Identi cation of Malassezia species isolated from patients with seborrhoeic dermatitis, atopic dermatitis, pityriasis versicolor and normal subjects

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1 Medical Mycology 2000, 38, Accepted 28 February 2000 Identi cation of Malassezia species isolated from patients with seborrhoeic dermatitis, atopic dermatitis, pityriasis versicolor and normal subjects A. NAKABAYASHI*, Y. SEI* & J. GUILLOT *Department of Dermatology, Sho wa University Fujigaoka Hospital, Y okohama, Japan; Parasitology -Mycology Unit, A lfort s National Veterinary School, Maisons-A lfort, France Introduction We identi ed Malassezia species isolated from 42 patients with seborrhoeic dermatitis, 17 patients with atopic dermatitis, 22 patients with pityriasis versicolor, 35 normal subjects and 73 healthy medical students. Regarding the prevalence of Malassezia species in the 35 normal subjects, the frequency of isolation of Malassezia globosa was 22%, M. sympodialis 10% and M. furfur 3%., M. pachydermatis, and M. obtusa were infrequently isolated from normal skin. Two different species were isolated coincidentally from seven samples. In the patients with atopic dermatitis, M. furfur was isolated more frequently from lesional skin (21%) than non-lesional skin (11%). However, there was no statistical signi cance. Therefore, this result, by itself, is insuf cient to prove that M. furfur should be considered to be an exacerbating factor of atopic dermatitis. In seborrhoeic dermatitis, M. furfur (35%) and M. globosa (22%) were isolated from lesional skin on the face at signi cantly high rates in comparison with the normal subjects. Therefore, M. furfur and:or M. globosa may be pathogens of seborrhoeic dermatitis. M. globosa was isolated at a frequency of 55% from lesional skin of pityriasis versicolor, while all other species were below 10%. These data suggest that the pathogenic species of pityriasis versicolor is M. globosa. Keywords dermatitis The fungal nature of pityriasis versicolor was recognized by Eichstedt in 1846, but the genus Malassezia, which was created by Baillon half a century later, has never been properly classi ed. Due to the absence of any clear differences at the time, the genus was limited to Malassezia furfur, a lipid-dependent species responsible for various cutaneous conditions in humans, and M. pachydermatis, a lipophilic species which is able to grow Correspondence: Atsuhiro Nakabayashi, Department of Dermatology, Showa University Fujigaoka Hospital, 1-30 Fujigaoka, Aobaku, Yokohama , Japan. Tel.: ; fax: ; a-nakaba@db3.so-net.ne.jp atopic dermatitis, Malassezia species, pityriasis versicolor, seborrhoeic in rich medium containing short-chain fatty acids and is considered to be restricted to animals [1]. The third edition of The Yeasts [2] listed Pityrosporum orbiculare and P. o ale, fungi considered to be M. furfur, and P. canis, now named M. pachydermatis. Simmons & Guého [3] subsequently added a new species to the genus Malassezia, namely M. sympodialis, which was differentiated from M. furfur by occasional sympodial budding and a low guanine plus cytosine content. In addition, in 1996 Guého et al. [4] reclassi ed the genus Malassezia on the basis of morphology, ultrastructure, physiology and molecular biology. They advocated adding M. globosa, M. obtusa, and to the genus Malassezia. This classi cation of these seven species is now recognized ISHAM

2 338 Nakabayashi et al. In recent years, the genus Malassezia has come to be considered important in the etiology of seborrhoeic dermatitis [5 7]. There are reports that Malassezia is an exacerbating factor of scalp, face and neck lesions of atopic dermatitis [8,9]. However, it is not yet clear which species might be the pathogens in Malassezia-associated diseases. The aim of this study was to examine the Malassezia species of the normal skin ora as well as the species isolated from patients with seborrhoeic dermatitis, atopic dermatitis and pityriasis versicolor according to the method devised by Guillot et al. [10]. Materials and methods