The Biology of Malassezia Organisms and Their Ability to Induce Immune Responses and Skin Disease (Pages 4–26)

8/9/2019 The Biology of Malassezia Organisms and Their Ability to Induce Immune Responses and Skin Disease (Pages 426)

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4 2005 European Society of Veterinary Dermatology

Veterinary Dermatology 2005,16, 426

BlackwellPublishing,Ltd.

Review

The biology of Malassezia organisms and their ability to induceimmune responses and skin disease

TAI-AN CHEN and PETER B. HILL

Division of Veterinary Clinical Studies, Royal (Dick) School of Veterinary Studies, The University of Edinburgh,UK

(Received 9 December 2003;accepted 11February 2004)

TABLE OF CONTENTS

Introduction 4History and taxonomy of the genus Malassezia 5Biological characteristics of Malassezia organisms 5Structure 5Reproduction 6Biochemistry 6Distribution of Malassezia organisms on the host 6Immunological and epidermal responses toMalassezia organisms 7The immune response toMalassezia organisms 7

Antigen release, penetration and presentation 8Cell-mediated immune responses 8IgG, IgM and IgA responses toMalassezia organisms 9IgE responses toMalassezia organisms 10Mast cell responses 11Epidermal responses associated withMalassezia dermatitis 12

Malassezia organisms as pathogens in humans andanimals 13Diseases associated withMalassezia spp. in humans 13Pityriasis versicolor 13Malassezia folliculitis 13Seborrheic dermatitis and dandruff 14Atopic dermatitis 14Malassezia fungaemia 14Diseases associated withMalassezia spp. in animals 15Malassezia dermatitis in dogs 15

Predisposing factors for overgrowth ofMalassezia pachydermatis 15Pathogenesis 16Clinical features 16Diagnosis 17Treatment 18Conclusions 19References 19

INTRODUCTION

The genusMalassezia is now known to include sevenspecies of yeast, many of which have been associatedwith various diseases in humans and dogs.Malassezia

pachydermatis is a lipophilic budding yeast that colo-nizes the skin and mucosal sites of healthy dogs.1,2Despite being part of the normal cutaneous micro-ora, it is now known that the yeast may become apathogen under certain circumstances.Malassezia der-matitis, an inammatory dermatosis associated withelevated populations ofM. pachydermatis on the skinof dogs, has been recognized with increasing frequency.

Its importance in canine dermatology can be illus-trated by the continuous rise in the number of articles

on this condition in the literature since it was rstdescribed by Dufait in 1983.3The interaction betweenMalassezia yeasts and their

host species has been studied extensively in humans butless widely in dogs. In this article, we describe the basictaxonomy and biology ofMalassezia yeasts and reviewcurrent knowledge on the interplay between the organ-ism and the skins protective responses. These includecutaneous and systemic immune responses and theprotective barrier afforded by the epidermis. Failure of these mechanisms to protect the host against prolifera-tion of the organism can lead to pathogenicity and var-ious disease states. In some cases, these responses areactually deleterious and can lead to hypersensitivityreactions or epidermal pathology. We end the review bydescribing the clinical aspects of diseases associatedwithMalassezia organisms in humans and animals and

Correspondence: P. B. Hill, Division of Veterinary Clinical Studies,Hospital for Small Animals, Royal (Dick) School of VeterinaryStudies, Easter Bush Veterinary Centre, Edinburgh EH25 9RG, UK.E-mail: [email protected]


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6 T-A Chen and PB Hill

2005 European Society of Veterinary Dermatology,Veterinary Dermatology , 16, 426

Around the cell wall there is an outer lamellar layer, thestructure of which varies depending on the lipidsources in the medium. It is suggested that this lamellarlayer contains lipid and may play a role in the adhesionprocess.26

The inner surface of the cell wall is corrugated,

which corresponds to invaginations of the closelyadherent plasma membrane.24,26,28 Cytoplasmicorganelles that have been described include a nucleus,vacuoles containing lipid, and mitochondria.24,28 It hasbeen shown that the number of mitochondria pos-sessed by round cells (P. orbiculare ) is different fromthat of oval cells (P. ovale ). The former cells containapproximately three mitochondria per cell, whichincrease in volume rather than number during growth.In contrast, there are approximately 23 mitochondriain an oval cell and they increase in number rather thansize during growth.27 However, electron microscopicobservations do not reveal noticeable differences inthe mitochondrial appearance betweenP. ovale andP. canis (M. pachydermatis ).24

ReproductionThe proliferation ofMalassezia yeasts is of clinical rel-evance because it is often overgrowth of the organismthat leads to disease. Yeasts of the genusMalasseziareproduce by unipolar or sympodial (M. sympodialis )budding and the budding process described forP.ovale , P. orbiculare and M. pachydermatis is very sim-ilar.19,24,25,27 However, the budding base ofM. pachy-dermatis (0.91.1m in diameter) is broader than thatof P. ovale and P. orbiculare (0.50.7m).25,27Nishimura et al. 25 precisely described the buddingprocess ofM. pachydermatis in stages. The buddingyeast rst forms an ear-like structure separating fromthe cell wall. This structure grows outwards followedby protrusion of the cytoplasm and dimpling of the cellwall. The cell wall of the daughter cell then becomesthicker, layered and serrated as it grows. When thedaughter cell is as long as the mother cell, a narrowlumen forms in the cross-wall and ssion takes place.The bud is then completely released leaving the ear-likestructure as a bud scar.

BiochemistryLipid-dependent Malassezia species have been re-ported to elaborate a range of enzymes. These enzymeshave been the subject of recent interest because theymay be involved in the pathogenesis of clinical diseasestates. Lipase activity has been demonstrated withinthe cell wall and at the membrane sites of the yeastin vitro using histochemical techniques and electronmicroscopy. This suggests that this enzyme is synthes-ized intracellularly and exported to the cell surface.29It has also been found that the fatty acid compositionof the cell varies depending on the lipid supplementa-tion present in the medium, indicating that fatty acidsare required for membrane synthesis.30 The lipoxy-genase activity of the yeast has been proposed to beinvolved in the pathological changes associated with

pityriasis versicolor.31,32 Increased levels of lipoperox-ides were detected in lipids from lesional but not non-lesional skin of patients with pityriasis versicolor.32The by-products of lipoperoxidation induced by lipoxy-genase activity of the yeast were considered to causedamage to melanocytes, resulting in the hypopigmen-

tation seen in pityriasis versicolor.31

In vitro , M. furfuralso produces phospholipases,33,34 the activity of whichwas demonstrated to cause the release of arachidonicacid from human epithelial cell lines, a mechanism bywhichMalassezia organisms may trigger inammationin the skin.35 In addition to the enzymes described above,Malassezia organisms produce azelaic acid in culturessupplemented with oleic acid.36 Azelaic acid is an inhi-bitor of tyrosinase, an enzyme involved in the produc-tion of melanin, indicating that the production of azelaicacid byMalassezia organisms may also play a role inthe pathological changes of pityriasis versicolor.36

