Malassezia infections: experimental answers for a medical conundrum

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ALASSEZIA INFECTIONS

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EXPERIMENTAL ANSWERS FOR

A MEDICAL CONUNDRUM

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Dissertação de candidatura ao grau de Doutor em Medicina apresentada à Faculdade de Medicina da Universidade do Porto Programa Doutoral em Medicina

O presente estudo decorreu no Serviço de Microbiologia da Faculdade de Medicina da Universidade do Porto e no Serviço de Dermatologia e Venereologia do Centro Hospitalar Universitário de São João EPE, Porto.

Orientação

Professor Doutor Acácio Agostinho Gonçalves Rodrigues

Co-orientação

Professora Doutora Carmen Maria Lisboa da Silva

Júri da Prova de Doutoramento em Medicina

Presidente: Professora Doutora Maria Leonor Martins Soares David, professora catedrática da Faculdade

de Medicina da Universidade do Porto.

Vogais

Professora Doutora Lidia Rudnicka, professor and chairman of the Medical University of Warsow, Poland, Department of Dermatology;

Professor Doutor Américo Manuel Costa Figueiredo, professor catedrático de Dermatologia e Venereologia da Faculdade de Medicina da Universidade de Coimbra;

Professora Doutora Teresa Maria Fonseca de Oliveira Gonçalves, professora associada do Instituto de Microbiologia da Faculdade de Medicina da Universidade de Coimbra;

Professor Doutor Acácio Agostinho Gonçalves Rodrigues, professor associado da Faculdade de Medicina da Universidade do Porto e orientador da tese;

Professora Doutora Cândida Manuela Ferreira Abreu, professora auxiliar da Faculdade de Medicina da Universidade do Porto;

Professor Doutor Luís Filipe Duarte Reino Cobrado; professor auxiliar da Faculdade de Medicina da Universidade do Porto.

Apoio financeiro da European Academy of Dermatology and Venereology (EADV)

PPRC-2017-7

ACKNOWLEDGMENTS /AGRADECIMENTOS

Agradeço ao meu orientador, Professor Doutor Acácio Gonçalves Rodrigues, por todo o apoio e motivação prestados durante o desenvolvimento da presente tese de doutoramento. Sem a sua valiosa ajuda e orientação este trabalho não seria possível desde o planeamento dos projetos de investigação até à sua execução e revisão crítica, a participação do Professor foi sempre oportuna e louvável. Foi também fundamental na indução de motivação e fortalecimento pessoais para levar a cabo este trabalho ao longo do tempo.

À minha co-orientadora, Professora Doutora Carmen Lisboa, muito agradeço toda a disponibilidade demonstrada ao longo destes anos, a ajuda imprescindível na organização, execução e correção dos trabalhos de investigação. A sua calma, paciência e perspicácia em inúmeras circunstâncias transmitiram uma serenidade capaz de fazer interpretar a ciência com um brilho no olhar de quem não perde a curiosidade de procurar sempre fazer mais e melhor. A nível pessoal, é inegável o carinho e amizade com que sempre me presenteou, que muito agradeço.

À Professora Doutora Isabel Miranda, agradeço a disponibilidade e ajuda no desenho e execução de múltiplas experiências científicas no decorrer do desenvolvimento desta tese de doutoramento. O seu conhecimento científico e noções práticas nos ensaios laboratoriais, nomeadamente na área da biologia molecular foram cruciais para o desenrolar dos trabalhos. A sua capacidade de otimizar a metodologia e enfrentar criticamente os resultados foi valiosa, para além da sua palavra amiga e motivacional.

À Dra. Filomena Azevedo, diretora do Serviço de Dermatologia e Venereologia do Centro

Hospitalar Universitário de São João EPE, Porto muito agradeço a possibilidade de ter realizado neste

Serviço o recrutamento de voluntários para a execução desta tese de doutoramento e a sua compreensão e apoio à investigação.

À Doutora Isabel Ramos, investigadora, agradeço o apoio na realização dos trabalhos laboratoriais, o seu rigor e metodologia científicas que muito me ajudaram na execução deste projeto, bem como a sua amizade e força motivacional.

À Mestre Joana Branco, investigadora e estudante de doutoramento no Serviço de Microbiologia, agradeço todo o apoio e disponibilidade demonstradas. Foi sempre incansável na procura das melhores soluções para levar a cabo os objetivos propostos.

Ao Paulo agradeço todos os dias o caminho que traçamos junto dos meninos, Dinis e Nuno, para os vermos crescer felizes alicerçados nos valores que cremos serem os melhores. A sua ajuda foi fundamental para a realização desta tese, não só com o seu espírito crítico, mas também com todo o apoio dado para prosseguir com os trabalhos, mesmo nas alturas mais difíceis, quando o ânimo enfraquece, esteve sempre pronto para contribuir para que tudo corresse pelo melhor. A sua força e o sorriso dos meninos foram a minha principal fonte de inspiração.

Aos meus Pais, Manuel e Maria do Céu, agradeço profundamente por tudo o que me ensinaram, por me motivarem a seguir os meus sonhos e por me apoiarem na sua concretização. Muito obrigada por estarem sempre presentes e dispostos a ajudar.

Ao Doutor Christian Pellevoisin, diretor científico da Episkin Academy, muito agradeço a colaboração que permitiu desenvolver um projeto de investigação que em muito valorizou a presente tese de doutoramento. A sua disponibilidade e a valorização do nosso trabalho foram muito importantes.

À Doutora Rita Teixeira, investigadora do Serviço de Microbiologia, agradeço a ajuda no tratamento estatístico dos dados e colaboração na análise crítica dos resultados. À Doutora Elisabete Ricardo e à técnica Isabel Santos agradeço toda a assistência técnica prestada a nível laboratorial no Serviço de Microbiologia da Faculdade de Medicina da Universidade do Porto. A todos os restantes elementos do Serviço de Microbiologia da Faculdade de Medicina da Universidade do Porto, nomeadamente à Professora Doutora Cidália Pina-Vaz, ao Professor Doutor Luís Cobrado, à Professora Doutora Sofia Costa, às Doutoras Ana Teresa Silva e Ana Isabel Dias agradeço a simpatia e hospitalidade com que me acolheram neste Serviço e a ajuda na discussão crítica de resultados. À D. Marta Garcês agradeço a ajuda e disponibilidade para a resolução de questões administrativas.

Agradeço à Professora Doutora Raquel Silva, investigadora da Universidade de Aveiro, o seu empenho, dedicação e apoio no estudo e identificação dos isolados clínicos.

Às Mestres Cláudia Mendes e Ana Almeida e à Drª Daniela Silva agradeço a ajuda na preparação, obtenção e análise das técnicas de imagem utilizadas para avaliação da formação de biofilmes.

À Academia Europeia de Dermatologia e Venereologia, agradeço o apoio com o financiamento do projeto que permitiu a execução de grande parte do trabalho investigacional.

