Journal de Mycologie Mdicale (2010) 20, 279282

ORIGINAL ARTICLE/ARTICLE ORIGINAL

Identification of Malassezia species isolated from

patients with seborrheic dermatitis, atopic
dermatitis, and normal subjects
es de patients atteints de
Identification des levures du genre Malassezia isole
dermatite se borrhe
ique ou de dermatite atopique et de sujets normaux

M. Saghazadeh a,*, S. Farshi b, J. Hashemi c, P. Mansouri b, A.R. Khosravi d

a
Department of Microbiology, Faculty of Science, Islamic Azad University. Qom Branch, 15, Khordad boulevard,
P.O. Box 37185 364, Qom, Iran
b
Department of Dermatology, Imam Hospital, Keshavarz boulevard, Tehran, Iran
c
Department of Mycology, Faculty of Hygiene, University of Tehran, Tehran, Iran
d
Department of Mycology, Faculty of Veterinary, University of Tehran, Tehran, Iran

Received 12 February 2010; received in revised form 14 August 2010; accepted 20 August 2010
Available online 23 October 2010

KEYWORDS
Atopic dermatitis;
Seborrhoeic dermatitis;
Malassezia yeasts
Summary
Objective. Aim of this study was to examine the Malassezia species of the normal skin flora as
well as the species isolated from patients with seborrheic dermatitis (SD) and atopic dermatitis
(AD).
Materials and methods. In this study, the subjects were 81 patients (34 with AD and 47 with
SD) plus 40 normal subjects. A direct microscopic examination and culturing were carried out on
the skin samples. The isolated yeasts were identified by their morphological features as well as
physiological characteristics.
Results. Fifty-six patients (69.1%) were female and the rest (30.9%) were male. The highest
prevalence of skin lesions was seen in patients with 2130 years of age (41.3%). Cultures yielded
positive results in 85.1% of patients with SD and 47.1% of patients with AD as well as 77.5% of the
normal subjects. The culture results showed a statistically significant difference among the
patients and normal subjects (x2, P = 0.001).
Conclusions. The results showed that M. globosa was the most frequent species isolated from
AD and normal subjects and M. furfur was the most prevalent species isolated from SD.
# 2010 Elsevier Masson SAS. All rights reserved.

* Corresponding author.
E-mail address: saghazadeh_99@yahoo.com (M. Saghazadeh).

1156-5233/$ see front matter # 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.mycmed.2010.08.003
280 M. Saghazadeh et al.
Resume
MOTS CLS Objectif. Le but de ce travail est didentifier les levures du genre Malassezia isoles chez des
Dermatite atopique ; sujets normaux et chez des patients atteints de dermatite sborrique (SD) ou de dermatite
Dermatite sborrhique ; atopique (AD).
Malassezia Patients et methode. Dans cette tude, 40 sujets normaux et 81 patients atteints daffections
cutanes (47 SD et 34 AD) ont t inclus. Un examen direct et une mise en culture des
prlvements cutans ont t mis en uvre. Les levures isoles ont t identifies par leurs
caractres morphologiques et physiologiques.
Resultats. La plus forte prvalence des lsions cutanes a t observe chez les patients dont
lge tait compris entre 21 et 30 ans (41,3 %). Les cultures ont t positives chez 85,1 % des patients
avec SD, 47,1 % des patients avec AD et 77,5 % des sujets normaux. Les rsultats des cultures
montrent des valeurs statistiques significatives entre les patients et les sujets normaux (x2 :
p = 0,001).
Conclusion. Les rsultats montrent que M. globosa est lespce la plus frquemment isole chez
les patients atteints de AD et chez les sujets normaux, tandis que M. furfur est lespce la plus
rpandue chez les patients atteints de SD.
# 2010 Elsevier Masson SAS. Tous droits rservs.