Subjects The subjects were 17 patients with atopic dermatitis who had intractable lesions on the face and:or neck (eight males, nine females; years old), 42 patients with seborrhoeic dermatitis (32 males, 10 females; years old), and 22 patients with pityriasis versicolor (16 males, six females; years old) who visited our outpatient clinic. Normal subjects consisted of 35 healthy volunteers (26 males, eight females; years old) and 73 medical school students (64 males, nine females; years old) without any serious basal disease or dermatosis of the examined regions. C ollection and culture of samples In the patients with atopic dermatitis or seborrhoeic dermatitis, and the normal subjects (excluding medical students), samples were collected by the swab method from scalp, face and trunk sites with and without skin lesions. The collection sites from patients with atopic dermatitis and seborrhoeic dermatitis, which do not present skin lesions, were regarded as non-lesional skin. In the pityriasis versicolor patients, samples were collected by the swab method from the lesional skin of the trunk. The collected samples were cultivated at 32 C in Dixon agar medium. Samples were collected from the healthy medical school student volunteers using the hairbrush method for the scalp and the tape method for the face. In the tape method, Scotch tape was af xed to the normal skin, and it was then transferred sticky-side down to Dixon agar medium. We identi ed Malassezia species according to the method of Guillot et al. [10] (Fig. 1). Isolates from the primary cultures were used for identi cation. M. pachydermatis is able to grow on Sabouraud agar ( a in Fig. 1). Additional tests were necessary for identi cation of other Malassezia species, especially the Tween assimilation test. Brie y, Malassezia yeast suspensions were mixed with Sabouraud agar, and the mixtures were plated. Four holes were made in the agar by means of a 3-mm diameter punch and lled with 5 ml each of Tween 20, 40, 60 and 80, respectively. The agar plates were cultivated at 32 C for 1 week. The growth of M. sympodialis is inhibited by the high concentration of Tween 20 ( b in Fig. 1). M. furfur exhibits similar growth with both Tween 20 and 80 ( c in Fig. 1). grows better with Tween 20 than with Tween 80 ( d in Fig. 1). M. globosa, M. obtusa and are unable to utilize any of the four Tween compunds. In the morphological ndings under a microscope, M. globosa ( e in Fig. 1) exhibits large and spherical cells, while M. obtusa ( f in Fig. 1) appears cylindrical. is the only lipiddependent species lacking catalase ( g in Fig. 1). Con rmation of the identi ed species Three stock strains of each species we isolated, except for M. obtusa, were sent to Dr Guillot to con rm the identi cation (macroscopic and microscopic examination, and Speci c identi cation Fig. 1 Identi cation of Malassezia species ISHAM, Medical Mycology, 38,

3 Identi cation of Malassezia species from different patients 339 molecular biology). Three primary strains of M. globosa were also sent. Statistical analyses The data of the patients with atopic dermatitis and seborrhoeic dermatitis were analyzed using Pearson s chisquare test. In addition, the difference between the isolates from lesional skin of seborrhoeic dermatitis and from normal subjects was evaluated using Fisher s exact method. Cases of unknown:contamination were excluded from the statistical analyses. Results Isolates from normal subjects The prevalence of Malassezia species in the normal subjects (excluding medical students; Table 1) was 22% (23: 105) for M. globosa, 10% (11:105) for M. sympodialis and 3% (3:105) for M. furfur. The isolated species from the trunk belonged to only two species: M. globosa (51%; 18:35) and M. sympodialis (26%; 9:35). Samples from the scalp and face frequently yielded negative cultures (rates over 60%), while M. globosa, M. furfur and M. sympodialis were isolated from a few cases. Two different species (M. globosa and M. sympodialis) were both isolated from each of three samples from the trunk. Isolates from healthy medical