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LIST OF PUBLICATIONS 13

Manuscripts 13

Abstracts from international scientific congresses 14

LIST OF ABBREVIATIONS 15

LIST OF TABLES AND FIGURES 17

List of Tables 17 List of Figures 18 ABSTRACT 21 RESUMO 23 INTRODUCTION 25 General considerations 27

Malassezia-related skin diseases 28

Pityriasis versicolor 29

Malassezia folliculitis 30

Seborrheic dermatitis and dandruff 30

Atopic dermatitis 31

Epidemiology 32

Mechanisms of pathogenicity 34

Isolation and identification methods 36

Antifungal susceptibility profile 40

Treatment 41

Prognosis and prevention 43

A medical conundrum 43

OBJECTIVES AND OUTLINE OF THE THESIS 45

Objectives 47

CHAPTER-1 COMPARISON OF CONTACT PLATE VERSUS ADHESIVE TAPE IN THE

RECOVERY OF MALASSEZIA SPECIES 49

Summary 51

Background 51

Material and methods 51

Results 52

Discussion 54

CHAPTER-2 SPECIES DISTRIBUTION AND SUSCEPTIBILITY PROFILE OF MALASSEZIA ISOLATES TO CLASSIC ANTIFUNGALS AND OVER-THE-COUNTER PRODUCTS 57

Summary 59

Background 60

Material and methods 60

Data collection and skin sampling 60

Yeast isolation and speciation 61

Antifungal susceptibility profile assessment 62

Data analysis 63

Results 63

Discussion 67

CHAPTER-3 MALASSEZIA SYSTEMIC INFECTIONS: REPORT OF THREE CASES

OCCURRING AT A PORTUGUESE UNIVERSITY HOSPITAL 71

Summary 73

Background 73

Material and methods 74

Results 74

Epidemiology 74

Pathogenesis 76

Diagnosis 77

Susceptibility profile and treatment 77

CHAPTER-4 MALASSEZIA INTERACTION WITH A RECONSTRUCTED HUMAN

EPIDERMIS: BIOFILM IMAGING AND KERATINOCYTE RESPONSE 81

Summary 83

Background 84

Material and methods 86

Yeast isolation and identification 86

Growth conditions 86

Biofilm development on RhE 86

Histological imaging by light microscopy 87

Wide-field fluorescence microscopy analysis 87 Scanning electron microscopy (SEM) imaging 88

RNA extraction and cDNA synthesis 88

Real-time quantitative polymerase chain reaction (RT-qPCR) 89

Lactate dehydrogenase (LDH) activity 90

Statistical analysis 90 Results 90 Pilot study 90 Biofilm imaging 92 Gene expression 96 LDH analysis 99 Discussion 99

CONCLUDING REMARKS AND FUTURE PERSPECTIVES 103

Concluding Remarks 106 Future Perspectives 109 REFERENCES 111 PUBLICATIONS 127 Publication I 129 Publication II 139 Publication III 143 Publication IV 153 Publication V 161 Publication VI 179 Abstracts 187

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LIST

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PUBLICATIONS

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I. Pedrosa AF, Lisboa C, Rodrigues AG. Malassezia infections: a medical conundrum. J Am Acad

Dermatol 2014;71(1):170-6.

II. Pedrosa AF, Lisboa C, Faria-Ramos I, Silva RM, Miranda IM, Rodrigues AG. Malassezia

species retrieved from skin with pityriasis versicolor, seborrhoeic dermatitis and skin free of lesions: a comparison of two sampling methods. Br J Dermatol 2018;179(2):526-7.

III. Pedrosa AF, Lisboa C, Faria-Ramos I, Silva RM, Ricardo E, Teixeira-Santos R, Miranda IM,

Rodrigues AG. Epidemiology and susceptibility profile to classic antifungals and over-the-counter products of Malassezia clinical isolates from a Portuguese University Hospital: a prospective study. J Med Microbiol 2019; 68(5):778-84.

IV. Pedrosa AF, Lisboa C, Rodrigues AG. Malassezia infections with systemic involvement:

figures and facts. J Dermatol 2018;45(11):1278-1282.

V. Pedrosa AF, Lisboa C, Branco J, Almeida AC, Mendes CS, Pellevoisin C, Leite-Moreira AF,

Miranda IM, Rodrigues AG. Malassezia interaction with a reconstructed human epidermis: imaging studies. Revised manuscript submitted to Mycoses.

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VI. Pedrosa AF, Lisboa C, Branco J, Pellevoisin C, Miranda IM, Rodrigues AG. Malassezia

interaction with a reconstructed human epidermis: keratinocyte immune response. Mycoses 2019. doi: 10.1111/myc.12965 [in press]

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A1. Pedrosa AF, Lisboa C, Faria-Ramos I, Silva RM, Teixeira-Santos R, Miranda IM, Rodrigues AG.

Malassezia recovered from skin with pityriasis versicolor, seborrheic dermatitis and skin free of lesions:

do previous exposures impact upon yeast recovery and its antifungal susceptibility profile? Presented at the 27th _{European Academy of Dermatology and Venereology Congress 2018, Paris, France. P2143.}

A2. Pedrosa AF, Lisboa C, Branco J, Mendes CS, Pellevoisin C, Miranda IM, Rodrigues AG.

Malassezia clinical isolates behavior on a reconstructed human epidermis. Presented at the 24th _World Congress of Dermatology in June 2019, Milan, Italy.

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LIST

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ABBREVIATIONS

AFLP Amplified Fragment Length Polymorphism AIDS Acquired Immune Deficiency Syndrome AMB Amphotericin B

AMP Antimicrobial Peptides

ARDS Acute Respiratory Distress Syndrome AT Anti-tuberculosis

cDNA Complementary Deoxyribonucleic Acid CHT Chemotherapy

CI Confidence Interval CICLO Ciclopirox Olamine CLIM Climbazole CLO Clotrimazole

CLSI Clinical and Laboratory Standards Institute CVC Central Venous Catheter

DGGE Denaturing Gradient Gel Electrophoresis DNA Deoxyribonucleic Acid

ECM Extracellular Matrix

EDTA Ethylenediamine Tetraacetic Acid EU European Union

FITC-ConA Fluorescein Isothiocyanate-Concanavalin A FLU Fluconazole

HAART Highly Active Antiretroviral Therapy HBD Human β Defensin

H&E Hematoxylin and Eosin

HIV Human Immunodeficiency Virus IGS Intergenic Spacer

IL Interleukin

ITS Internal Transcribed Spacer ITZ Itraconazole

l-AMB Amphotericin B Liposomal LDH Lactate Dehydrogenase LNA Leeming-Notman Agar

- 16 - LSU Large Subunit Region

MALDI-TOF MS Matrix Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry

MIC Minimal Inhibitory Concentration mRNA Messenger Ribonucleic Acid

NCBI Nacional Center for Biotechnology Information NK Natural Killer

NY New York

OD Optical Density

OR Odds Ratio

PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction PIRO Piroctone Olamine

POS Posaconazole PV Pityriasis Versicolor

RAPD Random Amplified of Polymorphic DNA RFLP Restriction Fragment Length Polymorphism RhE Reconstructed Human Epidermis

RPMI Roswell Park Memorial Institute rRNA Ribosomal Ribonucleic Acid

RT-qPCR Real-Time quantitative Polymerase Chain Reaction SD Seborrheic Dermatitis

SEM Scanning Electron Microscopy

SIRS Systemic Inflammatory Response Syndrome SPSS Statistical Package for Social Sciences TER Terbinafine

tFLP Terminal Fragment Length Polymorphism TGF Transforming Growth Factor

TNF Tumor Necrosis Factor UK United Kingdom USA United States of America VCZ Voriconazole

WFFM Wide-Field Fluorescence Microscopy YPD Yeast Peptone Dextrose

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LIST

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TABLES

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FIGURES

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INTRODUCTION

Table 1. Distribution of Malassezia species recovered from healthy skin, cutaneous and systemic

conditions, according to different studies.

