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Abstract
Identification of Malassezia species
in skin of different clinical variants of
Seborrhoeic dermatitis
Thesis submitted for
partial fulfillment of master degree in Dermatology and Andrology
By
Amira A. I. Issa
M.B.B.CH
Oct.2001
Physician at general medical services department
Suez Canal University
Faculty of Medicine
Suez Canal University
2007
Supervisors
Atif I. Al Akhras Rabie Abo El Magd Abo Bakr
Prof. of Dermatology and Andrology Assistant Prof of Dermatology and Andrology
Faculty of Medicine Faculty of Medicine
Suez Canal University Suez Canal University
Gehan Sedeik El-Hadidi
Assistant Prof of Microbiology
Faculty of Medicine
Suez Canal University
Acknowledgment
First and always, my deep gratefulness and indebtedness is to ‘Allah’ the most beneficial, unlimited and continuous support to me.
I wish to introduce my deep, sincere gratitude and utmost thanks to my advisor, Prof. DR. Atef Ibrahim El-Ahkras Prof. Of Dermatology and Andrology, Faculty of Medicine, Suez Canal University, for his sincere great encouragement and constant support, which contributed to the accomplishment of this work. Without his great effort, this work would not be possible.
I would like expressing my great gratitude to DR. Rabie Abo El- Maged Abo Bakr, Assistant Prof. Of Dermatology and Andrology, Faculty of Medicine, Suez Canal University, for his guidance of this work, suggesting the line of search of this thesis, active supervision and great encouragement.
Much more than thanks are dedicated to Dr. Gehan Sedik El-Hadedy, Assistant Prof. Of Microbiology, Faculty of Medicine, Suez Canal University, for her kind active supervision, valuable advices, continuous encouragement and cooperation during this study.
I would like to express my gratitude to all members of Dermatology clinic, Suez Canal University Hospital for the facilities they offered this work.
Finally, I would like to thank my family for their support. I am greatly indebted to my Husband for his understanding and support.
Index
Introduction………………………………………… 1
Seborrhoeic dermatitis……………………………… 4
Definition…………………………………………...4
Etiology……………………………………………..5
Skin Flora…………………………………………….7
Microbial aspect of Seborrhoeic Dermatitis………….12
Pityrosporum and Malassezia species………………..12
Taxonomy of Malassezia species…………………… 13
Serologic identification of Malassezia species………..17
Antigenicity and Morphology of Malassezia…………17
Isolation and cultural identification of Malassezia……18
Structure, physiology and biochemistry of Malassezia.21
Commensalism………………………………………..25
Seborrhoeic Dermatitis and Malassezia………………27
Immunological responses to Malassezia in Seborrhoeic Dermatitis patients versus healthy individuals………..28
Humoral immune responses………………………...28
Cellular immune responses………………………….29
Clinical features and manifestations of Seborrhoeic Dermatitis……………………………………………...31
Clinical Variants……………………………………...32
Materials and Methods……………………………… 37
Results…………………………………………………45
Discussion……………………………………………. 54
Summary and conclusion……………………………...60
References……………………………………………62
Appendices…………………………………………..75
Arabic summary……………………………………...79
List of tables and figures
Table: 1…………………………………………………15
Table: 2…………………………………………………17
Table: 3…………………………………………………43
Table: 4…………………………………………………50
Table: 5…………………………………………………53
Fig: 1…………………………………………………….43
Fig: 2a…………………………………………………...45
Fig: 2b…………………………………………………..45
Fig: 2c…………………………………………………..45
Fig: 2d…………………………………………………..45
Fig: 2e…………………………………………………..45
Fig: 3………………………………………………….. 46
Fig: 4a………………………………………………….46
Fig: 4 b…………………………………………………46
Fig: 5……………………………………………………47
Fig: 6……………………………………………………48
Fig: 7……………………………………………49
Fig: 8……………………………………………51
Abbreviations
Ag: age group.
Ah: aryl hydrocarbon.
C 3: complement 3.
C.: Candida.
ELISA: enzyme Liked immunosorbent assay.
GI: gastrointestinal.
HLA: human leukocyte antigen.
IgA: immunoglobulin A.
IgG: immunoglobulin G.
IgM: immunoglobulin M.
IIF: indirect immunoflurescence.
ISD: infantile Seborhoeic Dermatitis.
M.: Malassezia.
NK 1: natural killer 1.
P.: Pityrosporum.
PV: pityriasis versicolor.
S.: staphylococci.
SD: seborhoeic dermatitis.
SG: sebaceous gland.
Introduction
Seborrhoeic dermatitis (S.D.) is a papulosquamous disorder (Selden, 1998) that causes flaking of the skin of the face and chest, and the creases of the arms, legs and groin (Mayser et al, 2003). It usually affects the scalp (Selden, 1998).
S.D. is among the most frequent diseases caused by Malassezia yeasts (Mayser et al, 2003), and with increased sebaceous gland secretions in a suspected individual due to genetic and immunologic tendency (Gupta& Bluhm, 2004).
The worldwide prevalence of S.D. is 3-5%. Dandruff, the mildest form of this dermatitis, is present in 15-20% of the population (Selden, 1998).
S.D. most often occurs in babies younger than 3 months of age and in adults from 30 to 60 years of age with a peak at age 40 years. It’s more common and worse in men than in women. It is less severe, but present, among older people. S.D occurs in all races (Mayser et al, 2003).
Various medications may induce or flare S.D. such as auranofin, aurothioglucose, buspirone, chlorpromazine, cimetidine, ethionamide, gold, griseofulvin, haloperidol, interferon alfa, lithium, methoxsalen, methyldopa, phenothiazines, psoralens, stanozolol, thiothixene, and trioxsalen (Selden, 1998).
The common etiology of S.D. is a convergence of three factors: (1) sebaceous gland (SG) secretions, (2) microfloral factors, and (3) individual susceptibility. Scalp flaking shows a strong correlation with SG activity, in the neonate it results in microfloral colonization and cradle cap. At puberty, the SG activity turns on under sex hormone control, that gives the low Malassezia population a new food source in the form of SG secretions and therefore it proliferates, resulting in the scalp itching and flaking which is common to greater than 50% of adults (Ro & Dawson, 2005).
Malassezia yeasts are opportunistic organisms. In 1995, Guillot et al, defined& named seven species of Malassezia: M.furfur, M. sympodialis, M. obtusa, M. slooffiae, M.sympodialis, M. globosa, M. restricta and M. pachydermatis, Subsequently after this taxonomic classification Japanese researchers found another four new species: M. dermatis (Sugita et al, 2002), M. japonica (Sugita et al, 2003), M. yamatoensis (Sugita et al, 2004), and M. nana (Hirai et al, 2004).
This Malassezia species distribution is not even at the surface of the stratum corneum. Rather, they are clumped on some corneocytes while other corneocytes in their vicinity are almost free of these microorganisms. The corneocytes heavily coated by yeasts suggest a cell-related defect in the mechanisms controlling the skin biocene including the natural antimicrobial peptides and nitric oxide. Most environmental factors influencing pityrosporoses indeed affect the natural human defenses against certain microorganisms (Celis & Cepro, 2005; Pierard et al, 2006).
Some authors suggested that S.D. is associated with normal levels of Malassezia organisms which are probably not the cause but are a cofactor linked to immunologic abnormalities (Pierard et al, 2006), also it is associated with increased sebum levels, and an activation of the alternative complement pathway (Seldon, 1998), others suggested that the colonization rate of Pityrosporum species was higher in the seborrhoeic dermatitis patients as in their research Pityrosporum yeast cells were seen microscopically in (74%), and the cultures were positive in (86%). Among the normal individuals yeast cells were seen microscopically in (46%) and the cultures were positive in (44%) (Mirza et al, 2005).
Antimycotics achieve clinical improvement, while recolonization leads to recurrent symptoms of seborrhoeic dermatitis (Mayser et al, 2003).
The pathogenic role of Malassezia in SD is focused on their lipid metabolism. Malassezia restricta and M. globosa require lipids. They degrade sebum, free fatty acids from triglycerides, consume specific saturated fatty acids, and leave behind the unsaturates. Penetration of the modified sebaceous secretions results in inflammation, irritation, and scalp flaking (Ro & Dawson, 2005).
The Tween assimilation test was used to identify Malassezia species isolated from patients with pityriasis versicolor and seborrhoeic dermatitis, and suckling infants with seborrhoeic dermatitis in Japan. In both adult and suckling infant cases of seborrhoeic dermatitis, M. globosa and M. furfur were isolated at high incidences compared with the healthy control subjects, indicating the possibility that one or both of these species are the causative organisms of these diseases (Nakabayashi et al, 2000).
S.D. is a common problem affecting almost all age groups and causes significant discomfort to its patients. The role of Malassezia in its pathogenesis is not yet resolved but supported in many researches, but are their levels and rate of colonization in the normal and lesional skin the same or different, are there specific species involved more in a clinical variant than the other these were the questions that we thought about finding their answer through this thesis.
Aim of the work
1-To identify the Malassezia species commonly associated with seborrhoeic dermatitis skin lesions.
2-To determine if there are certain Malassezia species identified in certain S.D. clinical variant.
Seborrhoeic dermatitis
Definition
Seborrhoeic dermatitis (SD) is a chronic papulosquamous disorder, usually causes the skin to look a little greasy and scaly or flaky. It has a distinctive morphology (red, sharply marginated lesions covered with greasy looking scales) and a distinctive distribution in hairy skin areas with a rich supply of sebaceous glands, namely the scalp, face and upper trunk, and the creases of the arms, legs and groin. In some cases the flexures are also involved, but this is not an essential diagnostic criterion, and is now believed to be at least in part due to over growth of the normal skin flora in the regions affected (Marks, 1993; Seldon, 1998).
Dandruff (pityriasis simplex capitii) is a common complaint generally characterized by the presence of flakes on the scalp and in the hair, and by itching. The symptoms can vary, and the severity can range from mild scaling, similar to dry skin, to severe scaling (Seldon, 1998). It appears to be the precursor of SD, as it may gradually progress through redness, irritation and increasing scaling of the scalp to true SD (Mayser et al, 2003).
The condition known, as SD of infancy is surrounded by controversy as some authors suggested that it is normally confined to the first months of life as it represents a minor and transient abnormality of desquamation from the scalp, others considered it to be a form of atopic dermatitis suggesting that cradle cap occurring in the newborn is probably not related to proper seborrheic dermatitis (Marks, 1993; Seldon, 1998) others consider it to be infantile seborrhoeic dermatitis that is confined to the scalp (Negan, 2006).
The cradle cap is an infantile S.D. that is confined to the scalp. The cause of cradle cap is not clearly defined but it is not caused by infection, allergy or poor hygiene. It is possibly related to overactive sebaceous glands in the skin of newborn babies, due to the mother’s hormones still in the baby’s circulation. The glands release a greasy substance that makes old skin cells attach to the scalp as they try to dry and fall off. There may be a relationship with Malassezia (Negan, 2006).
Etiology
Dry scalp flaking, dandruff, and SD are chronic manifestations of similar etiology differing only in severity, they are more than superficial stratum corneum disorders, including alteration of the epidermis with hyperproliferation, excess lipids, interdigitation of the corneal envelope, and parakeratosis (Seldon, 1998).
The common etiology of SD is a convergence of four factors: (1) individual susceptibility, (2) sebaceous gland (SG) secretions (3) microbial factors specially Malassezia which is normally present on the skin in small numbers, but sometimes its numbers increase, resulting in skin problems and (Ro and Dawson, 2005), (4) immunologic abnormalities, and activation of the complement system (Seldon, 1998).
1-Individual susceptibility
Generally, the make up of the normal flora depends upon various factors, including genetics since different people have different physiological properties that can affect microbial growth, age sex, stress, nutrition, and diet of the individual also personal hygiene also affects the normal flora
(Paustian, 2006).
Regarding Malassezia specifically it was suggested that there is genetically determined host susceptibility for a greater liability for heavy colonization by Malassezia, or for the infectivity by heterochonous yeast flora (Roberts, 1969; Faregmann et al, 1991).
This can be supported by the finding of HLA-DR expression on Keratinocytes of the SD lesional skin in few cases (Bergbrant et al, 1991).
Also there was a research that studied cellular immune responses specific to Malassezia in SD patients and controls and found that patients with SD were generally more responsive to Malassezia than controls were (Ashbee et al, 1995).
Another study which studied the results of stimulation of PBMC with Malassezia obtained from healthy controls and SD patients and they found that it was different with, higher levels of IL-2 and gamma interferon (IFN- ) in controls, and higher levels of IL-10 (that interfere with the development of protective cell immunity against Malassezia) in SD patients than in controls (Neuber et al, 1996; Donnarumma et al, 2004).