M. pachydermatis has also been demonstrated toproduce various enzymesin vitro , including alkalineand acid phosphatase, chondroitin-sulphatase,esterase, esterase lipase, galactosidase, glucosidase,hyaluronidase, leucine arylamidase, lipase, lecithinase,peroxidase, phosphoamidase, phospholipase, phos-phohydrolase, protease and urease.1,2,3740 Lipase wasfound to be predominantly associated with the cellmembrane ofM. pachydermatis , whereas protease issecreted by the yeast.40 No signicant difference wasfound in the production of chondroitin-sulphatase,hyaluronidase, phospholipase and protease byM.

pachydermatis strains isolated from dogs with otitis ordermatitis.37 When cultured in liquid media, the activ-ities of alkaline phosphatase and esterase lipase in theculture supernatants fromM. pachydermatis were sig-nicantly greater than that in the cell suspension. Fur-thermore, signicantly greater activity of C4 esterasewas detected in cell suspensions ofM. pachydermatisstrains obtained from healthy dogs compared to thosefrom dogs with dermatitis.2

DISTRIBUTION OF MALASSEZIA ORGANISMS ON THE HOST

Unlike many bacteria and other fungi,Malasseziayeasts are rarely found in the environment.1,14 Theirhabitat is primarily the skin and mucosae of mammalsand birds.1,14 Lipid-dependentMalassezia organismsare frequently isolated from human skin. In a studyinvolving 200 healthy volunteers, Bandhaya41 isolatedMalassezia furfur from 10 different skin sites in everyindividual, with the highest counts being on the headand upper trunk.M. pachydermatis was only isolatedfrom 12% of the subjects.41

In healthy dogs,M. pachydermatis can be isolatedfrom the ear canal, anus, rectum, oral cavity and, lesscommonly, the nose and vagina.4245 On normal canineskin, carriage of the yeast is most common in the inter-digital areas and around the mouth but uncommonon the axilla, groin or dorsum.45,46 Interestingly, the


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The biology and cutaneous responses toMalassezia organisms 7

frequency of isolation and colony counts from the skinand mucosal sites of Basset hounds, a breed predis-posed to developingMalassezia dermatitis, are signic-antly higher than on other breeds.47 M. pachydermatisis thought to adhere to the skin surface via trypsin-sensitive protein adhesion molecules.48

In other species,Malassezia organisms have beenrecovered from the skin of healthy cats, ferrets, foxes,bears, pigs, horses, birds and rhinoceroses.1,4954

IMMUNOLOGICAL ANDEPIDERMAL RESPONSES TOMALASSEZIA ORGANISMS

The skin is continually challenged by a variety of envir-onmental hazards. The rst line of defence and protec-tion is provided by the epidermis, the outermost skinlayer.55 The epidermis protects the body from water loss,UV damage, mechanical injury, microbiological inva-sion, and immunological insults. The predominant celltype of the epidermis, the keratinocyte, provides mech-anical protection from parasites and micro-organisms,and it also forms the rst line of immunologicaldefence against environmental or microbiological anti-gens and allergens.56

The skin tends to respond to environmental insultsby activating the skin immune system and increasingthe thickness of the skin. The former mechanismresults in cutaneous inammation; the latter resultsfrom increased proliferation of cells in the basal layerof the epidermis, leading to epidermal hyperplasia,hyperkeratosis and lichenication. Inammation andlichenication are both generally seen in the skin of dogs withMalassezia dermatitis, indicating that bothof these protective mechanisms are involved in thedisease process.

The immune response to Malassezia organismsThe skin immune system consists of cells residing in theskin including keratinocytes, Langerhans cells, dermaldendrocytes and mast cells, as well as skin-seeking Tlymphocytes and endothelial cells of cutaneous post-capillary venules.57 It functions in conjunction with

humoral substances, such as secretory immunoglobu-lins and cytokines, to deal with challenges from bothexogenous and endogenous antigens, to mount effectorresponses of various types, and to balance the responsein order to minimize tissue damage whilst eliminatingthe original insult.57 Defects in both innate andacquired immune responses may lead to the develop-ment of skin diseases.

Based on information from existing immunologicalliterature related to other organisms or antigens, wecan formulate a hypothesis to explain the mechanismsby which Malassezia pachydermatis might interactwith the cutaneous immune system in dogs (summar-ized in Fig. 1). In this scheme, it is postulated thatM.

pachydermatis on the skin surface would produce anti-gens that could penetrate the skin and be captured byepidermal Langerhans cells or dermal dendritic antigen-presenting cells. These cells would then migrate toregional lymph nodes and present the antigen to a Tlymphocyte via a major histocompatibility complex(MHC) class II molecule, which, in co-operation withdifferent environmental cytokines, would stimulate Thelper (Th) 0 precursor cells to differentiate into Th1cells and/or Th2 cells. A cytokine environment domin-ated by IL-12 would favour Th1 cell development,whereas IL-4 and IL-13 would stimulate the develop-ment of Th2 cells. T helper cells would activate Blymphocytes and stimulate them to differentiate intoantibody-forming plasma cells. By secreting IL-2 andIFN- , Th1 cells would promote IgG production,whereas IL-4 and IL-13 from Th 2 cells would promote

Figure 1. Possible pathways forimmunological responses stimulated byMalassezia organisms.Malassezia organisms

on the skin surface might release antigens(Ag) that penetrate the skin and be capturedby an antigen-presenting cell (APC). TheAPC would then present the antigens to aT lymphocyte. The cytokines present in theenvironment determine which T cell subset isproduced from Th0 cells. IL-12 stimulatesdevelopment of T helper 1 (Th1) cells. IL-4and IL-13 favour the production of T helper2 (Th2) cells. Th1 cells secrete IL-2 and IFN- that induce IgG responses.Malassezia -specic IgG might be protective or activatecomplement, provoking inammation.Th2 cells secrete IL-4 and IL-13 that induceallergen-specic IgE responses. The IgE

antibodies bind to mast cells (MC) and uponsubsequent encounter with allergens, triggerdegranulation leading to inammation andclinical features of Type I hypersensitivityreactions.


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immunoglobulin class switching to IgE. It is importantto remember that development of Th subsets into twopolarized subgroups with divergent cytokine responsesdoes not necessarily occur in immunological reactionsand both mechanisms can occur concurrently. Theproduction of Malassezia -specic IgG antibodies

could potentially provide a degree of protective immun-ity againstMalassezia organisms. Alternatively, theseantibodies might activate the complement system caus-ing epidermal damage and inammation. The develop-ment of allergen-specic IgE antibodies could lead tosensitization of cutaneous mast cells. Subsequentexposure to Malassezia allergens could cross-linksurface-bound IgE and trigger the release of a variety of inammatory mediators, resulting in the clinical signsof Type I hypersensitivity reactions (Fig. 1). To date,only certain parts of the mechanisms described in theabove hypothesis have been investigated in dogs. Theseinclude cell-mediated immune responses, humoralimmune responses, and mast cell responses toM. pachy-dermatis . The evidence in the literature to support var-ious aspects of the proposed pathways described aboveis reviewed in the following sections.

