Diagnostic Microbiology and Infectious Disease 76 (2013) 450–457

Contents lists available at SciVerse ScienceDirect

Diagnostic Microbiology and Infectious Disease

journal homepage: www.elsevier.com/locate/diagmicrobio

Mycology

Epidemiological characteristics of Malassezia folliculitis and use of the

May-Grünwald-Giemsa stain to diagnose the infection☆
Murat Durdu a, Mümtaz Güran b, Macit Ilkit b,⁎
a
Department of Dermatology, Faculty of Medicine, Başkent University Adana Hospital, Adana, Turkey
b
Division of Mycology, Department of Microbiology, Faculty of Medicine, University of Çukurova, Adana 01330, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: Various bacterial, fungal, parasitic, and viral pathogens can cause folliculitis, which is often mistakenly
Received 27 February 2013 treated with antibiotics for months or even years. A laboratory diagnosis is required before therapy can be
Received in revised form 27 March 2013 planned. Here, we describe the prevalence and risk factors, as well as the clinical, cytological, and
Accepted 2 April 2013
mycological characteristics, of patients with Malassezia folliculitis (MF) in Adana, Turkey. We also report the
Available online 22 May 2013
treatment responses of the MF patients and describe the Malassezia spp. using culture-based molecular
Keywords:
methods. Cytological examinations were performed in 264 folliculitis patients, 49 of whom (18.5%) were
Acne vulgaris diagnosed with MF. The positivity of the May-Grünwald-Giemsa (MGG) smear was higher (100%) than that
Cytology of the potassium hydroxide test (81.6%). Using Wood's light, yellow-green fluorescence was observed in
Itraconazole 66.7% of the MF patients. Identification using the rDNA internal transcribed spacer region revealed that
Pityriasis versicolor Malassezia globosa was the most common species, followed by Malassezia sympodialis, Malassezia restricta,
rDNA and Malassezia furfur. The MF patients were treated with itraconazole capsules (200 mg/d) for 2 weeks.
Wood’s light Complete recovery was observed in 79.6% of the patients. These novel findings help improve our current
understanding of the epidemiological characteristics of MF and establish MGG as a practical tool for the
diagnosis of MF.
© 2013 Elsevier Inc. All rights reserved.

1. Introduction Culture-dependent (1996–2002) and culture-independent

Lipophilic yeasts of the genus Malassezia are common skin
commensals. However, they may cause infections in the presence of
predisposing factors, such as environmental changes and/or alter-
ations in the host's defense mechanisms (Batra et al., 2005; Gupta et
al., 2004a). In current taxonomy, 14 Malassezia spp. have been
identified, with 9 described in humans (i.e., Malassezia furfur,
Malassezia sympodialis, Malassezia globosa, Malassezia obtusa, Malas-
sezia slooffiae, Malassezia restricta, Malassezia dermatis, Malassezia
japonica, and Malassezia yamatoensis) and 5 in animals (Malassezia
pachydermatis, Malassezia caprae, Malassezia equina, Malassezia nana,
and Malassezia cuniculi) (Batra et al., 2005; Cabaňes et al., 2011; Gupta
et al., 2004a). Malassezia spp. are associated with various dermato-
logical diseases, such as pityriasis versicolor (PV); seborrheic
dermatitis (SD); atopic dermatitis (AD); Malassezia folliculitis (MF);
dandruff; psoriasis; and, rarely, onychomycosis (Batra et al., 2005;
Gupta et al., 2004a).
☆ Conflict of interest: The authors report no conflicts of interest. The authors alone are
responsible for the content and the writing of the paper.
⁎ Corresponding author. Tel.: +90-532-286-0099; fax: +90-322-457-3072.
E-mail address: milkit@cu.edu.tr (M. Ilkit).
(2003–present) methods have been used to identify Malassezia
spp. (Guého et al., 1996; Makimura et al., 2000; Sugita et al., 2003).
Questionable results have been obtained using conventional iden-
tification techniques because of the continual increase in novel
species (Batra et al., 2005; Guého et al., 1996). Gaitanis et al.
(2009a) discussed the pros and cons of each molecular typing
method and highlighted the potential scarcity of epidemiological
data. Briefly, these methods involve targeting the polymerase chain
reaction (PCR) amplification of selected sequences and subsequently
searching for mutations (Gaitanis et al., 2006; Sugita et al., 2003;
Tajima et al., 2008), random PCR amplifications of polymorphic DNA,
or minisatellites within the genome (Gaitanis et al., 2009b; Gupta
et al., 2004b; Makimura et al., 2000). In 1 study, an analysis of
cutaneous Malassezia microbiota in 770 healthy Japanese individuals
using real-time PCR revealed that M. globosa and M. restricta
together accounted for more than 70% of the Malassezia spp. (Sugita
et al., 2010). Consistent with this finding, studies including
molecular analysis data have reported that M. globosa is mainly
responsible for PV, whereas AD, SD, and MF are principally caused
by M. globosa and M. restricta (Akaza et al., 2009; Sugita et al., 2006;
Tajima et al., 2008).
Although MF is the most common type of fungal folliculitis, little is
known about its epidemiological characteristics. Clinically, MF is