2-Sebaceous gland secretions and seborrhoeic dermatitis:
An early research debated the role of increased sebaceous gland excretions in the pathogenesis of SD as they found that many young adult SD patients’ sebum excretions rate was normal in male patients and significantly reduced in female patients, but there were no data to support this (Burton et al, 1983).
In contrast there was a more recent research that suggested a strong correlation between SD and SG activity based on the observation that there is a significant relation between the age and the incidence of SD, as they found that the SG are active at birth and this early activity in the neonate results in microfloral colonization and cradle cap. After maternal hormonal control subsides, there is a little SG activity until puberty as the SG turns on under sex hormone control, the SG activity increases, the present but low Malassezia population has a new food source and proliferates, resulting in SD and the scalp itching and flaking common to greater than 50% of adults (Seldon, 1998).
Other studies demonstrated that however that the amount of sebum production does not correlate with the severity of the disease, implying that individuals with excessive sebum production might be free of SD and vice versa, there were qualitative abnormalities in the composition of sebum, mild abnormalities in the surface lipids were relevant for this disease as elevation of triglycerides and cholesterol, decrease of free fatty acids and squalene, which could result from the ineffective keratinization which is often demonstrated histologically in SD
( Pye et al, 1977; Seldon, 1998).
Also Tollesson et al in their research supported that there are abnormalities in surfaces lipids as they found that the levels of Essential Fatty Acid 18:1w9 in Infantile SD were increased and levels of 18:2w6 were decreased, whereas 20:4w6 levels remained normal, the rare fatty acid 20:2w6 was found in significant amounts in infants with active Seborrheic dermatitis and this was only barely detectable levels in the controls and that the normalization of the deviation in Infantile SD paralleled the recovery at any age it occurred (Tollesson et al, 1993).
Skin Flora
The average human adult has two-meter square of skin surface. The chemical composition and moisture of skin varies and dictates what type of bacteria will grow on it and how much. The surface of skin (epidermis) is not a favorable place for microbial growth because it is often dry, salty, and has low pH. Most microorganisms are associated with sweat glands and hair follicles because of the moist and nutritious environment. Urea, amino acids, salts, lactic acids, and lipids are secreted through the skin and provide microorganisms with what they need to grow (Kerr and McHale, 2003).
Skin provides good examples of various microenvironments. Skin regions have been compared to geographic regions of Earth: the desert of the forearm, the cool woods of the scalp, and the tropical forest of the armpit. The composition of the dermal microflora varies from site to site according to the character of the microenvironment. A different bacterial flora characterizes each of three regions of skin: (1) axilla, perineum, and toe webs; (2) hand, face and trunk; and (3) upper arms and legs. The varied environment of the skin sites with partial occlusion (axilla, perineum, and toe webs) harbor more microorganisms due to their high moisture content than do less occluded areas (legs, arms, and trunk), These quantitative differences may relate to increased amount of moisture, higher body temperature, and greater concentrations of skin surface lipids. The axilla, perineum, and toe webs are more frequently colonized by Gram-negative bacilli than are drier areas of the skin (Marples et al, 1972; Todar, 2002).
The number of bacteria on an individual’s skin remains relatively constant; bacterial survival and the extent of colonization probably depend partly on the exposure of skin to a particular environment and partly on the innate and species-specific bactericidal activity in skin. Also, a high degree of specificity is involved in the adherence of bacteria to epithelial surfaces. Not all bacteria attach to skin; staphylococci, which are the major element of the nasal flora, possess a distinct advantage over streptococcus viridans in colonizing the nasal mucosa. Conversely, streptococcus viridans are not seen in large numbers on the skin or in the nose but dominate the oral flora, however Staphylococcus aureus is found on the face and hands, particularly in individuals who are nasal carriers. (Todar, 2002).
The microbiology literature is inconsistent about the density of bacteria on the skin; one reason for this is the variety of methods used to collect skin bacteria. The scrub method yields the highest and most accurate counts for a given skin area. Most microorganisms live in the superficial layers of the stratum corneum and in the upper parts of the hair follicles. Some bacteria, however, reside in the deeper areas of the hair follicles and are beyond the reach of ordinary disinfections procedures. These bacteria are a reservoir for recolonization after the surface bacteria are removed. The flora changes near the various orifices of the body (e.g., mouth, nose and anus). Not surprisingly, the bacteria will be similar to those found within the orifice (Maibach and Aly, 1981; Paustian, 2006).
Host Infection by Elements of the Normal Flora
Opportunistic microorganisms are those organisms, which when given the right circumstances, can become pathogenic. Examples of opportunistic microorganisms of the skin include any of the normal flora of the human body that gain entry into the body through a breach in skin or the mucous membranes producing diseases (Maibach and Aly, 1981).
In addition, impairment of the host defenses (due to immunosuppression, chemotherapy, or irradiation) may result in failure of the normal flora to suppress transient pathogens or may cause members of the normal flora to invade the host themselves. In either situation, the host may die (Maibach and Aly, 1981; Tannock, 1995).
Opportunistic Skin flora
Diptheroids- pleomorphic Gram-positive rods, ex. Propionibactrium acnes (anaerobic, live in hair follicles & grow on sebum), Corynebacterium xerosis (aerobic); Yeast- Pityrosporum species (Malassezia) and Candida; Staphylococci- S. epidermidis & other coagulase negative species, (Staphylococcus aureus- in some individuals).
All of them are bacteria, except for Pityrosporum and Candida, which are yeast like fungi.
Generally speaking, because of the antimicrobial properties of sweat and sebaceous glands secretions, the numbers of bacteria actually found on the skin are not great. Organisms that do thrive on the skin are generally resistant to both drying and high salt concentrations (Marples et al, 1972).
Bacteria:
Staphylococcus epidermidis
Staphylococcus epidermis is a major inhabitant of the skin, and in some areas it makes up more than 90 percent of the resident aerobic flora. It is non-motile, gram-positive coagulase-negative cocci, arranged in irregular clusters, this bacteria is an opportunistic pathogen that does not cause problems unless it enters the bloodstream via cuts, catheters, or needles (Marples et al, 1972).
Staphylococcus aureus
Staphylococcus aureus (S. aureus) is gram positive, forms clusters, non-motile, non-spore forming, and is a facultative anaerobe. This bacteria tests positive for coagulase, Catalase, and forms yellow colonies on agar (Maibach and Aly, 1981). It is prevalent (67%) on vulvar skin. S. Aureus is extremely common (80% to 100%) on the skin of patients with certain dermatological diseases such as atopic dermatitis, but the reason for this finding is unclear. Also it can cause or at least be involved in the etiology of boils, styes, and furuncles (Kerr and McHale, 2003).
Gram-Negative Bacilli
Gram-negative bacteria make up a small proportion of the skin flora. In view of their extraordinary numbers in the gut and in the natural environment, their scarcity on skin is striking. They are seen in moist intertriginous areas, such as the toe webs and axilla, and not on dry skin. Desiccation is the major factor preventing the multiplication of Gram-negative bacteria on intact skin. Enterobacter, Klebsiella, Escherichia coli, and Proteus species are the predominant Gram-negative organisms found on the skin. Actinobacter species also occurs on the skin of normal individuals and, like other Gram-negative bacteria, is more common in the moist intertriginous areas.
Micrococci
They are frequently present on normal skin. Micrococcus luteus, the predominant species, usually accounts for 20 to 80 percent of the Micrococci isolated from the skin (Kerr and McHale, 2003).
Micrococcus luteus is a strict anaerobe that produces yellow to cream-white water insoluble pigment on agar. This bacterium is nitrogen reductase negative and oxidase positive (Tannock, 1995).
Due to its tendency to colonize the sebaceous rich areas it was suspected in the etiology of SD. But it was not proven to be an etiological factor, as a study held in university of Leeds, U.K., measured the population densities of micrococci on the chest, back, forehead, left and right cheeks of SD, PV patients and age and sex matched controls, and they found that the densities did not vary at a given site between patients and corresponding control subjects (Ashbee et al, 1993).
Diphtheroids (Coryneforms)
The term diphtheroid denotes a wide range of bacteria belonging to the genus Corynebacterium which is it is gram positive, aerobic, non-motile, tests positive for catalase, belongs to the Mycobacteriacae family, and is rod-shaped (Kerr and McHale, 2003).
Classification of diphtheroids remains unsatisfactory; for convenience, cutaneous diphtheroids have been categorized into the following four groups: lipophilic or nonlipophilic diphtheroids; anaerobic diphtheroids; diphtheroids producing porphyrins (coral red fluorescence when viewed under ultraviolet light); and those that possess some keratinolytic enzymes and are associated with trichomycosis axillaris (infection of axillary hair). Lipophilic diphtheroids are extremely common in the axilla, whereas nonlipophilic strains are found more commonly on glabrous skin (Marples et al, 1972; Tannock, 1995).
Anaerobic diphtheroids are most common in areas rich in sebaceous glands, so it was suspected in the etiology of SD. But it was not proven to be an etiological factor, as a study held in university of Leeds, U.K., measured the population densities of probionobacteria on the chest, back, forehead, left and right cheeks of SD, PV patients and age and sex matched controls, and they found that the densities did not vary at a given site between patients and corresponding control subjects (Ashbee et al, 1993).
Although the name Corynebacterium acnes was originally used to describe skin anaerobic diphtheroids, these are now classified as Propionibactrium acnes and as P. granulosum. P. acnes is seen eight times more frequently than P. granulosum in acne lesions and is probably involved in acne pathogenesis. Children younger than 10 years are rarely colonized with P. acnes. The appearance of this organism on the skin is probably related to the onset of secretion of sebum at puberty. P avidum, the third species of cutaneous anaerobic diphtheroids, is rare in acne lesions and is more often isolated from the axilla (Tannock, 1995).
Streptococci
Streptococci, especially B-hemolytic streptococci, are rarely seen on normal skin. The paucity of B-hemolytic streptococci on the skin is attributed at least in part to the presence of lipids on the skin, as these lipids are lethal to streptococci. Other groups of streptococci, such as a-hemolytic streptococci, exist primarily in the mouth, from where they may, in rare instances, spread to the skin.
Yeast:
Malassezia
Yeasts of the genus Malassezia is an anthropophilic dimorphic fungus which can grow in a yeast phase as well as in a mycelial phase, on normal human skin, the yeast phase predominates and it changes to the mycelial form under the influence of exogenous and endogenous factors (Faergemann, 1993).
In the mycelial form it causes several cutaneous diseases, such as pityriasis versicolor, Malassezia (Pityrosporum) folliculitis, seborrhoeic dermatitis and dandruff, atopic dermatitis, and less commonly is involved in the etiology of other dermatologic disorders such as confluent and reticulated papillomatosis, onychomycosis, and transient acantholytic dermatosis, and can cause the provocation of psoriatic lesions. Also it can contribute or cause systemic diseases in immunocompromised individuals including pneumonia, catheter-associated sepsis and peritonitis (Schmidt, 1997; Rincon et al, 2005).
Microbial aspect of Seborrhoeic Dermatitis
A recent study compared the colonization rate of Pityrosporum yeast in the SD patients with normal healthy individuals found that in SD patients Pityrosporum yeast cells were seen microscopically in 74% of cases, and the cultures were positive in 86%. Among the normal individuals, the yeast cells were seen microscopically in 46% and the cultures were positive in 44% of them, and this lead them to the conclusion that the colonization rate of Pityrosporum species was higher in the SD patients and that it might be playing a causative role in the etiology of this disease (Mirza et al, 2005).
Although Malassezia was more numerous and made up a larger proportion of the flora in patients with dandruff or SD, the authors felt that these data presented here cannot be constructed to argue for or against an etiologic role for bacteria or yeast-like organisms in dandruff (Mayser et al, 2003).
Pityrosporum and Malassezia species
Yeasts of the genus Malassezia is an anthropophilic dimorphic fungus which can grow in a yeast phase as well as in a mycelial phase, On normal human skin, the yeast phase predominates and it changes to the mycelial form under the influence of predisposing factors such as high temperature, high relative humidity or endogenous factors such as greasy skin, sweating, heredity, immunosuppressive treatment or disorders (Faergemann, 1993).
In the mycelial form it causes several cutaneous diseases, such as pityriasis versicolor, Malassezia (Pityrosporum) folliculitis, seborrhoeic dermatitis and dandruff, and less commonly is involved in the etiology of other dermatological disorders such as confluent and reticulated papillomatosis, onychomycosis, and transient acantholytic dermatosis, and can provocate psoriasis and atopic dermatitis. Also it can contribute or cause systemic diseases in immunocompromised individuals including pneumonia, catheter-associated sepsis and peritonitis (Schmidt, 1997; Rincon et al, 2005).
Ashbee in 2006 suggested that in normal skin they may down regulate the inflammatory response, allowing them to live as commensal. In contrast, in atopic/eczema dermatitis syndrome and psoriasis, they may elicit an inflammatory response that contributes to the maintenance of lesions.
