Pityrosporum species as a cause of allergy and infection
Jan Faergemann MD, PhD, Department of Dermatology, Sahlgrenska University Hospital, S-413 45 Gothenburg, Sweden
Molecular biology using rRNA sequence analysis and nDNA comparisons, as well as GC (guanine/CYTOSINE) ratios in extracted DNA, has clearly divided the genus Pityrosporum or Malassezia into seven different species (1, 2). M. pachydermatis, the nonlipophilic member of the genus, is isolated primarily from animals. M. furfur, M. sympodialis, M. globosa, M. obtusa, M. restricta, and M. slooffiae are the lipophilic members of the genus. The genus name Malassezia has been used by these researchers, but I still prefer the name Pityrosporum. According to guidelines for taxonomy, the name used by the person who first describes a microorganism has priority. In 1874, Malassez described yeast-like cells in the stratum corneum of patients with various skin diseases, and the name Malassezia furfur was proposed by Bailon. However, Malassez was never able to culture the microorganism. In 1913, Castellani and Chalmers cultured the organism and characterized its growth properties ( 3). They introduced the name Pityrosporum ovale, and their description of the organism is based on the description of the fungus both in skin scales and in the laboratory ( 3). The name Pityrosporum ovale will be used in the rest of this paper.
P. ovale is part of the normal skin flora (3, 4). However, it is not always a harmless saprophyte but may also be an opportunistic pathogen. It is the etiologic agent of or plays an important role in pityriasis versicolor, Pityrosporum folliculitis, seborrheic dermatitis, and atopic dermatitis (3, 4). Even systemic infections with this yeast have been reported (3, 4).
Pityriasis versicolor is a chronic superficial fungal disease usually affecting the upper trunk, neck, or upper arms (3[4, 5]–6). Lesions are slightly scaling and papular or nummular. They may coalese to involve greater parts of the body and may vary in color from red to brown to white. The spores and short hyphae of P. ovale can be detected by microscopy. Under the influence of predisposing factors, P. ovale changes in pityriasis versicolor from the round blastospore form to the mycelial form. The most important exogenous factors are high temperature and high relative humidity, factors which probably explain why pityriasis versicolor is more common in the tropics. The most important endogenous factors are greasy skin, hyperhidrosis, hereditary factors, corticosteroid treatment, and immunodeficiency.
Pityriasis versicolor has a worldwide distribution. In tropical areas, it has been reported in 30–40% of the population ( 6). The incidence is much lower in temperate climates (1–4%). Pityriasis versicolor is generally a disease of postpubertal and mature ages, when the sebaceous glands are most active, and is generally seen in otherwise healthy people. It is found on skin where sebaceous glands are present, and it is most often localized to the sebaceous areas of the trunk, often occluded by clothing. However, in the tropics, it is more often localized to the face. Lesions are slightly scaling, papular, nummular, or confluent and vary from red or brown to white. Without treatment, pityriasis versicolor is a chronic disease, and, after treatment, recurrence is an outstanding problem, affecting 60% of subjects 1 year after treatment and 80% after 2 years.
The diagnosis is primarily based on the typical clinical picture in combination with bright-yellow fluorescence under Wood's light examination and direct microscopy (5, 6). Direct microscopy is of major importance. Potassium hydroxide (20%) wet smears can be used, but the tape method is very easy. Thin layers of stratum corneum containing the fungus can be obtained by stripping with tape. The section of the tape containing the fungi can be stained with methylene blue (1%, 1 min). The round budding cells and hyphae (“spaghetti and meatballs”) can easily be identified.
There are numerous ways of treating pityriasis versicolor topically, with many different formulation alternatives (5, 6). However, depigmentation will remain for several months after treatment. The patients should treat the whole trunk, neck, arms, and legs down to the knees, even when small areas are involved. Systemic therapy is primarily indicated for extensive lesions, for lesions resistant to topical treatment, and for frequent relapse. However, with short-term treatment, the risk of side-effects with systemic therapy may be minimized, and oral antifungals may therefore be used even for other indications (7–9). The high rate of recurrence, affecting 60% of patients in 1 year and 80% after 2 years, is an outstanding problem in pityriasis versicolor. Recurrence is due to the presence of predisposing factors, which may be difficult to eradicate. Therefore, a permanent cure is difficult to achieve, a fact which explains the chronicity. Consequently, a prophylactic treatment regimen is necessary to avoid recurrence ( 10).
