Malassezia: Zoonotic Implications, Parallels and Differences in Colonization and Disease in Humans and Animals

Malassezia spp. are commensals of the skin, oral/sinonasal cavity, lower respiratory and gastrointestinal tract. Eighteen species have been recovered from humans, other mammals and birds. They can also be isolated from diverse environments, suggesting an evolutionary trajectory of adaption from an ecological niche in plants and soil to the mucocutaneous ecosystem of warm-blooded vertebrates. In humans, dogs and cats, Malassezia-associated dermatological conditions share some commonalities. Otomycosis is common in companion animals but is rare in humans. Systemic infections, which are increasingly reported in humans, have yet to be recognized in animals. Malassezia species have also been identified as pathogenetic contributors to some chronic human diseases. While Malassezia species are host-adapted, some species are zoophilic and can cause fungemia, with outbreaks in neonatal intensive care wards associated with temporary colonization of healthcare worker’s hands from contact with their pets. Although standardization is lacking, susceptibility testing is usually performed using a modified broth microdilution method. Antifungal susceptibility can vary depending on Malassezia species, body location, infection type, disease duration, presence of co-morbidities and immunosuppression. Antifungal resistance mechanisms include biofilm formation, mutations or overexpression of ERG11, overexpression of efflux pumps and gene rearrangements or overexpression in chromosome 4.


Introduction
Malassezia are small thick-walled ovoid, ellipsoid or cylindrical commensal yeasts of warm-blooded vertebrates. Their genome of approximately 10 Mb is almost half the size of Cryptococcus, another basidiomycete of medical and veterinary importance [1,2]. The mycelial phase of Malassezia spp. has been observed naturally in some skin lesions and induced in specialized culture media incubated at 30 • C [2][3][4][5][6]. Malassezia species reproduce asexually by unipolar broad-based budding. The sexual form has not been detected, although the mating-type locus region has been identified [7].
An important characteristic of all Malassezia is their dependence on lipids for growth due to an absent fatty-acid synthetase gene and consequent inability to synthesize longchain fatty acids. Although one species, M. pachydermatis, can readily grow on Sabauraud's dextrose agar (SDA), a medium without lipid supplementation, it is still lipid dependent and its growth in this medium is due to the use of lipid fractions within the peptone, a component of SDA [2,[8][9][10][11].
M. furfur was first identified on human skin in 1846 [12], but recently the genus has received more attention, not only because of its association with dermatological diseases in animals (dermatitis, otitis externa) and humans (pityriasis versicolor, atopic dermatitis,
Malassezia species colonization of the skin starts directly after birth, increases until around 12 months of age, and then remains relatively static until puberty, when another significant quantitative increase in colonization occurs, associated with increased sebaceous gland activity and changes in the lipid composition of the skin [6,77]. After puberty, Malassezia species comprise 50 to 80% of the human mycobiome [78][79][80]100]. The limited data currently available about cutaneous mycobiomes in preterm and term neonates shows that Malassezia species distribution on the skin of neonates and children varies between studies, but M. globosa, M. furfur, M. sympodialis and M. restricta are the most prevalent species described [14].
Climate and ethnicity also impact the carriage of Malassezia species [94,95]. In a study by Leong et al. in 2019, people in Singapore of four different ethnicities (Chinese, Malay, Indian and Caucasian) carried a higher number of Malassezia species and showed greater species diversity and evenness than Caucasians in Switzerland. The predominant species (isolated by culture from the skin of the side of the nose) in the latter were M. restricta and M. sympodialis, while M. globosa was absent. In contrast, sampling from the same site in the four ethnic groups in Singapore showed M. globosa, M. furfur and M. restricta were the dominant species. Caucasians from the two locations showed different species distributions, with M. restricta being twice as common among those in Singapore, while M. globosa was absent in Swiss Caucasians. The same study associated the lower temperature and humidity of Switzerland compared to Singapore with a lower positive culture rate and lower species diversity [95] (Figure 1). From other studies, it can be concluded that M. restricta plays a dominant role as a skin commensal in Europe, whereas M. globosa comparatively dominates in Asia [13,96]. distributions, with M. restricta being twice as common among those in Singapore, while M. globosa was absent in Swiss Caucasians. The same study associated the lower temperature and humidity of Switzerland compared to Singapore with a lower positive culture rate and lower species diversity [96] (Figure 1). From other studies, it can be concluded that M. restricta plays a dominant role as a skin commensal in Europe, whereas M. globosa comparatively dominates in Asia [13,97]. Several studies have shown that sex and body site also influence the species of Malassezia species present on the skin and their abundance [1,89,92,[98][99][100][101]. Site-specific species include M. restricta, which favors colonization of the external ear canals, retroauricular crease and forehead and M. globosa, which is most commonly isolated from the back, occiput and groin [100,102].
A Japanese study in 2010 quantified Malassezia colonization of the skin of the cheek using real-time PCR and determined associations with gender and age in 770 healthy individuals [100]. Total Malassezia DNA in males stayed constant from age 0 until around 9 years of age, with a progressive increase each year thereafter until the age of 16 to 18. In females, total Malassezia DNA increased until the age of 12, decreased between the ages of 19 and 22, and then increased again between the ages of 30 and 39. In both genders, there was a gradual decrease in Malassezia species abundance over the course of life. Overall, males tended to have more abundant Malassezia DNA than females, and M. globosa and M. restricta were the dominant species for both for all ages.
Malassezia species carriage at different skin locations was investigated using culturebased methods. No significant differences between the genders were found. While M. restricta dominated the scalp and M. sympodialis dominated the trunk, M. globosa was about equally common at both locations [103]. Several studies have shown that sex and body site also influence the species of Malassezia species present on the skin and their abundance [1,88,91,[97][98][99][100]. Site-specific species include M. restricta, which favors colonization of the external ear canals, retroauricular crease and forehead and M. globosa, which is most commonly isolated from the back, occiput and groin [100,101].
A Japanese study in 2010 quantified Malassezia colonization of the skin of the cheek using real-time PCR and determined associations with gender and age in 770 healthy individuals [99]. Total Malassezia DNA in males stayed constant from age 0 until around 9 years of age, with a progressive increase each year thereafter until the age of 16 to 18. In females, total Malassezia DNA increased until the age of 12, decreased between the ages of 19 and 22, and then increased again between the ages of 30 and 39. In both genders, there was a gradual decrease in Malassezia species abundance over the course of life. Overall, males tended to have more abundant Malassezia DNA than females, and M. globosa and M. restricta were the dominant species for both for all ages.
Malassezia species carriage at different skin locations was investigated using culturebased methods. No significant differences between the genders were found. While M. restricta dominated the scalp and M. sympodialis dominated the trunk, M. globosa was about equally common at both locations [102].
Other factors that may influence the colonization of Malassezia species include host factors (immune response, body secretion, skin occlusion), other skin inhabitants (e.g. parasites, other microbes) and environmental parameters, including exposure to ultraviolet light [96]. Even the birth process itself has a significant impact. If a baby is born via natural delivery, its skin microbiota resembles the mother's vaginal communities, but if delivered via caesarian section, it represents the mother's skin surface population [103][104][105][106][107]. In addition, vaginal birth is associated with a higher abundance of Malassezia [108,109].
Malassezia species were previously thought to be commensals of the skin only. Although the skin is the primary ecological niche, more recent data demonstrate that these yeasts also colonize the mucosa of the sinonasal and oral cavities, as well as the gastrointestinal and lower respiratory tract [110][111][112][113][114][115]. Malassezia species are dominant members of the mycobiome of the sinuses, with M. restricta and M. sympodialis most frequently detected [116]. Malassezia also comprise 30% of the gastrointestinal mycobiome, with three species detected-M. globosa, M. restricta and M. pachydermatis [117]. The fungal burden in the lungs of healthy people is relatively low. In one study, using a metagenomic approach, the lung mycobiome was characterized by a high proportion of basidiomycetes, including M. restricta and M. globosa [118], while in another ascomycetes, including Candida species, were most abundant [119] (Figure 1).

