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 ~ Results
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 ~ Conclusion
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ORIGINAL ARTICLE
Year : 2020  |  Volume : 38  |  Issue : 3  |  Page : 351-356
 

Epidemiological pattern of Malassezia, its phenotypic identification and antifungal susceptibility profile to azoles by broth microdilution method


1 Department of Microbiology, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India
2 Department of Dermatology, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India
3 Department of Microbiology, Madras Medical College, Chennai, Tamil Nadu, India

Date of Submission15-Mar-2020
Date of Decision29-Aug-2020
Date of Acceptance25-Aug-2020
Date of Web Publication4-Nov-2020

Correspondence Address:
Dr. Anupma Jyoti Kindo
Department of Microbiology, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai – 116, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_20_106

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 ~ Abstract 


Background: Malassezia though known for its cutaneous infections can potentially cause invasion. The skin infections caused by Malassezia have poor patient compliance due to its chronicity and recurrent nature of the disease. There is also a lack of standardised antifungal susceptibility profile for Malassezia due to its complex growth requirement. Objective: This study was performed to understand the epidemiological pattern of disease and to study the antifungal susceptibility testing (AFST) profile so as to choose the appropriate drug/drugs to treat the infections caused by Malassezia. Materials and Methods: Samples were collected and processed, species were identified by conventional method and AFST was done by broth microdilution method. Results: The epidemiological pattern showed adolescent females commonly affected in torso. The most common lesion was pityriasis versicolor. The systemic antifungal of choice was itraconazole with the lowest minimum inhibitory concentration (MIC) of 0.125–1 μg/ml. The best topical drug with the lowest MIC value was clotrimazole 0.03–0.5 μg/ml. Conclusion: AFST is important as it will help the dermatologist to choose the appropriate antifungal agents for the patient and thereby reduce the chronicity of the disease with good patient compliance


Keywords: AFST by broth microdilution, epidemiology, fluconazole resistant, Malassezia, phenotypic speciation


How to cite this article:
Romald PN, Kindo AJ, Mahalakshmi V, Umadevi U. Epidemiological pattern of Malassezia, its phenotypic identification and antifungal susceptibility profile to azoles by broth microdilution method. Indian J Med Microbiol 2020;38:351-6

How to cite this URL:
Romald PN, Kindo AJ, Mahalakshmi V, Umadevi U. Epidemiological pattern of Malassezia, its phenotypic identification and antifungal susceptibility profile to azoles by broth microdilution method. Indian J Med Microbiol [serial online] 2020 [cited 2020 Nov 28];38:351-6. Available from: https://www.ijmm.org/text.asp?2020/38/3/351/299808





 ~ Introduction Top


Malassezia, lipophilic basidiomycetes yeast, are natural habitat of stratum corneum of animals and humans. Initially, Malassezia was known to cause a number of cutaneous infections such as pityriasis versicolor (PV), seborrhoeic dermatitis, Malassezia folliculitis and atopic dermatitis. But now, the scenario has changed as it has been implicated in systemic and bloodstream infections, especially in immunosuppressed patients. It has also been isolated from a patient with Crohn's disease. Many neonatal intensive care unit cases on outbreaks of invasive Malassezia infections, particularly in neonates and infants receiving total parenteral nutrition, have also been reported. This has led to the importance of Malassezia in the field of medical mycology.

The treatment of the infections caused by Malassezia has always been a challenge to the dermatologist owing to its chronicity and recurrence. The topical and systemic antifungal drugs used to treat these infections were not satisfactory. Adding to it, the increase in incidence of severe dermatological and systemic infections caused by this genus also emphasised the need to know the susceptibility profile of this yeast in order to choose a specific and accurate drug for treatment. Standardised assay to determine thein vitro susceptibility of the yeast to any antifungal is also not available due to its complex nutritional requirement. Hence, we undertook the study on antifungal susceptibility pattern to decide on the appropriate antifungal agent for infections caused by Malassezia.


 ~ Materials and Methods Top


A cross-sectional study was done in a tertiary care hospital in Tamil Nadu, from August 2016 to October2018. After obtaining institutional ethical clearance, hyper/hypopigmented skin samples were collected from patients attending the dermatological outpatient department by skin scrapings and swabs. They were subjected to microscopic examination with 10% KOH.

Culture

Samples were inoculated into Sabouraud's dextrose agar (SDA), SDA with olive oil overlay (SDA-O) and modified Dixon's agar (MDA) containing chloramphenicol and incubated at 32°C up to 3 weeks.

Phenotypic identification

Phenotypic identification was done by colony growth characteristics, Gram staining, urease test and catalase test, bile esculin with Tween 60 hydrolysis and tween assimilation.