Immune response to P. ovale in healthy subjects and patients with pityriasis versicolor
Serum IgG antibodies against P. ovale are found in sera from both healthy adults and patients with pityriasis versicolor ( 11). Titers are very low in children and are low in 80-year-old as compared with 30-year-old subjects ( 12). Antibodies against the organism are produced when an individual becomes colonized by the yeast, and serum antibodies are found in the same titers in sera from both healthy adults and patients with pityriasis versicolor ( 11). However, studies have shown an increase in antibody titers in patients (13, 14). In most studies, the indirect immunofluorescence technique with whole P. ovale cells has been used. However, the ELISA method is much more sensitive ( 12). With this technique, it would also be easier to study different antigens.
A cell-mediated immune response to P. ovale has been demonstrated in patients and healthy individuals (15[16, 17]–18). In a study by Sohnle & Collins-Lech, both patients with pityriasis versicolor and healthy controls demonstrated positive lymphocyte transformation responses, but the response was significantly lower in patients than in controls ( 16). Lymphocytes from patients with pityriasis versicolor produced significantly less lymphocyte migration factor when stimulated with P. ovale extract ( 16). Later, Wu & Chen found a higher response in patients with pityriasis versicolor than in healthy controls ( 17), However, Ashbee et al. found no difference between patients and controls ( 18).
A recent study by Kesavan et al. found that Pityrosporum could significantly decrease the release of the proinflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) by human peripheral blood mononuclear cells from healthy individuals ( 19). Removal of the lipids from the cell wall has been shown to reverse the suppressive effect on IL-1 and IL-6 ( 20).
A review paper by Ingham & Cunningham proposed the following theory regarding the interaction of P. ovale with nonspecific immune defense mechanisms ( 21). P. ovale may act as an adjuvant by stimulating free and fixed macrophages. Secondly, azelaic acid, produced by P. ovale, has been shown to decrease formation of oxygen in radicals by neutrophils in vitro. If P. ovale is normally killed by phagocytic cells, the humoral immune defenses (complement opsonization and antibody opsonization) will play only a minor role in limiting the spread of P. ovale in tissues. Richardson & Shankland have shown that only 5% of phagocytosed Pityrosporum was killed by neutrophils after stimulation ( 22). It has also been shown that Pityrosporum can activate both the alternative and the classical complement pathway ( 23).
In pityriasis versicolor, exogenous and endogenous predisposing factors are probably of major importance in the disease and its recurrence. The transformation from blastospore to hyphal form of the organism is equivalent to that seen in cutaneous Candida infections.
In biopsies from pityriasis versicolor, fungi are observed in the stratum corneum, and a moderate cell infiltrate in both the epidermis and the dermis has been described ( 24). Most of the infiltrating perivascular cells were CD4-positive cells; pityriasis versicolor is not a simple overgrowth of the fungus in the stratum corneum, but is accompanied by infiltrating immunocompetent cells in both the epidermis and the dermis.
Pityrosporum folliculitis is characterized by follicular papules and pustules localized to the back, chest, upper arm, sometimes the neck, and less often the face. It is often associated with troublesome itching (5, 25, 26). Under the influence of predisposing factors, Pityrosporum folliculitis may be explained by an extensive growth of P. ovale in the hair follicle. Local occlusion may play an important role. The inflammation may be due both to products of the yeast and to free fatty acids produced as a result of the lipase activity of the fungus.
Pityrosporum folliculitis is more common in tropical countries, and one study from the Philippines has shown a prevalence of 16.5% in patients visiting a dermatologic clinic. In temperate climates, the disease is more common during the summer months. Acne aestivalis, which has a clinical picture corresponding to Pityrosporum folliculitis, was originally described as a condition characterized by heavy sun exposure. The disease is also seen as a complication in patients receiving oral corticosteroids or immunosuppressive treatment. It is also a complication seen more often in AIDS patients.