Malassezia Species and Their Role as Commensals in Companion Animals
Using culture-based techniques, Malassezia species have been identified as the most common yeast colonizing healthy canine skin [120,121]. Metagenomic approaches reveal that, in contrast to humans, Ascomycota, especially Alternaria and Cladosporium species, are the most abundant fungal species on the skin of healthy dogs and cats [122,123].
Other factors that may influence the colonization of Malassezia species include host factors (immune response, body secretion, skin occlusion), other skin inhabitants (e.g. parasites, other microbes) and environmental parameters, including exposure to ultraviolet light [97]. Even the birth process itself has a significant impact. If a baby is born via natural delivery, its skin microbiota resembles the mother's vaginal communities, but if delivered via caesarian section, it represents the mother's skin surface population [104][105][106][107][108]. In addition, vaginal birth is associated with a higher abundance of Malassezia [109,110].
Malassezia species were previously thought to be commensals of the skin only. Although the skin is the primary ecological niche, more recent data demonstrate that these yeasts also colonize the mucosa of the sinonasal and oral cavities, as well as the gastrointestinal and lower respiratory tract [111][112][113][114][115][116]. Malassezia species are dominant members of the mycobiome of the sinuses, with M. restricta and M. sympodialis most frequently detected [117]. Malassezia also comprise 30% of the gastrointestinal mycobiome, with three species detected-M. globosa, M. restricta and M. pachydermatis [118]. The fungal burden in the lungs of healthy people is relatively low. In one study, using a metagenomic approach, the lung mycobiome was characterized by a high proportion of basidiomycetes, including M. restricta and M. globosa [119], while in another ascomycetes, including Candida species, were most abundant [120] (Figure 1).