0732-8893/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.diagmicrobio.2013.04.011
 M. Durdu et al. / Diagnostic Microbiology and Infectious Disease 76 (2013) 450–457 451

difficult to distinguish from bacterial folliculitis and acne vulgaris.
Therefore, it may be treated with topical and/or systemic antibiotics
for months or even years. To differentiate MF from other types of
folliculitis and to reach a reliable diagnosis, samples for cytology,
culture, and molecular analyses should be collected before treatment
(Durdu and Ilkit, 2013). Few studies have performed a molecular
analysis of the Malassezia spp. responsible for MF (Akaza et al., 2009;
Ko et al., 2011). We aimed to determine the prevalence and associated
risk factors and the clinical, cytological, and mycological features of
MF. We also studied the treatment responses in MF patients and
described the Malassezia spp. in both lesional and nonlesional areas
using culture-based molecular methods.
2. Materials and methods
2.1. Patients
This prospective study was conducted between May 2012 and
August 2012 at the Department of Dermatology, Başkent University
Faculty of Medicine, Adana Hospital, Adana, Turkey. MF patients

Fig. 1. Clinical presentation of a patient with MF. (A) Presence of follicular papules or pustules on the back and (B) resolution of these lesions with treatment; (C) cystic lesions on the
chin and (D) resolution of these lesions with treatment; (E) numerous excoriations on the back; (F) id reaction caused by MF. Follicular papules or pustules on the extensor surface of
the arm and scaling on the fingers.
452 M. Durdu et al. / Diagnostic Microbiology and Infectious Disease 76 (2013) 450–457

whose diagnoses were confirmed by cytological examination, fungal 2.6. Treatment

culture, and molecular analysis were included in the treatment arm of
this study. The exclusion criteria included current pregnancy or
lactation, liver failure, antifungal drug allergy, systemic antifungal
treatment during the past 3 months, topical antifungal treatment
within the previous 4 weeks, and the use of medications that might
interact with the study drugs. This study was reviewed and approved
by the Institutional Review Board at the University of Başkent, Ankara,
Turkey. The Declaration of Helsinki protocols were followed, and the
patients provided written informed consent.
2.2. Study design
The folliculitis patients were examined, and 2 scraping smears
were collected using a scalpel blade (No. 15). The first smear was
stained with May-Grünwald-Giemsa (MGG), and the other was
stained with potassium hydroxide (KOH). Subsequently, the cytolog-
ical samples were microscopically evaluated. If the MGG or the KOH
smears were positive in terms of numerous budding spores and short,
curved hyphae, then additional samples were taken from pustular
lesions and nonlesional regions for fungal culture. A Wood's light
examination of these lesions was also performed. The patients were
questioned regarding provoking factors, concomitant and predispos-
ing diseases, id reactions, subjective complaints, and lesion duration. If
concomitant PV or SD was observed, then clinical samples were also
taken from those lesions.
2.3. Fungal culture
All the samples were inoculated onto modified Dixon (MDA;
Batra et al., 2005) and Leeming-Notman agars (Leeming and
Notman, 1987). Both media contained 0.05% chloramphenicol
(Sigma, Steinheim, Germany) and 0.05% cycloheximide (Sigma,
Germany). The plates were incubated at 32 °C and examined daily
for 20 days.
2.4. DNA extraction, PCR, and molecular analysis
The identification of the fungal isolates was confirmed using
molecular analysis. DNA extraction and PCR amplification were
performed as described previously (Turin et al., 2000). rDNA
sequences spanning the internal transcribed spacer (ITS) 1 region
were amplified using the universal fungal primers ITS1 and ITS4 on an
ABI PRISM 3130XL genetic analyzer at Refgen Biotechnologies
(Ankara, Turkey). The CAP contig assembly software, which was
included in the BioEdit Sequence Alignment Editor 7.0.9.0 software
package, was used to edit the obtained sequences (Hall, 1999). The
assembled DNA sequences were examined using the BLAST (nucle-
otide-nucleotide) software on the National Center of Biotechnology
Information database.
Patients with MF confirmed by cytological examination, fungal
culture, and molecular analysis were treated with oral itraconazole
capsules (200 mg/d) for 2 weeks. Those patients who could not
continue because of adverse effects were treated with topical
ketoconazole creams for 4 weeks. Their treatment responses were
evaluated after 1 month when the clinical examination was repeated.
At this stage, patient compliance was determined, and adverse
events were recorded. The patients' treatment responses were
assessed using the following categories: i) complete recovery (no
visual evidence of disease), ii) moderate improvement (mild residual
lesions, but no visual evidence of active disease), iii) partial
improvement (active disease with some improvement), and iv) no
improvement or deterioration.
2.7. Statistical analysis
Fisher's exact tests were used to test the differences in the illness
duration, disease recurrence, pruritus, pigmentation type, hyperhi-
drosis (presence/absence), and fluorescence on Wood's light exam-
ination among the patients who were infected with various Malassezia
spp. The software SPSS for Windows version 17.0 was used for the
statistical analysis. Statistical significance was defined as a P value of
less than 0.05.
3. Results
3.1. Patients
Over a 4-month period, 1225 new patients were examined.
Cytological examinations were performed in 264 folliculitis patients
who were admitted to our dermatology clinic. Forty-nine (18.5%) of
these patients (18 males and 31 females) were diagnosed with MF
and included in the study. The mean age of the MF patients was 26
years (range, 12–62). The patients lived in the cities of Adana and
Hatay (Iskenderun town), which are 2 hot, subtropical cities located
south of the Mediterranean region of Turkey. The average tempera-
tures range from 30 to 43 °C, and the humidity ranges from 30 to 92%.
3.2. Clinical features
Follicular papules or pustules measuring 2 to 4 mm in diameter were
detected in all the MF patients (Fig. 1A). Cystic lesions were observed in
1 patient who had received intermittent topical and/or systemic
antibiotic treatments for 10 years (Fig. 1C). The mean duration of the