Taxonomy of Malassezia species (Pityrosporum versus Malassezia)
The first official taxonomic classification placed them in the genus Pityrosporum and defined two species P. ovale and P. pachydermatis, associated with animals (Lodder et al, 1952), but it can occasionally be isolated from human (Schmidt, 1997). Gordon in 1952 then added another species, P. orbiculare, differentiating this species on the basis of its round cell shape.
Slooff in 1970 stated that there was a relationship between the yeast form and the mycelial form, conversion between them had never been demonstrated. In 1977 Dorn et al, Nazzaro-porro et al independently succeeded in inducing the yeast to produce hyphae in vitro using a variety of culture conditions. Also Salkin & Gordon in 1977 observed that both the round and oval yeast forms and hyphae were simply stages in the life cycle of a single organism.
The ability to induce the yeast to form mycelial elements paved the way for the unification of the two genera in 1986, with the acceptance of the species names Malassezia furfur (including P.orbiculare, P. ovale, and Malassezia furfur) and Malassezia pachydermatis (including P. Pachydermatis) (Cannon, 1986). Despite this, many workers maintained the use of the names P. ovale and P. orbiculare and continued to differentiate strains on the basis of cellular and colonal morphologies (Faergemann, 1993).
In the early 1990s the taxonomy of the genus Malassezia was still chaotic, this chaos was finally resolved in a publication in by (Guillot et al, 1995). They defined, and named seven species of Malassezia: M.furfur, M. sympodialis, M. obtusa, M. slooffiae (the last were previously named and known as P. Ovale and also M. sympodialis was known as M. furfur serovar A), and the fifth is M. globosa which was previously known as P. orbiculare or M. furfur serovar B, and the sixth M. restricta which was known as M. furfur serovar C, and the seventh is M. pachydermatis which was previously known as P. pachydermatis (Gueho et al, 1996). The clinical significance of each of these species was yet not understood clearly (Mark, 2002).
Other subsequent molecular studies have confirmed this classification and taxonomic grouping (Kano et al, 1999; Gupta et al, 2000).
The characteristics of these seven different Malassezia species explained before are summarized in table (1)
Table one:
Characteristics or tests M.
Furfur M. Sympodialis M. Pachydermatis M. Globosa M. Slooffiae M. Restricta M. Obtusa
Colony morphology and texture Umbonate, usually smooth soft friable Flat smooth, shiny, soft Pale convex, smooth, soft friable Rough course, brittle Finely folded, brittle Dull smooth hard, and brittle Smooth, flat, sticky
Colony color Cream Cream to buff Cream Cream to buff Cream to buff Cream Cream
Cell shape and size Elongated, oval or spherical, 6 micrometer Ovoid globosa, 2.5-5 micrometer Cylindrical, 2.5-4 micrometer long Spherical, 6-8 micrometer in diameter Cylindrical 1.5-3.5 micrometer long Spherical oval 2-4 micrometer Cylindrical, 4-6 micrometer
Budding pattern Broad bud base Some sympodial budding Broad bud base, pronounced bud scar Narrow bud base Broad bud base Narrow bud base Broad bud base
G + C content (%) 66.4 62.2 55.6 53.5 68.7 59.9 60.7
Catalase reaction Positive Positive Variable Positive Positive Negative Positive
Urease reaction Positive Positive Positive Positive Positive Positive Positive
Growth at 37 C Good Good Good Poor Good Poor Poor
Max growth temp (C) 40-41 40-41 40-41 38 40-41 38 38
Use as lipid source
Tween 20 Positive Negative Positive Negative Positive Negative Negative
Tween 40 or 60 Positive Positive Positive Negative Positive Negative Negative
Tween 80 Positive Positive Positive Negative Negative Negative Negative
Ability to split esculin Negative Positive Variable Negative Negative Negative Positive
(Walters et al, 1995).
Subsequently after this taxonomic classification Japanese researchers found another four new species: M. dermatis (Sugita et al, 2002), M. japonica (Sugita et al, 2003), M. yamatoensis (Sugita et al, 2004), and M. nana (Hirai et al, 2004).
M. Japonica was isolated and its name was proposed by a group of Japanese researchers from a healthy female (Sugita et al, 2003). M. Dermatis was isolated from skin lesions of atopic dermatitis patients (Sugita et al, 2002).
The characteristics of M. Japonica, M. Dermatis are summarized in table 2
Table 2:
Characteristics or tests M.
japonica M.
dramatis
Colony morphology and texture Sympodial to dull, wrinkled, butyrous. Has an entire to lobed margin Convex and butyrous and had an entire or lobed margin.
Colony color Pale yellowish Yellowish white, semi shiny to dull
Cell shape and size Spherical, oval, or ellipsoid 2-5 by 2-7 Spherical, oval or ellipsoidal 2-8 by 2-10 micrometer
Budding pattern Sympodial budding Filaments sometimes formed at the area of the origin of the bud
Catalase reaction Positive Positive
Growth at 37 C Good Good
Max growth temp (C) 40-41 40-41
Use as lipid source
Tween 20 Negative Positive
Tween 40 or 60 Positive Positive
Tween 80 Negative Positive
Serologic identification of Malassezia species:
Various investigations have examined the serological relationship between cultural and micro morphological variants of M. furfur. Early studies demonstrate antigenic similarities between oval yeast cell (P. ovale) round yeast cell (P. orbiculare) and the mycelial phase of M. furfur (Faergemann et al, 1982).
The micro morphological and culturally different variants of M. furfur share antigenic determinants, although serological differences between variants can be demonstrated. Theses serological defined variants are unlikely to affect the taxonomic position of M. furfur. However, the difference in surface antigen expressed by M. furfur variants may be important in colonization.
Antigenicity and Morphology of Malassezia:
In earlier literature; Malassezia described the mycelial phase of Malassezia furfur only; the yeast phase was divided into two distinct species on microscopic morphology (Sloof, 1970).
Pityrosporum Orbiculare had had round yeast cells budding from a narrow neck, whilst Pityrosporum ovale cells were ovale and budded from a wide base but the characteristics of cell shape were reported to be unstable as morphological variants have been observed in strains following successive subculture (Maheswari Amma & Painker, 1982).
Antigenic identity was suggested for P. oval, P. orbicular and M. furfur by Bruneau et al in 1984 using Quantitative immunoelectrophoretic techniques as they found up to 63 antigenic differences and two common antigens between them, lending support to the idea that they were all stages in the life cycle of the same organism.
In 1986, the weight of evidence led to the unification of the different morphological form within the species M.furfur (Cannon, 1986). However, the ability to grow very distinct colony variants from the same site on human skin led workers to define three serovars of M. furfur, which were designated A, B and C, and were distinguished on the basis of growth characteristics, colony morphology, and specific surface antigens which were both serovars-specific and common surface antigens (Cunningham et al, 1990; Cunningham et al, 1992).
In 1989, Midgley reported that morphologically stable variants of M. furfur yeast cells can be maintained in vitro and using molecular work it was found that these species are sufficiently different to be classified as separate species.
Malassezia appears to be an antigenically complex organism, which alters the antigens expressed throughout its growth cycle.
Variety of antigens from Malassezia, ranging from low- to high-molecular-mass proteins and also high-molecular-mass carbohydrates have been defined, many of the antigens have similar masses and may be identical. Although the proportion of each may vary during the growth cycle, the protein antigens are likely to be cell wall or cytoplasmic components that can easily be detected in immunoblotting and are present in the early phase of growth. The carbohydrate antigens, probably mannans or mannoproteins, are less easy to detect by immunoblotting and are maintained throughout the growth cycle (Johansson et al, 1991)
Saadatzadeh in 1998 found that all the Antigens identified from the mycelial form are also can be identified from the yeast form of Malassezia, which was confirmed by Ashbee et al, in 2006 who stated that mycelium-specific antigens were not present on the mycelia and so the mycelia did not have any phase-specific antigen, at least not on the cell surface.
Isolation and cultural identification of Malassezia
Malassezia species has been surrounded by controversy because of their fastidious nature in vitro, and relative difficulty in isolation, cultivation, and identification (Rincon et al, 2005).
In 1939, Benham demonstrated the lipophilic nature of this organism, as exogenous long-chain fatty acids were absolutely required for in vitro growth of all the Malassezia species With the exception of M. pachydermatis.
There is little doubt that the medium used for M.furfur cultivation influence M.furfur yeast cell micromorphology (Leeming & Notman, 1987; Midgley, 1989).
Early worker utilized basal nutrient media overlaid with a layer of olive oil that favored oval yeast cells (Gordon, 1952, Leeming & Notman, 1987).
In 1964, Van Abbe isolated four colonial variants using a medium formulated by Dixon in which the lipid source (Ox bile; Tween 40 and Glycerol monoleate) was incorporated into malt extract agar). This medium could be used for quantitative study of M.furfur. Previous media could not, as the colonies coalesced under the oil overlay. Using this medium, described four M.furfur variants.
Three oval variants and one rounded variant had different colonial morphologies P. ovale form one had domed colonies with fine radial groves P.ovale form two had smooth dull colonies whereas the colonies of form three were smooth and shiny. Filaments were occasionally observed in culture of P. orbiculare and P. ovale form two (Midgley, 1989).
In 1987, Leeming and Notman assessed several media for their ability to recover M.furfur from normal skin. The most successful medium was modified and retested until 164 variants had been assessed. The final medium, which incorporated Ox bile, Glycerol monostearate, Tween 60 and cows whole fat milk yielded M.furfur recovery comparable with microscopic counts.
The superiority of M.furfur isolation has been confirmed by an independent group (Korting et al, 1991).
Using this medium Cunningham et al placed M.furfur isolates into one of three cultural groups based on colonial morphology and growth characteristic in lipid medium. Group A samples had round yeast cells and large (5mm), circular, cream, raised, smooth dentate colonies. Group B samples were also round, colonies were circular, (2-3), flat with pointed button center friable and crenated. Group C samples had oval cells, their colonies being (2-3), and circular Umbonate and entire (Cunningham et al, 1990).
The M.furfur isolate was different to those observed in this later. It is most likely that difference in culture conditions account for variation in colony and yeast cell morphology (Midgley, 1989).
The phenomenon of yeast-mycelial transformation was shown to be influenced by the culture medium studied 22 samples of P. orbiculare isolated from pityriasis versicolor and three types samples of P.ovale. Yeast cell micromorphology was stable on an M.furfur growth medium. Cultivation in filamentation medium (described as starvation medium containing glycine) produced hyphae in 20/22 round yeast samples but no oval samples (Dorn et al, 1977).
Nazzaro et al in 1977 included cholesterol and cholesterol esters into a Bacto yeast morphology agar and were able to induce both round and yeasts to produce hyphae.
Many other authors have confirmed these findings (Maheswari & Painker, 1982; Faergemann et al, 1983).
In the mycelial phase, M.furfur possesses curved and controlled hyphae with either round or oval spores. Several groups used serology to determine whether there were any antigenic relationships between yeasts of different cell shapes and the yeast and mycelial phases, Sternberg et al in 1961 made use of sera from patients with PV to examine the cross reaction between M.furfur and P. orbiculare. Using tape strips o f M.furfur from PV lesions and smears of P.orbiculare from cultures, they obtained florescence of ”equal brilliance” when the serum was applied to the cells. from this, they concluded that the yeast and hyphal forms had common antigens.
Structure, physiology and biochemistry of Malassezia
Malassezia is able to exist in both yeast and mycelial forms, with the yeast being most commonly associated with normal skin. The yeast form also predominates in culture, although hyphae may be seen with some species (Gueho et al, 1996).
Several groups have succeeded in including mycelial formation in vitro using a variety of media. Although not all isolates of Malassezia are able to undergo this transformation (Dorn et al, 1977).
Malassezia species undergo asexual reproduction by monopolar, enterblastic budding from a characteristic broad base. The mother and daughter cell are divided by a septum, and the daughter cell separates by fission, leaving a bud scar or collaret through which successive daughter cells will emerge (Ahearn et al, 1998).
Structure
The cell wall of the genus Malassezia is poorly characterized. It is very thick in comparison with other yeasts (about 0.12 micrometer) and constitutes 26 to 37% of the cell volume (Keddie et al, 1972).
The major components o f the cell wall are sugars (70%), protein (10%), and lipids (15-20%), with small amounts of nitrogen and sulfur (Thompson et al, 1970).
Several workers suggested that the cell wall consisted of two layers with indentations on the inner layer (Keddie, 1966; Breathnach et al, 1976), while other workers have found multiple layers within the wall (Simmons et al, 1987).
The most recent worker in the cell wall has confirmed the presence of an outer lamellar layer was noted, which may explain the previous reports mentioned by others but never investigated (Mittag, 1995).
The lamellar layer was ”membrane –like ” with an electron-transparent middle enclosed by two electron-dense lines. The structure of the layer varied with different lipid sources in medium and stained with Nile blue sulfate, suggesting that it contained lipid. The lamellar layer may play a role in adhesion of the organism to both human skin and indwelling catheters (Mittag, 1995).