The typical lesions are small follicular papules and pustules (5, 25). Pruritus is often the main complaint, and there is generally a discrepancy between the small lesions and the often troublesome itching. Distribution of lesions predominantly on the trunk, itching, and lack of comedones distinguish the condition from acne. However, the two diseases may often coexist, with nonitching acne lesions on the face and itching lesions of Pityrosporum folliculitis on the trunk and upper arms.
The diagnosis is based on a typical clinical picture of itching papules and pustules as the predominant symptoms, direct microscopy and culture in combination with histopathology, and the effect of antimycotic treatment. If a biopsy is serial cut and MS stained, black, round, budding yeast cells and sometimes even hyphae will be found in a dilated follicle.
The effect of antimycotic treatment is often dramatic (5, 25). Most cases respond well to topical treatment. In patients with extensive lesions or in those who do not respond to topical treatment, oral ketoconazole or the triazoles fluconazole or itraconazole may be effective alternatives ( 5). Lesions and itching recur in most patients if treatment is not maintained intermittently. Therefore, a prophylactic treatment schedule, such as topical treatment once or twice a week, is mandatory.
Immune response to P. ovale
Pityrosporum folliculitis may be explained by an overgrowth of the yeast in the follicle. The inflammation may be due to products released such as free fatty acids and the complement activity of the yeast. However, the mechanism that predisposes some patients to develop Pityrosporum folliculitis and others to pityriasis versicolor is still not clearly understood.
By the indirect immunofluorescence technique, circulating antibodies against P. ovale were found to be present in higher titers in patients with Pityrosporum folliculitis than in healthy controls or in patients with pityriasis versicolor ( 26). When high numbers of yeast cells are present deep in the follicle, as in the case of Pityrosporum folliculitis, antibody production may be more stimulated than when the yeast cells are present primarily in the stratum corneum, as in pityriasis versicolor (5, 26). The cellular response in skin lesions was also more pronounced and the cell infiltrate was greater than in pityriasis versicolor ( 26). Most of the infiltrating cells were CD4-positive cells. The number of Langerhans' cells was also increased. HLA-DR-expressing keratinocytes have also been described, indicating that a cellular immune reaction was present ( 26).
Several patients with Pityrosporum folliculitis produced a flare reaction around the lesions, and a wheal after scratching suggesting histamine release. However, prick test results with P. ovale protein extract were negative or only weakly positive, indicating that type I hypersensitivity to the yeast cannot explain the wheal-and-flare reaction in this disease ( 26).
There are now many studies indicating that P. ovale plays an important role in seborrheic dermatitis (5, 27–29). Many of these are treatment studies which describe the effectiveness of antimycotics, paralleled by a reduction in the number of P. ovale, and recolonization leading to a recurrence of seborrheic dermatitis (5, 27, 28). Other studies indicate that disorders of the immune function are important. The increased incidence of seborrheic dermatitis in patients with immunosuppressive disorders suggests that the relationship between P. ovale and the immune system is of importance (29, 30). HIV-positive patients, especially patients with AIDS, have an increased incidence of seborrheic dermatitis. Patients with neurologic disorders such as Parkinson's disease, multiple sclerosis, stroke, and even mood depression also have an increased incidence of seborrheic dermatitis.
Seborrheic dermatitis is characterized by inflammation and desquamation in areas with a rich supply of sebaceous glands; namely, the scalp, face, and upper trunk. Dandruff is the mildest manifestation of the disease. Seborrheic dermatitis is a common disease, with a prevalence range of 2–5% in various studies ( 29).
The skin lesions are distributed on the scalp, eyebrows, nasolabial folds, cheeks, ears, presternal and intrascapular regions, axilla, and groin. The lesions are red and covered with greasy scales. Itching is common in the scalp. Complications include lichenification, secondary bacterial infections, and otitis externa. The course of seborrheic dermatitis tends to be chronic with regular flare-ups. In temperate climates, it improves during the summer period. The disease flares during periods of mental and physical stress.
Antifungal therapy for P. ovale is effective in treating most cases of seborrheic dermatitis, and prophylactic treatment with antifungal drugs reduces the recurrence rate much more than with corticosteroids (5, 27, 28).