Antigen release, penetration and presentationIt has been suggested that entry through the skin is themost likely mechanism by whichMalassezia organismsstimulate the immune system in human patients withatopic dermatitis.58 A recent study demonstrated thatM. furfur was able to invade human keratinocytesand resist phagolysosome fusion.59 It has also beenreported that the rst clonedMalassezia allergen (Mals 1), a major allergen that is localized to the cell wall,can be released into the culture medium when grownin vitro .58 These ndings support the hypothesis thatMalassezia organisms can penetrate the epidermalbarrier and probably release allergens in the skin,where both whole organisms and allergenic compon-ents would come into contact with Langerhans cellsin the epidermis. Uptake of wholeM. furfur yeast cellsand various allergenic components from the yeast,includingM. furfur extracts, recombinantM. furfurallergen 5 (Mal f 5) andM. furfur mannan, has been

demonstrated in vitro using immature monocyte-derived dendritic cells (MDDCs), which reect Langer-hans cells in the skin.60 These results suggest thatsensitization of atopic patients toM. furfur can bemediated by immature dendritic cells in the absence of IgE in the skin. The internalization was shown to occurvia binding to the mannose receptor (other receptorsmay also be involved) or pinocytosis.60 The presenceof M. furfur was also shown to induce maturation of immature MDDCs by up-regulation of CD83 expres-sion, and increase in expression of the costimulatorymolecules CD80 and CD86.61 Mature dendritic cellsare poor at antigen uptake, but excellent at presentingantigens. They would therefore efciently present anti-gen-derived peptides on MHC molecules to T cells.62

There is some evidence that suggests the interactionbetweenMalassezia antigen-bearing antigen-presenting

cells (APCs) and T cells takes place in the skin. An inl-tration of CD4+ T cells has been detected at 24 h atpatch test sites inP. orbiculare patch test-positive,atopic dermatitis patients, but was more pronounced at72 h.63 The expression of intercelluar adhesion mole-cule (ICAM)-1 and human leucocyte antigen (HLA)-

DR in the dermis of these patients was also up-regulated. At 24 h post-test, the former correlated withthe scale of the dermal CD3+ lymphocytic inltrates,with the majority being CD4+.63

To date, the release, penetration and presentation of Malassezia antigens in the skin of dogs has not beenstudied. However, it has been demonstrated that appli-cation ofM. pachydermatis suspensions on healthy dogskin can induce skin lesions similar to those observedin naturally occurringMalassezia dermatitis.64 Thisindicates thatMalassezia antigens and/or organismsmay be able to penetrate into the skin, thus inducingpathogenic effects. Also, there are some data providingindirect evidence for a transepidermal route of antigenpenetration in dogs with atopic dermatitis. Highernumbers of Langerhans cells have been detected inlesional atopic skin compared to clinically normalatopic or normal control canine skin.65,66 These cellswere present in clusters in lesional skin of dogs withatopic dermatitis.67 Expression of surface IgE has alsobeen observed on epidermal Langerhans cells inlesional atopic canine skin,66 and these cells are respon-sible for allergen capture and presentation.67 More-over, eosinophils can be seen below the stratumcorneum in lesional atopic canine skin, but not in clin-ically normal atopic skin.68 Canine atopic skin alsoexhibits hyperplasia of T lymphocytes expressing thegamma-delta T-cell receptor.68 Furthermore, transepi-dermal penetration of staphylococcal antigens hasbeen demonstrated in dogs.69 Taken together, thesendings indicate it is likely thatM. pachydermatis maybe able to release antigens that would penetrate theskin of dogs, particularly those suffering from atopicdermatitis, where they are captured by epidermalAPCs and thus initiate the process of antigen presenta-tion to T cells and a cascade of immunologicalresponses.

Cell-mediated immune responsesT-cell-mediated immunity is important in the pre-vention and recovery from fungal infections.70 Adeciency in cell-mediated responses could thereforepredispose the host to overgrowth ofMalasseziaorganisms.70 Cell-mediated immune responses toMalassezia organisms have been investigated in bothhumans and dogs using various assays. In order to gaina clearer picture of cell-mediated immunity toM. fur-

fur , Ashbee and Evans7 collated data from human subjects in various studies, ranging from 8 to 61 years of age. They concluded thatMalassezia organisms couldelicit signicant cell-mediated immune responses inhealthy individuals as measured by lymphocyte trans-formation assays or a leucocyte migration inhibitionassay. Also, the responses were similar in different age


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mycelium of the yeast. One study reported that healthyadults had detectable concentrations of IgM, IgG, IgGsubclasses and IgA to mycelial antigens ofMalasseziaorganisms in their sera, with the highest titres beingfound for IgG.7

No signicant difference inMalassezia -specic IgG

concentrations was found between adult patients withatopic dermatitis and healthy individuals as measuredby in vitro serological tests.63,73 However, one studyreported signicantly elevatedMalassezia -specic IgGconcentrations in young adult patients with atopicdermatitis aged between 16 and 21 years.86 The invest-igators proposed that this probably reected increasedexposure to the organisms through atopic skin and atendency for IgG to follow IgE production. No corre-lation has been found betweenMalassezia -specicserum IgG levels and APT responses to the yeast inpatients with atopic dermatitis.63 It is therefore con-sidered that determination ofMalassezia -specic IgGconcentrations has little value in the diagnosis of Malassezia sensitization in atopic human patients.58,63

Bondet al. 79 studied the humoral immune responsesto M. pachydermatis in healthy dogs and dogs withMalassezia dermatitis using ELISA. Serum titres of Malassezia -specic IgG and IgA in seborrhoeic Bassethounds with high cutaneous populations ofM. pachy-dermatis and affected dogs of various breeds werefound to be signicantly greater than those of healthyBasset hounds and healthy beagles. The investigatorsconcluded that high serum titres of IgG and IgA do notprevent seborrhoeic dermatitis associated withM.

pachydermatis in either Basset hounds or other breeds.Using western immunoblotting to detect IgGresponses to extracts ofM. pachydermatis , fourproteins of 219, 110, 71 and 42 kDa were shown to berecognized mainly by nonatopic dogs withMalasseziadermatitis, compared to healthy dogs.87

Atopic dogs, with or without cytological evidence of M. pachydermatis overgrowth, had signicantly higherserum titres ofMalassezia -specic IgG than healthydogs as measured by ELISA.88 However, there was nosignicant difference between atopic dogs with or with-out Malassezia overgrowth.88 By comparing the IgG

response toM. pachydermatis antigens using westernimmunoblotting, a protein of 25 kDa was identied inthe majority of atopic dogs withMalassezia dermatitis,but in only a few atopic dogs withoutMalassezia over-growth and in none of the normal dogs, suggesting thatthis protein may have some clinical relevance.89

In summary, it is clear that IgG responses toMalas-sezia yeasts are common in both healthy humans anddogs. This probably reects exposure of the immunesystem to antigens produced by commensal organisms.However, enhanced IgG responses can be seen in dogswith Malassezia dermatitis and in humans and dogswith atopic dermatitis. The role of this IgG response inthe pathogenesis of skin disease is currently unclear,both in humans and dogs. IgG antibodies are known tobe able to act as opsonins coating micro-organisms andto activate phagocytes, which in turn ingest and destroy

extracellular pathogens.90 This could in theory provideprotection for the host. However, as overgrowth withMalassezia organisms does not appear to be a self-resolving condition, it seems likely that these antibodiesare not protective. Alternatively, IgG antibodies couldactivate the complement system, as has been demon-

strated withPityrosporum ovale and P. orbiculare ,91,92

and exacerbate the inammatory response.90 A nalpossibility is that IgG responses to the yeast are merelyan epiphenomenon and neither contribute to, norinhibit the ongoing disease process. Further studies aretherefore required to determine the precise role playedby these antibodies inMalassezia -induced skin disease.

