doi: 10.1111/1346-8138.

14120 Journal of Dermatology 2017; : 1–4

CONCISE COMMUNICATION
Utility of Gram staining for diagnosis of Malassezia folliculitis
Wei-Ting TU,1 Szu-Ying CHIN,1,2 Chia-Lun CHOU,1,3 Che-Yuan HSU,1 Yu-Tsung CHEN,1
Donald LIU,1 Woan-Ruoh LEE,1,3,4 Yi-Hsien SHIH1,3,4
Departments of 1Dermatology, 2Pathology, Taipei Medical University Shuang Ho Hospital, New Taipei City, 3Department of
Dermatology, School of Medicine, 4Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei,
Taiwan

ABSTRACT
Malassezia folliculitis (MalF) mimics acne vulgaris and bacterial folliculitis in clinical presentations. The role of
Gram staining in rapid diagnosis of MalF has not been well studied. In our study, 32 patients were included to
investigate the utility of Gram staining for MalF diagnosis. The final diagnoses of MalF were determined according
to clinical presentation, pathological result and treatment response to antifungal agents. Our results show that
the sensitivity and specificity of Gram staining are 84.6% and 100%, respectively. In conclusion, Gram staining is
a rapid, non-invasive, sensitive and specific method for MalF diagnosis.

Key words: acne vulgaris, bacterial folliculitis, Gram staining, Malassezia folliculitis, Pityrosporum folliculitis.

INTRODUCTION CASE REPORT

Patients and study design
Malassezia folliculitis (MalF), a prevalent inflammatory skin
All patients with clinical suspicion of MalF who presented to
disease in tropics and subtropics,1–5 associates with coloniza-
our institution from June 2015 to January 2016 were included
tion and overgrowth of Malassezia in hair follicles.6,7 The typical
in this study. The pustule content of all patients was collected
clinical presentation is numerous erythematous, pruritic
for Gram staining. Patient demographics, clinical presentations,
papules and pustules on the trunk and proximal extremities.1–
5,8 photographic records, Gram staining results, skin biopsy
MalF sometimes mimics or coexists with acne vulgaris and
results and treatment responses were collected by retrospec-
bacterial folliculitis.3,8,9 However, MalF does not present with
tive chart reviews and telephone interviews.
comedones, and it responds to antifungal agents instead of
traditional acne treatments or antibiotics.6–8,10
For MalF diagnosis, skin biopsy typically shows round
Smear preparation and Gram staining
For each patient, one to five pustules were sampled to ensure
spores, blastospores or short hyphae in the inflamed hair folli-
there were adequate specimens for microscopic examination.
cle or perifollicular dermis.5 The use of periodic acid-Schiff
Gram staining was performed following the standard method.12
(PAS) stain further increases diagnostic accuracy.11 Neverthe-
The result of Gram staining was interpreted as follows: (i) if
less, skin biopsy is invasive and time-consuming. Alterna-
there were more than 30 blastospores or short hyphae in more
tively, direct microscopic examination of pustule content in
than three oil-immersion fields, the smear was marked as
hair follicles serves as a rapid, easily performed and non-inva-
“MalF”; (ii) if there were numerous bacteria, regardless of their
sive method for Malassezia detection.6–8,10 Multiple stains,
morphology, the smear was interpreted as “bacterial folliculi-
including blue/black Parker ink, May–Grunwald–Giemsa
tis”; (iii) if both criteria were met, the smear was categorized as
(MGG) stain, methylene blue, lactophenol blue and calcofluor
“mixed folliculitis”; and (iv) if no criteria was met, the examina-
white have all been used to visualize Malassezia.1–4 Gram
tion result was “no diagnosis”.
staining is widely used to visualize bacteria and has been
used to identify Malassezia in one very early study,12 in which
demonstration of Malassezia was presumed to be diagnostic Skin biopsy and histopathological diagnosis
of MalF. However, the study design was simple and crude, Skin biopsy was performed in standard fashion. Under micro-
focusing mainly on the clinical characteristics and epidemiol- scopic examination, the presence of suggestive pathogens in
ogy of MalF, rather than the diagnostic accuracy of Gram inflamed hair follicles or perifollicular dermis led to histopatho-
staining. Therefore, we aim to investigate the utility of Gram logical diagnosis. Importantly, presence of Malassezia in the
staining for MalF diagnosis. epidermis or near the follicular opening did not constitute the

Correspondence: Yi-Hsien Shih, M.D., Department of Dermatology, Taipei Medical University Shuang Ho Hospital, 291 Zhongzheng Road,
Zhonghe District, New Taipei City 23561, Taiwan. Email: 13263@s.tmu.edu.tw
Received 7 March 2017; accepted 5 October 2017.