2.5. Clustering Malassezia isolates

The sequences were aligned with ClustalW (Tamura et al., 2011)

and compared by neighbor-joining phylogenetic tree analyses using
MEGA version 5.05 (http://www.megasoftware.net). The clustering
indications of the Malassezia spp. were taken from Gupta et al.
(2004b). The CBS deposition numbers and GenBank accession
numbers of the analyzed strains were as follows: M. globosa CBS
7966, type strain (ITS accession no: AY387132) (Cluster 1) and M.
globosa CBS 7874 (AY387133) (Cluster 2); M. sympodialis CBS 7222,
type strain (AY387157); M. furfur CBS 1878, neotype of Pityrosporum
ovale (AY387100) (Cluster 1) and M. furfur CBS 7860 (AY387114)
(Cluster 2); and M. restricta CBS 7877, type strain (AY387143) Fig. 2. Diffuse green fluorescence on the face caused by topical tetracycline cream in a
(Cluster 1) and M. restricta CBS 8747 (AY387144) (Cluster 2). patient with MF.
 M. Durdu et al. / Diagnostic Microbiology and Infectious Disease 76 (2013) 450–457 453

lesions was 10 months (range, 2 weeks to 10 years). In 15 (30.6%)
patients, pustular lesions had occurred previously (range, 2–10 years).
In most cases (71.4%), the lesions were found in more than 1 region.
The most common locations of the lesions were the face (57.1%), back
(53%), extensor side of the arms (38.8%), chest (36.7%), and neck
(18.3%). The facial lesions were localized on the forehead, chin, and
sides of the face in which contrast to the central facial location of acne
vulgaris. Thigh and gluteal involvements were observed in 1 patient
with pemphigus. Hyperpigmented macules developed in 31 (63.3%)
patients. Thirty-nine (79.6%) patients complained of pruritus. Five
(10.2%) patients presented with itching and numerous excoriations
(Fig. 1E). Two patients had scaling and itching on the hands (Fig. 1F).
The KOH examination was negative in these hand lesions, which
improved in response to antifungal treatment. The European standard
patch test and the skin-prick allergy test for common inhalant and
food allergens were negative. Thus, these patients were diagnosed
with scaling form dyshidrotic id reactions caused by MF. Urticarial
lesions were present in 1 MF patient. The folliculitis lesions resolved
with the systemic itraconazole treatment, but the severity of the
urticarial lesions did not change. Therefore, an urticarial id reaction
caused by MF was not considered. Six (12.2%) of the MF patients
exhibited coexisting acne vulgaris, and 5 (10.2%) had PV.

Fig. 3. Cytological findings of patients with MF. (A) Abundance of footprint-shaped spores (arrows); (B) hyphae (black arrows) and spores (red arrows) detected in some patients
with PV; (C) bacilli (black arrows) and spores (red arrows) present in a patient with acne vulgaris; (D) acantholytic cells (red arrows) accompanied by spores (black arrows) in a
patient with pemphigus vulgaris; (E) foreign body–type giant cells (red arrow) observed in a specimen obtained from cystic lesions on the jaw; (F) numerous eosinophils (black
arrows) observed in a specimen obtained from a patient with severe pruritus.
454 M. Durdu et al. / Diagnostic Microbiology and Infectious Disease 76 (2013) 450–457