Table 2. Distinct sampling and diagnostic procedures methods and corresponding Malassezia species.

CHAPTER 1

Table 1. Recovery of Malassezia species from pityriasis versicolor and seborrheic dermatitis patients, and

from controls without skin lesions, according to the sampling method.

CHAPTER 2

Table 1. Distribution of the Malassezia species according to the source of the isolates.

Table 2. Susceptibility profile of Malassezia clinical isolates to antifungal drugs.

Table 3. Susceptibility profile of Malassezia clinical isolates to non-antifungal agents.

CHAPTER 3

Table 1. Clinical features of patients with Malassezia furfur systemic infections.

CHAPTER 4

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Figure 1. Clinical aspect of pityriasis versicolor. a) hypopigmented macules and patches on the torso; b)

hyperpigmented and erythematous macules and patches on the pre-sternal area.

Figure 2. Clinical aspect of Malassezia folliculitis with monomorphic papules and pustules on the torso.

Figure 3. Clinical aspect of seborrheic dermatitis. a) Erythematous and scaly patches on the eyebrows,

and b) on the pre-sternal area.

Figure 4. Macro and microscopic morphologic aspect of Malassezia furfur Culti-Loops® _{ST 8036 type}

strain. Macroscopic aspect of colonies grown on CHROMagar Malassezia® _{medium and microscopic} aspect of the yeasts after being stained with methylene blue.

CHAPTER 1

Figure 1. Colonies of Malassezia sympodialis retrieved from skin with pityriasis versicolor using two

distinct sampling methods: (a) contact plate and (b) adhesive tape placed at the surface of the chromogenic culture medium.

CHAPTER 2

Figure 1. Odds ratio (OR) for minimal inhibitory concentration (MIC) in mg/L of distinct antifungals

against Malassezia isolates with a confidence interval (CI) of 95%; referring to participants exposed during the previous year to (a) topical corticosteroids and to (b) topical selenium disulphide shampoo.

CHAPTER 4

Figure 1. Malassezia sympodialis yeast aggregates at the surface of SkinEthicTM _{RHE small (aged 17}

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Figure 2. Representative SEM images at 24 hours of incubation. (a) Malassezia sympodialis and (b)

Malassezia furfur yeast aggregates at the surface of the SkinEthicTM _{RHE small (aged 17 days) exhibiting} rudimental extracellular matrix formation.

Figure 3. Light microscopy (H&E) images of the RhE colonized with (A) M. furfur and (B) M.

sympodialis at 96 hours of incubation; both unveiling yeast aggregates on the top of the stratum corneum

clustered in an eosinophilic amorphous structure.

Figure 4. Representative images of wide-field fluorescence microscopy obtained following 48, 72 and 96

hours of incubation of M. sympodialis and M. furfur on RhE. Calcofluor white staining is represented in blue and FITC-ConA staining in red. Scale bar: 20µm.

Figure 5. SEM images obtained, at the same magnification (20000x), with yeast suspension in PBS.

Figure 6. SEM images obtained with yeast suspension in modified RPMI medium. M. furfur biofilm

exhibiting a thicker ECM encasing the yeasts.

Figure 7. Progression of M. furfur aggregate formation overtime and accordingly to the medium of the

yeast suspension: (a,b,c) M. furfur suspended in modified RPMI medium, and (d,e,f) suspended in PBS (magnification 400x).

Figure 8. Energy dispersive spectroscopy (EDS) of the (A) extracellular matrix surface and of the (B)

yeast surface unveiling a distinct pattern of elemental constituents. C: carbon; N: nitrogen; O: oxygen; Na: sodium; Os: osmium tetroxide; S: sulphur; Cl: chlorine; Ca: calcium.

Figure 9. SEM images representative of M. furfur and M. sympodialis biofilm formed at the surface of

the RhE following 96 hours of incubation; (a,b) yeast suspension in PBS (magnification 10000x) and in (c,d) modified RPMI medium (magnification 20000x).

Figure 10. Variation of gene expression in the RhE incubated with M. sympodialis and M. furfur

following 6, 24 and 48 hours. Each bar represents the mean value of gene expression ± standard deviation from 3 independent experiments. Each experiment was run in triplicate. Values represent fold-change in gene expression relative to the cells of RhE incubated without yeasts. * P < 0.05.

Figure 11. Light microscopy (hematoxylin and eosin) of the SkinEthicTM _{RHE small colonized with M.}

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ABSTRACT

Malassezia yeasts are lipophilic organisms members of the healthy cutaneous microbiome

despite being often involved in numerous skin diseases, such as pityriasis versicolor and seborrheic dermatitis.

Our project started by reviewing the literature about Malassezia-related skin diseases focusing on clinical manifestations, epidemiology, pathogenesis, sampling and diagnostic methods, antifungal susceptibility testing, treatment, prognosis and prevention.

After such revision we were prompt to explore Malassezia species involvement in skin diseases, namely in pityriasis versicolor and seborrheic dermatitis, and in healthy skin through the development of a prospective study. A total of 182 volunteers were enrolled, 86 Malassezia isolates were retrieved by culture. Two methods of sampling were used (contact plate and adhesive tape) in each half of the participants, based on the assumption that adhesive tape might recover more yeasts deeply located in the stratum corneum. Actually, we have concluded that contact plate sampling seems to be a more convenient and easier method to collect skin samples for culture.

The following study aimed to provide insights whether the Malassezia isolates recovered from patients with pityriasis versicolor, seborrheic dermatitis and healthy volunteers (control group), recruited at the clinical grounds, differed in recovery rate and species distribution. We have found out that the recovery rate of Malassezia yeasts was similar among the three study groups and that M. sympodialis predominated as the most frequent species. It is known that Malassezia species distribution varied accordingly to the studies from distinct geographic regions. From this study important clinical and demographic data were obtained as well as information regarding previous treatment. These results, together with the susceptibility profile of clinical isolates to antifungal and non-antifungal agents led us to conclude that previous topical corticotherapy associated to a significant increase of the minimal inhibitory concentration values of fluconazole and terbinafine against Malassezia species. Furthermore, terbinafine exhibited in vitro rather low minimal inhibitory concentration values against Malassezia organisms, a fact that may deserve attention when selecting alternative drugs to azoles, the latter being frequently used in long-term preventive therapeutic regimens.

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Considering the ability of Malassezia organisms to colonize the skin and eventually to invade, causing systemic bloodstream infection in immune suppressed patients, we have reviewed the cases of

Malassezia systemic infection occurring at our University Hospital during a 2-year period. Three

unrelated cases of Malassezia furfur bloodstream infection were reported; one afflicting a child with a congenital immunodeficiency and the other two involving adult severely immunosuppressed patients. The isolated species in all three cases, M. furfur, was in accordance with most studies reporting such invasive infections. Malassezia systemic infections, although rare, exhibit a high mortality rate associated, in part, to the underlying predisposing conditions and, presumably, also related to an erratic response to the commonly prescribed antifungal drugs.

The subsequent work involved the use of a reconstructed human epidermis, recreating in part epidermal skin in vivo conditions. The interaction of Malassezia clinical isolates (one selected strain each of M. synpodialis and M. furfur) with this substrate was assessed. Herein, we have shown by imaging techniques that the abovementioned yeasts were capable of producing a variable amount of biofilm, with an intricate architecture at the epidermal surface, which to the best of our knowledge had not yet been demonstrated. We have also detected differences in the biofilm structure between the two tested

Malassezia species.