Introduction seborrheic dermatitis (SD) (28 female, 19 male; mean

Yeasts of the genus Malassezia have been recognized as mem-
bers of the microbiologic flora of the skin for over a century.
Undercertainconditions,theycancausesuperficialinfectionof
the skin and associated structures such as pityriasis versicolor,
seborrhoeicdermatitis,atopicdermatitis,pityriasiscapitis,the
folliculitis, blepharitis and they can become an opportunistic
pathogen in patients with catheters [1,12]. Although lots of
studieshavebeenconductedonMalasseziaspeciesinthe recent
years, this genus of fungi is not still fully known. Recently
molecular studies defined 13 Malassezia species:
M. pachydermatis, M. furfur, M. sympodialis, M. slooffiae,
M. globosa, M. obtusa, M. restricta, M. dermatis, M. japonica,
M. nana, M. yamatoensis, M. caprae, M. equina [1,9,10,23].
The difficulties of isolation and preservation of these yeasts
have long-delayed their identification and taxonomic position.
The culture of these microorganisms has been made possible
after the demonstration of their lipophilic character [1].
Different identification methods have been developed and
various epidemiologic studies conducted for their pathogen-
esis and immunologic aspects interpretations. Malassezia
species have been found causative role in various superficial
dermatoses as well as how they develop on the surface of
healthy skin has been examined. In recent years, the genus
Malassezia has come to be considered important in the etiol-
ogy of seborrheic dermatitis [4,8,17,20]. Furthermore, there
are reports that Malassezia is an exacerbating factor of lesions
of psoriasis and at opic dermatitis, but it is yet to be clear
which Malassezia species are pathogenic and may have the
causative role in the aforementioned diseases [3,17].
This study has been carried out in order to isolate and
identify different Malassezia species as well as examine their
relation with atopic dermatitis and seborrheic dermatitis
using morphological, biochemical and physiological criteria.
Patients and methods
Subjects
The subjects were 34 patients with atopic dermatitis (AD) (28
female, six male; mean age  SD: 28.6  11.4) and 47 with
age  SD: 27.4  10.1) whom were visited by dermatologists
of the dermatology ward of Imam Hospital; the process of
sampling and examinations took 15 months. A number of 40
normal subjects (27 female, 13 male; mean age  SD:
27.6  7.4) consisted of healthy volunteers without any
serious disease or dermatosis and were matched with the
patients as far as their age and sex were concerned. Before
sampling, a questionnaire including personal data and med-
ical condition of the subjects, has been prepared and filled
out by the patients.
Collection and culture of samples
In the patients with AD or SD, samples were collected by
scrapping and in the normal subjects tape method was used.
In the tape method, Scotch tape was affixed to the normal
skin, and it was then transferred sticky-side down to Dixons
agar.
Specific identification
Malassezia yeasts were identified on the basis of microscopic
observation of cells and physiological properties such as
presence of catalase, urease; the ability to utilize Tween
compounds and Cremophor EL (castor oil) as a single-lipid
supplement. Small portion of the scales isolated from the
patients were observed by direct microscopic examination
and the rest were cultured in the laboratory. Whereas the
Malassezia yeasts, excluding M. pachydermatis, are lipophile
and need extra lipid to grow, the medium innovated by Van
Abbe (1964), i.e. a Dixons agar, was used [6,13,14]. Then,
the cultivated samples were incubated at 32 8C for two
weeks. Microscopic slides of colonies were prepared and
examined. Morphological observation includes examination
of macroscopic (i.e., colonies shape, texture and colour)
and microscopic appearance (i.e., cell morphology and bud-
ding location). Samples with positive results found on the
slides were cultivated in Dixons agar and incubated at 32 8C
for complementary and differentiation tests.
We identified Malassezia species according to Guillot et
al. [7]. Isolates from the primary cultures were used for
Identification of Malassezia species isolated from patients with dermatitis 281