3.3. Predisposing factors
The majority of the patients had excessive sweating (51%).
Approximately one-third (34%) of the patients were involved in
sports, and the lesions in 5 patients occurred shortly after participat-
ing in sports. Four (8.1%) patients were new to the area and were
unaccustomed to a hot climate. In 3 of these patients, the pustular
lesions appeared after arriving in Adana. Three patients, 2 who had
pemphigus vulgaris and 1 who had alopecia areata, had used systemic
corticosteroids. Five patients had used oral antibiotics for acne
vulgaris. As the MF had been misdiagnosed as acne, 2 patients had
received systemic isotretinoin treatment. Three patients had been
treated with steroid creams and oral antihistamine because of severe
itching. Eight patients had used topical antibiotics because the MF was
misdiagnosed as bacterial folliculitis.
3.4. Findings of Wood's light examination
Using Wood's light examination, yellow-green fluorescence was
observed in MF (65.3%) and PV (100%). This fluorescence disappeared
after antifungal treatment. Diffuse green fluorescence was also
observed on the faces of 3 patients because of topical tetracycline
cream (Fig. 2). Orange-red fluorescence was detected in the
comedonal regions of 6 (12.2%) MF patients along with acne vulgaris
(Table 2).
3.5. Cytological findings
The KOH examination revealed numerous blastospores in 40
(81.6%) MF patients. Abundant footprint-shaped spores adhering to
keratinocytes were observed in all the MGG-stained samples (Fig. 3A).
Hyphae were detected in some of the patients (8.2%) (Fig. 3B). Bacilli
and spores were present in all 6 patients with acne vulgaris (Fig. 3C).
Acantholytic cells were accompanied by spores in the 2 patients with
pemphigus vulgaris (Fig. 3D). Foreign body–type giant cells were
observed in 4 specimens (8.2%), one of which was obtained from
cystic lesions on the jaw and the others from excoriated papules after
squeezing (Fig. 3E). In the specimens obtained from molluscum-like
papules, inflammatory cells were usually absent, but abundant
neutrophils and eosinophils were observed in the cystic lesions and
in the erythematous papules and pustules. Eosinophils were predom-
inant in 7 (14.3%) samples obtained from patients with severe
pruritus (Fig. 3F).
3.6. Fungal culture
Both MDA and Leeming-Notman agars were positive within 7–10
days in samples taken from all patients.
3.7. Clusters of Malassezia spp.
In the lesional samples, M. globosa was the most frequently (69.4%)
isolated species (Tables 1 and 2). Other species that were isolated
from pustular lesions were M. sympodialis (14.3%), M. restricta
(10.2%), and M. furfur (6.1%). M. globosa, M. restricta, and M. furfur
isolates were classified into 2 clusters by molecular phylogenetic
analysis using the ITS1 region. In the nonlesional samples, the
Malassezia spp. were isolated in approximately one-half (51%) of
the MF cases, with M. globosa the most frequently (64%) isolated
fungus. Other species recovered from the nonlesional regions were M.
sympodialis (28%), M. restricta (4%), and M. furfur (4%). In the lesional
and nonlesional samples, the same Malassezia spp. were present in
most cases (72%), but different species were isolated from the lesional
and nonlesional samples in 7 (28%) patients. In these samples, the
same species but different clusters were identified in 3 (12%) of the
MF patients. Molecular identification was possible in 3 of the 5
samples from the PV patients, and the same Malassezia spp. as that
obtained from the MF cases were found in 2 of these. In this
investigation, GenBank ITS accession numbers for each Malassezia
spp. and clusters (presented in parenthesis) were as follows: M. furfur
JX993621 (Cluster 1); M. furfur JX993620 (Cluster 2); M. globosa
JX993623 (Cluster 1); M. globosa JX993622 (Cluster 2); M. restricta
JX993625 (Cluster 1); M. restricta JX993624 (Cluster 2); and M.
sympodialis JX993626.
The statistical analyses revealed no significant differences
between the Malassezia spp. distribution and the patients' age or

Table 1
Distribution of Malassezia spp. based on clinical characteristics.

Clinical characteristics n [cluster 1/cluster 2] (%)