Based on the ability of keratinocytes to initiate an immediate immune response whenever in contact with microorganisms and given that the biomarker profile induced by Malassezia yeasts is still under investigation, we have used this reconstructed human epidermis to assess the expression of cytokines and antimicrobial peptides following exposure to Malassezia organisms. We have noticed a distinct expression profile evinced by the epidermal model after contact with clinical isolates of M.

sympodialis and of M. furfur. Moreover, no significant expression and often even downregulation of

pro-inflammatory cytokines and antimicrobial peptides was found following 24 and 48 hours of incubation, highlighting the prompt keratinocyte response against Malassezia with subsequent tolerance to the yeast presence.

This dissertation highlights the complex interplay between Malassezia yeasts with its virulence factors and the host susceptibility which seems to be crucial for disease development.

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RESUMO

Malassezia são leveduras lipofílicas, membros do microbioma cutâneo, embora estejam

implicadas em inúmeras patologias cutâneas, tais como a pitiríase versicolor e a dermite seborreica.

Inicialmente procedemos à revisão da literatura sobre infeções cutâneas relacionadas com

Malassezia com particular enfoque nas manifestações clínicas, epidemiologia, patogénese, métodos de

amostragem e diagnóstico, avaliação de suscetibilidade antifúngica, tratamento, prognóstico e prevenção. Em seguida tivemos como objetivo explorar o envolvimento das espécies de Malassezia em doenças cutâneas, nomeadamente na pitiríase versicolor e dermite seborreica, e na pele saudável, através do desenvolvimento de um estudo prospetivo. Foi incluído um total de 182 voluntários o que levou à recuperação por cultura de 86 isolados de Malassezia. Foram utilizados dois métodos de amostragem (placa de contacto e fita adesiva) em cada metade dos participantes, baseados na premissa de que a fita adesiva seria capaz de recuperar mais leveduras localizadas profundamente no estrato córneo. De facto, concluímos que a amostragem por placa de contacto revelou-se um método mais conveniente e fácil para obter amostras cutâneas para cultura.

Seguidamente, pretendeu-se caracterizar os isolados de Malassezia recuperados de doentes com pitiríase versicolor e dermite seborreica e de voluntários saudáveis (grupo controlo), recrutados a nível hospitalar, relativamente a taxa de isolamento e distribuição das espécies. A taxa de isolamento de leveduras de Malassezia foi sobreponível nos três grupos em avaliação e M. sympodialis foi a espécie predominante. A distribuição das espécies de Malassezia varia de acordo com estudos provenientes de regiões geográficas distintas. A partir deste estudo foi possível obter importantes dados clínicos e demográficos, assim como informação relativamente a tratamentos prévios. Estes resultados em associação com o perfil de suscetibilidade dos isolados clínicos de Malassezia a agentes antifúngicos e não-antifúngicos permitiram-nos concluir que a terapia tópica prévia com corticoesteróides associou-se significativamente com um aumento dos valores de concentração inibitória mínima do fluconazole e da terbinafina relativamente às espécies de Malassezia. Adicionalmente, a terbinafina demonstrou in vitro valores relativamente reduzidos de concentração inibitória minima contra Malassezia, o que pode merecer especial atenção na seleção de fármacos alternativos aos azoles, estes últimos frequentemente utilizados em regimes terapêuticos de prevenção a longo-prazo.

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Face à capacidade de Malassezia colonizar a pele e de, eventualmente, invadir provocando infeções sistémicas em doentes imunossuprimidos, realizámos uma revisão dos casos de infeção sistémica por Malassezia que ocorreram no Centro Hospitalar Universitário de São João EPE, Porto durante um período de 2 anos. Verificaram-se três casos, não relacionados, de infeção sistémica por Malassezia

furfur; um dos casos correspondeu a uma criança com uma imunodeficiência congénita e os restantes dois

envolveram doentes adultos profundamente imunossuprimidos. A espécie isolada nos três casos, M.

furfur, foi concordante com a maioria dos estudos que reportaram infeções invasivas. Estas infeções

sistémicas por Malassezia, embora raras, resultaram invariavelmente numa elevada taxa de mortalidade, parcialmente associada às condições predisponentes subjacentes e, presumivelmente, também relacionada com uma resposta errática aos fármacos antifúngicos habitualmente prescritos.

O trabalho subsequente foi realizado utilizando epiderme humana reconstruída para recrear, em parte, as condições cutâneas epidérmicas in vivo. A interação dos isolados clínicos de Malassezia (uma estirpe selecionada de M. sympodialis e de M. furfur) com este substrato foi avaliada. Demonstrámos através de técnicas de imagem que as leveduras acima referidas foram capazes de produzir uma quantidade variável de biofilme, com uma arquitetura intrincada na superfície epidérmica, o que, à luz dos nossos conhecimentos, não tinha ainda sido demonstrado. Detetámos, adicionalmente, diferenças na estrutura do biofilme entre as duas espécies de Malassezia testadas.

Tendo em conta a capacidade dos queratinócitos de desencadearem uma resposta imune imediata quando em contacto com microrganismos e dado que o perfil de biomarcadores induzido pelas leveduras de Malassezia está ainda sob investigação, utilizámos a epiderme humana reconstruída para pesquisar a expressão de citocinas e péptidos antimicrobianos após exposição às duas espécies de Malassezia. Evidenciámos um perfil distinto de expressão de citocinas e péptidos antimicrobianos pelo modelo epidérmico após contacto com isolados clínicos de M. sympodialis e M. furfur. Não houve expressão significativa e, frequentemente, ocorreu mesmo regulação negativa de citocinas pró-inflamatórias e péptidos antimicrobianos após 24 e 48 horas de incubação, realçando a resposta imediata dos queratinócitos contra Malassezia com subsequente tolerância à presença da levedura.

Esta dissertação realça a complexa interação entre Malassezia, os seus fatores de virulência e a susceptibilidade do hospedeiro, que parece ser um aspeto crucial para o desenvolvimento de doença

Introduction

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G

Formerly called Pityrosporum, with only three species recognized for a long time – P.

orbiculare, P. ovale and P. pachydermatis – this genus was revised in the 80’s with the acceptance of the

name Malassezia. In 1995, Guillot & Guého [1] included seven species in Malassezia genus, based in molecular analysis (M. furfur, M. obtusa, M. globosa, M. slooffiae, M. sympodialis, M. pachydermatis and M. restricta). Currently, the genus includes seventeen species isolated from humans and animals, of which M. sympodialis, M. globosa, M. restricta, and M. furfur are most commonly associated with humans.[2] With the possible exception of M. pachydermatis, a common feature among Malassezia organisms is the inability to synthesize lipids due to the absence of a fungal fatty acid synthase gene; as a consequence, these organisms mainly colonize sebum rich-areas of the skin such as the scalp, face, and trunk.[3-5]

The members of the genus Malassezia are opportunistic yeasts of increasing medical importance, capable of causing localized dermatologic and systemic diseases in humans, despite being members of the indigenous cutaneous microbial population from the early beginning of life.[5, 6]

The classic skin diseases caused by Malassezia yeasts include pityriasis versicolor (PV) and