Immune response to P. ovale
The increased incidence of seborrheic dermatitis in patients with immunosuppressive disorders suggests that the relationship between P. ovale and the immune system is important. In peripheral blood from a high number of patients with seborrheic dermatitis, we found an increase in the number of natural killer cells and decreased phytohemagglutinin and concanavalin A stimulation (29, 30). Secondly, we found low P. ovale-specific serum IgG antibody titers in patients compared to controls. Other studies have found raised antibody titers ( 31) or no differences in titers ( 32). Two reports found a reduced lymphocyte stimulation reaction when lymphocytes from patients with seborrheic dermatitis were stimulated with P. ovale extract (33, 34). However, another study found an increase in both lymphocyte transformation response and leukocyte migration inhibition assay ( 18). As with pityriasis versicolor, the preparation of the antigen extract may, explain the difference in results. Neuber et al. found that IL-2 and interferon-gamma (IFN-γ) production by lymphocytes from patients with seborrheic dermatitis was markedly depressed and IL-10 synthesis was increased after stimulation with P. ovale extract (34). The lipid amount on the skin in patients with seborrheic dermatitis was significantly higher than in controls ( 29). In conclusion, a defect in immune response (specific or nonspecific) may facilitate fungal survival in the skin. The inflammatory response to P. ovale products was not downregulated; therefore, an increased inflammatory response occurred and the dermatitis was triggered.
There have now been many reports clearly indicating the role of P. ovale in atopic dermatitis, especially atopic dermatitis in adults in the head and neck area (35[36-50]–51). P. ovale can be cultured from patients with atopic dermatitis at the same frequency as from healthy individuals ( 39).
Skin prick test
The first published report of a role of P. ovale in atopic dermatitis was in 1983 ( 35). In this paper, Clemmensen & Hjorth described a positive effect of oral ketoconazole in the treatment of adult patients with atopic dermatitis in the head and neck area and positive skin prick tests to P. ovale ( 35). In 1985, Waerstad & Hjorth investigated 741 patients with atopic dermatitis and found positive P. ovale skin prick tests only in patients with active eczema, and most frequently among patients with head and neck dermatitis (28%) ( 36). Patients with other atopic manifestations such as rhinitis or asthma, but without atopic dermatitis, were negative in skin prick tests to P. ovale. In a paper published in 1990, Kieffer et al., in collaboration with our group in Gothenburg, Sweden, reported positive skin prick test to P. ovale in 79% of adult patients with atopic dermatitis in the head and neck area ( 37). Some 45% of patients with atopic dermatitis, but without the head and neck distribution, were positive, and patients with seborrheic dermatitis were negative. These results are much higher that the results obtained by Waersted & Hjorth. However, their material included several children. The P. ovale extract used in this and the two earlier papers is a protein extract prepared by ALK Laboratories, Copenhagen, Denmark. The same extract has also been used for a histamine-release assay (Lucotest-HR, Lundbeck Diagnostics, Copenhagen, Denmark) in basophils after stimulation with the antigen. The results of histamine release after stimulation with P. ovale extract paralleled the results obtained with the prick test ( 37). In another paper, Rokugo et al. found that the results of prick tests vary according to the age of the patients ( 38). Among patients with atopic dermatitis younger than 10 years, 39% were positive in the prick test, while 64% of patients older than 10 years were positive ( 38). Wessels et al. found positive skin prick tests to P. ovale in 84% of adult patients with atopic dermatitis ( 41). In a study by Broberg et al., children with atopic dermatitis, rhinitis, and asthma, without atopic dermatitis, and healthy controls were investigated ( 39). A total of 60 children in each category were included, and the children were divided into four groups: 0–5 years, 6–10 years, 11–15 years, and 16–21 years, with 15 children in each group. Skin prick tests to P. ovale were positive in 23% of patients with atopic dermatitis, 0% of patients with rhinitis and/or asthma, and 8% (three subjects) of healthy controls. Among the patients with atopic dermatitis, 1/15 patients in the age group 0–5 years was prick test positive compared to 6/15 or 40% in the age group 16–21 years ( 39). In summary, positive skin prick tests to P. ovale are primarily found in adult patients with atopic dermatitis, especially in adult patients with the head and neck distribution of atopic dermatitis.