IgE responses to Malassezia organismsUsingin vitro serological tests such as ELISA, the radio-allergosorbent test (RAST) and western immuno-blotting, Malassezia -specic IgE has been detectedin human atopic patients for over a decade. Krgeret al. 76 studied the effect ofMalassezia extracts on IgEproduction by PBMCsin vitro . They found that IgEsynthesis by PBMCs from atopic dermatitis patientswith specic IgE forMalassezia organisms (RAST+)was signicantly higher than with RAST() atopicdermatitis patients or normal controls as measured byELISA. Also, stimulation withMalassezia extractsand IL-4 led to a dose-dependent increase in IgE syn-thesis from PBMCs only in RAST(+) atopic patients,indicating a Th2-type skewed response towardsMalas-sezia organisms in these patients.76 Several studiescomparing the titres of IgE specic toMalasseziaorganisms have revealed similar results. Patients withatopic dermatitis were found to have signicantlyhigher levels ofMalassezia -specic IgE in their seracompared to those with other atopic diseases orhealthy individuals.73,9395 Two studies investigatingIgE antibodies againstMalassezia organisms in chil-dren and young adults (up to 21 years of age) alsoshowed that these antibodies were detected signic-antly more frequently in those with atopic dermatitisthan the other two groups.86,96 TheMalassezia -specicIgE, but not the total IgE in serum, has been found tocorrelate with the degree of response to APT toMalas-

sezia extracts at 48 h post-test in atopic dermatitispatients.63By using western immunoblotting to detect

Malassezia -specic IgE, and by using the criteria thatreaction of more than 50% of patients sera repres-ents a major allergen, a number of major IgE-bindingproteins in the range of 9110 kDa have now beendocumented in humans.97101 Some antigens dened asmajor allergens in one study have been cited as minorallergens by other investigators.7 This is likely to bebecause of the disparity between methods and antigenpreparations used in different studies. Of the numerousantigens documented, a limited number of antigenshave been further characterized. Nine allergens of Malassezia furfur (Mal f 1Mal f 9) have recently beensequenced and expressed as recombinant proteins.102106However, the strain used in some of these studies has


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now been re-assigned to the speciesM. sympodialis ,58and the current nomenclature of puriedMalasseziaallergens is summarized in Table 1. It is important tonote that many of the studies described above wereperformed using the old classication ofMalasseziaspecies, and therefore might reect the characteristicsof other, more recently describedMalassezia species. Astudy investigating IgE binding components in ve of the seven species currently classied under theMalas-sezia genus, includingMalassezia furfur , M. globosa ,M. restricta , M. sloofae , and M. sympodialis , showedthat both species-specic and common antigenic com-ponents were present between species. The molecularweights of bands most frequently recognized using thesera of AD patients were 6772 (28%), 4550 (80%),3540 (39%), 4346 (20%) and 1922 kDa (43%),respectively.107 Zargari et al.108 also showed that sevenMalassezia species shared allergenic determinants to agreat extent, but also contained species-specic aller-gens, by measuring species-specic IgE antibodies andperforming inhibition immunoblotting. It is likely thatfurther studies comparing different species and the useof molecular techniques will dene more precisely the

identity of major allergens ofMalassezia species.Recently, specic IgE antibodies toM. pachyderma-tis in dogs have been investigated. Signicantly higherconcentrations ofMalassezia -specic IgE, measuredby ELISA, were detected in atopic dogs with or with-out Malassezia dermatitis and/or otitis than eitherhealthy dogs or nonatopic dogs with clinical evidenceof Malassezia overgrowth in the skin and/or earcanals.88 However, the difference between the atopicgroups was not signicant.88 By using western immu-noblotting, proteins from M. pachydermatis withmolecular weights of 45, 52, 56, and 63 kDa were dem-onstrated to be major allergens, recognized by IgE inmore than 50% of the sera from atopic dogs withMalassezia overgrowth.109

Hence, as with IgG responses, humans and dogswith atopic dermatitis can develop enhanced IgE

responses to allergens derived from the yeast. TheMalassezia -specic IgE antibodies in human andcanine atopic patients could play a key role in enhance-ment of immune responses. In addition to their rolein mast cell-mediated inammation (see below), theallergen-specic IgE antibodies could bind to Langer-hans cells in the skin, thus enhancing their allergencapturing and presentation capacity upon a secondencounter with the allergen.110,111

Mast cell responses

The mast cell response toMalassezia antigens has beeninvestigated with intradermal tests (IDT) or skin pricktests (SPT) in human atopic patients. One study com-paring the two test methods withMalassezia extractsshowed that a higher percentage of patients with atopicdermatitis reacted positively in IDT than in SPT.95However, positive IDT reactions toMalasseziaextracts were also seen in some patients with otheratopic diseases, whereas all the atopic controls gavenegative results in SPT.95 Studies using SPT to detecthypersensitivity toMalassezia organisms have shownan increased sensitivity in patients with generalized

atopic dermatitis72,86,95

or those with lesions predomin-antly on the head and neck.100,112,113 The SPT resultshave also been found to correlate with levels of Malassezia -specic IgE in the serum,86,114 and withresults of basophil histamine release tests,112 but notwith the severity of atopic dermatitis.114

Positive IDT results toMalassezia extracts have alsobeen reported in atopic dogs. Immediate hypersensitiv-ity responses to intradermal injections ofM. pachy-dermatis extracts at concentrations which caused noreaction in healthy dogs have been observed in atopicdogs withMalassezia dermatitis, although they werealso seen in some atopic dogs withoutMalassezia der-matitis.115 Nevertheless, the reactivity to the extracts inatopic dogs with cytological evidence ofMalasseziaovergrowth was signicantly higher than that inatopic dogs without.115 Bond et al. 116 investigated the

Table 1. Three allergens ofMalassezia furfur and six allergens ofMalassezia sympodialis that have been sequenced and expressed as recombinantproteins

Allergen nameMW(kDa)

Percentage of ADpatients reacting Identity

Mal s 1 37 70 No homology to known proteinsMembrane or secreted cell wall protein

Mal f 2 21 72 Homology to peroxisomal membrane proteins of Candida boidinii and Aspergillus fumigatus (Asp f 3)

Mal f 3 20 70 Homology to peroxisomal membrane proteins of Candida boidinii and Aspergillus fumigatus (Asp f 3)

Mal f 4 35 83 Showing 57% homology to mitochondria malatedehydrogenase fromSaccharomyces cerevisiae

Mal s 5 18.2 48 No homology to known proteinsMal s 6 17.2 48 Showing 82% homology to cyclophilin from

Schizosaccharomyces pombeMal s 7 16.2 40 No homology to known proteinsMal s 8 19.2 40 No homology to known proteinsMal s 9 14 24 No homology to known proteins

AD, atopic dermatitis.