Malassezia folliculitis. Malassezia is also known to be involved in seborrheic dermatitis (SD) and

dandruff, due to the facts that antifungals are effective in their treatment and that improvement of such diseases is associated with a reduction in Malassezia levels.[6, 7] Notably, many aspects of the physiopathology of such diseases and the role of specific species still remain to be completely elucidated. Yet under-explained is also the high frequency of these diseases registered among acquired immune deficiency syndrome (AIDS) patients,[7, 8] and also in patients with Parkinson’s disease.[9] Malassezia have been also associated with psoriasis, confluent and reticulate papillomatosis[3, 10] and atopic dermatitis, specifically the head and neck dermatitis subtype.[11, 12]

Invasive infections caused by Malassezia species, namely indwelling catheter-related invasive infections are increasingly reported, notably among immunocompromised hosts and premature infants during lipid infusion administration.[13]

Given the difficulty to isolate and maintain these yeasts in culture in order to proceed with speciation and antimicrobial susceptibility tests, such procedures are most often not performed by routine laboratories and the cutaneous infections are most often treated empirically. The recurring nature of the

Introduction

- 28 -

superficial skin infections and the threat of systemic infections raise the need of faster and more sensitive techniques to achieve identification and assessment of antimicrobial susceptibility profile. A review about the latest available data concerning Malassezia-related skin diseases and recent developments of diagnostic methods, virulence mechanisms and antifungal susceptibility assessment is herein presented.

M

-

The predilection of certain skin disorders for precise anatomical locations of similar biogeography suggests close interactions between the skin and its microbiota, as well as the microbial ability to modify skin health and induce disease.[14]

The roles of microbial causality in disease have historically been defined by Koch’s postulates:[15]

1. The microorganism must be found in all organisms with disease, but not in healthy organisms;

2. The microorganism must be isolated from a diseased organism and grown in pure culture; 3. The cultured microorganism should cause disease when introduced into a healthy

organism;

4. The microorganism must be re-isolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.

Modern methods of isolation, speciation and characterization of microorganisms opened a Pandora’s box of complexity, including, among others, “unculturables”, inter-kingdom interactions, host-microbe communication, and the concept of individual host susceptibility. Fungal infections and opportunism do often complicate Koch’s postulates: Malassezia may be commensal or symbiotic and at other times may cause disease.[14]

Pityriasis versicolor is the only human skin disease in which the causative role of Malassezia is fully established; in other skin diseases, such as Malassezia folliculitis, SD and atopic dermatitis, the pathogenic roles of these yeasts remains less clear, and the transition of yeast cells to their hyphal form

Introduction

- 29 -

cannot be clearly demonstrated.[5, 8] There is currently no conclusive evidence that any given species of

Malassezia is responsible for a specific skin disease.[5]

P

Pityriasis versicolor represents one of the most common human skin diseases and the one wherein the causative relationship with Malassezia yeasts appears more obvious.[7, 16] On biopsy specimens the entire stratum corneum is filled by budding yeasts and short hyphae could be found.[17] The invasion causes a disruption of the structure of the stratum corneum which leads to an increased fragility of the affected skin area.[3] This superficial fungal infection is characterized clinically by hyper and hypopigmented, round to oval, flaking lesions, most commonly found on the trunk and upper aspects of the arms. Most patients are asymptomatic, but these lesions can sometimes be pruritic. It is most commonly found among adolescents and young adults, in whom the sebaceous gland activity is maximal.[6, 7] Clinical examples of pityriasis versicolor lesions are showed in Figure 1.

Figure 1. Clinical aspect of pityriasis versicolor. a) hypopigmented macules and patches on the torso;

Introduction

- 30 -

M

Malassezia folliculitis is an inflammatory skin disorder presenting as erythematous,

monomorphic, follicular papules and pustules on the torso, neck and arms, that may be asymptomatic or pruritic.[7, 18] Predisposing factors include occlusion, sweating, and increases in temperature, as well as immunosuppression, diabetes mellitus, and the use of broad-spectrum antibiotics, which may be responsible for the imbalance between the Malassezia species and the host response, predisposing towards disease occurrence.[19] Diagnosis is based on the clinical picture, microscopy, and a favorable response to antifungal therapy. Histological examination shows the colonization by budding yeasts of hair follicles.[5, 7] A clinical example of Malassezia folliculitis is shown is Figure 2.

Figure 2. Clinical aspect of Malassezia folliculitis with monomorphic papules and pustules on the torso

S

Seborrheic dermatitis and dandruff are common chronic relapsing skin diseases causing flaking and itching, considered by some authors as differing severity manifestations of a common etiology.[7, 20] Dandruff is restricted to the scalp, displaying loosely adherent oily flakes without overt inflammation. In SD inflammation is observed with superimposed greasy and yellowish flakes, affecting predominantly the scalp, nasolabial folds, ears, eyebrows and the chest; most commonly affects male adolescents and young

Introduction

- 31 -

adults (Figure 3).[6, 7] Most evidences support a causal role of Malassezia in SD and dandruff.[16, 21, 22] These conditions are often found in AIDS patients (30–85% compared with 3–5% of the general population);[23] its occurrence was considered to be an early marker of the evolutionary trend of human immunodeficiency virus (HIV) infection.[7, 23] Another medical condition frequently associated with SD and dandruff is Parkinson’s disease.[7, 9, 24]

Figure 3. Clinical aspect of seborrheic dermatitis. a) Erythematous and scaly patches on the eyebrows,

and b) on the pre-sternal area.

A

Atopic dermatitis is a multifactorial skin disease characterized by frequent exacerbations.

Malassezia organisms have been implicated in a particular subtype of atopic dermatitis – the head and

neck dermatitis – with high levels of specific IgE antibodies detected against Malassezia.[11, 25] In addition, it has also been shown that a subset of these patients, recalcitrant to conventional therapy, responded to topical and systemic antifungal agents.[26, 27] A review by Darabi et al.[28] concluded that

Malassezia organisms seem to participate in this disease through the induction of specific antibodies

contributing to a subset of cases of refractory head and neck dermatitis. Interestingly, the rate of recovery of Malassezia organisms from the lesional skin was found to be rather low comparatively to other

Malassezia-related skin diseases, and the prevalence of positive Malassezia cultures did not correlate with

Introduction

- 32 -

E

The distribution of Malassezia species isolated from the skin varies considerably among different medical conditions and also in healthy skin. Such differences in the epidemiology of these yeasts remain to be elucidated. Some authors speculated that the geographical and ethnic origins may affect interactions of the yeast with the skin microenvironment or reflect climatic differences.[32] Interestingly, others proposed that the composition of the Malassezia microbiota might be under the influence of age-related changes in sebaceous gland activity and in the fatty-acid composition of the sebum or even age-related to differences in lifestyle habits of the subjects under study.[5, 33, 34] However, it should be taken into account that these studies employed distinct methods to achieve speciation (culture and non-culture based), which certainly had considerable impact in the species recovered. Table 1 displays an overview of the distribution of different Malassezia species isolated from distinct conditions.