The cytoplasmic membrane adheres closely to the inner surface of the cell wall and follows the indentations present, differing between the round and oval cell shapes (Keddie et al, 1972).
The nucleus has a well-defined limiting membrane surrounded by a granular homogenous nucleoplasm. Vacuoles present in the cell contained lipid and varied in size according to the age of the cell (Barfatani et al, 1964).
Physiology
The physiology of Malassezia species is poorly understood because problems with reliably culturing and maintaining the organism have hindered progress in this area. In 1939, Benham noted that Malassezia was unable to ferment sugars.
The organism can use lipid as the sole source of carbon, does not require vitamins, trace elements, or electrolytes, and preferentially uses methionine as the sole sulfur source, but it can also use cystine or cysteine (Brotherton, 1967; Mayser et al, 1998).
It is able to use many amino acids, as well as ammonium salts, as nitrogen sources (Mayser et al, 1998). Although the organism is normally grown in vitro under aerobic conditions, it is also able to grow under microaerophilic and anerobic conditions (Faergemann et al, 1981).
The growth requirement of Malassezia for lipid was first noted in 1939 but was not studied in detail until Shifrine et al in 1963 demonstrated the inability of the organism to form long-chain fatty acids due to a block in de novo synthesis of myristic acid, requiring the addition of preformed fatty acids.
Subsequent work showed that the addition of most fatty acids with a carbon chain length greater than ten supported growth and that it did not matter weather odd or even numbered carbon chain lengths were used (Wilde & Stewart, 1968).
The lipid source used during growth affects the fatty acid composition of the organism, suggesting that the fatty acids are not used as energy sources but, rather, are incorporated directly into cellular lipids without being further metabolized (Caprilli et al, 1973).
Wilde and Stewart in 1968 further found that the lipids present on normal human scalps were able to fulfill the lipid requirement of the organism.
Biochemical characteristics
Malassezia species elaborate a range of enzymes and metabolites that might be important virulence factors, favoring host tissue invasion (Giusiano, 2006).
They have lipolytic activity both in vitro and in vivo indicating the production of a lipase. The lipase is located in the cell wall and /or membrane sites in the cytoplasm (Catterall et al, 1978).
Ran et al in 1993 found the PH optimum was 5 and that lipase production was greatest during the logarithmic phase of growth, perhaps demonstrating its importance in the hydrolysis of lipids for cell growth.
In contrast, Plotkin et al in 1998 found the PH optimum to be 7.5 but also found that lipase activity was greatest during active cell growth and correlated with substrate concentration. They concluded that there were at least three separate lipases in Malassezia, which were essential for cell growth.
Mayser et al in 1995 studied the lipolytic activity of Malassezia on fatty acid esters and found that it had only minor substrate specificity, with the degree of hydrolysis being determined by the alcohol moiety.
In vitro, Malassezia species also produce a phospholipase, which its activity is able to cause the release of arachidonic acid from HEp-2 cell lines (Plotkin et al, 1998).
Since arachidonic acid metabolites are involved in inflammation in the skin, this has been suggested as a mechanism by which Malassezia species may trigger inflammation (Greaves et al, 1988).
Phospholipases is considered to be contributing to the Malassezia virulence as it is suggested to probably be one of many factors involved in the complex interaction between the yeast and host leading to the development of skin lesions caused by Malassezia yeast species (Plotkin et al, 1998).
Malassezia species produce an enzyme with lipoxygenase activity, as demonstrated by its ability to oxidize free and esterified unsaturated fatty acids, squalene, and cholesterol (Nazzaro-Porro et al, 1987).
The resultant production of lipoperoxides may damage cell membranes and consequently interfere with cellular activity – a mechanism that has been proposed to cause the alterations in skin pigmentation associated with PV (De Luca et al, 1996).
Another research suggested that this alteration is because that M. furfur is able to convert tryptophan into a variety of indole alkaloids that causes apoptosis of melanocytes. Also malassezin, previously isolated from the Malassezia furfur is an agonist of the aryl hydrocarbon (Ah) receptor therefore; contribute to the marked depigmentation observed (Kramer et al, 2005)
Culture of Malassezia produces a characteristic ”fruity” smell, first described by Van Abbe in 1964.
Gas chromatographic-mass spectrometric analysis of the gas from the culture headspace of Malassezia grown in lipid containing medium showed it to consist volatile gamma lactones. This characteristic was unique to Malassezia and was suggested as a possible way to differentiate this genus from others (Labows et al, 1979).
Another metabolite produced is azelaic acid, a C 9 decarboxylic acid. It is produced when Malassezia is grown in the presence of oleic acid and is a competitive inhibitor of tyrosinase, an enzyme involved in the production of melanin (Nazzaro_Porro et al 1987).
In addition to having antibacterial and antifungal activity, azelaic acid inhibits the proliferation of several tumor cell lines and decrease the production of reactive oxygen species in neutrophils by inhibiting cell metabolism (Brasch et al, 1993).
Commensalism
Distribution of Malassezia species on normal skin
Malassezia species are members of the normal human cutaneous commensal flora and can be isolated from the sebaceous-rich areas of the skin, particularly the chest, back, and head region (Leeming et al, 1989).
Many studies have examined carriage rates in different populations and different age groups. However, early studies often found low carriage rates due to the limitations in sampling techniques and culture media (Spoor et al, 1954).
An extensive study of the distribution of Malassezia species at various sites on adults was carried out by Leeming et al in 1989, using an optimized culture medium and a sampling method known to recover 98% of the surface skin flora (Williamson et al, 1965).
They examined clinically normal skin at twenty different sites over the entire body surface. Malassezia species were recovered from every subject from the chest, medline back, scalp, ear and upper inner thigh. The highest mean population densities occurred on the chest, ear, upper back, forehead and cheeks but it was also significantly higher in the chest compared with the forehead, arm and thigh (Lee et al, 2006).
Some differences in carriage rates were noted between females and males, with higher population densities from the lower trunk and upper thigh of males by different studies, using the same medium (Bandhaya et al, 1993; Lee et al, 2006).
Bergbrant et al in 1988 found that the density of Malassezia species on the skin decreased with increasing age, which was probably due to a reduction in the level of lipid on the skin. Therefore, 30-year-old subjects had significantly greater numbers of Malassezia species than did any other age group from 40-80 years old; this was also supported by another study that found that the population density of Malassezia yeast was significantly higher in the age group (AG) of 21-30 years compared with other AGs.
M. Pachydermatis is occasionally isolated from human skin, but its presence is transitory and it is not a human commensal (Bandhaya et al, 1993).
Other several recent studies have examined the distribution of the newly defined species of Malassezia on healthy adult human skin. The findings, summarized, very significantly among studies, and there are two possible explanations for this. First, there are genuine differences in the distribution of species on the skin of individuals in different countries (Midgley, 2000).
However, even the two studies carried out in Spain show very different results. A second explanation is that the use of swabbing, a no quantitative and relatively insensitive method, is simply not able to produce the quantitative data needed to determine which species predominate at the different sites studied (Lee et al, 2006).
Colonization rates in children are the subject of some controversy. As with adult, the colonization rate reported are partly a reflection of the sensitivity of the sampling method and culture medium used. Particularly in young children or newborns, swabs may be the only practical sampling method available since more disruptive techniques are unethical (Bergbrant et al, 1994).
One study of 60 healthy children, who ranged from 2 months to 14 years of age, yielded no positive specimens for Malassezia (Abraham et al, 1987).
This contrasts with other studies that colonization rates recorded range from 37% (Powell et al, 1987) to 100% (Leeming et al, 1995) in premature and full- term hospitalized neonates.
Other studies on healthy children have found carriage rates of 74% on the scalp (Noble & Midgley, 1978), 93% on the back (Garcia, 1976), and 87% on the forehead (Bergbrant et al, 1994).
Factors such as young gestational age, low birth weight, and extended periods of hospitalization may predispose to colonization in this group (Bell et al, 1988; Ahtonen et al, 1990).
In general, carriage of Malassezia appears to increase around puberty, correlating with the increase in sebaceous gland activity seen at this time (Cunningham et al, 1992).
Seborrhoeic Dermatitis and Malassezia
He was Malassez in 1874 who first associated Malassezia with scaling scalp conditions (Malassez, 1874), and the debate about its role in SD and dandruff has continued unabated since then.
Innumerable studies have sought to resolve the issue, focusing either on the microbiology of the condition or on the therapeutic efficacy of various antifungal preparations. In the early 1950s, both Martin-Scott and Spoor et al. expressed reservations about the association of Malassezia with SD, since it was found on normal scalps as well as in patients with SD (Martin-Scott, 1952; Spoor, et al, 1954).
But Shuster has criticized this suggesting a pityrisporal etiology, relying on that the organism is found in the disease, it can be cultured, and infection with the organism can experimentally induce the disease (Shuster, 1984).
Supporting the earlier study that assessed the population densities of Malassezia on the scalps of normal subjects, patients with dandruff, and patients with SD finding that Malassezia made up 46% of the microbial flora in normal subjects, 74% of the flora in patients with dandruff, and 83% of the flora in patients with SD (McGinley et al, 1975).
Wyk et al in 1976 studied the effect on dandruff with inhibiting either the yeast or bacterial populations on the scalp and found that reduction of the yeast population correlated with a decrease in dandruff while inhibition of the bacterial population did not.
Also another several subsequent studies have supported the role of Malassezia in SD and dandruff; demonstrating parallel decreases in the number of organisms and the severity of the condition (Salkin & Gordon, 1977; Pierard et al, 1998).
Despite this there was a study suggested that there is lack of correlation between Malassezia numbers and the presence and severity of dandruff (McGinley et al, 1969). And there was another experiment that found that suppression of the Malassezia population on the scalp, either alone or in combination with suppression of the bacterial flora, had no effect on dandruff production. They concluded that dandruff was merely an excessive desquamation of the scalp and was totally unrelated to the microbial flora (Leyden et al, 1976).
But the finding that treatment of seborrhoeic dermatitis with an antifungal agent not only resulted in clinical improvement but also reduced the number of Malassezia yeasts on the skin while recolonization leads to recurrent symptoms resulted in a resurgence of interest in the Malassezia yeasts and adds considerable weight to the role of Malassezia in SD and dandruff (Danby et al, 1993; Mayser et al, 2003).
Although simple overgrowth of Malassezia is unlikely to be the cause of SD and dandruff, the balance of evidence suggests that the organism is very important in their etiology as it acts as a cofactor linked to increased sebum levels (Walters et al, 1995) and an altered relationship between these skin commensals and the host (Gupta and Bluhm, 2004), and an abnormal host immunological response to the Malassezia yeasts (Gupta and Nicol, 2004) as activation of the alternative complement pathway and T-cell depression (Seldon, 1998).
Immunological responses to Malassezia in Seborrhoeic Dermatitis patients versus healthy individuals.
Many studies have examined the immunological responses in these patients and compared them to the responses in healthy individuals to determine whether patients had particular immunological predisposing factors.
Humoral immune responses
In the commensal state, Malassezia usually occurs as yeast cells, although mycelium may also be seen (Midgley, 1989), Immunoglobulins specific to the yeast phase of Malassezia can readily be detected in normal individuals even from a relatively young age with no history of skin disease, Antigen is presented to the immune system over a sufficient period to initiate both naive (IgM) and anamnestic (IgG) responses. Levels of IgA are generally low; suggesting that mucosal sensitization by Malassezia is not an important route (Midgley, 1989).
Humoral response to mycelium in normal individuals., was addressed by Saadatzadeh in 1998 who induced the mycelial phase and used whole mycelial antigens in Indirect immunoflurescence. All the classes of immunoglobulins were detected, with the highest titers being found for IgG. Appreciable levels of IgM, IgA, and the IgG subclasses were also found suggesting that despite the limited amount of mycelium on normal skin, the immune system recognizes and responds to mycelial antigens. This may be due to the presence of common antigens, shared either with the yeast phase of Malassezia or with other commensal organisms.
In seborhoeic dermatitis
Many studies examining the humoral immune response to Malassezia in patients with SD, and generally, most studies have not demonstrated significant differences in antibody levels between patients and controls, although one found lower levels in patients (Neuber et al, 1996), and two found increased levels (Silva et al, 1997; Parry et al, 1998).
Faeregmann, 1983 reported that antibody levels to M.furfur whole cells (serovar A) was lower in children and elderly individuals than young and middle aged adults. Sohnle et al in 1983 failed to find any differences in IgG titres specific to a homogenized extract of M.furfur (a round variant) between a group of young adults and elderly individuals. The levels of IgM were however lower in the elderly individuals. Low levels of M.furfur specific IgA were present in both groups.
Cellular immune responses.