Catalase test

Catalase test was performed using 3% hydrogen per oxide to detect the only catalase-negative species Malassezia restricta.[1]

Test for enzyme production – Urease

The organism was tested for production of enzyme urease using Christensen's urea agar containing 2% urea.

Tween 60 bile esculin

A loop full of fresh yeast was inoculated deep in the Tween 60 esculin agar and incubated for 5 days at 32o C. The splitting of esculin is revealed by darkening of the medium. This test is used to distinguish Malassezia furfur, Malassezia sympodialis, Malassezia slooffiae, Malassezia cuniculi from other Malassezia species.[2]

Tween assimilation

Malassezia can be speciated depending on their ability to utilise tween compounds (Tween 20, Tween 40, Tween 60 and Tween 80).[3]

Sterile SDA (16 ml) was melted and allowed to cool to 50°C, 2 ml of Malassezia yeast suspension about 105 colony-forming units (CFU)/ml was mixed with SDA, and the mixtures were plated by pour plate method. Four wells were made in the agar by means of a 2-mm diameter punch, one in each quadrant, and filled with 5 μl Tween 20, Tween 40, Tween 60 and Tween 80. The plates were incubated for 1 week at 32°C. Utilisation of tween was assessed by the degree of growth and/or reaction.

Antifungal susceptibility testing

Antifungal susceptibility testing (AFST) was performed by the broth microdilution method, according to the Clinical and Laboratory Standards Institute (CLSI) guidelines M27-A3 (2008).

Systemic antifungal agents used were as follows – fluconazole (FLU), itraconazole (ITR) and voriconazole (VOR). Topical antifungal agents used were as follows – clotrimazole (CLO), miconazole (MCZ), sertaconazole (SER), luliconazole (LUL).

Since Malassezia are lipid-dependent yeast, Roswell Park Memorial Institute (RPMI) 1640 medium was supplemented with glucose, peptone, ox bile, malt extract, glycerol, Tween 80, Tween 40 and chloramphenicol.[4]

Inoculum preparation

Malassezia isolates grown on MDA for 72 h at 32°C ± 2°C were used. Antifungal stock solutions of drugs were prepared and stored at −70°C.

Quality control

Each microtitre plate included growth control well with inoculum and supplemented RPMI-1640 medium without the antifungal agent. Candida krusei ATCC 6258 was used as a quality control strain for AFST.

Incubation and reading results

The microtitre plates were incubated at 32°C. Results read after 24 h for Candida isolate and after 72–96 h for Malassezia isolates. The minimum inhibitory concentration (MIC90) (reduction of 90% growth compared to growth control well) and MIC50(50% reduction of growth compared to growth control well) were recorded.


 ~ Results Top


Age distribution

The most common age affected was 20–30 years. The extremes of age showed lower incidence [Figure 1]b.
Figure 1: The epidemiological result (a) Site of prevalence, (b) Age distribution, (c) Sex distribution

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Sex distribution

The incidence among males and females was almost equal, showing little female predominance [Figure 1]c.

Site of predilection

Among the total 151, the most common site was back – 47 samples, followed by neck and shoulder – 31 samples and scalp –27 samples [Figure 1]a.

Skin lesions

Both hypo- and hyperpigmented skin lesions were seen. However, more of hypopigmented lesions were noted in our study [Figure 2].
Figure 2: (a) Skin lesions: A1, A2, A4 and A5 showing hypopigmented skin lesions. A3 and A6 showing hyperpigmented lesions, (b) microscopic KOH picture - meatball and spaghetti appearance, (c) Gram stain pictures: C1 – cylindrical yeast cell, C2 – globosa-shaped yeast cell, C3 – Gram stain picture of Malassezia furfur

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Microscopic examination

It showed meatball and spaghetti appearance [Figure 2]b.

Phenotypic speciation

Among the 151 samples collected, 95 isolates grew on both SDA-O and MDA [Figure 3]. Growth on MDA was faster than SDA-O. All isolates were urease positive belonging to Basidiomycetes family [Figure 3]b and catalase negative. M. furfur, M. sympodialis, Malassezia globosa and Malassezia obtusa were the four species isolated [Table 1]. The most common species was M. sympodialis (33), followed by M. globosa (30), M. furfur (21) and M. obtusa (4) [Figure 3].
Figure 3: (a): A1, A2 and A3 showing growth on modified Dixon's agar, (b) test for urease enzyme-pink colour positive, (c) growth on Sabouraud's dextrose agar with olive oil overlay, (d) Tween 60 bile esculin test-positive shows blackening, (e) tween assimilation (Tween 20, Tween 40, Tween 60, Tween 80 and cremaffin oil): E1 – assimilation of tweens (20, 40 and 60) and precipitation of Tween 80. E2 – assimilation of all four tweens. E3 - All tweens not assimilated