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frequency of IDT reactivity toM. pachydermatis extractsin atopic dogs and reported similar results. In thisstudy, atopic dogs were not grouped according to theircytological ndings ofMalassezia populations. How-ever, the frequency of positive reactivity to the extractin atopic dogs was found to be signicantly greaterthan in healthy beagles.116 In contrast, the frequency of immediate hypersensitivity responses toM. pachyder-matis extracts in nonatopic dogs withMalasseziadermatitis is low. One recent study investigating IDTreactivity toM. pachydermatis in eight healthy bassethounds, 17 basset hounds withMalassezia dermatitis,and 19 healthy beagles, reported that only two affectedbasset hounds and one healthy beagle showed immedi-ate hypersensitivity reactions.117 Additionally, a recentreport has demonstrated positive immediate hypersen-sitivity reactions to extracts fromM. pachydermatisusing PrausnitzKstner tests.118 Clinically normal dogsreceived pooled sera from atopic dogs withMalasseziadermatitis that were IDT positive toMalassezia ex-tracts and serum from an atopic dog withMalasseziadermatitis exhibiting high levels of anti-MalasseziaIgE on an ELISA assay. Positive IDT responses were

observed in the recipients following subsequent injec-tion of the yeast extract, indicating that anti-Malassezia IgE antibodies are functional in Type Ihypersensitivity reactions.118 Taken together, thesendings suggest that mast cell-mediated hypersensitiv-ity responses toM. pachydermatis allergens may beinvolved in the pathogenesis, and contribute to the clin-ical signs, in some cases of canine atopic dermatitis.

Epidermal responses associated with Malassezia dermatitisThe skin acts as a barrier to prevent invasion of micro-organisms and its barrier function is largely providedby the epidermis. The process by which the stratumcorneum is continually renewed by keratinization of the epidermal cells provides a defence against cutane-ous micro-organisms, including supercial fungi. The

renewing process results in continuous shedding of the stratum corneum, which may remove fungalmicro-organisms.119

Similar to other chronic inammatory dermatoses,Malassezia dermatitis is often associated with epider-mal hyperplasia, which is a protective mechanism seenin the skin in response to a variety of environmentalinsults. Mild epidermal hyperplasia has also beenobserved at the sites of application ofMalasseziaorganisms to the skin surface of laboratory beagles.64The mechanism by which epidermal hyperplasiaoccurs inMalassezia dermatitis is however not com-pletely understood. A number of hypotheses can bedrawn up to explain the hyperplasia and lichenicationseen in clinical cases of skin disease (Fig. 2). Theorganism could secrete proteins capable of acting asgrowth factors, and/or interact with surface moleculesof keratinocytes, which could convey signals to stimul-ate their proliferation. Alternatively, the organismmight play an indirect role and the epidermal hyper-plasia could be caused by immune responses stimulatedby the yeast or other underlying diseases commonlyassociated withMalassezia dermatitis. Many of these

underlying diseases can cause inammation and self-trauma, which could then lead to the formation of epidermal hyperplasia (Fig. 2).

Little is known about the direct interaction betweenMalassezia organisms and keratinocytes, either inhumans or in dogs. In a preliminary report, a signi-cant increase in a cellular proliferation marker wasdemonstrated in canine keratinocytes cocultured withM. pachydermatis compared to control cellsin vitro .120This could indicate thatMalassezia organisms mightbe able to activate the proliferative cell cycle of caninekeratinocytes. Recently, the epidermal dysplasia in twoWest Highland white terriers with cytological evidenceof Malassezia overgrowth was reported to be reversibleafter antifungal therapy,121 suggesting a possible patho-genic role ofM. pachydermatis in the epidermal hyper-plasia associated withMalassezia dermatitis. However,

Figure 2. Possible mechanisms for theformation of epidermal hyperplasia inMalassezia dermatitis.Malassezia organismsmight secrete proteins or interact withkeratinocytes, which could have a directeffect on their proliferation (upper pathway).Alternatively,M. pachydermatis might be anindirect factor in the proliferation ofkeratinocytes. In this case, the formation ofepidermal hyperplasia could be induced byimmune responses stimulated by theorganism, or inammation and self-traumacaused by other underlying diseasescommonly associated withMalassezia dermatitis.


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in a study published more recently, we have shown thatextracts or culture supernatants fromM. pachyderma-tis failed to stimulate the proliferation of canine kera-tinocytesin vitro , regardless of whether or not proteaseinhibitors were present.122 We have also shown thatcoculture of whole, viableM. pachydermatis organisms

with cultured canine keratinocytesin vitro did notinduce the cells to proliferate.123 These results suggestthat M. pachydermatis has no direct effect on keratino-cyte proliferation (upper pathway in Fig. 2). However,it must be borne in mind that these werein vitro studiesin which the keratinocytes were isolated from their nor-mal environment. It is possible thatM. pachydermatismight be able to stimulate keratinocyte proliferationdirectly in a living epidermis that had a blood supplyand a functional immune system. An alternative possi-bility is thatM. pachydermatis has no direct effect onkeratinocyte proliferation eitherin vitro or in vivo, andthe epidermal hyperplasia seen inMalassezia dermati-tis is a result of the inammatory response stimulatedby the organism itself or the underlying diseases (lowerpathway in Fig. 2).123 Future studies using living epi-dermis (skin explants) or experimental dogs are there-fore required to further investigate the mechanisms bywhich overgrowth ofM. pachydermatis might causeepidermal hyperplasia in dogs.

MALASSEZIA ORGANISMS ASPATHOGENS IN HUMANS ANDANIMALS

AlthoughMalassezia organisms can be found on nor-mal human skin, they have been implicated in a rangeof both cutaneous and systemic diseases. They aremost frequently associated with pityriasis (tinea) versi-color, which is one of the most common disorders of pigmentation seen in human dermatological clinicsworldwide. Diseases associated withMalassezia spp. inanimals have also been widely reported. In dogs, a der-matitis caused by overgrowth ofM. pachydermatis onthe skin surface is recognized with increasing fre-quency. The pathogenic role ofMalassezia spp. in vari-

ous diseases is therefore a continued topic of interest inhuman and veterinary medical literature.

Diseases associated with Malassezia spp. inhumans

Pityriasis versicolor. Pityriasis versicolor is a chronicsupercial fungal infection of the skin caused byMalassezia spp. organisms.124,125 Since the differenti-ation of the newMalassezia species, several studies onthe mycology of pityriasis versicolor have been docu-mented. The species that have been isolated frompatients with pityriasis versicolor includeM. furfur , M.

globosa , M. restricta , M. sloofae and M. sympodialis ,and more than one species can be found in somepatients.126129 Although some investigators have sug-gested that M. globosa was the causative agent of

pityriasis versicolor because of its predominant pres-ence in populations of affected patients,127 it remains tobe determined whetherM. globosa is also the predomin-ant species in the lesions of individuals. Pityriasisversicolor most often occurs on the trunk, neck andproximal extremities. It is characterized by scaly hypo-or hyperpigmented macules and patches with minimalpruritus (Fig. 3).130 The diagnosis can be made by

potassium hydroxide preparations of skin scrapingsor tape strippings, which reveal typical clusters of yeasts with hyphae.124,125 There are several therapeuticoptions for treating pityriasis versicolor, such astopical treatment with lotions or creams containingselenium, sodium thiosulphate or specic antifungalagents, or oral medication with ketoconazole, ucona-zole or itraconazole. However, relapse is very commonand prophylactic treatment may be required.125

Malassezia folliculitis. Malassezia folliculitis is char-acterized by follicular papules and pustules localized tothe trunk, upper arms, neck, and, less often, the face(Fig. 4). These lesions are generally pruritic.124 Dia-gnosis is based on clinical signs, cytology and culture incombination with histopathology. Budding yeasts and,rarely, hyphae can be found in cytological samples and

Figure 3. Pityriasis versicolor in an adult human. Note themultifocal patches of hypopigmentation over the trunk. Photographcourtesy of Dr Gina Kavanagh, University of Edinburgh.