Relatively few studies have examined cellular immunity in SD. One mechanism postulated to be involved in the pathogenesis of SD is contact sensitization to the antigens of Malassezia. Application of killed Malassezia cells resulted in lesions similar to SD in an early study (Moore et al, 1936) and scaling of rabbit skin (Rosenberg et al, 1980).
Faeregmann et al in 1991 who studied the humeral and cellular immune status in patients with SD found that there is an intermittent T-cell depression.
Cellular immunity is known to be of major importance in the host defense against fungal infection (Casadevall et al, 1998).
The higher incidence of Malassezia-associated dermatoses in patients with cellular immunodeficiency suggests that cellular immunity is also important in maintaining the organism as a commensal and the incidence of SD is very high in patients with AIDS (Smith et al, 1994).
Nicholls et al in 1990 carried out patch testing on 11 patients with SD by using various concentrations of cell wall and cytoplasmic antigens from Malassezia. They tested untreated skin and skin that had been repeatedly stripped with adhesive tape to remove loosely adherent skin squames and so improve the sensitivity of the test. However, only one patient had an irritant reaction to the antigens.
In summary, most studies of cellular immunity in SD have demonstrated either increased reactivity to Malassezia in patients or no differences between patients and controls.
Clinical features and manifestations of seborhoeic dermatitis
Intermittent, active phases manifest with burning, scaling, and itching, alternating with inactive periods. Activity is increased in winter and early spring, with remissions commonly occurring in summer. Active phases may be complicated by secondary infection in the intertriginous areas and on the eyelids. Candidal overgrowth is common in infantile napkin dermatitis. Such children may have a diaper dermatitis variant of Seborrheic dermatitis or psoriasis. Generalized Seborrheic erythroderma is rare (Seldon, 1998).
Skin lesions manifest as branny or greasy scaling over red, inflamed skin. Hypopigmentation is seen in blacks. Infectious eczematous dermatitis, with oozing and crusting, suggests secondary infection (Shuster and Blatchford, 1988).
Distribution follows the oily and hair-bearing areas of the head and the neck, such as the scalp, behind the ears, the glabella, skin the forehead the nasolabial folds, the medial sides of eyebrows (supraorbital regions), the beard, the lash line, and an extension to submental skin can occur. Trunk can be involved especially presternal and interscapular area more frequently than flexural involvement. Upper and lower extremities can also be affected. Otitis externa can also occur accompanying postauricular involvement or alone. Also localized forms of SD can occur involving the occipital areaof the scalp, non scaling intertrigo of the umbilicus, inframammary and areolar area in women, anogenital area, blepharitis of the eye lids (Shuster and Blatchford, 1988), it also can occasionally occur as generalized scaling (Lawrence, 1985; Seldon, 1998)
Clinical variants:
There are several clinical variants of SD in adults related to the different sites:
The scalp: it is the least severe but by far the commonest phase, it may present itself in two forms:
1- (pityriasis sicca), or Dandruff- it manifests itself as perifolicular redness and scaling and a dry, flaky, branny desquamation, beginning in small sharply marginated patches and rapidly involving the entire surface with a profuse amount of fine powdery scales commonly known as dandruff.
2-An oily type, pityriasis steatoides, at times accompanied by erythema and an accumulation of thick crusts (Ackerman & Kligman, 1969).
Hair in the affected areas tends to fall out, characteristically beginning on the vertex and frontal regions and progressively receding. SD is probably the commonest cause of premature baldness in men (Domonkos et al, 1982).
3- Configurate patches of psoriasiform eruptions is another type of SD of the scalp, which is a more severe form and is manifested by greasy, scaling, exudation and crusting. The disease usually spreads beyond the hairy scalp to the forehead, ears, postauricular regions and neck, in these areas the patches have convex borders and a reddish yellow or yellow color. In the extreme cases the entire scalp covered by a greasy, dirty crust with an offensive odor (Rook et al, 1980).
Behind the ears: it can occur as
1- only greasy scaling, but a crusted fissure often develops in the fold and adherent masses of sticky scale and crusts may extend into the adjacent scalp.
Both sides of the pinna, the periauricular region and the sides of the neck may be involved.
2- may be accompanied by Otitis externa, which is irritable and intractable (Stewart et al, 1978).
The face SD here is less well defined than on the scalp. Areas of erythema and scaling occur predominantly in the rosaceous area in association with involvement of the scalp and eyelids.
It can manifest itself by one or more or all of the following :
1- Papular eruption on the cheeks, nose, and forehead. Persistent erythema in the alar –malar angle is called dyssebacea, on the supraorbital regions, flaky scales are seen in the eyebrows and pruritic or show yellowish scaling patches (Domonkos et al, 1982).
2- Involvement of the edges of the lids which may be erythematous swollen and granular, and the lid margins show yellowish pink, fine scales, the borders of which are usually indistinct (marginal blepharitis), it may be accompanied with styes. Yellow crusts may form and separate to leave small ulcers healing to form scars with destruction of lash follicles (Shuster and Blatchford, 1988).
3- Involvement of the conjunctivae can occur in as injection and crustation sometimes in the morning from overnight exudation (Seldon, 1998).
4- Involvement of the glabella, with cracks in the skin in the wrinkles at the inner end of the eyebrow accompanying the fine scaling on an erythematous patch (Domonkos et al, 1982).
5-Involvement of the nasolabial creases and on the ala nasi, with yellowish or reddish yellow scaling macules, sometimes with fissures. In men, folliculitis of the upper lip may occur (Stewart et al, 1978).
6- Beard involvement, which can assume one of two forms.
A- Barbers rash, which is the common form the pilosebaceous orifices are slightly reddened and edematous and may be covered by small, brownish crusts. This superficial folliculitis is most common in clerical workers. It may be associated with other forms of SD and is often very persistent.
B-The second form is much less common. Diffuse redness and greasy scaling of the moustache or the beard area is associated with pustulation involving the whole depth of certain follicles, leading eventually to their destruction and to scarring. The progressive involvement of contiguous follicles over the years can lead to much disfigurement (Shuster & Blatchford, 1988).
7-The lips and mucosa are not usually involved, but sometimes the changes on the lips are pronounced, resulting in chelitis exfoliativa. The vermilion surfaces may be persistently dry, red and fissured (Domonkos et al, 1982).
The trunk, it can be manifested in forms of
1-The commonest form is the petaloid form (so-called because the lesions are petal shaped). This is often in men on the front of the chest and in the interscapular region. The initial lesion is a small red brown follicular papule, covered by a greasy scale. Some patients have a wide spread eruption of lesions which don’t progress beyond this stage. More often, extension and confluence of the follicular papules gives rise to a figured eruption, consisting of multiple cercinate patches, with a fine branny scaling in their center, and with dark red papules with larger greasy scales at their margin.
2- a rare form, involving the trunk and limbs is the so-called pityriasiform type. This is a generalized erythematosquamous eruption, somewhat similar to, but more extensive than, pityriasis rosea. In particular it involves the neck up to the hair margin. It is not particularly pruritic, and it resolves spontaneously, though somewhat more slowly than does pityriasis rosea.
3- in some patients the lesions may become psoriasiform (Seldon, 1998).
The flexures. In the flexure, notably in the axilla, the groin, the anogenital and inframamary regions, and the umbilicus and all pendulous folds of the neck and trunk of obese patients seborrheic dermatitis can presents as
1-an intertrigo with diffuse, sharply marginated erythema and greasy scaling.
2- a crusted fissure develops in the folds, and with sweating, secondary infection and inappropriate treatment, a weeping dermatitis may extend far beyond them.
3-The genetalia of both sexes may be involved and the lesions show the usual range from minimal erythema and scaling to severe crusted dermatitis, and also can occasionally be manifested as a chronic, thickened, dull red, scaly patches of psoriasiform features (Lawrence, 1985).
The upper and lower extremities: it can manifest itself as
1-eczematous patches, which often lack the characteristic color and fineness scale.
2- on the palms and soles it takes one of two forms
A- pompholyx-like vesicular eruption
B- diffuse thickening and scaling even the nails may be involved, showing longitudinal and transverse ridges, and brittleness and grayish discoloration. In such cases it could mimic inverse psoriasis (Seldon, 1998).
The severity and course of seborrheic eruptions are very variable, all show a tendency to chronicity and to recurrence (Kaaman & Torssander, 1983).
Severe and extensive forms may be complicated by eczematous reactions remote from the sites initially involved, especially by pompholyx and discoid eczema. The pattern and course of the disease may also be modified by contact dermatitis and provoked by an infective bacterial dermatitis (Kaaman & Torssander, 1983).
Atopy can Modify the clinical features and the course of seborrheic dermatitis (Peck, 1950), Sebopsoriasis is a term of clinical and histopathological overlap between SD and Psoriasis and the condition can be terminated to any of them (Seldon 1998).
Localized forms of seborrheic dermatitis
Otitis externa
Seborrhoeic dermatitis may sometimes persist in special localized forms that offer difficulties in respect to both diagnosis and treatment. Seborrheic dermatitis of the external auditory canal is the most common underlying factor in Otitis externa (Seldon, 1998).
The ear involvement may simply be apart of a more generalized condition or it may be a localized expression of such a condition. The inaccessibility of the region for every day cleansing makes treatment somewhat more difficult. Moisture retention and maceration are inevitable due to its anatomic construction and, whatever the initial cause of irritation, healing is impaired. Swelling further narrows the canal, serous exudates provide a culture milieu that is highly inviting to the growth of bacteria (Seldon 1998).
In acute inflammations, therefore, bacterial proliferation is greatly enhanced, and this infection may obscure the original condition. In less acute conditions, particularly if cerumen is retained, saprophytic fungi may colonize the debris and accumulate in the form of a plug of fungal elements (Seldon 1998).
SD can be manifested as acute or chronic otitis externa:
Acute otitis externa, may be mild or severe in the severe form, pus formation is prominent, chronic otitis externa, is an eczematous condition with varied and ill-defined causes, the ear canal generally shows varying degrees of scaling and erythema. Pruritis is the chief symptom (Lawrence, 1985).
Localized dermatitis of the occipital area of the scalp (nuchal or suboccipital dermatitis) may have its onset with seborrheic dermatitis. The two conditions are frequently seen together, and sometimes are found with psoriasis as well.
Seborhoeic dermatitis of the umbilicus: may have its genesis in the seborrheic dermatitis. When the umbilicus is involved, manifest itself as sharply marginated erythema and greasy scaling. Fissuring and secondary infection develop almost inevitably and then with sweating, and inappropriate treatment, a weeping dermatitis may occur (Lawrence, 1985).
Seborhoeic dermatitis of the areolar area of the breasts in women may be a manifestation of seborrheic dermatitis. If it is unilateral and persistent biopsy should be performed to differentiate it from paget’s disease (Seldon, 1998).
.
Anogenital Seborrhoeic dermatitis: the localization in the crural folds and gluteal cleft in both males and females has been mentioned. In males, seborrheic dermatitis may persist in localized patches on the penis, particularly on the glans or under the prepuce (Lawrence, 1985).
Seborrhoeic dermatitis of the upper eyelids: may be a manifestation of seborrheic dermatitis. With involvement of this type, it is important to examine the scalp and other areas. The disorder may occasionally be associated with a severe and resistant folliculitis of the beard. In blepharitis particularly, treatment of seborrheic dermatitis of the scalp is essential if the inflammation of the lid margin is to be brought under control
(Domokos et al, 1982).
In a classical case the diagnosis is easy, but in some cases the diagnosis can be difficult, partly because of the lack of well-defined diagnostic criteria. The diagnosis is often made too freely (Seldon, 1998).
Infantile variants:
Cradle cap is characterized by greasy, yellow scaly patches over the scalp. In some cases a thick scaly layer may cover the whole scalp. Over time the scales may become flaky and rub off easily. The condition is usually not itchy and in most cases babies are unaware of the problem (Seldon, 1998; Negan, 2006).
No particular baby is more at risk than any other. Cradle cap is a very common not serious and temporary condition that usually appears within the first 6 weeks of life. In some cases the condition will slowly resolve itself over a few weeks whilst in others it may continue for 6 to 9 months. Seborrhoeic dermatitis may then occur again after the child reaches puberty (Seldon, 1998).
Infantile seborrhoeic dermatitis may also affect other areas of the body such as behind the ears, in the creases of the neck, armpits and diaper area (Seldon 1998; Negan, 2006).
Leiner’s disease
It is a form of infantile erythrodermia with scaling, it is also known as erythema desquamativium occurs chiefly in nursing infants from age of six to twenty weeks as a generalized exfoliative dermatitis with marked erythema and scaling usually suggesting severe type of SD (Seldon, 1998).