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Table 1: Phenotypic speciation of Malassezia

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Antifungal susceptibility pattern

The antifungal susceptibility pattern to the oral and systemic azoles is summarised in [Table 2]. ITR (0.125–1 μg/ml) and VOR (0.25–2 μg/ml) showed the lowest MIC values among oral drugs. FLU, the commonly used oral drug for Malassezia infection, showed resistant pattern in our study with high MIC values (16–64 μg/ml). Among the topical drugs, CLO (0.03–0.5 μg/ml) showed the lowest MIC values compared to MCZ, SER and LUL. M. sympodialis showed a sensitive pattern to most of the drugs used in contrast to M. furfur which showed higher MIC values among the species isolated.
Table 2: Minimum inhibitory concentration (50% reduction of growth compared to growth control well MIC50 and reduction of 90% growth compared to growth control well MIC90) values of oral and topical antifungal agents

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 ~ Discussion Top


In our study for a period of 2 years in dermatological patients, we found that 36% of people affected were adolescents (20–30 years). This was similar to many studies done,[5] but there is also a report showing the highest prevalence from the third to fourth decade. Adolescants are commonly affected because hormonal changes and increase in sebum secrection occurs in this age group. Malassezia are lipophilic organism that requires lipids for their growth as they cannot synthesis their own. They have the ability to split the lipids in the sebum and utilise it for their growth.

The incidence of Malassezia showed little female predominance (52%) than male (48%). Extensive studies have shown variable male-to-female ratios, but they appear to be nearly equal. Since South India is a tropical country, hormonal changes, sebum secretion, high precipitating work and easy access to hospital could have attributed females to be little predominant in our study compared to males. Since our study was done in a coastal area in South India, high humidity caused increased sweating.[6] Sweat retention due to high moisture content in the summer season aggravates and facilitates the rapid growth of these fungi resulting in high incidences. We also reported an increased incidence of cases during summer for the same reason.

Malassezia cause various skin lesions, among which PV is the most common and worldwide in occurrence. Majority of the patients in our study also suffered from PV in par with other studies.[7]

Hypopigmented lesions were seen in 76% and hyperpigmented lesions in 24% of patients. Other studies from India also showed predominance of hypopigmented lesions in PV.[8] The very nature of PV to cause hypopigmented in dark-skinned individuals and hyperpigmented in fair-skinned individual was disproved by Aljabre et al. Studies on pigmentary changes in patients showed that there is no correlation between pigment variation and the type of skin, sex and age of patient and site of lesion. The pathogenesis behind hypopigmentation caused by this fungus was due to the production of dicarboxylic acids, azelaic acid. This acid acts through competitive inhibition of DOPA tyrosinase and has a direct cytotoxic effect on hyperactive melanocytes. The pathogenesis behind hyperpigmentation is not fully understood but could be due to increased thickness of the keratin layer along with pronounced inflammatory cell infiltrate in these individuals which act as a stimulus for the melanocytes.

Distribution of PV lesions on different sites of the body showed that back (34%) was the most common site involved, followed by neck and shoulder (22%), scalp (20%), axilla (7%), face (7%), hip and inner thigh (5%). Some patients showed involvement of two or more sites, neck, arm and axilla (10%). Similar findings were also noted by many studies.[9] Some studies have shown chest and trunk being higher because of the distribution of sebaceous glands.[10] These areas are covered with clothes favouring the development of lesions, according to the theory that occlusion of glands plays a role in this disease. The recurrence of PV lesions could be attributed to the idiosyncratic nature of the condition and the fact that local factors such as humidity and high temperature remain unchanged.

Among the 151 samples, 95 (62%) yielded growth on both SDA with olive oil and MDA. None of them showed any growth on SDA without olive oil. The growth on SDA with olive oil appeared after 8–10 days of incubation and on MDA within 5 days of incubation. The isolation rate of Malassezia species in patients with PV in the current study is 70%, which is comparable to other studies.

Among the 95 samples grown, we found that the predominant species was M. sympodialis – 33 (34%), followed by M. globosa – 30 (31%), M. furfur – 21 (23%) and M. obtusa – 4 (4%). The distribution of Malassezia species varies with different geographical locations. Few studies done in India also showed[11]M. sympodialis to be the most common species similar to our result. In contrast, studies from North-Central India[9] showed that 54% of isolates belonged to M. globosa. The next frequent species isolated in our study was M. globosa almost equal in incidence with M. sympodialis. M. furfur is responsible for PV, especially in tropical countries.[9] Studies have reported M. furfur to be the second most frequent species isolated from PV lesions. However, in the present study, it is the third most common agent.