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in dilated follicles of biopsy sections.6,124 Although ithas been suggested that follicular occlusion was theprimary cause ofMalassezia folliculitis with over-growth of Malassezia organisms as a secondaryevent,131 colonization of normal pilosebaceous unitsby these yeasts can also be heavy.132 The exact role of Malassezia organisms inMalassezia folliculitis there-

fore awaits further elucidation.Malassezia folliculitisresponds rapidly to antifungal therapy. It can betreated with topical antifungal agents or with oralazole antifungal drugs for patients who do not respondto topical treatment. As with pityriasis versicolor,recurrence tends to be a common problem.133

Seborrheic dermatitis and dandruff. Seborrheic derma-titis is characterized by inammation and desquama-tion in areas that are rich in sebaceous glands suchas the scalp, face and upper trunk (Fig. 5), whereasdandruff is a noninammatory scaling condition of thescalp. It is now generally considered that the latter isthe mildest form or a variant of seborrheic dermati-tis.124,133,134 The importance ofMalassezia organismsin these two conditions has been supported by studiesdemonstrating parallel decreases in the number of

organisms and the severity of the diseases.135,136 Thespecies that have been isolated from patients with seb-orrheic dermatitis includeM. furfur , M. globosa andM. sympodialis , with the rst two species showinghigher frequency.129 Seborrhoeic dermatitis can betreated with topical antifungal agents, which can alsobe used prophylactically to reduce the recurrencerate.124,133

Atopic dermatitis. Atopic dermatitis is a chronic,multifactorial, inammatory skin disease associatedwith abnormal immunological regulation (Fig. 6). Asdiscussed in detail above, allergens fromMalasseziaorganisms have been implicated in its pathogene-sis.58,137 The IgE response to the organism has beeninvestigated extensively using several diagnostic meth-ods such as the intradermal test, skin prick test, andELISA. SixMalassezia species have been isolated frompatients with atopic dermatitis, includingM. furfur , M.

globosa , M. restricta , M. sloofae , M. sympodialisand M. dermatis . However, the most frequently iso-lated species reported varies with different investiga-tors.128,129,138 The respective pathogenic role of

differentMalassezia spp. in atopic dermatitis remainsto be claried. For atopic patients with a hypersensitiv-ity response toMalassezia spp., antifungal therapyshould be included in the treatment regime.124

Malassezia fungaemia. Systemic bloodstream infec-tion with Malassezia organisms has been recognizedfor about two decades. It is related to administration of lipids through intravenous catheters, especially toinfants in intensive care units.M. furfur and M. pachy-dermatis are the only two species that have beenreported to cause systemic disease, and the latter isconsidered to be transferred from a household pet as itis rarely isolated from normal human skin.139142 How-ever, otherMalassezia spp. might also be involved butwere not recognized before the new taxonomy wasadopted. Many blood culture systems do not effectively

Figure 4. Malassezia folliculitis on the upper back of an adulthuman. Note the multiple papules and pustules.Malassezia organisms can be found on cytological specimens. Photographcourtesy of Dr Gina Kavanagh, University of Edinburgh.

Figure 5. Seborrhoeic dermatitis on the neck of an adult human.Note the erythema and scaling around the hair margin.Malassezia organisms were found on stained tape strips.

Figure 6. Atopic dermatitis on the exural surface of the elbow in anadult human. These lesions can be colonized byMalassezia organisms, resulting in a hypersensitivity response to allergens fromthe yeast.


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support the growth of lipid-dependentMalasseziaspp. and this may also hinder the identication.143 Thecatheter has to be removed if antifungal therapy isunsuccessful, possibly because of incomplete penetra-tion of drugs to organisms that are embedded in thecatheter.4,7

In addition to the diseases described above, the iso-lation of Malassezia spp. from a range of other humanskin conditions including psoriasis, otitis and acne hasalso been reported.7,143,144

Diseases associated with Malassezia spp. inanimalsAn Indian rhinoceros with exfoliative dermatitis wasthe rst case of skin disease that was reported to beassociated withM. pachydermatis .16 More recently,similar lesions have been observed in a southern whiterhinoceros.145 Malassezia organisms have also beenfound in a variety of other animals with diseases.Nonlipid-dependentMalassezia yeasts were recentlyisolated from a horse showing an erythematous patchof alopecia on the face, which responded well to a mico-nazole/chlorhexidine shampoo.146 M. pachydermatis -associated dermatitis has also been reported in sealions.147,148 In addition to dogs and cats, otitis externacaused byM. pachydermatis has been described in fer-rets, fennecs, pigs and dromedaries.1,149 In contrast, thespecies that have been isolated from cattle with otitisare M. globosa , M. sympodialis , M. furfur and M.sloofae , with higher frequencies seen with the rst twospecies.150,151 A case of noncutaneous disease has beendocumented in a macaw with an ulcer in the crop fromwhichM. pachydermatis was isolated.152

In contrast to dogs, in whichMalassezia dermatitisand otitis are frequently diagnosed, these conditionsare less common in cats. The clinical signs in catsinclude pruritus, erythema, self-excoriation, and lesscommonly lichenication (Fig. 7).153 M. pachyder-matis has also been associated with feline chin acne.154Three lipid-dependentMalassezia spp. (M. furfur , M.

globosa and M. sympodialis ) have been isolated fromhealthy cats4952 to date, and M. sympodialis and M.

pachydermatis have been reported to be associated with

otitis externa in cats.155,156

Malassezia overgrowth hasalso been reported in cats with thymoma-associateddermatitis and paraneoplastic alopecia.157159

Malassezia dermatitis in dogs. Malassezia dermatitisin dogs was rst reported in 1983 by Dufait.3 To date,the potentially important role ofM. pachydermatis inthis condition has gained widespread acceptance dueto the consistent recovery of elevated populations of M. pachydermatis from the skin of affected dogs andthe favourable therapeutic response that occurs to anti-fungal therapy.

Predisposing factors for overgrowth of Malasseziapachydermatis. The predisposing factors forMalas-sezia overgrowth on the skin of dogs are still a focus of research and debate. Two mechanisms that have been

suggested to trigger overgrowth of the yeast are altera-tions in host defence mechanisms and changes in thecutaneous microenvironment.70,160 By causing thesechanges, various diseases have been suggested asunderlying causes ofMalassezia dermatitis.

As described earlier, a disrupted epidermal barrierrenders the skin more prone to bacterial and yeastinfections. Diseases that can cause a decrease in cuta-neous barrier function and are commonly associated

with Malassezia dermatitis are hypersensitivity dis-eases (especially atopic dermatitis), parasite infestationand keratinization disorders.160,161 Alterations in theimmune system caused by hypersensitivity and endo-crine diseases are also thought to predispose toMalas-sezia dermatitis.160

The cutaneous microenvironment is generallyconsidered to be important in controllingMalasseziapopulations. Malassezia dermatitis seems to be morecommon in warm, humid climates and seasons, and incertain anatomic sites such as skin folds, suggestingthat increased cutaneous humidity favours yeastgrowth.161 Additionally, changes in lipids on the skinsurface, resulting in increased availability of nutrientsand growth factors forMalassezia organisms, may pro-mote their proliferation. The diseases that can causechanges in sebum production and that are associated

Figure 7. (a) Malassezia overgrowth in a cat at the site of extensionap surgery, used to close a wound. The area was inamed, pruriticand exudative. Large numbers ofMalassezia organisms were seen onstained tape strips. (b) The same area after 7 days of topicalantifungal therapy with 2% chlorhexidene/2% miconazole shampoo(Malaseb, Leo Animal Health, Denmark). The small ulcer at thecentre of the picture is an area of granulation tissue surrounding asuture.