The disease usually begins in the perianal and inguinal areas, then appears on the scalp, intertriginous areas, particularly the anogenital region, trunk, and extremities. At first there is a diffuse inflammatory erythema that may cover the whole body. Latter the skin becomes covered with grayish white scales that may be fine and branny. As the process develops there may be general exfoliation, cracking, and thickening of the skin. The scalps always thickly crusted and the nails are destroyed. There is glandular enlargement. Nikolsky’s sign is absent (Shuster and Blatchford, 1988).
The infants with Leiner’s disease are usually in poor general condition, they may be in state of marasmus, and diarrhea is commonly present. This disease is believed to be a systemic metabolic disorder (Seldon, 1998).
Materials and Methods
Study design
This study is descriptive cross sectional study.
Study site
This study was carried out in Suez Canal university hospital dermatology clinic, Ismailia governorate.
Subjects
The study was carried out on 28 patients attending the clinic with different clinical variants of seborrhoeic dermatitis.
All of these patients were selected according to eligibility criteria, which were:
Inclusion criteria:
Both sexes,
Any age group,
With acceptance to participate in the study.
Exclusion criteria:
Patients receiving recent oral antifungal treatment,
patients using local antifungal treatment,
patients with other dermatological conditions with Malassezia species involved in its etiology as Pityriasis versicolor, Malassezia folliculitis, atopic dermatitis, confluent and reticulated papillomatosis, onychomycosis, and transient acantholytic dermatosis.
Samples were taken from scalp, back, chest, flexures of normal individuals skin of both sexes, any age group with acceptance to participate, who were not receiving recent oral antifungal treatment and with no apparent dermatological disease.
Statistical analysis:
Data obtained were analyzed statistically by means of one-way analysis of chi square test. The P value was considered significant at P<0.05.
All statistical analysis was performed using SPSS soft ware (version 11.0).
Methods
History:
All patients were asked to fill in a specifically designed questionnaire that covers the most important aspects of a full medical history (see appendix: 1).
General examination:
All patients were assessed clinically for general or systemic disease.
Local examination:
Local skin examination was carried out for each patient to diagnose SD and to identify the SD clinical variant and distribution of lesions.
Identification of causative species
Specimen collection:
For isolation and identification of Malassezia species, all the randomly selected patients participated in the mycological study. The chosen lesion was cleaned with alcohol and scrapped with a sterile scalpel.
The scrapping was collected in a sterile container for microscopic examination and culture on Dixon agar medium.
Microscopic examination:
The scales were put on a slide with an identification number and were mounted in two DROPs of 15% KOH mixed with two DROPs of 5% lactophenol cotton blue (see appendix (2) for composition of lactophenol blue), for better visualization of the causative organism (Mark, 2002).
The slide was covered with a cover slip then heated to increase the effectiveness of KOH to dissolve the keratin and was examined under the microscope by power 10x and 40x.
Culture on Dixon agar medium:
All samples were cultured onto Dixon agar medium (see appendix (3) for composition of Dixon agar medium) and incubated at 32 C, for one week, all petri dishs were labeled with an identification number.
Identification of different species was done according to the growth characteristics and colony morphology (see tab: 3 for colony Morphology), (see fig: 2a, b, c, d, e for colony morphology of M. globosa, M.restricta, M globosa mixed with obtusa and M. furfur, M. pachydermatis respectively) (Hammer et al, 2000).
Culture on Sabouraud agar:
For identification of M. Pachydermatis, isolates from primary cultures onto Dixon agar medium for each sample were subcultured onto Sabouraud agar (see appendix (4) for Sabouraud agar composition), as M. Pachydermatis was able to grow on Sabouraud agar (see Fig: 3 for positive M. Pachydermatis growth on Sabouraud agar).
Tween assimilation test:
Malassezia species were identified according to the method of (Guillot et al, 1996) (see Fig. 1).
Additional tests were necessary for identification of other Malassezia species, especially the ‘Tween assimilation test’. Malassezia yeast suspensions were mixed with Sabouraud agar, and the mixtures were plated. Four holes were made in the agar by means of a 3-mm diameter punch and filled with five micron each of Tween 20, 40, 60 and 80 respectively. The agar plates were incubated at 32 degree centigrade for one week. The growth of Malassezia sympodialis is inhibited by the high concentration of Tween 20. Malassezia furfur exhibits similar growth with both Tween 20 and 80 (see Fig: 4a). M. slooffiae grows better with Tween 20 than with Tween 80, Malassezia globosa, Malassezia obtusa and Malassezia restricta are unable to utilize any of the four Tween compounds (see Fig: 4b) and were identified by Catalase reaction.
Catalase reaction:
Catalase reaction was made to differentiate Malassezia globosa, Malassezia obtusa and Malassezia restricta. One colony of Malassezia emulsified in a DROP of distilled water on a slide then adding one DROP of hydrogen peroxide to see the bubbles, this means positive reaction. If no bubbles appear this means negative reaction, as the catalase is an intracellular soluble enzyme capable of splitting hydrogen peroxide into water and oxygen.
M. restricta was catalase negative, so M. Restricta was the only lipid dependent species lacking catalase reaction, While Malassezia globosa, Malassezia obtusa were catalase positive.
M.globosa and M. obtusa were differentiated by the morphological findings under the microscope; M.globosa exhibits large and spherical cells, while M. obtusa appears cylindrical cells.
Fig (1):
Table (3):
Colony morphology of seven species of Malassezia
Species Colony morphology Colony color
M.Globosa Rough, course, brittle Cream to buff
M.Furfur Umbonate, usually smooth, smooth, soft, friable Cream
M. Pachydermatis Pale convex, smooth, shiny, soft Cream
M.Sympodialis Flat, smooth, shiny, soft Cream to buff
M.Restricta Dull, smooth, hard, and brittle Cream
M.Slooffiae Finely folded, brittle Cream to buff
M.Obtusa Smooth, flat, sticky Cream
Fig: 2a Fig: 2b
M. globosa growth M. globosa Mixed with
on dixon agar M. obtusa growth on
Dixon agar
Fig: 2c Fig: 2d
M. restricta growth M. furfur growth
on dixon agar on dixon agar
Fig: 2e
M. pachydermatis growth
on dixon agar.
Fig: 3
M. pachydermatis growth on Sabouraud agar
Fig: 4a
Tween Assimilation Test Negative Tween Assimilation Test
showing M. furfur pattern of assimilation.
.
Results
Regarding the age of the patients there were 4 patients (14.3%) between 1-4 years who were mainly in the first 2 years of their life, 6 patients (21.4%) between 5-9 years, 3 patients (10.7%) between15-19 years, 7 patients (25%) between 20-24 years, 2 patients (7.1%) between35-39 years, 2 patients (7.1%) between 40-44years, 1 patient (3.6%) between 45-49 years, 2 patients (7.1%) between50-54 years, 1patient (3.6%) between 55-59 years.
Regarding the gender of the patients there were 21 female patients (75%), and 7 male patients (25%).
A positive history of psychological stress was reported among 15 patients (53.6%) and was reported among 7 of the control (25%). A difference that was statistically significant (P=0.029). (See Fig: 5).
Fig: 5:
Regarding the clothes fabrics most commonly used by the subjects included in the study it was reported that 14 SD patients (50%) used the synthetic fabrics most often and the other 14 patients (50%) used the cotton fabrics, but among the normal subjects only 4 of them (14.3%) used synthetic fabrics while the other 24 (85.7%) used the cotton fabrics most often, A difference that was statistically significant (P=0.004). (See Fig: 6)
Fig: 6
A positive family history of SD was reported in 22 of the SD patient (78.6), and was reported in 11 normal subjects (39.3%). A difference that was statistically significant (P value= 0.003).
Regarding the seasonal exaggeration of SD symptoms, 5 patients (17.9%) reported that their disease occurs more frequently with exaggerated symptoms in summer, 20 patients (71.4%) reported that their disease occurs more frequently with exaggerated symptoms in winter, and 3 patients (10.7%) of the patients noticed no seasonal variation in their symptoms. (See Fig: 7)
Fig: 7
Isolation and identification of different Malassezia species
28 SD patients were included in this study. They were randomly selected from patients attending the dermatology clinic at the Suez Canal university hospital’ with clinically diagnosed SD, with age ranging from5 month to 65 years none of the patients was receiving medication for at least 2 weeks previously. 28 normal healthy individuals with matched age and gender who had no previous history of SD and without any dermatological disease who were not taking antimicrobial therapy at time of sampling were included as control subjects. Samples from lesional skin were taken and samples from identical sites in the control subjects’ skin were also sampled.
One scrapping was taken from every patient, unless the patient has more than one clinical variant, a scrapping was taken from each different lesion. Accordingly we had 33 scraping from 28 patients of different age groups as 3 patients had more than one clinical variant (as one had generalized SD and 2 had dandruff and postauricular SD lesions).
Regarding the distribution of the scrapings taken from the SD patients: Out of 28 patients with definite diagnosis of Seborrhoeic dermatitis, 9 patients (32.1%) had dandruff, 7 of them (25%) had non-inflammatory dandruff, and 2 of them (7.1%) had inflammatory dandruff, 6 patients (21.4%) had flexural “postauricular”, 1 patient (3.6%) had petaloid trunk lesion, 1 patient (3.6%) had follicular lesion of the trunk, 4 patients (14.3%) had cradle cap, 2 patients (7.1%) had blepharitis, 2 patients (7.1%) had face SD lesions one from the medial side of the eye brow and one from the nasolabial fold, 1 patient (3.6%) had pityriasiform lesion from the trunk of a child, 1 patient (3.6%) had flexural SD lesion of the trunk of a child.
(See table: 4).
Table: 4
Frequencies of SD clinical variant
Frequency Percent
Dandruff 9 32.1
Flexural ”postauricular” 6 21.4
Trunk ”petaloid” 1 3.6
Trunk ”follicular” 1 3.6
Cradle cap 4 14.3
Face ”blepharitis” 2 7.1
Face ”eyebrow & nasolabial fold” 2 7.1
Trunk “ pityriasiform “ child 1 3.6
Trunk ”flexural” of child 1 3.6
Trunk of child 1 3.6
Total 28 100.0
Twenty eight of SD lesional samples out of the 33 collected samples and 10 of the normal subjects, were KOH smear-positive, where microscopic examination demonstrated the presence of characteristic thick-walled spherical or oval yeast forms (meatballs shape) and coarse thick septate mycelium often broken up into short filaments (spaghetti shape).
Out of the 28 KOH smear-positive scrapings from SD patients, 25 showed positive Malassezia growth on Dixon agar culture (primary isolates). And out of the 10 KOH smear-positive scrapings from normal subjects, 7 showed positive Malassezia growth on Dixon agar culture (primary isolates) was obtained; this difference was statistically significant (P < 0.001).
The SD patients’ scrapings primary isolates were as follows: Samples from non inflammatory dandruff (7) yielded 7 (100%) primary isolates; Samples from inflammatory dandruff (2) yielded 1 (50%) primary isolates; Samples from cradle cap (4) yielded 3 (75%) primary isolates; Samples from flexure of the child (1) yielded 1 (100%) primary isolates; Samples from trunk of the child (1) yielded 1 (100%) primary isolates; Samples from flexures of the trunk of adult (1) yielded no primary isolates; Samples from postauricular region (6) yielded 5 (83.3%) primary isolates; Samples from blepharitis (2) yielded 2 (100%) primary isolates; Samples from nasolabial fold region (1) yielded 1 (100%)primary isolates; Samples from eye brows (2) yielded 2 (100%) primary isolates; Samples from trunk: petaloid lesion (1) yielded 1 (100%) primary isolates; follicular lesion (1) yielded no primary isolates, pityriasiform lesion (1) yielded 1 (100%) primary isolate. (See Fig: 8).
Fig: 8
Each species was recognized by applying the scheme of species identification, according to its growth on sabouraud agar, the results of the Tween assimilation test, the colony morphology (see table 3) and the microscopic examination. And the order of frequency of species isolation, regarding the SD clinical variants, was also defined.
from the samples of dandruff: M. globosa was identified in 2 samples (one in inflammatory and one in the non inflammatory dandruff lesion), M. restricta was identified in 5 samples (all were non inflammatory), and M. obtusa was identified mixed with M.globosa in a non-inflammatory lesion. from the samples of postauricular (flexural) lesions: M. pachydermatis was identified from 5 samples. from the samples of trunk: M. restricta was identified from the one petaloid lesion, and M. restricta was identified in the one-psoriasiform lesion, no Malassezia was identified from the follicular lesion. from the samples of the face: from blepharitis lesions M. globosa was identified from the 2 samples, from the medial side of the eye brow M.globosa was identified from one sample, from the nasolabial folds M. pachydermatis was identified from one sample. from the cradle cap lesions: M. globosa was identified from two samples; M. furfur was identified from one sample. from the sample taken from the flexure of the trunk of children: M. restricta was identified from one sample. from the sample taken from the pityriasiform lesion of the trunk of the child: M.restricta was identified from one sample. from the sample taken from the flexure of trunk of a child M. restricta was identified. from the sample taken from the trunk of a child M. restricta was identified. These differences were statistically insignificant as (P=0.126)
Regarding the Malassezia identified from the SD patients and from the normal skin samples: revealed in SD lesion, M. globosa in 8 (28.6%) cases, M. pachydermatis was identified in 6 (21.4%) cases, M. restricta was identified in 8 (28.6%) cases, M.obtusa was identified in 1 case mixed with M. globosa, M. furfur was identified in 2 (7.1%) cases, while from the normal skin samples, M. Globosa was identified in 5 (17.9%) cases, M. restricta was identified in 2 (7.1%) cases, while the other Malassezia species were not identified. These differences were statistically non significant (P=0.327).