Antifungal susceptibility

The susceptibility pattern of Malassezia was done either by agar[11] or broth dilution methods.[12] There was no standardised method of susceptibility for these lipid dependant organism which allowed for the integration of different assays.[13] Molecular method of identification revolutionised the taxonomy of Malassezia by adding more species to this genus. As of late 17 species of genus Malassezia is known. Contrary to it, susceptibility pattern of Malassezia species were published even before the recognition of these new species identified by molecular methods. This questioned the AFST studies made from the isolates identified by conventional method that failed to differentiate all species of Malassezia.

The major drawback in AFST of Malassezia was its growth requirement. Hence, we supplemented RPMI 1640 with malt extract, ox bile and tween.[14] The four Malassezia species tested gave suitable growth and the supplementations added did not interfere with the readings since only minimal quantities were added.[4]

Another controversy was that the reading of MIC values was species dependant in many studies. In some studies, they incubated for M. furfur isolates for 48 h and other species for 72 h.[15] In another, they incubated M. globosa for 96 h because they were slow growers. In our study, we used an inoculum size (0.5–2.5 × 105 CFU/ml) larger than in CLSI M27-A3 in order to achieve uniform reading times for all four species tested. The different incubation times could be due to the different sizes of final inoculum. Hence, we increased the inoculum size and found that all four species provided suitable growth at 72 h of incubation.

The MIC working breakpoints for Malassezia species and the correlation betweenin vitro andin vivo results have not been established for any antifungal agent. In our study, we used the breakpoints of CLSI M27-A3 and species-specific clinical breakpoints CLSI M27-S4 supplement for species of Candida. The oral antifungal drugs tested were FLU, ITR and VOR.

FLU showed resistance pattern with high MIC (16-64 μg/ml) values similar to many studies done.[16],[17] We categorised them as resistant when the MIC value is ≥8 μg/ml (CLSI M27-A3). In contrary, studies with similar methodology have shown low MIC values also,[4],[15] but it was for M. furfur while species such as M. sympodialis and M. globosa showed high MIC values.[15] In our study also, we saw that M. furfur showed high MIC values (16–64 μg/ml) than M. globosa and M. obtuse (16–32 μg/ml). The high MIC values and wide MIC ranges obtained from many studies make it not to choose this drug for treating infections caused by Malassezia. Almost all our patients were treated with oral FLU which could be the reason of treatment failure and recurrence. The high MIC values against Malassezia isolates emphasise the importance of performingin vitro susceptibility testing for these yeasts.

ITR and VOR showed low MIC values against all four species, but VOR had MIC values higher than those obtained for ITR.[16] The MIC values of VOR were between 0.25 and 2 μg/ml. According to M27-S4 supplemented, the MIC value for VOR of ≥0.25 μg/ml is considered as susceptible dose-dependent strains for Candida species. These breakpoints are not applicable for Malassezia species, but our study results also showed MICs of ≥ 0.25 μg/ml for VOR. This further added importance to set clinical susceptibility breakpoints for this genus. ITR with low MIC values for all four species was concluded as the best oral antifungal agent to treat infections caused by Malassezia in our study group.

Among the topical drugs used, most of our patients were treated with MCZ. Reports on effectiveness of this drug against Malassezia are limited. We did obtain resistance to MCZ among M. furfur isolates but not so with other three species. Similar results were obtained in some studies[17],[18] questioning the effectiveness of this drug against Malassezia. Therefore, it is important to obtain more information about its activity.

The other topical acting azoles tested showed CLO with the lowest MIC values (0.03 μg/ml) compared to other two recent azoles tested – SER (0.125–1 μg/ml) and LUL (0.125–1 μg/ml). Although both recent azoles showed effectiveness in treatment of Malassezia infection, their MIC values were not as low as for CLO and also the cost and availability of the drug comes to play while choosing the best drug. Only few studies were carried out using recent drugs SER[19] and LUL.

Hence, we concluded that ITR was the most active oral drug and CLO is the most active topical drug against Malassezia in our population group, with low MIC values and limited variation in susceptibility among all four isolates. Similar results were obtained by others under comparable conditions[15] or using other methods. M. sympodialis is considered one of the most susceptible species to antifungal drugs. Variations in susceptibility pattern among the species M. furfur, M. sympodialis, M. globosa and M. obtusa emphasise the need to identify the species and to do the antifungal susceptibility profile before choosing the drug to treat the infection caused by major pathogenic Malassezia species.