Table 1. Distribution of Malassezia species recovered from healthy skin, cutaneous and systemic

conditions, according to different studies

Condition _{Malassezia species}

_*

Healthy skin M. sympodialis [17, 29, 34, 35] M. globosa [34-36] M. restricta [34, 36, 37] Pityriasis versicolor M. globosa [17, 32, 38] M. sympodialis [39-41] Seborrheic dermatitis M. restricta [17, 21, 42, 43] M. sympodialis [17, 29] M. globosa [17, 30, 44] M. obtusa [29] Atopic dermatitis M. sympodialis [29-31] M. globosa[42, 45] M. furfur [42, 45] M. japonica [42, 46] M. slooffiae [42] M. dermatis [42] Bloodstream infections M. furfur [47-49] M. pachydermatis [50, 51] M. sympodialis [49]

Introduction

- 33 -

Its relative prevalence also varies with age, body site and geographical location. A study that investigated these differences in healthy subjects in Canada using culture-dependent methods found that

M. globosa was most common in younger individuals (under 14 years), while M. sympodialis was most

common in adolescents and adults,[35] similarly to what a Korean study reported, with the exception that in the latter the most frequent isolate in individuals over the age of 41 was M. restricta.[34] Independently of the age, Gupta et al.[35] detected more often M. globosa in the scalp and forehead and less frequently in the trunk, where M. sympodialis was the predominant isolate. Conversely, the Korean group recovered

M. restricta more frequently from the scalp, forehead and face and M. globosa particularly from the

chest.[34] Malassezia sympodialis was reported to be the most common isolate from healthy skin in Spain,[17] as well as in Sweden;[29] however, among healthy Japanese, M. restricta predominated.[37] A study from the United States of America (USA), sequencing of the fungal skin inhabitants of healthy volunteers also demonstrated that specific Malassezia species predominated in different skin-sites; M.

restricta predominated in external auditory canal, retroauricular crease and glabella, while M. globosa

predominated on the torso, occiput and inguinal crease.[36]

Specifically related to PV, M. globosa has been found as the most frequent isolate in a variety of studies from distant geographic locations, including Greece,[32] Spain[17] and Iran,[38] which had used different methods of speciation. Conversely, in a Canadian study, M. sympodialis was the most common PV isolate,[39] similarly to studies from South America.[40, 41]

Multiple studies using cultural and phenotypical typing methods resulted in contradictory data about the relative proportion of Malassezia species isolated in SD cases.[17, 29, 30] Whereas studies using molecular quantitative techniques demonstrated M. restricta as the most common isolate in SD.[21, 42, 43] A report from China unveiled that the majority of SD lesions showed co-colonization with two or more Malassezia species, mainly M. restricta, M. sympodialis and M. globosa.[29, 44]

The co-isolation of two or more Malassezia species from the same lesion was recorded in multiple studies referring to PV and SD cases.[32, 43, 52]

In systemic infections and unlike the cutaneous disorders, the most common isolates were invariably M. furfur,[47-49] M. pachydermatis[50, 51] and M. sympodialis [49] which may be related to a distinct pathogenic potential exhibited by such species. Notably, M. pachydermatis presence on human

Introduction

- 34 -

skin is usually rare and transient, transmission most possibly occurring from pet animals and environmental sources.[5]

M

Malassezia virulence attributes and their relation to clinical manifestations remain yet to be fully

elucidated as well as the mechanisms underlying its shift from a commensal to a pathogenic organism.[14] Malassezia strains associated with the abovementioned clinical conditions were presumably derived from commensal strains, but this has not yet been experimentally proven.[53]

The pathogenicity of the Malassezia organisms at the skin niche is presumed to develop along the initial leniency of the immune system allowing the yeasts to grow, being generally asymptomatic. The yeasts might become pathogenic when the immune balance is disturbed.[54] The diversity of the clinical presentations of the abovementioned Malassezia-related skin diseases makes even more difficult the elucidation of solid pathogenic pathways.[16] Furthermore, the variable pathogenic potential exhibited by the same species isolated from healthy and diseased skin remains yet to be explained, although some studies highlighted genetic variability among implicated strains.[32, 42]

The emerging complexity of Malassezia-host interaction revealed that both the host and the microbe are components of pathogenicity – fungal toxins and direct fungal activities can obviously cause host damage, but aberrant host responses such as hyperinflammation, allergic sensitization, or “cytokine storms” also evoke host injury.[14]

The high lipid content of Malassezia cell wall provides mechanical stability and the associated mannoproteins promote cellular surface hydrophobicity which had been shown to favor Malassezia adhesion to host cells,[55] to protect the yeast from phagocytosis and to promote a downregulation of the inflammatory response.[4] In vivo, hyphae are sometimes observed in individuals with hyperactive sebaceous gland activity, since the excessive sebum seems to induce hyphae formation. Such phenotype confers yeasts promoted ability to actively penetrate the host tissues.[4, 56] The hyphal stage is thought to play a role in the pathogenesis of PV as hyphae are often present in scales taken from lesions.[4, 57] The specific involvement of hyphae in the generation of immune stimulatory molecules by Malassezia still needs to be clarified.[56] In the pathogenesis of PV secondary metabolic pathways are also involved

Introduction

- 35 -

resulting in the production of a broad spectrum of indole compounds which are potent ligands of the aryl hydrocarbon receptor implicated in the mediation of ultraviolet radiation damage and melanogenesis. Such mechanisms of pathogenesis may be responsible for the pigment changes occurring in PV. [4, 16, 58]

The density of yeasts in lesional versus non-lesional skin in SD patients is not straightforward,[30, 59] a quantitative positive relationship between Malassezia and SD presence or severity being often absent.[60] However a molecular-based study described higher densities of

Malassezia at lesional sites than at non-lesional sites,[42] which might suggest that the overgrowth of Malassezia organisms might be important in individuals who are immunologically predisposed towards

the development of SD.[5, 6] Faergemann et al. [61] when attempting to characterize the cellular profiles in skin biopsies of SD and Malassezia folliculitis found an increased number of natural killer (NK) 1 and CD16 cells as well as complement activation in lesional-skin and speculated that an irritant non-immunogenic stimulation of the immune system is involved in both diseases, caused by products released by Malassezia. Nevertheless, it is currently known that keratinocytes are able to develop an efficient innate immune response producing pro- and anti-inflammatory cytokines after stimulation with

Malassezia, which appears to be species-specific.[3, 62, 63] Yeast lipases exhibit broad spectrum activity,

hydrolyzing most of the cutaneous triglycerides into free fatty acids; unsaturated free fatty acids may act as irritant and immune stimulatory molecules in susceptible individuals,[56] who should enclose a specific unidentified defect in skin barrier function,[64] as demonstrated by the flaking elicited with application of oleic acid to human scalp in individuals with a disruption in epidermal integrity.[20] The role of Malassezia lipases and phospholipases as virulence attributes in SD was enhanced by the demonstration of significant increased expression of lipase genes in isolates from SD patients with and without HIV infection.[21] Similarly among Parkinson disease patients, high Malassezia density was found in seborrheic skin areas, associated to high phosphatase and lipase activity of the isolates in

vitro.[24] These findings were further supported by the in vitro induction of phospholipase activity of Malassezia SD isolates by β-endorphin, another factor often increased in inflammatory skin

conditions.[65] As abovementioned, SD is more prevalent in HIV infected and AIDS patients, a fact which may be related to the CD4 T cell lymphopenia allowing proliferation of Malassezia organisms as suggested from a T cell receptor transgenic animal model of SD-like disease.[66]

Introduction

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A further health threat by these pathogens is their propensity to develop biofilm on the skin and at the surface of indwelling medical devices. Biofilm represents a serious medical problem since it promotes resistance to antifungal drugs and acts as a reservoir for the seeding of infectious organisms at distant places.[67] Some strains of M. pachydermatis were able to form a three-dimensional biofilm on plastic catheters commonly used in clinical practice; these biofilms showed an heterogeneous architecture that consisted of a network of unipolar budding yeasts with collarets and extracellular matrix, without hyphae.[68] More recently, this finding was confirmed and expanded using M. pachydermatis isolates from dogs with and without Malassezia dermatitis; the authors unveiled that the quantity of biofilm production was directly proportional to the phospholipase activity and suggested that both could act synergistically, exacerbating lesions at topical and/or systemic level.[69]

I

There are no standardized methods for isolation and speciation of Malassezia. Whenever the diagnosis of Malassezia infection is suspected and the clinical situation requires speciation, which generally is only considered in systemic infections,[47] the yeast can be characterized by cultural and non-cultural methods.