Regarding the Malassezia species identified in relation to the different body sites; from scalp, M. globosa was identified in 8 samples, M. restricta was identified in 6 samples, M. furfur was identified in 1 sample, M.obtusa was identified in 1 samples mixed with M. globosa. from the face, M. globosa was identified in 3 samples; M. pachydermatis was identified in 1 sample. from the samples taken from the trunk M. globosa was identified in 1 sample, M. restricta was identified in 3 samples. from the flexures M. Globosa was identified in 1 sample, M. restricta was identified in 1 sample; M. pachydermatis was identified in 5samples. These differences were statistically significant (P= 0.019), (see tab: 5).
Tab: 5
Malassezia species identified in relation to the different body sites
Site of the sample taken M. globosa M. pachydermatis M. restricta M. furfur M.
obtusa
Scalp 8 0 6 1 1
Face 3 1 0 0 0
Trunk 1 0 3 1 0
Flexural 1 5 1 0 0
Total 13 6 10 2 1
Regarding the Malassezia species identified in all the study groups in relation to SD lesional skin taken from the different body sites. from the cases taken from scalp, M. globosa was identified in 5 cases, M. restricta was identified in 4 cases, M. furfur was identified in 1 case, M. obtusa was identified in 1 case mixed with M. globosa. from the cases taken from face, M. globosa was identified in 2 cases; M. pachydermatis was identified in 1 case. from the samples taken from the trunk, M. restricta was identified in 3 cases. from the samples taken from flexures, M. globosa was identified in 1 case, M. restricta was identified in 1 case, and M. pachydermatis was identified in 5 cases. These differences were statistically significant (P= 0.043).
Regarding the differences in the Malassezia species identified from the SD patients and the normal control subjects in relation to the different body sites.
In the scalp of SD patients M. globosa was identified in 5 cases, M. restricta was identified in 4 cases, M. furfur was identified in 1 case, M. obtusa was identified in 1 case mixed with M. globosa, while from the control, M. globosa was identified in 3 cases, M. restricta was identified in 2 cases. A differences were statistically non significant (P= 0.785).
In the face of SD patients, M. globosa was identified in 2 cases, M. pachydermatis was identified in 1 case, while from the control, and M. globosa was identified in 1 case. A difference that was statistically non significant (P= 0.505).
In the trunk of SD patients, M.restricta was identified from 3 cases; M.furfur was identified from 1 case, while from normal subjects M.globosa was identified from 1 case. A difference that is statistically insignificant (P=0.082).
In the flexures of postauricular and trunk areas of SD patients, M. globosa was identified from 1 case, M. pachydermatis was identified from 5 cases; M. restricta was identified from 1 case. While no Malassezia species were identified from the flexures of the normal subjects. This difference was statistically significant (P=0.007).
Discussion
In this study it was found most of the patients were in the adult group, which can be explained by another study that found that the population density of Malassezia yeast was significantly higher in the age group (AG) of 21-30 years compared with other AGs (Seldon, 1998). In this study most of the patients who were in the adult group, the onset of SD started sine puberty and also this can be explained by the research that supposed that the cause of transformation of the yeast phase of Malassezia to the mycelia form is presumably to be changes in the composition of fatty acids of the SG secretions, and the increase in SG activity due to the increase androgen concentration (Seldon, 1998).
The questionnaire in our study revealed that emotional stress was significantly related to SD, also Mayser et al in 2003 observed that SD is aggravated by emotional stress. This can be explained by the fact that prolonged psychological stress inhibits many aspects of the immune response including innate immunity (e.g. natural killer cell lyses), T-cell responses, as it induces CD8 cell division, and thus increasing their number and suppressing the immune function, and inhibits antibody production (Rabin, 1999). Also adrenergic stress hormones alter the synthesis and release of cytokines by white blood cells (leukocytes), which is responsible for stimulating cellular release of specific compounds involved in the inflammatory response (Rassnick et al, 1995). The longer the stress, the more the immune system shifted to negative changes, first at the cellular level and later in broader immune function (Rabin, 1999). And since that the cellular immunity is known to be of major importance in the host defense against fungal infection (Casadevall et al, 1998) and as it was found that it is also important in maintaining Malassezia as a commensal (Smith et al, 1994).
Also we found a significant relation between occurrence of SD and family history which might be explained by that there is a genetically determined host susceptibility and liability for heavy colonization by Malassezia, or for the infectivity by heterochthonous yeast flora (Faregmann, 1993), but this family history was not only in parents and siblings but also in husbands and wives which may be due to the same environmental conditions shared by the same family, or that infection may be transmitted from another individual through heterochthonous mycelia, but this was against the work of Mayser et al in 2003, who claimed that SD is not contagious.
In this study it was found that most of the patients experienced exaggeration of their symptoms in winter. Seldon in 1998 also observed this and stated that SD activity is increased in winter and early spring with remissions commonly occurring in summer. But also 5 of this study cases experienced exaggeration of their symptoms in summer. This might be explained by the claiming of Mark in 2002, that hot humid environmental conditions favor the heavy colonization of Malassezia on the skin surface, also the increased frequency of washing in summer especially if accompanied with aggressive brushing may alter the bacterial / yeast flora ratio on the skin in favor of Malassezia. Seldon in 1998, also observed that in SD patients, Malassezia were more numerous and made up a larger proportion of their flora. Also excessive brushing represents trauma that was claimed by Mark in 2002 to aggravate SD. While 3 of this study group noticed no seasonal variation in their symptoms, which is probably related to imbalance between Malassezia and the host defense mechanisms.
Although SD was the subject of many valuable studies there are still many unresolved aspects in regards to its pathogenesis, causative pathogen(s) that was supposed to be Malassezia, with no precise pathomechanisms completely elucidated so far (Pierard et al, 2006).
The expansion of the genus Malassezia has generated interest in epidemiological investigations of the distribution of Malassezia species in a range of dermatoses including SD on which variable results have been reported from different geographical regions (Rincon et al, 2005), but we could not reach any study regarding this point from Egypt.
In this study cultures of SD lesional samples were positive for Malassezia growth in 89.3%, while among samples taken from the normal individuals it was positive in 25%. This difference was highly significant, suggesting that Malassezia might play a role in the etiology of SD, which also can explain the positive correlation found between the number of yeasts on direct examination and the clinical severity of lesions in SD patients (Rendic et al, 2003).
Many other researchers also cultured the Malassezia from SD lesional skin and samples taken from healthy volunteers and different results were obtained. Of these that agreed with this current study were Gupta & Bluhm in 2004 who found that the colonization rate of Malassezia species was higher in the seborrhoeic dermatitis patients, and Mirza et al in 2005, as they found that cultures for Malassezia growth were positive in 86% of SD patients, While among the normal individuals the cultures were positive in (44%).
But also there were a study which their results were against the results found in this study, as, Rendic et al in 2003 who found the yeast Malassezia in 76% of SD patients and in 82% of subjects without skin lesions.
In this study it was found that the most commonly identified species from SD lesional skin were M. Globosa & M. Restricta, followed by M. Pachydermatis then M.Furfur, whereas M.Obtusa was identified in just one sample mixed with M. Globosa. This agreed with another study held in Japan, that found the species most commonly detected in SD patients were M. Globosa and M. Restricta (Tajima, 2004).
Other studies revealed that M. globosa was the most frequently isolated species from lesional skin of SD patients followed by M furfur and M sympodialis in two independent studies, one of them was held by Hernandez et al in 2003 in Mexico, & the other by Rendic et al in 2003 in Spain. Also In a study held in Greece, M. globosa was the prevalent species isolated from SD lesional skin (Gaitanis et al, 2006). While M. restricta and M. furfur were the most commonly isolated species from lesional skin of SD patients in a study held in Colombia (Rincon et al, 2005).
There were other studies held in different countries that observed different results regarding the frequency of Malassezia species isolated from lesional skin of SD, as Gupta& Bluhm in 2004 who held their study in Canada & Rincon et al in 2005 who held their study in Mexico, they both independently found that the more frequently isolated species were: M. sympodialis and M. slooffiae.
In a study held in Sweden both M. sympodialis and M. obtusa were the more frequently isolated species (Sandstrom et al, 2005).
The differences noted in the results obtained from different studies can be owed to that there are genuine differences in the distribution of the species on the skin of individuals in different geographical areas (Midgley, 2000; Rincon et al, 2005).
from all the above it was noticed that the most commonly involved species in most of the studies was M. globosa, this might be explained by the results of a study held by Rincon et al in 2005 which observed that M. globosa was involved at high frequency in patients with dermatological pathologies, suggesting a higher pathogenicity of this species.
The frequent high rates of identification of M. globosa & M. restricta from SD lesional skin in our study can be owed to that, among all other Malassezia species, they have the highest lipase, phospholipase and esterase activity which will produce lipolysis of epidermal lipids. This degradation of sebum, free fatty acids from triglycerides, is required for their growth as they consume specific saturated fatty acids, leaving behind unsaturates, which when penetrate the skin result in inflammation, irritation and flaking of the skin (Cafarchia and Otranto, 2004; Ro and Dawson, 2005).
In this study it was found that from the apparently healthy skin samples, M. globosa was the most frequently identified species followed by M. restricta, while the other Malassezia species were not identified.
Also M. globosa was the most prevalent species isolated from the apparently healthy skin from a Mexican population in a study Hernandez et al in 2003, but it was followed by M sympodialis, M slooffiae and M furfur. Also two independent studies, one of them was done in Japan by Nakabayashi et al in 2000, and the other was carried out by Rincon et al in 2005 in Colombia, on apparently healthy skin, both found that the more prevalent species were M.globosa, followed by M. sympodialis then M. furfur.
Regarding the Malassezia identified from the SD patients in comparison to these, which were identified from the normal skin samples in our study, there were no statistically significant differences between them. Hernandez et al in 2003, Sandstrom et al in 2005 and Rincon et al in 2005 also observed this result. No other study was against this point.
This might be explained by the suggestion of Walters et al in 1995 who stated that the simple overgrowth of Malassezia is unlikely to be the cause of SD, although it mostly acts as a cofactor linked to other factors. These factors include the increase of SG secretion with the change in its fatty acids composition (Kayoma et al, 2004), and an altered relationship between commensal yeast phase of Malassezia and the host leading to its change into the pathologic mycelial form (Gupta and Bluhm, 2004), together with an abnormal host immunologic response to the Malassezia (Gupta and Nicol, 2004). All in a genetically determined individual leads to a heavy colonization by Malassezia (Faregmann in 1993).
In this study we found that, from control subjects, the isolated species from the scalp belonged only to two species: M. globosa & M. restricta. But from the face & trunk the only isolated species was M. globosa. While no Malassezia species were identified from the flexures of the normal subjects. from SD lesional skin, the isolated species from the scalp belonged to: M. globosa & M. restricta, followed by M.furfur. from the face, the isolated species were M. globosa & M. pachydermatis. from the trunk, they were M. restricta & M. furfur. from the flexures they were M. pachydermatis, followed by M.globosa & M. restricta. from this it was observed that there was no significant difference in the species isolated from the different sites of the body when comparing SD and control results, but generally there was a significant relation between the M. species and the different body sites.
There were many researches which identified Malassezia species in SD patients, and some of them compared this with normal volunteers not only quantitavily but also qualitatively, in respect to their sites on the body. Cunningham et al in 1990, who held their research in Leeds University, UK on normal individuals, they found that M. sympodialis were most frequently isolated from the back and chest and M.globosa and M.restricta strains were most commonly isolated from head region. These results were supported by another research also held in UK at the same year (Ashbee et al, 1990).