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withMalassezia dermatitis include endocrine diseases,bacterial skin diseases (in which the bacteria couldrelease lipase), and keratinization disorders such asseborrheic dermatitis.160162

Genetic predisposition appears to be important incertain breeds, especially West Highland white terriers,

Basset hounds, dachshunds, cocker spaniels, ShihTzus, and English setters.161163 Furthermore, the useof certain medications such as long-term glucocorti-coid therapy may also be predisposing factors.161 Someinvestigators have suggested that antibiotic treatmentis associated with increasedMalassezia populations,162whereas others do not support this point of view.160

Pathogenesis. The pathogenesis ofMalassezia derma-titis in dogs has not been fully elucidated. In additionto the immunological factors described earlier in thisreview, there are several other mechanisms by whichM. pachydermatis might cause pathological changesin the skin of dogs. Zymogen (an inactive pro-enzyme)in the yeast cell wall is capable of activating thecomplement system. This could result in damage tokeratinocyte integrity, leading to epidermal spongiosis,inammation and pruritus. A defective epidermalwater barrier, caused either by direct keratinocyte dam-age or by underlying atopic dermatitis, could lead to anincrease in humidity on the skin surface, thus favouringyeast proliferation.70 Additionally, the disrupted epi-dermal barrier could permit the skin immune system tobe exposed toMalassezia antigens and products, elicit-ing inammatory and/or hypersensitivity reactions.161

As proteases are believed to be the mediator of itchat free nerve endings in the skin, the proteases releasedbyMalassezia organisms could also contribute to pru-ritus.164 Malassezia organisms also produce lipases,which alter sebum production and produce free fattyacids on the skin surface. Released lipids can be usedby yeasts for nutrition, and free fatty acids would pro-vide protection by inhibiting other organisms.165

Clinical features. Malassezia dermatitis occurs in dogsof any age, sex and breed, but is more often diagnosedin dogs between 1 and 3 years of age.166 Some breeds

also appear to be predisposed (listed above). The der-matitis often begins in the summer or in humidmonths, which also corresponds to the allergy season,and then persists into winter. There is a second spike of cases in early spring.70,161

Skin lesions may be localized or generalized.Regional dermatitis commonly occurs in the externalear canal (Fig. 8), or on the face, ventral neck (Fig. 9),axillae (Fig. 10), groin, interdigital skin or intertrigi-nous areas. Skin lesions are characterized by erythema,alopecia, greasy exudation and varying degrees of scal-ing. Chronic cases can have marked hyperpigmenta-tion and lichenication (Fig. 11). Pruritus varies frommild to extremely severe. Dogs with generalized lesionsoften have an offensive, rancid or yeasty odour.Malas-sezia paronychia may occur with or without more gen-eralizedMalassezia dermatitis. In these cases, there is

reddish-brown staining of the claws or hair, withinammation of the surrounding soft tissue (Fig. 12).In some cases, a localized area ofMalassezia over-growth can occur following persistent licking (Fig. 13).Most dogs withMalassezia dermatitis have concurrentdermatoses, especially hypersensitivity disorders,ectoparasitic infestation, bacterial pyoderma, endo-crinopathies, or keratinization defects.70,153,161,166,167

Figure 8. Malassezia otitis in a Golden retriever secondary tounderlying atopic dermatitis. Note the typical brown and waxydischarge.

Figure 9. Malassezia dermatitis on the ventral neck of a Bassethound. The skin is erythematous and the hair is stained by a browndiscoloration. This is a predilection site in a predisposed breed.


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However, it is important to remember that in somecases, especially in predisposed breeds, there is no iden-tiable underlying cause and the dogs skin diseaseresolves completely with antifungal therapy.

Diagnosis. The criteria required for the diagnosis of Malassezia dermatitis have not been denitively estab-lished. It has been proposed that a diagnosis of Malas-sezia dermatitis is appropriate when a dog withelevated M. pachydermatis populations on lesionalskin shows a good clinical and mycological response toappropriate antifungal therapy.47 The diagnostic toolsused to identify elevated populations ofMalasseziayeasts on the skin include cytological, cultural andhistopathological techniques.

Cytological examination is the most useful tech-nique that allowsMalassezia populations to be rapidlyassessed. A variety of methods have been used tocollect cytological samples for the evaluation of theorganisms. These include pressing a piece of clear ace-tate tape onto lesional skin several times, vigorouslyrubbing a cotton swab on the skin surface, performinga supercial skin scraping, and directly pressing a glassslide onto lesional skin.70,161 In many clinicians hands,the acetate tape technique appears to be the most use-ful clinically, especially for sampling between the toesor dry or greasy areas of skin. Cotton swabs are typic-

ally used to obtain material from the ear canal. Directimpression with a glass slide is possible if the skin sur-face is at and greasy.153,160,161 It is difcult to deter-mine which sampling method is the best as each has itsown benets and disadvantages. It has been shown thatswabs, skin scrapings and impression smears gave sim-ilar results in normal dogs.46 Using dogs with elevatedcutaneousMalassezia populations, Bond and Sant168observed relatively higher numbers ofMalasseziaorganisms using tape stripping and dry scraping thanwith damp swabs. Some investigators have reportedthat direct impression with a glass slide was the mostreliable technique for producing uniform cytologicalpreparations.162

The samples collected are transferred to a glass slideand stained with a suitable cytological stain suchas Diff-Quik (Dade AG, Dudingen, Switzerland),

Figure 10. Malassezia dermatitis in the axilla of a West Highlandwhite terrier, secondary to underlying atopic dermatitis. Note theerythema, scaling, lichenication and hyperpigmentation, indicatingchronicity. The papular eruption indicates concurrentstaphylococcal infection.

Figure 11. Severe and chronicMalassezia dermatitis on the ventralneck of a Shar pei. Note the extreme lichenication andhyperpigmentation.

Figure 12. Brown staining of the proximal claws in a dog withMalassezia paronychia.Malassezia organisms can often be found inscrapings of this material.

Figure 13. Localized area ofMalassezia dermatitis followingpersistent licking. The dog had started to lick the medial thighfollowing stie surgery. Large numbers ofMalassezia organismswere found on stained tape strips and the condition responded

completely to topical antifungal therapy.