Malassezia Species and Their Role as Commensals in Companion Animals
Using culture-based techniques, Malassezia species have been identified as the most common yeast colonizing healthy canine skin [121,122]. Metagenomic approaches reveal that, in contrast to humans, Ascomycota, especially Alternaria and Cladosporium species, are the most abundant fungal species on the skin of healthy dogs and cats [123,124].
In dogs, similar to cats, M. pachydermatis dominates the healthy ear canal, although other species, such as M. sympodialis and M. obtusa, can also be detected [142,149,150,153] ( Figure 2). In diseased canine ears, the colonization rate increases, but M. pachydermatis remains most prevalent, followed by M. sympodialis, M. furfur, M. obtusa, M. globosa and M. restricta. This has mostly been shown by cultural and biochemical evaluation methods [142,150,153,154].
Allergic dogs have a higher abundance of Malassezia species, especially of M. pachydermatis, compared to healthy dogs [140,148] as well as a decreased overall diversity [155].
In particular, in dogs with atopic dermatitis, the isolation of M. sympodialis associated with M. pachydermatis and/or M. furfur has also been reported [156]. The coexistence of M. pachydermatis with other Malassezia species makes the pathogenic role determination of a single Malassezia species challenging.
Other body sites where Malassezia species are commensals have not been well characterized in dogs and cats. Low numbers of M. pachydermatis are present on the corneal surfaces of healthy dogs [120].
In a recent study, the mycobiome of the healthy canine oral cavity was found to be dominated by Malassezia species and Cladosporium species. Malassezia arunalokei, M. restricta, M. pachydermatis and M. globosa have all been detected in the oral cavity of healthy and diseased dogs (Figure 2), although no correlation was found between these Malassezia species and periodontal disease [76].
In the gastrointestinal tract of dogs, Ascomycota and Basidiomycota are the most numerous, with Candida as a major genus [157][158][159][160]. Malassezia species have not yet been detected. In cats, even less data are available, but Ascomycota seem to be dominant [158].
The urinary mycobiome of healthy dogs was recently characterized, and reads of several fungal genera in very low abundance were detected, including M. restricta [161].

Malassezia Species in Other Animals
Malassezia species have been isolated from multiple mammalian and avian species (Table 1). In pigs, Malassezia species, especially M. sympodialis and M. slooffiae, were isolated from 73% of healthy ear samples but not from multiple skin sites [162]. M. pachydermatis has been isolated from pigs with otitis externa and from the skin of healthy farmed pigs [163,164]. One study [165] compared Malassezia species detection rates and species from healthy porcine ears among different porcine breed and age groups, showing that, similar to humans and companion animals, genetic factors and age can impact Malassezia abundance and diversity [2,85,95,141,145,152,166]. Overall, Malassezia were isolated from 22.5% of sampled pigs, including M. pachydermatis, M. furfur and M. sympodialis. M. pachydermatis was found in all breeds but not in adults of large breeds, whereas M. furfur and M. sympodialis were only present in adult pigs of large breeds [165].
Several Malassezia species were isolated from multiple skin sites of 12 to 60% of healthy cattle using culture-based techniques [149,167] (Table 1). One study showed a clear difference in the species isolated in cases of otitis externa depending on the time of the year, with thermotolerant M. sympodialis dominating in summer and less thermotolerant M. globosa being predominant in winter [168].
A variety of Malassezia species have been isolated from the skin of horses, sheep and goats (Table 1). In goats, skin infections have been associated with M. pachydermatis and M. slooffiae [169,170].
Among rabbits, M. cuniculi is commonly detected in healthy skin and ears [30,171]. In one study, rabbits bred for meat consumption were more frequently colonized with Malassezia species compared to pet rabbits [171]. In contrast to humans, Malassezia species were more commonly present in young rabbits (<3 months of age), and diet impacted Malassezia species prevalence.  125,172]. M. sympodialis has also been commonly found in diseased combs of adult chickens [173].

Zoonotic and Reverse Zoonotic Transmission of Malassezia Species
There is now ample evidence that different Malassezia species are shared between humans and animals (Table 1). However, some genotypes within a species might be host adapted or linked to a particular host site location or skin disorder [27,30,31,74,110,174]. In particular, sequence analyses of the LSU rDNA showed distinct Malassezia species subtypes on different host species [110]. Sequence analysis of IGS1 distinguished specific M. globosa, M. restricta, and M. pachydermatis variants in seborrheic dermatitis and atopic eczema and on the healthy skin of humans and animals [85,174]. Among M. pachydermatis, eight IGS1 subtypes were identified and subtype 3D was mainly associated with skin lesions [175]. Additionally, M. pachydermatis, frequently isolated from cats and dogs [176][177][178][179][180], but rarely from human skin [62,181,182] was known to cause fungemia in people, especially in neonates [34, 63,[183][184][185][186][187][188][189][190][191]. However, newborn babies have skin colonization by M. sympodialis and M. globosa, but not by M. pachydermatis [78][79][80]100]. Thus, the ease with which these yeasts can be transmitted from one body site to another [192] or between animals and their owners [182] makes us hypothesize that zoonotic and reverse zoonotic transmission of these yeast species can occur.
In particular, the carriage of M. pachydermatis in healthy and diseased dogs with allergic dermatitis or otitis externa was compared to healthy human owners [182]. M. pachydermatis DNA was identified on the palms of over 90% of pet owners, regardless of the disease state of their dogs. Based on culture results indicating the relative abundance of Malassezia species, owners of affected dogs were 11 times more likely to be culture positive than owners of healthy dogs [182].
The zoophilic potential of M. pachydermatis was first postulated by Dr. Gueho [193] but was clearly confirmed ten years later when an outbreak of neonatal fungemia caused by M. pachydermatis was investigated [184]. The strain implicated in the outbreak was isolated from a health care worker's hands, from contaminated equipment and from dogs belonging to three health care workers working in the involved intensive care nursery unit. One or several healthcare workers likely contaminated the nursery environment and their patients after transient colonization of their hands by the organism. After optimizing hand hygiene, no further cases were reported and all cultures from staff members tested negative [184].
Other studies have also demonstrated that hospitalized infants can be colonized by Malassezia species, especially M. pachydermatis and M. furfur, via contact with their parents or healthcare workers or indirectly via incubator surfaces [16,77,183,184,[194][195][196]. Healthcare workers can then further transmit the organism from one infant to another via their hands. Through this mechanism, several Malassezia species outbreaks have occurred in the past [184,197,198].
Carriage of M. pachydermatis in humans was detected in low numbers on the scalp and palms of 12% of healthy individuals in one study [181], and on the skin of 5% of healthy medical students in another [62]. In other studies it was not detected at all in healthy individuals, and overall appears to be a rare, transient colonizer of human skin [6,102]. Similarly, other studies have found no causal associations between M. pachydermatis and human Malassezia-associated skin conditions, including seborrheic dermatitis and pityriasis versicolor [62,199].
While there is evidence that M. pachydermatis can be transmitted between dogs and humans, further investigations into the genotypes involved, and the strain characteristics are warranted [184,[188][189][190]200]. The relatively recent discovery of M. pachydermatis as a commensal of the human gut introduces another potential reservoir of infection in humans by this species [117].
There is phenotypic and phylogenetic evidence that species with high host diversity, such as M. furfur, are undergoing diversification to enable successful adaptation to different hosts [201]. Strains from different animal species remain closely genetically related, but the extent and frequency of zoonotic or reverse zoonotic transmission have not been investigated.