M. globosa M. sympodialis M. restricta M. furfur

Region
Lesional 34 [29/5] (69.4) 7 (14.3) 5 [5/-] (10.2) 3 [1/2] (6.1)
Nonlesional 16 [11/5] (64) 7 (28) 1 [-/1] (4) 1 [-/1] (4)
Pruritus
Yes 26 [22/4] (66.7) 6 (15.4) 4 [4/0] (10.2) 3 [1/2] (7.7)
No 8 [7/1] (80) 1 (10) 1 [1/0] (10)
Pigmentation
Yes 20 [15/5] (64.5) 6 (19.4) 3 [3/0] (9.7) 2 [1/1] (6.4)
No 14 [14/-](77.8) 1 (5.5) 2 [2/0] (11.1) 1 [-/1] (5.5)
Exercise
Yes 13 [12/1] (76.5) 3 (17.6) 1 [1/0] (5.9)
No 21 [17/4] (65.6) 4 (12.5) 4 [4/0] (12.5) 3 [1/2] (9.4)
Hyperhidrosis
Yes 15 [11/4] (68.2) 4 (18.2) 2 [2/0] (9.1) 1 [-/1] (4.5)
No 19 [18/1] (70.4) 3 (11.1) 3 [3/0](11.1) 2 [1/1] (7.4)
Wood's light (fluorescence)
Yellow-green 22 [19/3] (68.8) 5 (15.6) 3 [3/0] (9.3) 2 [1/1] (6.3)
Orange-red 4 [3/1] (66.6) 1 (16.7) 1 [1/0] (16.7)
Diffuse green 2 [2/-] (66.7) 1 (33.3)
Negative 6 [5/1] (66.7) 1 (11.1) 1 [1/0] (11.1) 1 [-/1] (11.1)
Episode of disease
First 22 [18/4] (64.7) 4 (11.8) 5 [5/-] (14.7) 3 [1/2] (8.8)
Recurrence 12 [11/1] (80) 3 (20)
Gender
Male 13 [9/4] (72.2) 2 (11.1) 2 [2/0] (11.1) 1 [-/1] (5.6)
Female 21 [20/1] (65.6) 6 (18.8) 3 [3/0] (9.4) 2 [1/1] (6.2)
 M. Durdu et al. / Diagnostic Microbiology and Infectious Disease 76 (2013) 450–457 455

Table 2
Potassium hydroxide, MGG, Wood's light, and molecular findings as well as the response to antifungal treatment in different MF patient groups.

Analysis Number of patients (%)a

MF only (n = 35) MF/Acne vulgaris (n = 6) MF/PV (n = 5) MF/Pemfigus vulgaris (n = 2) MF/Alopecia areata (n = 1)

Potassium hydroxide 20 (57.1) 5 5 1 1

MGG
Footprint-shaped spores 35 (100) 6 5 2 1
Hyphae 4
Bacilli 6
Acantholytic cells 2
Foreign body–type giant cells 3 (8.6) 2 1
Abundant eosinophils 5 (14.3) 1 1
Wood's light (fluorescence)
Yellow-green 23 (65.7) 2 5 1 1
Diffuse green 3 (8.6)
Orange-red 6
Negative 9 (25.7) 1
Molecular data [cluster 1/cluster 2]
M. globosa 24 [21/3] (68.6) 4 [3/1] 3 [3/-] 2 [1/1] 1 [1/-]
M. sympodialis 5 (14.3) 2
M. restricta 4 [4/-] (11.4) 1 [1/-]
M. furfur 2 [1/1] (5.7) 1 [-/1]
Response to treatment
Systemic treatment (n = 47) Complete-33 (70.2) Moderate-6 Complete-5 Partial-2 Complete-1
Topical treatment (n = 2) Moderate-2
a
Stated if the total number was higher than 20.