The sampling procedures to recover Malassezia from clinical specimens vary among studies; there are reports about the use of contact plates,[29, 35] skin scrapings,[70, 71] adhesive tape,[42, 44, 72] swabbing[59] and detergent scrub techniques.[43] Contact plates filled with culture media enable quantitative culturing.[29, 35]

Regarding culture media, Sabouraud agar covered with sterile olive oil was initially used, but it did not allow enumeration of colonies since they coalesced.[8] Alternative media such as Leeming-Notman and modified Dixon agar are frequently employed.[8, 35] However, there was a need to develop a selective and differential medium, easy to prepare, that allows recovery of clinically relevant

Malassezia species.[72-74] Following the modification of the chromogenic Candida medium with some

lipid components (CHROMagar Malassezia®_{, Paris, France), Kaneko et al. [72] were able to support the} growth of up to 9 distinct Malasssezia species.

Introduction

- 37 -

Figure 4. Macro and microscopic morphologic aspect of Malassezia furfur Culti-Loops® _{ST 8036 type}

strain. Macroscopic aspect of colonies grown on CHROMagar Malassezia® _{medium and microscopic}

aspect of the yeasts after being stained with methylene blue .

In 1996, Guého et al.[75] attempted to identify seven Malassezia species based upon conventional laboratory procedures, such as morphological and physiological features, namely the production of catalase, tolerance to 37ºC and the ability to utilize certain concentrations of Tween as a source of lipid.

However, as the results of such tests were not always easy to read, molecular methods were developed in order to overcome such difficulties. These methods also provide the possibility to identify

Malassezia directly from the skin samples without the need to cultivate, giving the demanding culture

conditions and different growth rate between Malassezia species.[42, 71] The performance of such molecular methods is mostly dependent of the sensitivity of nucleic acid extraction procedure.[71] Considering molecular procedures to achieve speciation, a wide variety of methods have been developed. A comparison between the amplified fragment length polymorphism (AFLP) and the sequencing of the internal transcribed spacer (ITS) plus the large-subunit regions (LSU) of nuclear ribosomal DNA was described by Gupta et al.[76] The authors found a complete agreement between both methods in identifying 8 Malassezia species. Afterwards, restriction fragment length polymorphism (RFLP) analysis was compared with a nested polymerase chain reaction (PCR) and up to 11 species could be differentiated

Introduction

- 38 -

by both methods. However, despite nested PCR being faster, it seemed to be less accurate than the RFLP method.[77]

Phenotypical yeast identification methods such as API ID32C (BioMeriéux, Marcyl’Etoile, France), Auxacolor (Bio-Rad, Hercules, CA), and Vitek 2 (BioMeriéux) were compared with sequencing;[78] the authors found a correct identification of 8 isolates of M. furfur by Vitek 2, but misidentifications with other species, namely with M. pachydermatis. More recently, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) included Malassezia species in its databases to allow a rapid and accurate identification of these yeasts. Notably, it does not obviate the need of obtaining a culture of Malassezia.[79-81]

Introduction

- 39 -

Table 2. Distinct sampling and diagnostic methods and corresponding Malassezia species

Sampling Culture Molecular Identified species Reference

Contact plate Leeming-Notman agar

PCR-RFLP

(LSU rRNA, ITS, β-tubulin and lipase gene)

M. furfur, M. restricta, M. globosa, M. slooffiae, M. sympodialis, M. obtusa

[35] Contact plate Leeming-Notman

agar plus biochemical features --- M. furfur, M. restricta, M. globosa, M. slooffiae, M. sympodialis, M. obtusa, M. pachydermatis [29]

Skin scraping and others not specified

Leeming-Notman agar

PCR-RFLP

(LSU rRNA, ITS, β-tubulin and lipase gene)

M. furfur, M. restricta, M. globosa, M. slooffiae, M. sympodialis, M. obtusa, M. pachydermatis [70] Skin scraping --- PCR-RFLP and nested PCR (ITS 1/ 4 and 3/ 4) M. furfur, M. restricta, M. globosa, M. slooffiae, M. sympodialis, M. obtusa, M. pachydermatis. [71] Skin swabbing --- tFLP (nested PCR of ITS 1 and 2) M. furfur, M. restricta, M. globosa, M. slooffiae, M. sympodialis, M. obtusa, M. pachydermatis [59] Detergent scrub technique Leeming-Notman agar PCR-RFLP M. furfur, M. restricta, M. globosa, M. slooffiae, M. sympodialis, M. obtusa [43] Adhesive tape on skin CHROMagar

Malassezia plus biochemical features ---M. furfur, ---M. restricta, ---M. globosa, M. slooffiae, M. sympodialis, M. obtusa, M. pachydermatis, M. japonica, M. dermatis [72]

Adhesive tape on skin

--- Real-time PCR and nested PCR M. furfur, M. restricta, M. globosa, M. slooffiae, M. sympodialis, M. obtusa, M. pachydermatis, M. japonica, M. dermatis [42]

Not specified Modified Dixon agar Sabouraud agar PCR-RFLP (LSU rRNA) M. furfur, M. restricta, M. globosa, M. slooffiae, M. sympodialis, M. obtusa, M. pachydermatis [82]

Not specified Modified Dixon agar Sabouraud agar

Sequencing ITS-1 M. furfur, M. restricta, M. globosa, M. slooffiae, M. sympodialis, M. obtusa, M. pachydermatis

[83]

Not specified Leeming-Notman agar

Fingerprinting

(AFLP, RAPD, DGGE)

M. furfur, M. restricta, M. globosa, M. slooffiae, M. sympodialis, M. obtusa, M. pachydermatis

[84]

Not specified Modified Dixon agar PCR-RFLP M. furfur, M. restricta, M. globosa, M. slooffiae, M. sympodialis, M. obtusa, M. pachydermatis, M. japonica, M. dermatis, M. yamatoensis, M. nana [85]

Not specified Leeming-Notman agar

MALDI-TOF MS M. furfur, M. restricta, M. globosa, M. slooffiae, M. sympodialis, M. obtusa, M. pachydermatis, M. japonica, M. dermatis, M. caprae, M.cuniculi, M. yamatoensis, M. equina, M. nana [81]

Not specified Modified Dixon agar MALDI-TOF MS M. furfur, M. restricta, M. globosa, M. slooffiae, M. sympodialis, M. pachydermatis

[79] AFLP: amplified fragment length polymorphism; DGGE: denaturing gradient gel electrophoresis; ITS: internal transcribed spacer; LSU rRNA: large subunit of ribosomal RNA gene; MALDI-TOF MS: matrix-assisted laser desorption ionization-time of flight mass spectrometry; PCR: polymerase chain reaction; RAPD: random amplified of polymorphic DNA; RFLP: restriction fragment length polymorphism; tFLP: terminal fragment length polymorphism.