Malassezia-Associated Dermatological Diseases in Humans
The most common Malassezia-associated skin diseases in human patients are pityriasis versicolor, seborrheic dermatitis, Malassezia folliculitis and atopic eczema [6,13,38,[202][203][204]. The skin sites and species involved in these diseases are shown in Figure 3. [6,103]. Similarly, other studies have found no causal associations between M. pachydermatis and human Malassezia-associated skin conditions, including seborrheic dermatitis and pityriasis versicolor [62,200].
While there is evidence that M. pachydermatis can be transmitted between dogs and humans, further investigations into the genotypes involved, and the strain characteristics are warranted [185,[189][190][191]201]. The relatively recent discovery of M. pachydermatis as a commensal of the human gut introduces another potential reservoir of infection in humans by this species [118].
There is phenotypic and phylogenetic evidence that species with high host diversity, such as M. furfur, are undergoing diversification to enable successful adaptation to different hosts [202]. Strains from different animal species remain closely genetically related, but the extent and frequency of zoonotic or reverse zoonotic transmission have not been investigated.

Malassezia-Associated Dermatological Diseases in Humans
The most common Malassezia-associated skin diseases in human patients are pityriasis versicolor, seborrheic dermatitis, Malassezia folliculitis and atopic eczema [6,13,38,[203][204][205]. The skin sites and species involved in these diseases are shown in Figure 3.

Pityriasis Versicolor
Pityriasis versicolor, sometimes called tinea versicolor, is a common disease worldwide, with a prevalence of up to 50% in hot and humid regions. There is no gender or ethnic predisposition [206,207]. The disease is most commonly seen in young adult to adult patients, correlating with increased sebaceous gland activity and altered lipid

Pityriasis Versicolor
Pityriasis versicolor, sometimes called tinea versicolor, is a common disease worldwide, with a prevalence of up to 50% in hot and humid regions. There is no gender or ethnic predisposition [205,206]. The disease is most commonly seen in young adult to adult patients, correlating with increased sebaceous gland activity and altered lipid composition of the skin around this time. The disease is clearly associated with Malassezia species, especially M. furfur, M. globosa and M. sympodialis. A combination of factors including genetics, warm and humid environment, immunodeficiency, pregnancy, oily skin or application of oily topical substances lead to a transformation of resident Malassezia species into a pathogenic filamentous form. Most patients have multiple affected areas characterized by well-demarcated, oval, hyper-or hypopigmented macules with a fine scaly surface. These lesions are variably pruritic and the neck, trunk and proximal extremities are commonly involved [207][208][209] (Figure 3). Diagnosis is usually made clinically, but in individual cases, Wood's lamp examination (coppery-orange fluorescence) or microscopic examination of fungal elements may be needed [6,210,211].

Seborrheic Dermatitis
Seborrheic dermatitis also occurs worldwide, with 'normal' and dandruff forms affecting around 5% and up to 50% of the population, respectively. There is also an HIVassociated form. There is no ethnic predisposition, but males are clearly predisposed. Disease is mainly seen in infants and adults [212][213][214]. The etiology is not completely clear but involves an interplay of skin flora, lipid composition on the skin surface, skin barrier integrity, immune response to Malassezia species and individual host factors. Increased sebaceous gland activity, immunodeficiency, neurological and psychological diseases, certain drugs and environmental factors such as low humidity and temperature are risk factors for seborrheic dermatitis [213,215,216]. M. restricta or M. globosa are typically isolated from active lesions and antifungal treatment usually leads to significant clinical improvement. Other species can be isolated, including M. furfur, M. sympodialis, M. obtusa and M. slooffiae [38,[217][218][219]. The scalp, face and chest are most commonly affected, although in infants, the diaper area, neck and axillae may also be involved (Figure 3). Skin lesions are often inflamed, pruritic and present at one or multiple locations. They include poorly defined follicular papules and plaques, fine white scales, and yellow crusts. In the mild dandruff form, no inflammation but a fine, mild scaling on the scalp and beard dominates [220,221].