school student volunteers The percentage of negative cultures (Table 2) decreased to 25% (17:68) for the scalp and 40% (25:63) for the face. Three species (M. globosa, M. furfur and M. sympodialis) were the most frequently isolated from the scalp and face, followed by M. pachydermatis. Four scalp samples each yielded two different species. Table 1 subjects Region Number of isolated Malassezia species from normal Scalp (%) Face (%) Trunk (%) Total (%) M. globosa 2 (6) 3 (9) 18 (51) 23 (22) M. furfur 1 (3) 2 (6) 3 (3) M. sympodialis 1 (3) 1 (3) 9 (26) 11 (10) M. pachydermatis M. obtusa 1 (3) 1 (1) 1 (3) 1 (1) Negative culture 23 (66) 22 (63) 8 (23) 53 (50) Unknown: 7 (20) 6 (17) 3 (9) 16 (15) contamination No. of subjects Table 2 Number of isolated Malassezia species from normal skin of medical students Region Scalp (%) Face (%) Total (%) M. globosa 14 (21) 5 (8) 19 (15) M. furfur 6 (9) 9 (14) 15 (11) M. sympodialis 12 (18) 8 (13) 20 (15) M. pachydermatis 5 (7) 1 (2) 6 (5) M. obtusa 1 (2) 1 (1) 2 (3) 2 (2) Negative culture 17 (25) 25 (40) 42 (32) Unknown:contamination 16 (24) 14 (22) 30 (23) No. of subjects Isolates from lesional and non-lesional skin of patients w ith atopic dermatitis The percentage of negative cultures (Table 3) was 46% for lesional skin and 33% for non-lesional skin. M. furfur was isolated more frequently from lesional skin than non-lesional skin. However, there was no statistically signi cant difference among the isolated species between lesional and non-lesional skin (x 2, P\ 0 05). The number of samples was too small for comparison with the normal subjects. Isolates from lesional and non-lesional skin of patients w ith seborrhoeic dermatitis M. globosa (21%; 10:48) and M. furfur (21%; 10:48) were abundantly isolated from the lesional skin of seborrhoeic dermatitis, whereas M. sympodialis isolates were isolated in small numbers (6%; 3:48) (Table 4). Three species, M. globosa, M. furfur and M. sympodialis, were isolated from non-lesional skin of patients with seborrhoeic dermatitis at high rates of 12, 26 and 12%, respectively. M. pachy- Table 3 Number of isolated Malassezia species from patients with atopic dermatitis Lesional Non-lesional skin (%) skin (%) Total (%) M. globosa 4 (14) 6 (33) 10 (22) M. furfur 6 (21) 2 (11) 8 (17) M. sympodialis 2 (7) 2 (4) M. pachydermatis M. obtusa 1 (4) 1 (2) Negative culture 13 (46) 6 (33) 19 (41) Unknown: 2 (7) 4 (22) 6 (13) contamination No. of subjects ISHAM, Medical Mycology, 38,

4 340 Nakabayashi et al. Table 4 Number of isolated Malassezia species from patients with seborrhoeic dermatitis Lesional skin (%) Non-lesional skin (%) Total (%) M. globosa 10 (21) 6 (12) 16 (16) M. furfur 10 (21) 13 (26) 23 (23) M. sympodialis 3 (6) 6 (12) 9 (9) M. pachydermatis 4 (8) 4 (4) M. obtusa 1 (2) 1 (1) 1 (2) 1 (1) Negative culture 15 (31) 10 (20) 25 (25) Unknown:contami- 10 (21) 9 (18) 19 (19) nation No. of subjects dermatis, M. obtusa and were also isolated from non-lesional skin (Table 4). When the lesional and non-lesional skin of the patients with seborrhoeic dermatitis were compared, there was no statistically signi cant difference in the isolation frequency of these species (x 2, P\ 0 05). The percentage of negative cultures was 26% (6:23) for the face, which is less than half the value recorded for the normal subjects (63%; 22:35) (x 2, P B0 05) (Table 5). Lesional skin of the face yielded M. furfur (35%) and M. globosa (22%). The rates of isolation of these two species from lesional skin were signi cantly higher than in the normal subjects (PB 0 05). In addition, M. sympodialis was isolated from only one sample. Isolates from lesions of pityriasis versicolor and the trunk of normal subjects M. globosa was isolated from 55% (12:22) of lesional skin specimens in pityriasis versicolor, while other species were below 10% (Table 6). However, the isolation of M. Table 5 Comparison of number of isolated Malassezia species between normal subjects (Table 1) and patients with seborrhoeic dermatitis (SD) on the face Normal subjects (%) SD (%) M. globosa 3 (9) 5 (22) M. furfur 2 (6) 8 (35) M. sympodialis 1 (3) 1 (4) M. pachydermatis M. obtusa 1 (3) Negative culture 22 (63) 6 (26) Unknown:contamination 6 (17) 3 (13) No. of subjects Table 6 Number of isolated Malassezia species from patients with pityriasis versicolor Trunk (%) M. globosa 12 (55) M. furfur 1 (5) M. sympodialis 2 (9) M. pachydermatis 1 (1) M. obtusa 1 (5) Negative culture 3 (14) Unknown:contamination 2 (9) No. of subjects 22 globosa from the trunk of normal subjects was nearly the same (51%; 18:35) (Table 1). Species con rmation Each species, in follow-up studies performed by one of our group (J. Guillot) had phenotypic and molecular characteristics in accordance with the primary identi cations made in the original diagnostic laboratory. M. globosa was identi ed only from primary isolates, and not from continuously maintained stock strains, a step made necessary by its poor longevity in culture. Discussion It has been expected that the pathogenic roles of Malassezia could be clari ed by reclassifying the genus Malassezia. Guého et al. [11] reported that M. globosa was found regularly in pityriasis versicolor and seborrhoeic dermatitis, and that both M. sympodialis and M. restricta were commonly isolated from both healthy and diseased skin of the scalp and face. Leeming et al. [12] reported the prevalence of three Malassezia subgroups in 20 adult subjects. Subgroups A (M. sympodialis) and B (M. globosa) were dominant on the back, while subgroup C () was dominant on the forehead. M. globosa and M. sympodialis were also frequently detected on the forehead. However, there have been few reports on the speci c relationships of the Malassezia species to such diseases as atopic dermatitis, seborrhoeic dermatitis and pityriasis versicolor. Thus, the relationships between the species of Malassezia and these diseases need to be clari ed with additional data. The method of Guillot et al. [11] depends on species differences in lipid dependency, the catalase reaction, utilization of various Tweens and morphological features. This method also has the advantage of convenience and simplicity, requiring neither special facilities nor dif cult 2000 ISHAM, Medical Mycology, 38,

5 Identi cation of Malassezia species from different patients 341 techniques. However, a problem is that may not be detected in samples in which a catalase reaction-positive Malassezia species is mixed. In our results, was isolated at a very low rate compared with the ndings of Leeming et al. [12]. Our results indicate the possibility that several species were mixed in the primary cultures. In fact, we identi ed two different species within one sample. When the Tween test did not correspond to any pattern, the strain was labeled as unknown. In our results from subcultures, several times M. globosa which had been detected in the primary culture disappeared, and only M. sympodialis remained. Therefore, we did not use those subcultures. To identify the pathogenic species, it is obligatory to establish a new method of identi cation which more accurately differentiates mixtures of species. The trunk of normal subjects yielded M. globosa at the highest rate (51%), followed by M. sympodialis (26%). These results are in agreement with the report of Leeming et al. [12]. These two species appear to be the main normal ora on the skin of the trunk. Cultures of samples collected from the scalp and face were often negative (over 60%). This might be due to inadequate sampling methods. Therefore, the prevalence of Malassezia on the scalp and face of healthy subjects was examined using a group of medical school students. Samples were collected by the hair-brush method from the scalp and by the tape method from the face. As a result, not only M. globosa but also M. furfur and M. sympodialis were isolated from the scalp and face of these healthy subjects. M. pachydermatis was also isolated from the scalp, but less frequently. Our results suggest that M. globosa, M. sympodialis and M. furfur are the main members of thenormal skin ora on the scalp and face. M. pachydermatis, and were isolated infrequently from