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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 to demonstrate the diagnostic accuracy of Gram staining for
smears in MalF diagnosis and the results were conflicting, MalF. We provided clear instructions about the number of
showing positive rates ranging 44–100%.1,4,5 Diagnosis of MalF sampled hair follicles and the definition of positive Gram stain-
by Gram staining was first reported by Lim et al.5,12 In their ing used in our study. In addition, we not only focused on clini-
epidemiological study, the authors claimed that the positive cal characteristics and epidemiological data, but also followed
rates of Gram staining and skin biopsy were 44% and 84%, treatment responses to exclude false-positive cases caused by
respectively. However, they did not mention patients’ treatment commensal Malassezia.
responses and thus false-positive cases caused by commensal Parker blue ink staining had been proposed for MalF diag-
Malassezia could not be totally excluded. Our study is the first 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 2 Durdu M, Guran M, Ilkit M. Epidemiological characteristics of Malas-
higher than what we observed in our study using Gram stain- sezia folliculitis and use of the May-Grunwald-Giemsa stain to diag-
nose the infection. Diagn Microbiol Infect Dis 2013; 76: 450–457.
ing. However, 10 out of 62 patients (16%) in their study had
3 Jacinto-Jamora S, Tamesis J, Katigbak ML. Pityrosporum folliculitis
only one single blastospore in the skin scrapings, which may in the Philippines: diagnosis, prevalence, and management. J Am
not be sufficient for the diagnosis of MalF, and only 24% Acad Dermatol 1991; 24: 693–696.
patients had skin biopsy proofs. A more recent study used 4 Levy A, Feuilhade de Chauvin M, Dubertret L, Morel P, Flageul B.
[Malassezia folliculitis: characteristics and therapeutic response in
MGG staining for MalF diagnosis.4 In this study, MGG stain-
26 patients]. Ann Dermatol Venereol 2007; 134: 823–828.
ing smear was more often positive than histology (89% vs 5 Lim KB, Giam YC, Tan T. The epidemiology of Malassezia
33%). Importantly, the end-points of both studies are the (Pityrosporon) folliculitis in Singapore. Int J Dermatol 1987; 26: 438–
treatment responses to different regimens for MalF, but not 441.
the sensitivity and specificity of special staining smears for 6 Gupta AK, Batra R, Bluhm R, et al. Skin diseases associated with
Malassezia species. J Am Acad Dermatol 2004; 51: 785–798.
MalF diagnosis. In our study, we compared the Gram staining
7 Harada K, Saito M, Sugita T, et al. Malassezia species and their
diagnoses to the final diagnoses made by standard diagnostic associated skin diseases. J Dermatol 2015; 42: 250–257.
criteria, and thus accurate sensitivity and specificity could be 8 Rubenstein RM, Malerich SA. Malassezia (pityrosporum) folliculitis. J
calculated. Clin Aesthet Dermatol 2014; 7: 37–41.
9 Akaza N, Akamatsu H, Numata S, et al. Microorganisms inhabiting
Interestingly, we were able to find eight cases of mixed folli-
follicular contents of facial acne are not only Propionibacterium but
culitis with coexistence of bacteria and Malassezia using Gram also Malassezia spp. J Dermatol 2016; 43: 906–911.
staining. The bacteria we found were Gram-positive bacilli or 10 Hald M, Arendrup MC, Svejgaard EL, et al. Evidence-based Danish
cocci in most cases. They were morphologically consistent guidelines for the treatment of Malassezia-related skin diseases.
with either Propionibacterium spp. or Staphylococcus spp. Acta Derm Venereol 2015; 95(1): 12–19.
11 Song HS, Kim SK, Kim YC. Comparison between Malassezia Folli-
Coexistence of Malassezia and Propionibacterium in acne
culitis and Non-Malassezia Folliculitis. Ann Dermatol 2014; 26: 598–
patients was reported recently by Akaza et al.9 In their study, 602.
they found that the severity of acne correlated with the number 12 Lim KB, Boey LP, Khatijah M. Gram’s-stained microscopy in the eti-
of Malassezia but not with that of Propionibacterium, suggest- ological diagnosis of Malassezia (Pityrosporon) folliculitis. Arch Der-
matol 1988; 124: 492.
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13 Prohic A, Jovovic Sadikovic T, Krupalija-Fazlic M, et al. Malassezia
In another Japanese single-center case series, approximately species in healthy skin and in dermatological conditions. Int J Der-
12% of acneiform eruptions were finally diagnosed as MalF.14 matol 2016; 55: 494–504.
In our study, many patients with mixed folliculitis under Gram 14 Suzuki C, Hase M, Shimoyama H, et al. Treatment Outcomes for
staining responded to antifungal agents significantly or at least Malassezia Folliculitis in the Dermatology Department of a University
Hospital in Japan. Med Mycol J 2016; 57(3): E63–E66.
partly.
Consequently, Gram staining is a rapid, simple and non-
invasive test for MalF diagnosis. Nevertheless, Gram staining SUPPORTING INFORMATION
does not reveal the location and invasion of pathogens as
clearly as histology does. The study is also limited by the lack Additional Supporting Information may be found in the online
of culture-based or molecular technique-based data. More version of this article:
research of larger sample size is required to validate the use of Data S1. Demographic characteristics and clinical presenta-
Gram staining for MalF diagnosis. tions of the included patients.

CONFLICT OF INTEREST: None declared.

REFERENCES
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(Malassezia) folliculitis in Saudi Arabia–diagnosis and therapeutic
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4 © 2017 Japanese Dermatological Association