Malassezia Folliculitis
Malassezia folliculitis is another common worldwide disease with a prevalence of 1 to 17%. It occurs more commonly in young to middle-aged adult males [222][223][224]. Follicular occlusion or a disturbance of the normal cutaneous flora leads to an abnormal proliferation of Malassezia species and the development of disease. Common associated species include M. globosa, M. restricta and M. sympodialis [6,202,[224][225][226][227][228]. Predisposing factors include hot and humid climate, excessive sweating, non-breathable clothing, application of make-up or sunscreens, certain drugs (antibiotics, glucocorticoids) and immunosuppression [6,224,229,230]. The disease typically involves the face, upper back, extensor surfaces of the arms, chest and neck (Figure 3). In almost 75% of cases, more than one location is affected. Lesions include small but pruritic follicular papules and pustules. This presentation is often mistaken for acne or bacterial folliculitis [223,224,231,232].

Atopic Dermatitis (Head and Neck Dermatitis)
Atopic dermatitis (AD) is a common, chronic, inflammatory and pruritic disease, affecting 10 to 25% of children and 1 to 2% of adults. Head and neck dermatitis (HND), a subtype of AD, mostly occurs in adolescence and adulthood in individuals with a history of IgE-mediated AD. There is no gender or ethnic predisposition [233][234][235][236]. The etiology is incompletely understood, but it is clear that Malassezia species play an important role in disease pathogenesis. The high activity of sebaceous glands at affected sites, together with the skin barrier disruption of the atopic disease, allow Malassezia species to proliferate, leading to increased exposure to the immune system, triggering a humoral and cell-mediated immune response [237][238][239][240][241][242][243]. Some involved Malassezia antigens have been well characterized (M. globosa-MGL_1304; M. sympodialis-Mala s 8; M. restricta-Mala r 8) and have been identified in the sweat of patients, leading to aggravated clinical signs, especially after intense sweating [244,245]. These antigens have also shown variable histamine-releasing properties [246]. Malassezia species isolated from disease-associated sites have included M. furfur, M. obtusa, M. globosa, M. restricta and M. sympodialis, but there was no significant difference in isolation compared to healthy individuals [240,247]. Others found a higher colonization rate by M. furfur, as well as a lower colonization rate by M. globosa and M. sympodialis, in affected AD patients [62]. Specific genotypes of M. globosa and M. restricta have also been identified as colonizing AD skin [65,174].
HND patients have erythema and erythematous plaques on the forehead, eyelids, perioral, neck and upper trunk together with variable pruritus (Figure 3). In severe cases, the whole face may be involved, leading to the term "red face". With the chronicity of the disease, lichenification and scaling can occur [245,248]. Wheal-like, edematous changes have also been described [245].
In dogs with environmental allergies, the clinical signs of Malassezia dermatitis often mimic, or even worsen, those of atopic disease [270]. It has been shown that affected patients show elevated levels of Malassezia-specific IgG and IgE in their serum [277]. In addition, immediate hypersensitivity reactions were observed in canine atopic patients in which M. pachydermatis extracts were intradermally injected or after passive transfer of atopic serum to healthy recipient dogs using the Prausnitz-Küstner (P-K) technique [278,279]. Together with the frequent isolation and higher colonization rate of Malassezia species on and from the skin of these patients, their relevance and contribution, especially M. pachydermatis, to disease pathogenesis has been demonstrated [140]. Four major allergens of M. pachydermatis with a size of 45, 52, 56 and 63 kDa were detected in more than 50% of atopic dogs in a study by Chen et al. in 2002 [280]. In dogs with environmental allergies, the clinical signs of Malassezia dermatitis often mimic, or even worsen, those of atopic disease [272]. It has been shown that affected patients show elevated levels of Malassezia-specific IgG and IgE in their serum [279]. In addition, immediate hypersensitivity reactions were observed in canine atopic patients in which M. pachydermatis extracts were intradermally injected or after passive transfer of atopic serum to healthy recipient dogs using the Prausnitz-Küstner (P-K) technique [280,281]. Together with the frequent isolation and higher colonization rate of Malassezia species on and from the skin of these patients, their relevance and contribution, especially M. pachydermatis, to disease pathogenesis has been demonstrated [141]. Four major allergens of M. pachydermatis with a size of 45, 52, 56 and 63 kDa were detected in more than 50% of atopic dogs in a study by Chen et al. in 2002 [282].
Malassezia-associated otitis externa in animals can be unilateral or bilateral and is associated with ear scratching, head shaking and brown to black, often malodorous discharge. The pinnae, especially near the orifice of the ear canal, are often also affected [2,141,252,300]. In a recent canine study, a painful, erosive to ulcerative form of otitis externa with a watery brown to black discharge caused by Malassezia species was described [301]. In contrast, otomycosis due to Malassezia spp. is considered rare in humans [302,303].

Miscellaneous Forms of Superficial Malassezia-Associated Diseases
Occasionally, Malassezia species can also infect the nails of humans and the claws of animals. In companion animals, paronychia with erythema, swelling and a waxy brown to black discharge is common, while in humans, subungual hyperkeratosis and onycholysis can be seen [85,141,[304][305][306].
Another potential site of superficial Malassezia infection is the cornea. There are sparse reports of keratomycosis in humans and dogs associated with M. furfur and M. restricta in humans and M. pachydermatis in dogs [307][308][309]. Interestingly, one affected dog [309] and a human patient [308] both had diabetes mellitus and in all described cases immunomodulatory or antibiotic drugs were used. These predisposing factors could have facilitated Malassezia species overgrowth. The burden of corneal colonization by Malassezia species significantly increases in cases of corneal ulceration [120,310]. Whether Malassezia species could have a primary pathogenic role in some cases of corneal ulceration requires further investigation.