healthy subjects. In adult Japanese patients with atopic dermatitis, intractable skin lesions, especially on the face, scalp and neck regions, are dif cult to treat. It has been reported that patients with atopic dermatitis lesions of the head and neck show a high positive rate for anti-p. o ale immunoglobulin (Ig)E antibody [9]. However, the percentage of negative culture for the lesional skin of patients with atopic dermatitis (46%; 13:28) was as high as that for the normal subjects (50%; 53:105). M. furfur was isolated more frequently from lesional skin (21%) than non-lesional skin (11%) in the patients with atopic dermatitis, but the difference was not statistically signi cant. Obviously, further studies are required to elucidate the exacerbating factors in atopic dermatitis. The patients with seborrhoeic dermatitis showed a low negative culture rate compared with the normal subjects. The species isolated from the lesional skin of the face were almost entirely limited to two species, M. furfur (35%; 8:23) and M. globosa (5:23, 22%). These rates were signi cantly higher than observed in normal subjects (M. globosa : 9%, 3:35; M. furfur: 6%, 2:35). Therefore, the possibility that M. furfur and:or M. globosa are pathogens causing seborrhoeic dermatitis cannot be ruled out. M. pachydermatis, M. obtusa and were isolated infrequently from non-lesional skin. The lesions of pityriasis versicolor yielded M. globosa more frequently than the other species. Yeast cells from pityriasis versicolor lesions were observed by direct microscopic examination and seen to be large orbiculare types, similar to M. globosa. The overall results suggest that the pathogenic species involved in pityriasis versicolor is M. globosa. However, M. globosa was also detected from the trunk of 51%of the normal subjects. These results indicate that over-growth by M. globosa, which is a member of the normal ora, causes the lesions of pityriasis versicolor. References 1 Martin AG, Kobayashi GS. Yeast infections: Candidiasis, Pityriasis (Tinea) versicolor. In: Freedberg IM, ed. Dermatology in General Medicine, 5th edn. New York: McGraw-Hill, 1999: Yarrow D, Ahearn DG. Malassezia Baillon. In: Kreger-van RIJ NJW, ed. The Yeasts, A Taxonomic Study, 3rd edn. Amsterdam: Elsevier Science Publishers BV, 1984: Simmons RB, Guého E. A new species of Malassezia. Mycol Res 1990; 94: Guého E, Midgley G, Guillot J. The genus Malassezia with description of four new species. Antonie an Leeuwenhoek 1996; 69: Faergemann J. Seborrhoeic dermatitis and Pityrosporum orbiculare: Treatment of seborrhoeic dermatitis of the scalp with miconazole-hydrocortisone(daktacort), miconazole and hydrocortisone. Br J Dermatol 1986; 114: Bergbrant I-M. Seborrhoeic dermatitis and Pityrosporum o ale: cultural, immunological and clinical studies. Acta Dermatol Venereol 1991; 71 (Suppl. 167): Sei Y, Hamaguchi T, Ninomiya J, Nakabayashi A, Takiuchi I. Seborrheic dermatitis: Treatment with anti-mycotic agents. J Dermatol 1994; 21: Waersted A, Hjorth N. Pityrosporum orbiculare-a pathogenic factor in atopic dermatitis of the face, scalp and neck? Acta Dermatol Venereol 1985; 114: Wessels MW, Doekes G, Van Ieperen-van Kijk AG, Koers WJ, Young E. IgE antibodiesto Pityrosporumo ale in atopic dermatitis. Br J Dermatol 1991; 125: Guillot J, Guého E, Lesourd M, et al. Identi cation of Malassezia species, A practical approach. J Mycol Med 1996; 6: Guého E, Boekhout T, Ashbee HR, Guillot J, Van Belkum A, Faergemann J. The role of Malassezia species in the ecology of human skin and as pathogens. Med Mycol 1998; 36 (Suppl. 1): Leeming JP, Sanson JE, Bruton JL. Susceptibility of Malassezia furfur subgroups to terbina ne. Br J Dermatol 1997; 137: ISHAM, Medical Mycology, 38,

Tween 40-based precipitate production observed on modified chromogenic agar and development of biological identification kit for Malassezia species

Medical Mycology May 2006, 44, 227231 Tween 40-based precipitate production observed on modified chromogenic agar and development of biological identification kit for Malassezia species TAKAMASA KANEKO*$,