identification. M. pachydermatis is able to grow on Sabour- SD and 47.1% of patients with AD as well as 77.5% of the
auds agar. Tween assimilation and catalase test were normal subjects. The culture results showed a statistically
applied for identification of other Malassezia species. significant difference among the patients and normal sub-
Briefly, Malassezia yeast suspensions were mixed with jects (x2, P = 0.001). The positive results of cultures as well
Sabouraud agar and the mixtures were plated. Four holes as direct microscopic examinations, in patients with SD were
were made in the agar by means of a 3 mm diameter punch more than patients with AD. A number of 57 subjects (70%)
and filled with 5 ml each of Tween 20, 40, 60 and 80, were patients with Malassezia; patients with AD and SD as
respectively. The agar plates, incubated at 32 8C for 1 week, well as the normal subjects and patients with AD were
were examined each day for the existence of any growth compared, the difference was statistically significant (x2,
around the wells that contained Tween compounds. For P < 0.05). Table 1 shows the frequency of Malassezia species
Catalase reaction test, One drop of hydrogen peroxide solu- isolated from patients with AD and SD, and normal subjects.
tion was applied onto a yeast colony, which was on a glass Unknown Malassezia species were not isolated.
slide. Production of gas bubbles was considered a positive The results showed that M. globosa was the most frequent
catalase reaction. For urease reaction test, some colonies species isolated from AD and normal subjects and M. furfur
are introduced into a medium containing urea and an indi- was the most prevalent species isolated from SD.
cator such as phenol red. The urease hydrolyzes urea to
ammonia, which raises the pH of the medium, and changes
the color of the specimen. In addition, esculin hydrolysis test Discussion
was conducted for isolated samples to show the species
which are able to hydrolyze esculin and darken the medium. According to the previous reports, M. sympodialis, M. globosa,
Characteristics table of Malassezia spp. were used as the and M. furfur were the most commonly isolated Malassezia
identification guidelines summarized by Ben Salah et al., species in normal subjects and in patients with diseases such as
Gueho et al. and Guillot et al. [1,5,6,7]. If the results of AD and SD [19]. However, there were differences with respect
physiological and morphological evaluation did not match to the species most commonly isolated, not only between
the characteristics table or if there was discrepancy between normal subjects and patients with various skin diseases but
physiological and morphological results, that particular spe- also as regards results from different countries. In our study
cies would be classified as unknown. and in the studies conducted by Nakabayashi et al. and Sugita
Statistical analysis was performed with the use of SPSS et al. from Japan [17,21], M. globosa was the dominant
software, version 11.5, Chicago, Illinois. A two tailed P-value species isolated from normal subjects; but in Sandstrom Falk
of 0.05 or less was considered to indicate significance. For et al.s study [19], the dominant species isolated from normal
comparison of results between AD, SD and healthy subjects subjects was M. sympodialis. In patients with AD, the domi-
x2 test was used. nant species was also M. globosa in our study, the same as in
the studies carried out by Sugita et al. from Japan [21] and
Results Sandstrom Falk et al. from Sweden [19], but in the study
conducted by Nakabayashi et al., the dominant species was
M. furfur [17]. As far as SD patients are concerned, in our study
The highest prevalence of skin lesions was seen in patients
and Nakabayashi et al.s study the dominant species was
with 2130 years of age (41.3%). The percentage of positive
M. furfur, but in a previous study conducted by Hedayati
direct microscopic examinations in patients with AD, SD and
et al. [9] from Iran, the dominant species isolated from SD
normal subjects were 47.1, 95.7 and 80%, respectively; the
patients was M. globosa. This difference may be attributable
difference among the patients, as far as their positive results
to the sampling and culture techniques as well as to geogra-
are concerned, is statistically significant (x2, P < 0.001).
phical differences.
Cultures yielded positive results in 85.1% of patients with
The specimens isolated from 47 patients with SD, 34
patients with AD, and 40 normal subjects were cultured;
Table 1 Frequency of Malassezia species isolated from the results showed positive culture in 40 (85.1%) SD patients,
patients with AD, SD, and normal subjects. 16 (47.1%) AD patients, and 31 (77.5%) normal subjects. The
quence des espe
Fre ` ces de Malassezia isole
es de patients patients with SD showed a low negative culture rate com-
moins ou atteints de AD ou de SD.
te pared with the normal subjects and patients with AD. This is
further proof for our observation that patients with AD
Malassezia species Atopic Seborrheic Normal harbour a lower number of Malassezia yeasts compared
dermatitis dermatitis subjects not only with patients with SD but also with normal subjects.
One reason may be the reduced amount of lipids in the
n % n % n %
skin of patients with AD compared to normal subjects and
Sympodialis 4 13.8 18 25.4 8 23.5 patients with SD [19]. Meanwhile, in the study conducted by
Globosa 13 44.8 17 23.9 18 52.9 Nakabayashi et al., positive culture was observed in 46% of
