological features of Malassezia were found with isolates recovered from NL, SD, SD/HIV and HIV individuals. Table 2 Age and gender distribution of study participants. Study populations SD NL SD/HIV HIV All individuals ( n 95) Gender Female 48% (11) 76% (19) 21% (4) 14% (4) Male 52% (12) 24% (6) 79% (15) 86% (24) Age group 30 48% (11) 88% (22) 26% (5) 18% (5) % (7) 0 63% (12) 64% (18) to 50 22% (5) 12% (3) 11% (2) 18% (5) SD, Seborrheic dermatitis patient; NL, Healthy individual; SD/HIV, Seborrheic dermatitis and HIV positive patient; HIV, HIV positive patient. (The value in parenthesis represents the number of individuals from the total population). Fig. 1 Distribution of SD/HIV and SD individuals by age group.

5 872 Amado et al. Fig. 2 Malassezia species in SD, NL, SD/HIV and HIV individuals. The 5.8S rdna and the ITS2 region, amplified from Malassezia species using the ITS3 and ITS4 primers, allowed us to identify anthropophilic Malassezia species because the amplicon lengths differed among species; M. furfur 500 bp, M. globosa and M. restricta 410 bp, M. sympodialis 400 bp, M. yamatoensis 649 bp and M. slooffiae 430 bp. Fig. 3 Inferred ITS2 RNA secondary structures of isolated Malassezia species. The four helices are numbered I IV and the secondary structure Gibbs free energy value according to mfold is shown for the secondary structure.

6 Malassezia species and aspects associated with seborrheic dermatitis 873 Primary sequencing has always been a helpful tool for phylogenetic studies but in this study, the primary sequence did not present a good phylogenetic resolution because isolates from the same species were not grouped together despite showing high bootstrap values (Supplementary Fig. 1, available online at doi/abs/ / ). In order to improve the resolution of this technique, a phylogenetic analysis from the ITS2 secondary structure was implemented. As a result, the ITS2 secondary structure predictions for Malassezia species corresponded to a 4-helicoidal ring model (Fig. 3), as in all eukaryotic organisms. Additionally, the ITS2 secondary structure grouped the majority of isolates from each species with high bootstrap support values and their topology confir med the morphological/ physiological identification of these isolates (Fig. 4), suggesting that this tool provides a good resolution for the discrimination of Malassezia species. Discussion This study revealed that the occurrence of SD might not be related to an individual s endogenous factors such as age and gender. However, previous studies found that SD was more frequent in men than in women, a factor reflected in this study [12,21]. The species of Malassezia most frequently involved in the development SD in the Colombian population is M. restricta. SD prevalence has been reported when sebaceous glands activity is high, causing a decrease of intracellular lipids Fig. 4 Phylogenetic reconstruction of the genus Malassezia based on ITS2 secondary structure. Branch lengths represent genetic divergence in nucleotide per site changes. The arrow represent isolates that are not currently assigned to the species that they group with by this analysis method. Sequence of Cryptococcus neoformans EF was used as outgroup to root the phylogram.

7 874 Amado et al. such as ceramides, fatty acids and cholesterol, as during the first months of life and early adolescence [40 42]. In our case, there was a high proportion of SD in individuals who were over 50 years old, which could be related to high sebum excretion at this stage of life, in contrast to nonlesion individuals [43,44]. This can also be associated to the possible opportunistic activity of Malassezia spp., which can use their lipid machinery (lipases and phospholipases) for the hydrolysis of triglycerides from human sebum, releasing fatty acids, and thus generating epidermis damage, which in turn may be related to the development of SD [41]. However, the role that Malassezia species play in the evolution of SD remains unknown and the analysis of other specific individual factors such as antifungal therapy and concomitant diseases could be determinants that would allow further learning about the disease [12,13]. The ecological diversity of the Malassezia species has been studied around the world [11]. M. globosa and M. restricta are the most common species isolated from samples of SD patients, as reported from Spain, Japan, Korea and France, and other countries [20,22,45,46]. In our study we found, similar to that noted in previous investigations, that M. restricta is the most frequently isolated species from SD patients in Colombia [18]. These results are similar to those found in Bosnia-Herzegovina, for isolates in immunocompromised individuals with SD [47]. Although a high rate of M. furfur isolation from SD individuals has also been reported before [19,20], it was unexpected to find a high rate of recovery of this species from HIV individuals, with and without SD, because earlier studies associated M. globosa with patients with HIV and SD/HIV in Colombia [18]. These results might be related to the importance of environmental factors and the prophylactic therapy with fluconazole, as well as concomitant infections such as toxoplasmosis, cryptococcosis and tuberculosis, which were common in the patients during the sampling processes. We demonstrated that Malassezia yamatoensis is present as part of the Colombian human skin microbiota in low proportions, as previously found in a Japanese population [6]. Only one of the total of 95 strains of Malassezia spp. proved to be this species and was isolated from a sample of a non-lesion patient. Although a large number of Malassezia species have been reported in our region, the isolation of this species demonstrated that further work is required to learn more about the geographic distribution and identification of possible Malassezia spp. In this study, we found that the primary DNA sequence of the 5.8S and ITS2 did not show sufficiently good resolution to differentiate Malassezia species. We found a better phylogenetic signal from ITS2 secondary structure compared to the primary sequence alone. This information provided alignment corrections because of the increased number of characters that are included in the morphometric analysis [27,28]. Our results suggest that it is necessary to use additional molecular information, such as secondary structures and other molecular markers (such as D1/ D2 LSU), to obtain appropriate species identification [13]. The identification of other molecular markers in clinically important fungi has also been widely discussed in the genus Fusarium, in which no molecular markers with strongly supported clades have, as yet, been found [48]. In conclusion, this study is an approach to gain a better understanding of the Malassezia epidemiology in Colombia and its relationship with SD development. It is the first time that M. yamatoensis has been found in Colombia and the second time it has been found outside Japan using a culturebased method. 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