gender (P N 0.05). The distribution of Malassezia spp. according to
clinical characteristics is presented in Table 1. There were no
significant differences in the illness duration, disease recurrence,
pruritus, pigmentation type, or fluorescence on Wood's light
examination among the patients who were infected with various
Malassezia spp. (P N 0.05). All the Malassezia spp. were related to
hyperpigmented lesions.
3.8. Response to treatment
Of the 49 patients, 47 (95.9%) completed the systemic antifungal
treatment, and 2 abandoned the drug treatment because of side
effects (stomach pain and diarrhea). These 2 patients were treated
with topical ketoconazole cream, which yielded moderate improve-
ment. After 14 days of systemic treatment, 39 (79.6%) patients had
completely recovered (Fig. 1B and D). Six (12.2%) of the patients with
acne vulgaris showed moderate improvement. Partial improvement
was detected in 2 (4.1%) patients who underwent systemic steroid
therapy for pemphigus vulgaris.
4. Discussion
In the present study, MGG detected the presence of fungi in all the
samples from Malassezia culture-confirmed patients and proved itself
as a practical tool for diagnosing MF. Additionally, Wood's light
examination revealed yellow-green fluorescence in 66.7% of the
patients. The results from these preliminary, inexpensive, and widely
available diagnostic tools were confirmed by fungal culture and
molecular data. We determined that M. globosa was the most common
species, followed by M. sympodialis, M. restricta, and M. furfur, in both
the lesional and nonlesional samples. The MF patients were treated
with oral itraconazole capsules (200 mg/d) for 2 weeks, and complete
recovery was observed in 79.6% of the patients.
Only 2 molecular studies have previously investigated the
epidemiological features of MF. In the first study, which used real-
time PCR, Akaza et al. (2009) from Japan investigated 32 patients with
MF and found that M. globosa was the most common fungal pathogen,
followed by M. restricta, M. sympodialis, M. dermatis, and M. furfur.
Remarkably, the authors observed that MF is caused by resident
cutaneous Malassezia spp. but not by specifically exogenous Malasse-
zia spp. (Akaza et al., 2009). Consistent with these findings, our results
indicated that M. globosa was the predominant Malassezia spp. in the
MF (69.4%) and nonlesional (64%) samples. The same species were
detected in the lesional and nonlesional samples in 72% of our MF
patients. Moreover, we identified different clusters in 12% of the
patients. However, we failed to identify M. obtusa in the MF lesions,
which may be attributed to the noncultivatable or relatively slow-
growing strains that are included in these species (Akaza et al., 2009;
Gupta et al., 2001). In the second study, Ko et al. (2011) verified 60 MF
cases using 26S-rDNA PCR-RFLP analysis in Korea. This study
identified M. restricta as the most common lipophilic yeast, followed
by M. sympodialis, M. globosa, M. furfur, and M. dermatis. The different
results from the 2 above-mentioned studies might be due to their
geographical locations.
In an earlier study, M. globosa displayed remarkable diversity in
the intergenic spacer (IGS) 1 region, which is located between 5S and
26S of the rRNA gene. This species was divided into 4 major groups,
which corresponded to the source of the clinical samples: 2 from AD
patients, 1 from healthy subjects, and 1 from both AD patients and
healthy subjects (Sugita et al., 2003). Subsequently, Gaitanis et al.
(2006) reported that M. globosa isolated from PV patients was divided
into 5 groups based on single-strand conformational analysis (SSCP)
of PCR products in the ITS1 region. Remarkably, 1 genotype of M.
globosa in that study appeared to be associated with extensive clinical
diseases (Gaitanis et al., 2006). In a study that examined SD patients
and healthy subjects, genetic analyses of the IGS sequences of M.
globosa and M. restricta indicated that these organisms were divided
into 8 and 2 clusters, respectively. However, none of the clusters
correlated with the clinical manifestations and the severity of SD
(Tajima et al., 2008). In our study, M. globosa, M. restricta, and M. furfur
isolates formed 2 clusters, both related to MF. Although the M.
restricta strains could be grouped into 2 clusters, Cluster 1 was
identified only in the MF lesions. Cluster 2 was recovered from
nonlesional skin in only 1 case.
Gaitanis et al. (2009b) and Zhang et al. (2010) reported on the
genetic diversity, host ethnicity, and geographic origin of clinical M.
furfur strains by PCR-fingerprinting minisatellite DNA analysis. They
suggested that M. furfur could be a candidate for many applications,
such as forensic biological analysis and phylogeographic studies.
Consistent with their findings, an M. furfur SD isolate from a Dutch
456 M. Durdu et al. / Diagnostic Microbiology and Infectious Disease 76 (2013) 450–457
male residing for 5 years in China displayed complete ITS sequences
that were identical to M. furfur CBS 7982 from the ear skin of a healthy
French individual (Ran et al., 2008). However, based on the ITS