Furfur 5 17.2 32 45.1 4 11.8 AD patients, 69% of SD patients, and 75% of normal subjects
Restricta 6 20.7 1 1.4 2 5.9 [17]. Sandstrom Falk et al.s study showed positive culture in
Obtosa 1 3.4 1 1.4 1 2.9 56% of patients with AD, 88% of patients with SD, and 84% of
Slooffiea 0 0 2 1.4 1 2.9 normal subjects [19]. Obviously, further studies are required
Total 29 100 71 100 34 100 to elucidate the exacerbating factors in AD.
Simple methods based on morphology and physiology
AD: atopic dermatitis; SD: seborrheic dermatitis.
evaluation, such as the ones employed in this study, are
282
more accessible to every laboratory [1,2,9,10,11,16,18]. This
method also has the advantage of convenience and simplicity,
requiring neither special facilities nor difficult techniques.
However, Biomolecular techniques are considered more accu-
rate methods to identify Malassezia spp.[15,22].
It is hoped further studies will be performed with more
accurate techniques to identify Malassezia spp. (biomole-
cular techniques) from SD and AD patients in the near future.
In conclusion, we found fewer individuals with positive
Malassezia culture in the AD group than in the SD group, or
among the normal subjects.
Conflict of interest statement
The authors had not any conflict of interest.
Acknowledgment
We are indebted to Marzieh Fazelnia for her kind help in
English editing of the article, furthermore, would like to
thank Dr Farid Safar, Dr Samad Rezaee Khiabanloo, Shahin
Mansouri and all personnel of dermatology department of
Imam Hospital for their help in performing this study.
References
[1] Ben Salah I, Makni F, Cheikhrouhou F, Neji S, Sellami H, Ayadi A.
Malassezia species: pathology, isolation and identification me-
dia. J Mycol Med 2010;20:5360.
[2] Ben Salah S, Makni F, Marrakchi S, Sellami H, Cheikhrouhou F,
Bouassida S. Identification of Malassezia species from Tunisian
patients with pityriasis versicolor and normal subjects. Mycoses
2005;48:2425.
[3] Boguniewicz M, Schmid-Grendelmeier P, Leung DY. Atopic
dermatitis. J Allergy Clin Immunol 2006;118:403.
[4] Dawson T, Gemmer C, DeAngelis Y, Kaczvinsky J. Dandruff and
seborrheic dermatitis likely result from scalp barrier breach and
irritation induced by Malassezia metabolites, particularly free
fatty acids. J Am Acad Dermatol 2005;52:49.
[5] De Hoog GS, Guarro J, Gene J, Figueras MJ. Atlas of clinical fungi.
Baarn and Delft:Centraalbureau voor Schimmelcultures; 2000.
[6] Gueho E, Midgley G, Guillot J. The genus Malassezia with
description of four new species. Antonie Van Leeuwenhoek
1996;69:33755.
[7] Guillot J, Gueho E, Lesourd M, Midgley G, Chevrier G, Dupont B.
Identification of Malassezia species a practical approach. J
Mycol Med 1996;6:10310.
[8] Gupta AK. Role of Malassezia yeasts in seborrheic dermatitis. J
Am Acad Dermatol 2004;50:410.
M. Saghazadeh et al.
[9] Hedayati MT, Hajheydari Z, Hajjar F, Ehsani A, Shokohi T,
Mohammadpour R. Identification of Malassezia species isolated
from Iranian seborrhoeic dermatitis patients. Eur Rev Med
Pharmacol Sci 2010;14:638.
[10] Karakas M, Tura-Bier A, Ilkit M, Durdu M, Seydaog lu G.
Epidemiology of pityriasis versicolor in Adana, Turkey. J Der-
matol 2009;36:37782.
[11] Krisanty RI, Bramono K, Made Wisnu I. Identification of Malas-
sezia species from pityriasis versicolor in Indonesia and its
relationship with clinical characteristics. Mycoses
2009;52:25762.
[12] Ljubojevic S, Skerlev M, Lipozencic J, Basta-Juzbasic A. The
role of Malassezia furfur in dermatology. Clin Dermatol
2002;20:17982.
[13] Midgley G. The lipophilic yeasts: state of the art and prospects.
Med Mycol 2000;38(1):916.
[14] Midgley G, Gueho E, Guillot J. Diseases caused by Malassezia
species. In: Ajello L, Hay RJ, editors. Topley & Wilsons
microbiology and microbial infections. London: Arnold; 1998.
p. 20111.
[15] Mirhendi H, Makimura K, Zomorodian K, Yamada T, Sugita T,
Yamaguchi H. A simple PCR-RFLP method for identification and
differentiation of 11 Malassezia species. J Microbiol Methods
2005;61:2814.
[16] Moniri R, Nazeri M, Amiri S, Asghari B. Isolation and identifica-
tion of Malassezia spp. In pytiriasis versicolor in Kashan, Iran.
Pak J Med Sci 2009;25:83740.
[17] Nakabayashi A, Sei Y, Guillot J. Identification of Malassezia
species isolated from patients with seborrhoeic dermatitis,
atopic dermatitis, pityriasis versicolor and normal subjects.
Med Mycol 2000;38:33741.
[18] Rincn S, Celis A, Sop L, Motta A, Cepero de Garca MC.
Malassezia yeast species isolated from patients with dermato-
logic lesions. Biomedica 2005;25:18995.
[19] Sandstrom Falk MH, Tengvall LM, Johansson C, Bartosik J, Back
O, Sarnhult T, et al. The prevalence of Malassezia yeasts in
patients with atopic dermatitis, seborrhoeic dermatitis and
healthy controls. Acta Derm Venereol 2005;85:1723.
[20] Sei Y, Hamaguchi T, Ninomiya J, Nakabayashi A, Takiuchi I.
Seborrhoeic dermatitis: treatment with anti-mycotic agents. J
Dermatol 1994;21:33440.
[21] Sugita T, Suto H, Unno T, Tsuboi R, Ogawa H, Shinoda T, et al.
Molecular analysis of Malassezia microflora on the skin of atopic
dermatitis patients and healthy subjects. J Clin Microbiol
2001;39:348690.
[22] Sugita T, Takashima M, Shinoda T, Suto H, Unno T, Tsuboi R,
et al. New yeast species, Malassezia dermatis, isolated from
patients with atopic dermatitis. J Clin Microbiol 2002;40:
13637.
[23] Trabelsi S, Oueslati J, Fekih N, Kammoun MR, Khaled S. Iden-
tification of Malassezia species from Tunisian patients with
pityriasis versicolor. Tunis Med 2010;88:857.