Introduction

- 40 -

A

Although reliable antifungal susceptibility tests have been developed in order to provide insights regarding the available therapeutic options in case of yeast infections, there is still no standardized protocol for Malassezia susceptibility testing.

The variety of antifungal susceptibility tests described included an urea broth microdilution method based on urease activity of Malassezia,[86, 87] a liquid medium assay using a colorimetric indicator for metabolic activity [88] and a modified broth and solid Roswell Park Memorial Institute (RPMI) 1640 (Sigma-Aldrich, St. Louis, MO, USA) media supplemented with glucose, oxoid, glycerol and Tween 20.[89] All of them remain too cumbersome to be used in clinical routine testing. More recently, Leong et al.[90] developed an optimized colorimetric broth microdilution method based in a modified RPMI broth medium supplemented with glucose, glycerol, Tween 60, oleic acid, esculin and resazurin enabling the rapid visual and spectrophotometric determination of minimal inhibitory concentration (MIC) values.

Nevertheless, the results of in vitro susceptibility studies have unveiled conflicting results about antifungal susceptibility profile of Malassezia species. Gupta et al.[91] evaluated in vitro susceptibility of

Malassezia isolates from skin and blood to three azoles and terbinafine using a modified Diagnostic

Sensitivity Testing (DST; Oxoid, UK) agar medium supplemented with glycerol in case of M. furfur, M.

sympodialis and M. slooffiae, Leeming-Notman agar for M. globosa, M. obtusa and M. restricta, and

Sabouraud agar for M. pachydermatis. The authors reported low MIC values of voriconazole, ketoconazole and itraconazole, and variable MIC values of terbinafine regarding M. furfur, M. globosa and M. obtusa, but not M. sympodialis. However, this method of antifungal susceptibility assessment may not be suitable for routine use due to the long incubation time required before adequate growth for estimation of the MIC, namely 4 to 5 days.

Velegraki et al.[89] performed in vitro susceptibility testing of Malassezia yeasts to azoles, terbinafine and amphotericin B using a modified RPMI media incubated at 32ºC for 48 and 72 hours. The authors showed low MIC values of all azoles and terbinafine for the Malassezia species tested, but high MIC of amphotericin B against M. furfur, M. globosa, M. restricta and M. slooffiae strains. Susceptibility profile to azoles was assessed by Miranda et al.[92] reporting higher fluconazole MIC values relatively to other azoles, namely ketoconazole, itraconazole and voriconazole. Fluconazole was considered to be very

Introduction

- 41 -

active against M. sympodialis and M. slooffiae, but less effective against M. globosa and M. restricta, whereas itraconazole displayed high activity against M. globosa.[93] Cafarchia et al.[94] found some strains of M. pachydermatis corresponding in vitro to high fluconazole MIC values. Consistently high MIC values and very wide MIC ranges of fluconazole have been disclosed.[95, 96] More recently, a study reported very high ketoconazole MIC values for a few strains of Malassezia;[90] which might become a topic for clinical concern regarding that proposed treatment regimens for PV and SD often include topical ketoconazole and oral fluconazole maintained for long periods in cases of recurrence.[97]

A Japanese research group[33] tested in vitro susceptibility to tacrolimus, ketoconazole and itraconazole, revealing that Malassezia species exhibited low MIC values of both azoles and that tacrolimus was highly active against approximately 50% of the isolates. Moreover, whenever tacrolimus was combined with one of the azoles, the azole MIC was substantially reduced, most probably corresponding to a synergistic effect.

Notably, issues regarding the discrepancy between results of susceptibility testing and the clinical outcome should be considered, as it remains a common finding with other pathogenic yeasts like

Candida,[98] for instance.

T

Nonetheless the attempt to control Malassezia yeasts with topical and systemic antifungals, the recurrence of clinical signs of disease is often noticed, especially concerning PV and SD.[7, 97] Hitherto, questions about the effects of antifungals, especially azoles, attending also to its intrinsic anti-inflammatory properties still remain to be fully elucidated.[7, 99] The anti-anti-inflammatory activity of these agents occurs via inhibition of 5-lipoxygenase, which blocks leukotriene B4 synthesis in the skin.[100]

Topical compounds such as tea tree oil (Melaleuca alternifolia),[101] tacrolimus,[33] honey[102] and cinnamic acid[103] have demonstrated activity in vitro and in vivo against Malassezia species, exhibiting often also anti-inflammatory properties.[104] In 2015 a Danish research group [97] published compiled guidelines about the management of Malassezia-related skin diseases and concluded that topical treatments for PV and SD, in particular topical ketoconazole, should be the first-line approach. Recently, topical ketoconazole was considered effective for the treatment of Malassezia-related

Introduction

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skin diseases, with a reported efficacy of 71-89% for PV and 63-90% for SD.[105] A short course of topical corticosteroids or calcineurin inhibitors is frequently employed in SD due to its anti-inflammatory effect. However, in case of widespread lesions or refractory lesions to topical treatments, oral antifungal therapy may be indicated namely fluconazole and itraconazole.[7, 9, 97] A meta analysis of clinical studies indicated that fluconazole and itraconazole seemed equally effective for the treatment of PV.[106] Although topical terbinafine was effective for the treatment of PV,[107] oral terbinafine is not considered a valuable option,[7, 97] which may be related to a more uneven distribution at the most superficial layers of the epidermis not reaching high enough concentrations to exhibit fungicidal activity against Malassezia organisms.[88, 108] Interestingly, oral terbinafine efficacy in moderate to severe SD cases was described in a randomized trial using a dose of 250mg/day for 4 weeks with the effect maintained 8 weeks after stopping treatment.[109] In contrast to azoles, oral terbinafine may not display in vivo anti-inflammatory activity.[110] A study evaluated oral itraconazole (200 mg/day for a week, followed by a maintenance therapy of a single dose of 200 mg every 2 weeks) in the treatment of mild to severe facial SD, reporting significant clinical improvement and a decrease of Malassezia organisms in the direct smear,[111] confirming and expanding the findings of Kose et al.[112] Nevertheless Gupta et al.[113] in a systematic review concluded that data about oral therapy for SD is sparse, lacking comparative studies to clarify the optimal dose and duration of treatment.

Malassezia folliculitis is usually treated with systemic antifungal treatment since it eradicates

more effectively – versus topical therapy – Malassezia organisms located in the hair follicle.[7, 97, 111] Itraconazole in a dose of 200 mg for 7 days resulted in a distinct and statistically significant improvement over placebo,[114] whereas the extended treatment for 3 weeks resulted in a higher response rate.[115] Combined systemic and topical treatment may be favorable according to the Danish guidelines.[97]

Concerning systemic, catheter-related Malassezia infections, treatment often includes catheter replacement, discontinuation of the lipid infusion, and administration of systemic antifungal therapy.[116] Irrespective of the in vitro results and based solely in clinical experience, intravenous amphotericin B is most often used empirically for the management of Malassezia bloodstream infections, with variable therapeutic success.[117, 118] An azole drug may be a valid alternative option, based on in vitro data rather than in clinical studies.[13] There are no reports about treatment of Malassezia infections with echinocandins.