Rokugo et al. found many patients with atopic dermatitis who were patch test positive to P. ovale extract ( 38). However, they used the chamber scarification method, and Kieffer et al., using a standard test procedure without scarification, were unable to reproduce these high numbers of positive results ( 37).
Specific IgE antibodies to P. ovale
The first paper describing specific IgE antibodies to P. ovale was published in 1991 ( 40). Specific antibodies were found especially in patients with atopic dermatitis and less commonly in patients with other atopic manifestations. This was later verified by other groups using various extracts from P. ovale(39[40-44]–45). A study by Broberg et al. found specific IgE antibodies in eight children with atopic dermatitis, five in the age group 16–21 years, and only one patient with rhinitis and/or asthma had specific IgE antibodies ( 39). Several IgE-binding components of P. ovale have been isolated ( 40–46, 48–53). Three major allergen components of P. ovale have later been identified with monoclonal antibodies ( 45). Two of them, a 67-kDa and a 37-kDa component, were proteins, and one, a 14-kDa component, was probably a carbohydrate. Other papers have found allergenic cross-reactivity between P. ovale and Candida albicans(43, 44, 51, 52). According to Zargari et al., the 14-kDa component may be found in both yeasts ( 45). Jensen-Jarolim et al. have also tried to characterize various components of P. ovale, and they described 9-kDa and 15-kDa components that probably are nonprotein carbohydrate components of the cell wall ( 42). However, Savolainen et al. have described what they think are protein components common to both P. ovale and C. albicans(51, 54). Recently, Lintu et al. have described IgE antibodies to both protein and mannan antigens of P. ovale in sera from patients with atopic dermatitis ( 52). They found other major components than did Zargari et al. ( 52). There are several explanations of the different results obtained. The technique used is important, and the strain of P. ovale used may also be important. Recently, it has been shown that the length of the culture period may be important ( 48). The protein content of the extract was highest after 2 days but gradually declined after day 4. On the other hand, the carbohydrate content remained fairly constant.
Schmidt et al. have identified the complete cDNA sequence and expression of one major alergen protein of P. ovale ( 50). According to the WHO/IUS nomenclature, this antigen is named Mal f 1. Recently, Yasueda et al. have isolated two additional major antigens named Mal f 2 and Mal f 3 ( 55).
Kröger et al. measured the synthesis of various lymphokines as well as the IgE synthesis from peripheral blood mononuclear cells after stimulation with P. ovale extract ( 47). They found that IL-4 and IL-10 synthesis was increased and IL-2 and IFN-γ synthesis decreased in patients with atopic dermatitis and with RAST-positive specific serum IgE antibodies compared to healthy controls. IgE synthesis in the supernatants was increased only in patients with atopic dermatitis and with RAST-positive specific serum IgE antibodies ( 47). In another study, T-cell clones obtained from both the blood and skin of one patient with atopic dermatitis responded significantly higher than controls to stimulation with P. ovale extract ( 49). The T cells had a Th2-like profile. These data indicate that P. ovale plays a role at least in one group of patients with atopic dermatitis.
Clemmensen & Hjorth have shown oral ketoconazole to be statistically more effective than placebo in the treatment of patients with atopic dermatitis ( 35). This has later been confirmed in an open study ( 56). The positive effect of itraconazole has also been mentioned ( 42). In an unknown number of patients with atopic dermatitis, topical treatment with bifonazole has also been effective ( 36). In an open study, the combination of hydrocortisone and miconazole was effective in the treatment of atopic dermatitis ( 58). However, in a double-blind, controlled study of hydrocortisone and miconazole compared with hydrocortisone alone, no statistically significant difference was found, and both treatments were effective ( 59). Oral treatment may be more effective because it more effectively eradicates P. ovale located deep in the follicle.
Our knowledge of the etiology, pathogenesis, and treatment of pityriasis versicolor, Pityrosporum folliculitis, seborrheic dermatitis, and atopic dermatitis has increased tremendously during the last 10 years. Predisposing factors play a major role in pityriasis versicolor and Pityrosporum folliculitis. In seborrheic dermatitis, an abnormal reaction to P. ovale leading to a nonspecific or specific immune response is a major factor. In some patients with atopic dermatitis, especially patients with a head and neck distribution, P. ovale may play a major role as an allergen, and treatment should include antifungal therapy.