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Giemsa, or methylene blue.160 The slides should beexamined under high power (400) or preferably withan oil immersion lens (1000) on a light microscope.Microscopic examination reveals round to oval yeastswith monopolar budding (Fig. 14). Yeasts are oftenseen in clusters or adhered to keratinocytes.161 As M.

pachydermatis can be found in a small number onhealthy dog skin, it is difcult to dene the amount of yeast that is pathogenic. It has been suggested thatpopulations should be considered elevated if the yeastis readily identied.167 Some investigators have alsosuggested that an elevated population is more likelywhen a certain number of yeasts are found.161 The vari-ous criteria proposed include greater than 10 organ-isms in 15 randomly chosen oil-immersion microscopicelds (1000) using tape stripping samples; an averageof greater than or equal to four organisms per oil-immersion microscopic eld; an average of greaterthan or equal to one organism per eld in 10 oil-immersion microscopic elds; and greater than twoorganisms per high power eld (400) with specimensobtained using any of the commonly used samplingtechniques. In view of the conicting data, the authorswould recommend, as a general guide, that nding one

organism per oil-immersion eld in the presence of clinical signs can usually be taken to indicate an over-growth ofM. pachydermatis .

Four methods have been described to cultureMalassezia organisms from the skin including cottonswabs,45,169 adhesive tapes,46,169,170 contact plates171and detergent scrubs.172 M. pachydermatis grows wellon both Sabourauds dextrose agar and modiedDixons agar at 3237C. However, an atmospherecontaining 510% carbon dioxide signicantlyincreases the frequency of isolation and colony countson Sabourauds dextrose agar, but not on modiedDixons agar.173 Nevertheless, modied Dixons agar, alipid-supplemented medium, may be advantageous fordiagnostic purposes because it supports the growth of more lipid-dependent variants ofM. pachydermatis 174and the lipid-dependentMalassezia spp. that may be

found on cats.4952 As Malassezia spp. are commensalorganisms and as elevated populations can be readilyfound on cytology, culturing is not usually necessaryand rarely used by most clinicians.160,161 However,quantitative culture has been reported to have diagnos-tic value by some authors.171,172

Malassezia organisms may also be demonstrated bymicroscopic examination of skin biopsy specimens(Fig. 15). The yeasts are typically located in the stra-tum corneum and are occasionally seen in the follicularinfundibulum. Because of the possible loss or disrup-tion of the stratum corneum during processing, skinbiopsy is generally considered to be less sensitive andreliable than other diagnostic tools. There are severalcharacteristic histological features in skin biopsysamples from dogs withMalassezia dermatitis, althoughnone of these are pathognomonic.121,161,175178 The epi-dermis is characterized by marked irregular hyper-plasia with formation of deep rete ridges (Fig. 15a).Hyperplasia can also be seen in the follicular infundib-ula. Orthokeratotic hyperkeratosis with focal parak-eratosis is frequently observed. Spongiosis is marked inactive lesions (Fig. 15b). The dermal inammation isusually supercial perivascular to interstitial withlymphocyte exocytosis and focal accumulations of neutrophils. Eosinophils are present occasionally. Linearalignment of mast cells at the dermoepidermal junc-tion can also be observed in some affected dogs.178,179Scott and Miller175 described a hyperplastic dermatosisassociated with secondaryM. pachydermatis infectionin West Highland white terriers. They proposed a term,epidermal dysplasia, to describe the epidermal hyper-plasia with round-bottomed rete ridges in these dogs.

Treatment. The treatment of canineMalassezia der-matitis is currently based on the use of topical and sys-temic antifungal therapy.153 A combination of topicaland systemic therapy may speed resolution of the dis-ease and increase efcacy160,161 although, in the UK,topical therapy alone would be the initial treatment of choice. Topical agents that can be used forMalasseziadermatitis include chlorhexidine, clotrimazole, enilco-nazole, ketoconazole, miconazole, nystatin and sele-

nium sulphide.70,161

It has been reported thatM. pachydermatis showed sensitivity, in decreasing orderof efcacy, to ketoconazole, econazole, clotrimazole,miconazole and nystatinin vitro ,180 although the meth-ods used in this study could be questioned because theydid not take into account the diffusion properties of the various drugs on the media. These agents are usedin various forms of topical antifungal products such assprays, ointments and shampoos. A double-blindedstudy demonstrated that a shampoo containing 2%chlorhexidine and 2% miconazole (Sebolyse Medic-ated Foam, Dermcare-Vet, Australia), when usedevery 3 days for 3 weeks, was effective for treatingMalassezia dermatitis in dogs. This was because of itsdegreasing, anti-Malassezia and antibacterial proper-ties.181 The commonly used systemic agents forMalas-sezia dermatitis in dogs are the azoles. Ketoconazole or

Figure 14. Cytological examination of a Diff-Quik-stained tapestrip obtained from the skin of a dog withMalassezia dermatitis(1000). Numerous round to oval or peanut-shaped budding yeastsare present.


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itraconazole are usually given at 510 mg kg1 per dayper os for 2130 days.153,160 However, adverse effectsincluding anorexia, vomiting, diarrhoea and hepato-toxicity have been reported.153 A recent study demon-strated that itraconazole given at 5 mg kg1 every 24 hper os on 2 consecutive days per week for 3 weeks

(pulse administration) was as effective as when given at5 mg kg1 every 24 h on a daily basis for 21 days intreating canineMalassezia dermatitis.182

Clinical improvement usually occurs within 7 14 days after the start of antifungal therapy. AsMalas-sezia dermatitis in dogs is often associated with anunderlying disorder, the dog should be evaluated forconcurrent diseases in order to prevent frequentrelapses. For frequently recurring cases, either due toprimary disease or uncontrollable underlying factors,shampoo therapy or pulse oral medication may be usedprophylactically.153,161,167

CONCLUSIONS

Advances in molecular techniques have led to a clearerclassication ofMalassezia yeasts. Certain specieswithin the genusMalassezia have been associated withskin diseases, both in humans and animals. Currentresearch has revealed thatMalassezia organisms caninduce immunological responses in normal and atopicindividuals. The identication of several major aller-gens ofMalassezia organisms in atopic humans anddogs has provided the potential for future developmentof immunotherapy for chronically affected patients.Although the characteristics of the dermatoses associ-ated with Malassezia spp. such as greasy scales andepidermal hyperplasia have been well recognized, theinteraction betweenMalassezia organisms and the epi-dermis remains incompletely understood. Future stud-ies aiming to identify important factors which mediatethe initiation of inammation or the formation of epi-dermal hyperplasia seen inMalassezia dermatitis, suchas cytokines and adhesion molecules, might also leadto novel approaches to prevent or better controlMalassezia dermatitis in humans and dogs.

ADDITIONAL NOTE

A new species ofMalassezia has recently beendescribed and namedMalassezia nana (nana is Latinfor a female dwarf, so named because of the organismscomparatively small cells). The organism was isolatedfrom the skin of a cat and from the ear canal of cows.183

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Figure 15. Histopathology of canineMalassezia dermatitis.(a) Irregular epidermal hyperplasia and hyperkeratosis (40).(b) The epidermis shows marked spongiosis and orthokeratotichyperkeratosis with focal parakeratosis (250).Malassezia yeasts arevisible in the stratum corneum (thin arrow). Some keratinocytesshow multiple nucleoli, indicating mitotic activity (bold arrows).(c) Budding yeasts showing characteristic morphology ofMalassezia spp. (thin arrows,400). Scale bars for (a), (b) and (c) are 100, 10 and10m, respectively. H&E stain. Section courtesy of Dr S. Rhind,University of Edinburgh.


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Documents

The Biology of Malassezia Organisms and Their Ability to Induce Immune Responses and Skin Disease (Pages 4–26)