Fungemia and Systemic Infections
Of the 18 Malassezia species, only three are known to cause fungemia-M. furfur, M. pachydermatis and M. sympodialis. In the former two, one specific genotype is involved [14,198,311,312]. In fungemic patients, M. furfur is most frequently isolated, followed by M. pachdermatis and M. sympodialis [14,16,313].
Since the first report of systemic infection by an unspecified Malassezia species in 1979 [314], systemic infections have been described with increasing frequency [14][15][16]18,198], likely due to growing recognition of the pathogenic potential of Malassezia species, as well as improved detection methods [14,110].
The skin plays a significant role in the development of fungemia as both a reservoir of Malassezia species and a portal of entry into the bloodstream by Malassezia species when it is compromised [14]. Predisposing factors for fungemia include premature birth, hospitalization and duration of stay in a neonatal intensive care unit, immunosuppression, peritoneal dialysis, presence of central venous catheter, total parenteral nutrition with lipid supplementation (especially in neonates), invasive surgical procedures, long-term or broad-spectrum antimicrobial administration, chronic illnesses and topical application of soybean oil containing products [16,184,185,315]. Parenteral lipids are not only favorable for Malassezia species growth but can also reduce the immune response of a patient by the generation of reactive oxygen species, which decrease neutrophil phagocytosis [183,184,316,317].
Pathogenic Malassezia strains associated with fungemia are either already present colonizing the patient's skin or are transmitted to the skin through interactions with healthcare worker's hands or contaminated medical devices, materials and/or parenteral solutions [14,16,77,183,184,194,196].
Severe illness, the administration of immunosuppressive, antifungal or broad-spectrum antimicrobial drugs or parenteral lipids, poor anatomic conformation and/or premature age lead to an impaired immune state. Different combinations of such factors enable invasion of the body at an entrance point, such as a surgical wound or an intravenous catheter site [14,38,194,327,328].
Hematogenous dissemination of Malassezia species can involve infection of the heart, lungs and, less commonly, the kidneys, pancreas, liver, spleen, brain and skin (multiple cutaneous pustules) [183,184,316,317]. Biofilm formation facilitates local replication and further shedding of the organism into the blood system [317,[329][330][331].
The diagnosis of Malassezia-associated fungemia is challenging due to its special needs for growth including lipid dependency. It is recommended to directly culture blood or central venous catheter tips on lipid-rich culture media via blood culture specimen tubes and not to use an automated blood culture system [14,313,349]. In addition, since human blood can have inhibitory and toxic effects on yeasts, the addition of 3% palmitic acid may favor positive detection [350].
Thus far, Malassezia-associated fungemia has not been reported in animals.

Chronic Diseases in Humans and Animals
In patients with HIV infection, the burden of the Malassezia species in the gut and on the skin of individuals with seborrheic dermatitis is significantly increased, associated with low numbers of CD4+ helper cells/Th17 cells. This overgrowth of Malassezia species is a risk factor for fungemia and other Malassezia-associated infections, including HIV-associated seborrheic dermatitis [115,[351][352][353].
In patients with inflammatory bowel disease (IBD), including Crohn's disease, Malassezia species dominate the gastrointestinal mycobiome [354][355][356]. M. restricta colonization, especially in the sigmoid colon, can increase disease severity by intensifying the inflammatory response [356]. This effect is strongly associated with the presence of the Crohn's disease risk allele altered caspase recruitment domain 9 (CARD9 S12N). CARD9 is an adapter protein of the CARD-CC family that mediates pattern recognition signaling and is essential for fungal defense [115,[354][355][356][357][358][359]. In mice models, the same authors showed the capability of M. restricta to cause significant changes to the colon, including colon shortening, mucosal erosion and crypt destruction [356].
It has been speculated that Malassezia species could have a pathogenic role in the development or progression of colorectal cancer since affected individuals have gastrointestinal mycobiome dysbiosis with a higher burden of Malassezia species compared to healthy individuals [115,[360][361][362]. However, whether this is an effect or a cause of cancer remains to be proven. Malassezia species have been found to play a causal role in pancreatic ductal adenocarcinoma (PDA) associated with migration from the gut to the pancreas [361]. In human and murine PDA, cancerous pancreatic tissue contained a 3000-fold higher burden of fungi compared to healthy pancreatic tissue and was specifically enriched for the Malassezia species. The oncogenic pathway was also identified as the activation of mannose-binding lectin, which drives the complement cascade and promotes oncogenesis [361].
A role for Malassezia species in neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, has been speculated due to their frequent detection in affected areas of brain tissue [363][364][365][366]. The source of Malassezia species is fungemia due to breaches of the cutaneous or gastrointestinal barriers. However, whether their presence reflects opportunistic colonization of damaged tissue or is causal has not been determined. Similar studies in veterinary medicine are lacking.