Different results were obtained by another study also held in UK found that M. sympodialis was the predominant isolate on both the chest and back, in contrast, with this the mean population densities of M.globosa and M.restricta did not differ significantly between sites. On the forehead and cheeks there was no difference between the mean population densities of the three serovars. And this was the distribution in both the SD patients and the control, but with higher frequencies in SD patients’ skin (Ashbee et al, 1993). Another research found that the isolated species from trunk belonged to only two species: M.globosa followed by M. sympodialis. from face and scalp M.globosa, M.furfur and M. sympodialis were frequently isolated, but M. pachydermatis was less frequently isolated from the scalp and face. These results in these sites are significantly higher in SD cases than in normal subjects (Nakabayashi et al, 2000)
Regarding the relation between clinical variants of SD and the Malassezia species identified from them. It was not significant. This might be owed to the suggestion of Ashbee et al in 1993, which was that it is not that, the simple overgrowth of a particular strain of Malassezia, which can contribute to the development of dermatoses, but rather it can be a cofactor. Sohnle in 1998 stated that the immune status of the host rather than the characteristics of the organism per se is thought to play the major etiologic role. Also Watanabe et al in 2001 found that Malassezia stimulates cytokine production by Keratinocytes and there are differences in cytokine production of Keratinocytes regarding the different dermatoses.
Summary and conclusion
Seborrhoeic dermatitis (S.D.) is a papulosquamous disorder that causes flaking of the skin of the scalp, face, chest, and the creases of the arms, legs and groin. The common etiology of SD is a convergence of four factors: (1) individual susceptibility, (2) sebaceous gland (SG) secretions (3) microbial factors specially Malassezia which is normally present on the skin in small numbers, but sometimes its numbers increase, resulting in skin problems and (4) immunologic abnormalities, and activation of the complement system.
Malassezia yeasts are dimorphic, lipophilic, opportunistic organisms. Nowadays, the genus Malassezia has expanded to include eleven species. The role of Malassezia in SD pathogenesis is not yet resolved but supported in many researches, their this study aimed at identifying the Malassezia species commonly associated with seborrhoeic dermatitis skin lesions, and to determine if there is an association between the S.D. clinical variant and specific Malassezia species. In order to do this, SD patients and control subjects answered a specially designed questionnaire and samples from them were included in a microbiological analysis by applying the scheme of identifying the Malassezia species according to Guillot et al, 1995.
It was found that SD was mainly affecting adults with its onset at puberty most of them noticed exacerbation of their symptoms in association with emotional stress and in winter. In this study the frequency of positive cultures for Malassezia growth obtained from SD lesional samples were significantly higher than that obtained from normal individuals. The most frequently identified Malassezia species in SD skin were M. Globosa and M. Restricta. Which might be due to a higher pathogenicity of these species, as they have the highest lipase, phospholipase and esterase activity which will produce lipolysis of epidermal lipids, leaving behind unsaturated fatty acids, which when penetrate the skin result in inflammation, irritation and flaking of the skin.
There was a significant relationship between the Malassezia species identified from each body site regardless that they are patients or the control subjects as the most frequent Malassezia species identified from the scalp were M. Globosa and M. Restricta. from the face, it was mainly M. Globosa. from the trunk, it was mainly M. Restricta. from the flexures, it was mainly M. Pachydermatis. Regarding the relation between clinical variants of SD and the Malassezia species identified from them. Which suggest that it is not that the simple overgrowth of a particular strain of Malassezia, which can contribute to the development of SD, but rather it, can be a cofactor, and that in disease status the immune status of the host rather than the characteristics of the organism per se is thought to play the major etiologic role.
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Appendix (1)
Questionnaire
Personal history
Name
Age
Residence
Occupation
Nutritional habits
Sun exposure
Present history
Complaint
First attack or a recurrence
Onset
Course
duration
Itching
Erythema
Scaling
Soreness
Redness
Seasonal variation
Sites affected
Duration
Course
Any treatment taken (duration and effect)
Association with:
Psychological stress
Endocrinal disorders:
Obesity
D.M
Dermatological diseases:
Severe acne
Rosacea
Psoriasis
Drug intake
Systemic corticosteroid therapy
Family history
Any member of the family affected with any dermatological disease.
Appendix (2)
Lactophenol cotton blue
It is used or the staining and microscopic examination of fungi.
Composition
0.05g cotton blue; 20g phenol crystals; 40ml glycerol; 20 ml lactic acid; 20 ml distilled water.
Method
-Dissolve the cotton blue in distilled water.
-Add the phenol crystals to lactic acid in a glass beaker.
-Add the glycerol.
Filter the cotton blue and distilled water solution into the phenol/glycerol/lactic acid solution. Mix and store at room temperature.
Appendix (3)
Dixon agar
Dixon agar contain the following per liter:
Malt extract 36 g, mycological peptone 6 g, bacteriological agar 15 g, Ox bile desicate20 g, Tween 40 10ml, and glycerol alpha mono-oleate 2.5 ml.
Preparation:
Agar was prepared by adding distilled water to the dry ingredient, heating to dissolve and then adding the Tween 40 and glycerol alpha mono-oleate. Agar was autoclaved at 121 degree centigrade for 15 min. after cooling; approximatly18 ml of agar was dispensed into 90 mm plastic petre dishes. Agar plates were stored in plastic bags at 4-degree centrigrade.
Appendix (4)
Sabouraud agar
It is a base for cultivation of the fungi.
Composition
Approximate formula per liter purified water.
- Pancreatic digest of casein 5.0g
- Peptic digest of animal tissue 5.0g
- Dextrose 40.0g
-Agar 15.0g
Preparation
Suspended 65 g. of the powder in one liter of the purified water. Mix thoroughly, heat with frequent agitation and boil for one minute to completely dissolve the powder, autoclave at 121degree centigrade for 15 min.
الملخص العربى
تتميز بالبثور الحرشوفيه التى تتسبب فى تقشر بفروة الرأس، بجلد الصدر، تجاعيد مرض زيادة افراز الغدد الدهنية ( السيلان الدهنى) هو من الامراض التى الذراعين، الارجل والمناطق الأربية.
ان هذا المرض منتشر حول العالم بنسبة من 3-5% و هو يصيب الاطفال دون الثلاثة شهور والبالغين غالبا ما بين 30- 60سنة.
هذا المرض يعزى الى التقاء ثلاثة عوامل بالشخص الذى لدية القابلية و العوامل الجينية للاصابة بالمرض الا و هى زيادة و اختلاف تركيب افرازات الغدد الذهنية خاصة تحت تأثير الهرمونات الجنسية، اختلال بجهاز المناعة، و عوامل خاصة بفطر الملاسيزيا الذي يعتبر فطر متعايش فى الجسم و انتهازى، مزدوج الهيئة، محب للدهون.
على الرغم من أن كثير من الأبحاث قد لاحظت أن فطر الملاسيزيا يتدخل بشكل ملموس في نشأة مرض زيادة افراز الغدد الدهنية، الا أن هذا الدور لم يتم تحديده بعد بشكل واضح. بعض الأبحاث قد اقنرحت أن فطر الملاسيزيا يتغذى على الدهون المشبعة التى تدخل في تركيب افرازات الجلد و الغدد الدهنية حيث تحللها لدهون غير مشبعة التى يؤدى اختراقها للجلد الى التهاب و تهيج و تقشر للجلد و فروة الراس، أخرى أقترحت ان الملاسيزيا تعتبر عامل مساعد فى ظهور المرض من خلال احداث اضطرابات بالجهاز المناعى خاصة عندما يتم التحور الطورى للفطر من صورة معايشة الى صورته الباثولوجية حيث يتكاثر تحت ظروف معينة يتعرض لها المريض.
الهدف من الدراسة
البحث عن ما اذا كان هناك زيادة بمعدل تواجد الملاسيزيا للجلد المصاب بمرض زيادة افراز الغدد الدهنية عنه للجلد السليم.
تحديد ما اذا كان هناك فصائل محددة من الملاسيزيا على ارتباط باحد الاشكال الاكلينيكية للمرض عن الاخرى.
خطة البحث
لتحقيق الهدف من الدراسة تم اجراء هذه الدراسةعلى شريحة من مرضى زيادة افراز الغدد الدهنية المترددين على عيادة الامراض الجلدية والتناسلية بالمستشفى جامعة قناة السويس، كما تضمنت الشريحة المتطوعين الأصحاء، و ذلك خلال ستة أشهر من للدراسة. حيث تم سؤال المرضى و الأصحاء و فحصهم فحصا جلدياو تم أخذ عينة من القشور الموجودة على الجلد فى المناطق المصابة بالأشكال الاكلينيكية المختلفة، و ذلك لعمل مزرعة للفطر حيث تم التعرف على الفطر بطريقة نموه و شكله.
أجريت هذه الدراسة على عدد 28 مريض زيادة افراز الغدد الدهنية بمختلف اشكاله الاكلينيكية و 28 من المتطوعين الاصحاء من مختلف الاعمار و من الجنسين شريطة الا يكونوا قد استخدموا علاجا مضادا للفطريات سواء عن طريق الفم او موضعيا خلال الفترة السابقة لاخذ العينة.
من هؤلاء
9 كانوا يعانون من قشر الرأس.
6 كانوا يعانون من زيادة افراز الغدد الدهنية بثانيا الجلد ( خلف الأذن).
أحدهم كان يعانى من زيادة افراز الغدد الدهنية وردى بالجذع.
أحدهم كان يعانى من وجود الطفح على ننابت الشعربالجذع.
4 كانوا يعانون منالنوع الذى يصيب الأطفال دون السنتين مع قشرة سميكة تغطى الرأس مثل القبعة.
2 كانوا يعانون من ألتهاب دهنى بجفن العين.
2 كانوا يعانون من زيادة افراز الغدد الدهنية من جلد الوجه أحدهم من منطقة الحاجب و الأخرى من الثنايا بين الأنف و الشفة .
أحدهم كان يعانى من زيادة افراز الغدد الدهنية نخالى الشكل بالجذع.
أحدهم كان يعانى من زيادة افراز الغدد الدهنية بثانيا الجلد بمنطقة الجذع.
و قد أجرى لكل من أفراد العينة الاتى:
أخذ التاريخ المرضى.
فحص موضعى للجلد لمعرفة نوع و مكان الاصابة.
أخذ عينة من القشور الموجودة على الجلد فى المناطق المصابة المختلفة وعينة من جلد المتطوعين.
فحص ميكروسكوبي باستعمال 15% بوتاسيوم هيدروكسيد و 5% لاكتيفينول الأزرق.
زرع القشور فى وسط ملائم ديكسون أجار (Dixon Agar medium).
تزريع الفطر الناتج على الصبارود أجار ((Sabouraud Agar medium و ذلك لفصل الملاسيزيا باكيديرماتيس.
لمعرفة باقى الأنواع من الملاسيزيا(Malassezia) المعتمدة فى نموها على الدهون تم عمل اختبار تمثيل التوين (Tween Assimilation test).
تم عمل اختبار كاتاليز و ذلك للتمييز بين ملاسيزيا رستريكتا ( كتاليز سالب)، و ملاسيزيا أوبنيوزا و جلوبوزا ( كتاليز موجب) و تم التمييز بين كل من ملاسيزيا أوبنيوزا و ملاسيزيا جلوبوزا بالفحص الميكروسكوبى.
و كانت النتائج كالاتى:
لقد وجد أن حدوث المرض تكون غالبا عند سن البلوغ، و قد لاحظ معظم المرضى أنهم يعانون من تفاقم أعراض مرضهم مع حلول فصل الشتاء و أثناء تعرضهم للضغوط النفسية. كما لوحظ أن استعمال الملابس المصنوعة من الألياف الصناعية يؤدى الى زيادة نسبة حدوث المرض.
كما أسفرت الدراسة عن أن تواجد فطر الملاسيزيا للجلد المصاب بالمرض أعلى بشكل ملحوظ عنه للجلد السليم.
وجد أن أكثر الفصائل الموجودة بجلد المرضى هى من فصيلة الملاسيزيا جلوبوزا و رستريكنا مما قد يعزى الى أن لديهم أعلى نسبة نشاط لانزيمات الليبيز، الفوسفوليباز، الاستيريز الذين يعملون على اذابة الدهون المشبعة ”الموجودة بافرازات الجلد و الغدد الدهنية” مخلفة وراءها الدهون غير المشبعة و التى يتسبب اختراقها للجلد فى تهيجه، التهابه و تقشره.
كما وجد من خلال الدراسة وجود علاقة بين فصائل الملاسيزيا و مناطق الجسم المختلفه و التى لم مختلفة فى المرضى عن المتطوعين. حيث كانت أكثر الفصائل تحديدا من فروة الراس تنتمى الى ملاسيزيا جلوبوزا و رستريكتا، من الوجه ملاسيزيا جلوبوزا، من الجذع ملاسيزيا رستريكتا و من الثنايا ملاسيزيا باكيدرماتيس.
لم توجد علاقة بين فصائل فطر الملاسيزيا و الأشكال الاكلينيكية المختلفة لمرض زيادة افراز الغدد الدهنية. هذا قد يعزى الى أنه ليس مجرد تواجد فصيلة معينة من فطر الملاسيزيا هو المسؤل عن الاصابة بالمرض و لكن فطر الملاسيزيا يساهم فى حدوثه بكونه عامل مساعد لحدوث اختلال بجهاز المناعة للشخص المعد جينيا للاصابة بهذا المرض.