© 2017 Japanese Dermatological Association 1

W.-T. Tu et al.

diagnosis. If routine hematoxylin–eosin staining failed to agents.13 Bacterial folliculitis and mixed folliculitis were diag-
demonstrate any pathogen, PAS staining and Gram staining nosed mutatis mutandis. If final diagnosis remained undeter-
were performed to increase sensitivity. mined, the case was excluded.

Evaluation of treatment response Statistical analysis

Responsiveness to antifungal or antibacterial therapy was con- Gram staining results were compared with final diagnoses
sidered as “consistent with MalF” or “consistent with bacterial made by the above-mentioned criteria, and the sensitivity and
folliculitis”, respectively. Rarely, patients improved sponta- specificity of Gram staining were calculated.
neously without treatment, or did not respond to any treat-
ment. These situations, in which the etiology could not be Clinical characteristics of patients
supported by treatment responses, were designated as A total of 32 patients were included in this study. The mean
“equivocal”. age was 33.8 years. The lesions mostly affected the trunk,
neck, proximal upper extremities and face. The proximal lower
Final diagnosis of Malassezia folliculitis extremities, distal upper extremities and distal lower extremi-
Final diagnosis of MalF was made in patients who met two out ties were uncommonly affected (Table 1). Among these 32
of three of the following criteria: (i) typical clinical presentation patients, 26 (81.3%) patients were diagnosed with MalF, two
with papulopustules mainly distributed on the trunk; (ii) sugges- (6.3%) with bacterial folliculitis and four (15.4%) with mixed
tive skin biopsy results showing Malassezia in the inflamed hair folliculitis (Table 2).
follicles; and (iii) compatible treatment responses to antifungal
Gram staining for Malassezia folliculitis diagnosis
Table 1. Clinical characteristic of all patients Gram staining correctly diagnosed 27 (84.4%) out of the 32
patients, including 22 patients with MalF, one patient with bac-
Patient
terial folliculitis and four patients with mixed folliculitis. Of the
Sex (male/female) 28/4 patients diagnosed with MalF, 22 out of 26 patients (84.6%)
Mean age (years) 33.8 (12-75)
had suggestive Gram staining findings. Of the six patients who
Inpatient 8 (25%)
were not diagnosed with MalF, Gram staining showed findings
Distribution of lesions
Trunk 31 (96.9%) other than MalF in all (100%) (Table 2). Representative images
Neck 21 (65.6%) from cases of MalF, bacterial folliculitis and mixed folliculitis
Face 8 (25.0%) are demonstrated in Figure 1.
Scalp 6.3 (2%)
Proximal upper extremities 13 (40.6%) Gram staining for detection of mixed folliculitis
Distal upper extremities 3 (9.4%) In our study, Gram staining showed mixed folliculitis in eight
Proximal lower extremities 3 (9.4%) patients. Among these eight patients, the final diagnoses were
Distal lower extremities 2 (6.3%) mixed folliculitis (four patients), MalF (three patients) and
Biopsy result
bacterial folliculitis (one patient). Interestingly, five out of these
Malassezia folliculitis 15 (46.9%)
eight patients responded to antifungal agents partially or
Bacterial folliculitis 2 (6.3%)
Mixed folliculitis 4 (12.5%) significantly regardless of the final diagnoses (Data S1).
No pathogen 5 (15.6%)
Not biopsied 6 (18.8%) DISCUSSION
Treatment response
Responded to oral/topical antifungal therapy 18 (56.3%) In this retrospective single-center series, we show that Gram
Responded to oral/topical antibacterial therapy 1 (3.1%) staining has good sensitivity and excellent specificity for MalF
Responded to antibacterial and antifungal 5 (15.6%) diagnosis. Gram staining is also favored over other staining
therapy methods because it identifies MalF, bacterial folliculitis and
Equivocal 7 (21.9%)
mixed folliculitis simultaneously, and assists treatment
Loss of follow up 1 (3.1%)
decisions in time.