Methodology
Usually, established testing concentrations are used as a reference for systemically applied drugs at their recommended doses [367,368]. Topically, much higher concentrations can be reached, important for topical therapy and thereby susceptibility testing methods would need to be adjusted [369][370][371]. Since Malassezia species are involved in common diseases and can potentially cause deep infections, fungemia or even death, susceptibility testing becomes a necessary and very important tool [14][15][16]18,110]. Even if there are standard proposed guidelines for testing the susceptibility profile of filamentous fungi and yeasts, it is difficult to implement these methods with Malassezia species due to their special needs and growth characteristics [372,373]. As a consequence, and due to the lack of standardization, different procedures were proposed with culture medium, inoculum size, incubation time, and criteria used to determine MIC endpoints largely vary among the studies, thus making it difficult to interpret the data in the literature [374]. The susceptibility of Malassezia species to antifungal compounds has been tested using different methods, including a modified Clinical and Laboratory Standards Institute (CLSI) broth microdilution protocol [375][376][377][378] and agar-based diffusion methods (Disk Diffusion -DD and the E test-ET) [379][380][381][382][383]. However, the agreement analysis between agar-based diffusion methods and modified CLSI standard reference procedures still needs to be better investigated. Overall, DD might not represent a valid alternative for determining the susceptibility of Malassezia yeasts to azoles and amphotericin B (AmB), and ET should be used with specific media and longer reading times and only for specific drugs [374].
A completely different approach has been described via corneofungimetry. Stratum corneum cells coated with olive oil form the basement of this testing process, mimicking an in vivo situation [384][385][386]. There is no comparison of this principle with commonly used methods.
Overall, clear international standard guidelines for susceptibility testing of Malassezia are urgently warranted to effectively compare and analyze data, but the authors consider the broth microdilution method the most suitable one and regard this as the gold standard.

Patterns of Antifungal Susceptibility
For clinical usability, not only the MIC distribution but also other factors such as serum concentration of the drug, pharmacodynamics, resistance mechanisms and clinical efficacy need to be considered [387][388][389]. These are encompassed by clinical breakpoint values established by the CLSI and EUCAST [372,[388][389][390]. These breakpoints are regularly updated and if not available, usually the ones for Candida, including C. krusei, C. parapsilosis, C. tropicalis and C. albicans, are considered [391].
Nevertheless, the final proof of resistance is through the detection of the underlying mechanism. For Malassezia yeasts, clinical breakpoint values are still not established, but proof of the underlying mechanisms of resistance has been verified for some Malassezia species (see below).
Overall, Malassezia species antifungal susceptibility profiles against azoles, AmB and terbinafine (TER) vary between species or intraspecies, regardless of the media or other conditions employed [374]. M. sympodialis and M. pachydermatis are reported to have lower MICs of antifungals AmB, TER and azoles, in general compared to M. furfur and M. globosa [38,195,378,[392][393][394].
MIC variation can also be seen within a given species, as shown for M. sympodialis, M. globosa and M. furfur [376,378,393,395]. Similar results are reflected in a canine study involving M. pachydermatis, indicating less variation within the same patient but more dissimilarity between different patients [394,396].
Not surprisingly, the duration of a disease influences the MIC, as reflected in a canine study on otitis externa, in which patients with chronic disease had higher MIC values associated with MIZ and CL than those with an acute form [394,405]. This could also be related to prior antifungal exposure.
Studies focusing on fungemia have shown a better efficacy of AmB against M. pachydermatis than against M. furfur [393,395,406]. For M. furfur, better effects can be achieved when using the liposomal version of the drug or when combined with FCZ [393,407]. TER works better for M. pachydermatis and M. sympodialis than for M. furfur [395][396][397]. Considering Malassezia species overall, ITZ and KZ are reported to be more effective than FCZ, VCZ or AmB [195,392,393,395].
Nevertheless, looking at various reports, it can be concluded that for M. pachydermatis, ITZ and PSZ show the highest activity compared to other antifungals, with an MIC 90 of mostly less than 0.5 µg/mL. On the other hand, CL (up to 16 µg/mL) and thiabendazole (up to 32 µg/mL) show relatively high values [382,393,396,408,409]. However, from a clinical perspective, MIC 90 (values at which the growth of 90% of the tested isolates is inhibited) warrants careful interpretation since tissue concentrations are not included in the calculation.