 ~ Conclusion Top


The speciation and antifungal susceptibility will go a long way in successful treatment of Malassezia species and relieve the patient of chronicity and compliance problems.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 ~ References Top

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Guého E, Midgley G, Guillot J. The genus Malassezia with description of four new species. Antonie Van Leeuwenhoek 1996;69:337-55.  Back to cited text no. 1
    
2.
Mayser P, Haze P, Papavassilis C, Pickel M, Gruender K, Guého E. Differentiation of Malassezia species: Selectivity of cremophor EL, castor oil and ricinoleic acid for M. furfur. Br J Dermatol 1997;137:208-13.  Back to cited text no. 2
    
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Guillot J. Identification of Malassezia species; a practical approach. J Mycol Med 1996;6:103-10.  Back to cited text no. 3
    
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Rojas FD, Sosa MD, Mariana SF, Cattana ME, Córdoba SB, Giusiano GE. “Anti-fungal susceptibility of Malassezia to azole drugs and amphotericin – B using broth microdilution method”. Med Mycol 2014;52:641-6.  Back to cited text no. 4
    
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Tarazooie B, Kordbacheh P, Zaini F, Zomorodian K, Saadat F, Zeraati H, et al. Study of the distribution of Malassezia species in patients with pityriasis versicolor and healthy individuals in Tehran, Iran. BMC Dermatol 2004;4:5.  Back to cited text no. 5
    
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Sharma R, Sharma G, Sharma M. Additive and inhibitory effect of antifungal activity of Curcuma longa (Turmeric) and Zingiber officinale (Ginger) essential oils against Pityriasis versicolor infections. J Medicinal Plants Res 2011;5:6987-90.  Back to cited text no. 6
    
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Archana BR, Beena PM, Kumar S. Study of the distribution of Malassezia species in patients with pityriasis versicolor in Kolar region, Karnataka. Indian J Dermatol 2015;60:321.  Back to cited text no. 7
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Chaudhary R, Singh S, Banerjee T, Tilak R. Prevalence of different Malassezia species in pityriasis versicolor in central India. Indian J Dermatol Venereol Leprol 2010;76:159-64.  Back to cited text no. 8
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Dutta S, Bajaj AK, Basu S, Dikshit A. Pityriasis versicolor: Socioeconomic and clinico-mycologic study in India. International J Dermatol 2002;41:823-4.  Back to cited text no. 9
    
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Giusiano G, Sosa Mde L, Rojas F, Vanacore ST, Mangiaterra M. Prevalence of Malassezia species in pityriasis versicolor lesions in northeast Argentina. Rev Iberoam Micol 2010;27:71-4.  Back to cited text no. 10
    
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Gupta AK, Kohli Y, Li A, Faergemann J, Summerbell RC.In vitro susceptibility of the seven Malassezia species to ketoconazole, voriconazole, itraconazole and terbinafine. Br J Dermatol 2000;142:758-65.  Back to cited text no. 11
    
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Hammer KA, Carson CF, Riley TV.In vitro activities of ketoconazole, econazole, miconazole, and Melaleuca alternifolia (tea tree) oil against Malassezia species. Antimicrob Agents Chemother 2000;44:467-9.  Back to cited text no. 12
    
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Hald M, Arendrup MC, Svejgaard EL, Lindskov R, Foged EK, Saunte DM, et al. Evidence-based Danish guidelines for the treatment of Malassezia-related skin diseases. Acta Derm Venereol 2015;95:12-9.  Back to cited text no. 13
    
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Nakamura Y, Kano R, Murai T, Watanabe S, Hasegawa A. Susceptibility testing of Malassezia species using the urea broth microdilution method. Antimicrob Agents Chemother 2000;44:2185-6.  Back to cited text no. 14
    
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Velegraki A, Cafarchia C, Gaitanis G, Iatta R, Boekhout T. Malassezia infections in humans and animals: Pathophysiology, detection, and treatment. PLoS Pathog 2015;11:e1004523.  Back to cited text no. 15
    
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Garau M, Pereiro M Jr, del Palacio A.In vitro susceptibilities of Malassezia species to a new triazole, albaconazole (UR-9825), and other antifungal compounds. Antimicrob Agents Chemother 2003;47:2342-4.  Back to cited text no. 16
    
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Carrillo-Muñoz AJ, Rojas F, Tur-Tur C, de Los Ángeles Sosa M, Diez GO, Espada CM, et al.In vitro antifungal activity of topical and systemic antifungal drugs against Malassezia species. Mycoses 2013;56:571-5.  Back to cited text no. 17
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2]



 

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