molecular typing and ITS1 PCR-SSCP screening methods, M. sympo-
dialis appears to be a homogenous species (Gaitanis et al., 2006;
Makimura et al., 2000). We confirmed this finding in our study of
lesional and nonlesional skin samples (Table 1).
Clinically, MF can mimic bacterial folliculitis and acne vulgaris.
Thus, 15 (30.6%) of our MF patients had been previously
misdiagnosed and treated with antibiotics or oral isotretinoin.
Another 3 patients had been misdiagnosed with pruritus and
eczema and treated unnecessarily with steroid creams (Fig. 1E).
Similar cases of pruritic folliculitis have been reported (Fearfield et
al., 1999; Helm and Lookingbill, 1993). To diagnose MF routinely in
a dermatology practice, a sample is usually taken for KOH staining
(Durdu and Ilkit, 2013). A cytological examination can also be
undertaken with MGG-, Papanicolaou-, Giemsa-, or methylene blue–
stained smears. MGG is a simple, reliable, rapid (20–25 seconds),
and inexpensive ($1 per test) method (Durdu et al., 2011).
Additionally, cytology can also reveal other bacterial, fungal, viral,
and parasitic causes of folliculitis (Durdu and Ilkit, 2013). Our study
revealed that the positivity of MGG-stained smears was higher
(100%) than that of KOH-stained smears (81.6%). Moreover, the
MGG-stained smears revealed several different features compared
with the KOH smears (Fig. 3A–F; Table 2).
MF is more common in hot, humid climates and flares with
increased episodes of sweating. In 1 study from the Philippines, the
prevalence of MF was reported to be 16.5% in patients visiting a
dermatological clinic, whereas the prevalence was 1–1.5% in China
(Bulmer et al., 2008; Jacinto-Jamora et al., 1991). In our study, the
prevalence of MF was 4% in patients visiting a dermatological clinic in
Turkey. Predisposing factors of MF include excessive sweating (51%),
topical (16.3%) or oral (10.2%) antibiotic use, topical (6.1%) or oral
(6.1%) corticosteroid use, and excessive exercise (34%). Remarkably,
however, we observed that these rates were also similar for patients
with other infectious folliculitis.
Vesicular or scaling form dyshidrotic id reactions are usually
caused by dermatophytic infections, particularly tinea pedis (Ilkit et
al., 2012). However, dyshidrotic id reactions caused by MF have not
been previously reported. We detected 2 scaling form dyshidrotic id
reactions, and the associated lesions disappeared after oral itracona-
zole treatment (Fig. 1F).
Various topical and systemic antifungal drugs can improve MF,
which usually responds well to oral antifungal medication. Topical
antifungals are less effective than systemic antifungals, but topical
antifungals are important for maintenance and prophylaxis (Ayers et
al., 2005). Complete recovery was reported in 100% of MF patients in a
randomized study that combined topical and systemic antifungal
treatments. Conversely, only a 75% clearance was achieved with
systemic antifungal treatment (Abdel-Razek et al., 1995). Oral
ketoconazole, fluconazole, and itraconazole have been successfully
used to treat MF systemically (Abdel-Razek et al., 1995; Parsad et al.,
1998; Rhie et al., 2000). Systemic itraconazole treatment was used in
this study because of its favorable in vitro activity against lipophilic
Malassezia, its safety, and its high concentration excreted in the sebum
(Faergemann, 1984; Kavanagh et al., 1993). Only 2 (4.1%) patients
could not use the drug in our study because of side effects. After
systemic treatment, complete or moderate improvement was ob-
served in 45 (91.8%) patients.
In conclusion, we described a diagnostic algorithm, including MGG
staining, which offers more insight into all cases of infectious
folliculitis. Regarding the prevalence, we suggest that MF is not
uncommon, particularly in Adana, Turkey. Our findings are consistent
with reports that M. globosa is increasingly being identified as an
etiological agent of MF, indicating the importance of reexamining the
pathogenicity of this organism.
Acknowledgments
We are grateful to Dr. George Gaitanis for sharing his valuable
work with us. We also thank Dr. Aylin Döğen for her kind and helpful
assistance in the molecular study.
References
Abdel-Razek M, Fadaly G, Abdel-Raheim M, al-Morsy F. Pityrosporum (Malassezia)
folliculitis in Saudi Arabia—diagnosis and therapeutic trials. Clin Exp Dermatol
1995;20:406–9.
Akaza N, Akamatsu H, Sasaki Y, et al. Malassezia folliculitis is caused by cutaneous
resident Malassezia species. Med Mycol 2009;47:618–24.
Ayers K, Sweeney SM, Wiss K. Pityrosporum folliculitis: diagnosis and management in 6
female adolescents with acne vulgaris. Arch Pediatr Adolesc Med 2005;159:64–7.
Batra R, Boekhout T, Guého E, et al. Malassezia Baillon, emerging clinical yeasts. FEMS
Yeast Res 2005;5:1101–13.
Bulmer GS, Pu XM, Yi LX. Malassezia folliculitis in China. Mycopathologia 2008;165:
411–2.
Cabaňes FJ, Vega S, Castellá G. Malassezia cuniculi sp. nov., a novel yeast species isolated
from rabbit skin. Med Mycol 2011;49:40–8.
Durdu M, Ilkit M. First step in the differential diagnosis of folliculitis: cytology. Crit Rev