Resistance Mechanisms
Antifungal resistance can be primary (intrinsic) or secondary (acquired) [410]. The former occurs naturally without previous exposure to antifungal drugs. Acquired resistance takes place after or during interactions with antimicrobials [410].
An early study in 1994 showed that resistant-induced mutant strains of M. pachydermatis exhibited significantly decreased levels of membrane sterols but increased amounts of fecosterol, indicating a possible evasion mechanism of polyene antifungals by replacement of sterol with a precursor product [411]. Mutations in the gene ERG11 (CYP51), encoding for lanosterol-14α-demethylase, which converts lanosterol to ergosterol, have been detected for induced KZ-resistant M. pachydermatis and for clinically resistant M. globosa strains. These mutations include missense mutations, amino acid alterations and tandem quadruplication and confer azole resistance [412,413]. Chromosomal rearrangements and gene overexpression, leading to tandem quadruplication of genes within chromosome 4, have been identified in some mutant-resistant strains. Since this region carries genes, including ERG 4 and 11, affecting ergosterol synthesis, azole resistance was conferred by this resistance mechanism [413].
Overexpression of ERG11 can also lead to resistance due to the overwhelming presence of the target protein, which has been demonstrated in clinical isolates of M. pachydermatis and M. restricta [413,414].
A different resistance mechanism affecting azole drugs involves efflux pumps. These overexpressed proteins can actively transport accumulated intracellular antifungal drugs out of fungal cells. Around 30 different proteins have been described either belonging to the ATP-binding cassette (such as CDR1, CDR2 or PDR10) or the major facilitator (such as MDR1) superfamily. Such mechanisms have been detected among isolates of M. pachydermatis, M. furfur and M. restricta (Pdr5) [393,[414][415][416]. Mitochondrial dysfunction in M. restricta strains involving ATM1, an iron-sulfur transporter, leading to the activation of the pleiotropic drug resistance (PDR) pathway, resulting in an increased expression of efflux pump transporters, has also been described [414]. Interestingly, by using a Malassezia species broth microdilution chequerboard analysis testing the in vitro efficacy of azoles in combination with drug efflux pump modulators (i.e., haloperidol-HAL, promethazine-PTZ, and cyclosporine), FCZ MIC = 128 µg/mL for M. furfur, FCZ MIC = 64 µg/mL for M. pachydermatis and VOR MIC = 4 µg/mL for both Malassezia species were proposed as cut-off values to discriminate suscep2tible and resistant strains [415].
Finally, biofilm formation can also significantly decrease antifungal sensitivity, as shown in studies of M. pachydermatis [321,394,409]. For topical therapy, preparations of chlorhexidine alone or in combination with an azole antifungal are mostly used [2,232,374]. For severe Malassezia-associated skin diseases or cases that do not respond to topical therapy alone, oral KZ or ITZ are favored in dogs [2,374,[417][418][419][420] and ITZ in cats [2,282,374,421,422]. Due to its high concentration and persistence within the stratum corneum, pulse therapy of ITZ is used with 7 days on, 7 days off, 7 days on, or twice weekly administration [421,423]. Terbinafine [423][424][425][426] and FCZ [427] have been prescribed in single case reports and clinical trials are warranted before treatment recommendations can be made. Even if clinical evidence indicates the efficacy of azole for the control of skin infections, the common recurrence of skin disorders requires the recognition of underlying diseases or the use of prophylaxis systems for the management of these infections in animals [2]. As maintenance therapy, plant-based compounds (i.e., essential oils and phenolic compounds) and peptides have achieved interesting results, but future studies need to be done in order to propose them for clinical use [374].

Treatment in Humans
Pityriasis versicolor-A combination of keratomodulating (sulfur, salicylic acid, selenium sulfide, zinc pyrithione) and antifungal (azoles, ciclopirox olamine, TER) shampoos, sprays or solutions is usually effective, but in widespread, severe, refractory or recurrent cases, systemic antifungal therapy with ITZ or FCZ may be required. Terbinafine is not effective [428,429]. Relapses are common, even after successful initial treatment and long-term management can be challenging [429][430][431] (Figure 3).
Malassezia folliculitis-There is some evidence that systemic treatment is the most efficient method, considering the location of the disease within the hair follicles [438]. Itraconazole and FCZ show good efficacy [222,224,[439][440][441]. Topical treatment (azoles, selenium sulfide and propylene glycol 50%) may be better used as a preventive measurement or for patients where systemic treatment is contraindicated [439][440][441][442]. Photodynamic therapy as an alternative treatment has also been mentioned [443,444] (Figure 3).
Atopic dermatitis (head and neck dermatitis, HND)-HND patients respond best to systemic antifungal treatment, especially when using ITZ or KZ [243,[445][446][447][448][449][450]. Affected individuals are often treated daily for one to two months and then twice weekly for maintenance [448]. Fluconazole can also be used, although some studies report that it would not be as effective as the latter two mentioned drugs [448,451]. Limited data exist for systemic TER [452]. Topical antifungal treatment has not been very promising, although ciclopirox olamine twice daily may be an option for selected cases [453] (Figure 3).

Treatment of Systemic Malassezia Infections
For systemic infections in humans, rapid organism identification, together with an aggressive systemic treatment approach, is essential [14,110,196,460]. Intravenous therapy with AmB is effective in infants and adults [14,16,18,38,72,187,188,191,315,347]. FCZ, PSZ and VCZ have been administered, but careful considerations are necessary since failure of the first two drugs are reported, especially due to reported or suspected reduced susceptibility [18,34,187,188,191,404,[461][462][463]. Flucytosine or echinocandins have no efficacy against Malassezia and should be avoided [18,185,191,464]. In addition to antifungal therapy, it is of fundamental importance to remove any indwelling devices, such as catheters and to temporarily stop parenteral lipid supplementation [14,18,110,191,196,229,460].

Conclusions
Malassezia species are among the most widespread fungi on our planet and it is expected that new species and hosts will be discovered. While some Malassezia species are host adapted, many are shared between animals and humans. There is evidence of zoonotic transmission, especially for M. pachydermatis, but more longitudinal data are needed for further elucidation. Malassezia species can be associated with many different skin diseases in companion, production, avian and exotic animals as well as in humans. In people, Malassezia fungemia and internal infections are increasingly recognized, especially in immunocompromised individuals. In addition, these yeasts are associated with certain chronic diseases, such as Crohn's disease, but also with some cancers, such as pancreatic ductal adenocarcinoma. Malassezia species need special culture media to grow and international standardization for susceptibility testing is urgently needed. In both human and veterinary medicine, topical treatment is preferred unless the type, severity or refractory state of the disease doesn't allow it. For systemic Malassezia species infections, AmB is typically used, while for other diseases, azole preparations dominate.