Table 2. Gram staining as a diagnostic tool for Malassezia folliculitis

Gram staining (n = 32)

Final diagnosis Malassezia folliculitis Bacterial folliculitis Mixed folliculitis No diagnosis Total
Malassezia folliculitis 22 (68.8%) 0 (0.0%) 3 (9.4%) 1 (3.1%) 26 (81.3%)
Bacterial folliculitis 0 (0.0%) 1 (3.1%) 1 (3.1%) 0 (0.0%) 2 (6.3%)
Mixed folliculitis 0 (0.0%) 0 (0.0%) 4 (12.5%) 0 (0.0%) 4 (12.5%)
Total 22 (68.8%) 1 (3.1%) 8 (25.0%) 1 (3.1%) 32 (100%)

2 © 2017 Japanese Dermatological Association

 Gram stain for Malassezia folliculitis

Figure 1. Representative photographs and microscopic images of patients with (a) Malassezia folliculitis (middle, Gram staining,
original magnification 91000; right, periodic acid-Schiff performed on a paraffin block section, 91000), (b) bacterial folliculitis
(middle, Gram staining, 91000; right, Gram staining performed on a paraffin block section, 91000) and (c) mixed folliculitis (middle,
Gram staining, 91000; right, Gram staining performed on a paraffin block section, 91000).

Few studies have evaluated the accuracy of special staining
smears in MalF diagnosis and the results were conflicting,
showing positive rates ranging 44–100%.1,4,5 Diagnosis of MalF
by Gram staining was first reported by Lim et al.5,12 In their
epidemiological study, the authors claimed that the positive
rates of Gram staining and skin biopsy were 44% and 84%,
respectively. However, they did not mention patients’ treatment
responses and thus false-positive cases caused by commensal
Malassezia could not be totally excluded. Our study is the first
to demonstrate the diagnostic accuracy of Gram staining for
MalF. We provided clear instructions about the number of
sampled hair follicles and the definition of positive Gram stain-
ing used in our study. In addition, we not only focused on clini-
cal characteristics and epidemiological data, but also followed
treatment responses to exclude false-positive cases caused by
commensal Malassezia.
Parker blue ink staining had been proposed for MalF diag-
nosis by Abdel-Razek et al.1 They reported 100% positive

© 2017 Japanese Dermatological Association 3

W.-T. Tu et al.
rate of skin scrapings in all MalF patients, which was much
higher than what we observed in our study using Gram stain-
ing. However, 10 out of 62 patients (16%) in their study had
only one single blastospore in the skin scrapings, which may
not be sufficient for the diagnosis of MalF, and only 24%
patients had skin biopsy proofs. A more recent study used
MGG staining for MalF diagnosis.4 In this study, MGG stain-
ing smear was more often positive than histology (89% vs
33%). Importantly, the end-points of both studies are the
treatment responses to different regimens for MalF, but not
the sensitivity and specificity of special staining smears for
MalF diagnosis. In our study, we compared the Gram staining
diagnoses to the final diagnoses made by standard diagnostic
criteria, and thus accurate sensitivity and specificity could be
calculated.
Interestingly, we were able to find eight cases of mixed folli-
culitis with coexistence of bacteria and Malassezia using Gram
staining. The bacteria we found were Gram-positive bacilli or
cocci in most cases. They were morphologically consistent
with either Propionibacterium spp. or Staphylococcus spp.
Coexistence of Malassezia and Propionibacterium in acne
patients was reported recently by Akaza et al.9 In their study,
they found that the severity of acne correlated with the number
of Malassezia but not with that of Propionibacterium, suggest-
ing Malassezia may participate in the pathophysiology of acne.
In another Japanese single-center case series, approximately
12% of acneiform eruptions were finally diagnosed as MalF.14
In our study, many patients with mixed folliculitis under Gram
staining responded to antifungal agents significantly or at least
partly.
Consequently, Gram staining is a rapid, simple and non-
invasive test for MalF diagnosis. Nevertheless, Gram staining
does not reveal the location and invasion of pathogens as
clearly as histology does. The study is also limited by the lack
of culture-based or molecular technique-based data. More
research of larger sample size is required to validate the use of
Gram staining for MalF diagnosis.
CONFLICT OF INTEREST: None declared.
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Data S1. Demographic characteristics and clinical presenta-
tions of the included patients.