Microbiol 2013;39:9-25.
Durdu M, Seçkin D, Baba M. The Tzanck smear test: rediscovery of a practical diagnostic
tool. Skinmed 2011;9:23–32.
Faergemann J. In vitro and in vivo activities of ketoconazole and itraconazole against
Pityrosporum orbiculare. Antimicrob Agents Chemother 1984;26:773–4.
Fearfield LA, Rowe A, Francis N, Bunker CB, Staughton RC. Itchy folliculitis and human
immunodeficiency virus infection: clinicopathological and immunological features,
pathogenesis and treatment. Br J Dermatol 1999;141:3-11.
Gaitanis G, Velegraki A, Alexopoulos EC, et al. Distribution of Malassezia species in
pityriasis versicolor and seborrheic dermatitis patients in Greece. Typing of the
major pityriasis versicolor isolate M. globosa. Br J Dermatol 2006;154:854–9.
Gaitanis G, Bassukas ID, Velegraki A. The range of molecular methods for typing
Malassezia. Curr Opin Infect Dis 2009a;22:119–25.
Gaitanis G, Velegraki A, Alexopoulos EC, et al. Malassezia furfur fingerprints as possible
markers for human phylogeography. ISME J 2009b;3:498–502.
Guého E, Midgley G, Guillot J. The genus Malassezia with the description of four new
species. Ant Leeuw J Microb 1996;69:337–55.
Gupta AK, Batra R, Bluhm R, Boekhout T, Dawson Jr TL. Skin diseases associated with
Malassezia species. J Am Acad Dermatol 2004a;51:785–98.
Gupta AK, Boekhout T, Theelen B, Summerbell R, Batra R. Identification and typing of
Malassezia species by amplified fragment length polymorphism and sequence
analyses of the internal transcribed spacer and large-subunit regions of ribosomal
DNA. J Clin Microbiol 2004b;42:4253–60.
Gupta AK, Kohli Y, Summerbell RC, Faergemann J. Quantitative culture of Malassezia
species from different body sites of individuals with or without dermatoses. Med
Mycol 2001;39:243–51.
Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis
program for Windows 95/98 NT. Nucl Acids Symp Ser 1999;41:95–8.
Helm KF, Lookingbill DP. Pityrosporum folliculitis and severe pruritus in two patients
with Hodgkin's disease. Arch Dermatol 1993;129:380–1.
Ilkit M, Durdu M, Karakaş M. Cutaneous id reactions: a comprehensive review of clinical
manifestations, epidemiology, etiology, and management. Crit Rev Microbiol
2012;38:191–202.
Jacinto-Jamora S, Tamesis J, Katigbak ML. Pityrosporum folliculitis in the Philippines:
diagnosis, prevalence, and management. J Am Acad Dermatol 1991;24:693–6.
Kavanagh GM, Leeming JP, Marshman GM, Reynolds NJ, Burton JL. Folliculitis in Down's
syndrome. Br J Dermatol 1993;129:696–9.
Ko JH, Lee YW, Choe YB, Ahn KJ. Epidemiologic study of Malassezia yeasts in patients
with Malassezia folliculitis by 26S rDNA PCR-RFLP analysis. Ann Dermatol 2011;23:
177–84.
Leeming JP, Notman FH. Improved methods for isolation and enumeration of Malassezia
furfur from human skin. J Clin Microbiol 1987;25:2017–9.
Makimura K, Tamuro Y, Kudo M, et al. Species identification and strain typing of
Malassezia stock strains and clinical isolates based on the DNA sequences of nuclear
ribosomal internal transcribed spacer 1 regions. J Med Microbiol 2000;49:29–35.
Parsad D, Saini R, Negi KS. Short-term treatment of Pityrosporum folliculitis: a double
blind placebo-controlled study. J Eur Acad Dermatol Venereol 1998;11:188–90.
Ran Y, He X, Zhang H, et al. Seborrheic dermatitidis flare in a Dutch male due to
commensal Malassezia furfur overgrowth. Med Mycol 2008;46:611–4.
Rhie S, Turcios R, Buckley H, Suh B. Clinical features and treatment of Malassezia
folliculitis with fluconazole in orthotopic heart transplant recipients. J Heart Lung
Transplant 2000;19:215–9.
Sugita T, Kodama M, Saito M, et al. Sequence diversity of the intergenic spacer region of
the rRNA gene of Malassezia globosa colonizing the skin of patients with atopic
dermatitis and healthy individuals. J Clin Microbiol 2003;41:3022–7.
Sugita T, Suzuki M, Goto M, et al. Quantitative analysis of the cutaneous Malassezia
microbiota in 770 healthy Japanese by age and gender using a real-time PCR assay.
Med Mycol 2010;48:229–33.
Sugita T, Tajima M, Tsubuku H, Tsuboi R, Nishikawa A. Quantitative analysis of
cutaneous Malassezia in atopic dermatitis patients using real-time PCR. Microbiol
Immunol 2006;50:549–52.
 M. Durdu et al. / Diagnostic Microbiology and Infectious Disease 76 (2013) 450–457 457

Tajima M, Sugita T, Nishikawa A, Tsuboi R. Molecular analysis of Malassezia microflora
in seborrheic dermatitis patients: comparison with other diseases and healthy
subjects. J Invest Dermatol 2008;128:345–51.
Tamura K, Peterson D, Peterson N, et al. MEGA5: molecular evolutionary genetics
analysis using maximum likelihood, evolutionary distance, and maximum
parsimony methods. Mol Biol Evol 2011;28:2731–9.
Turin L, Riva F, Galbiati G, Cainelli T. Fast, simple and highly sensitive double-rounded
polymerase chain reaction assay to detect medically relevant fungi in dermato-
logical specimens. Eur J Clin Invest 2000;30:511–8.
Zhang N, Zhang R, Ran Y, et al. Genetic polymorphisms of Malassezia furfur isolates from
Han and Tibetian ethnic groups in China using DNA fingerprinting. Med Mycol
2010;48:1034–8.