Update on the genus Malassezia.

Malassezia yeasts are commensals of normal human skin, but also cause pityriasis versicolor, seborrhoeic dermatitis and evidence is accumulating that they play a significant role in atopic eczema/dermatitis syndrome (AEDS; formerly atopic dermatitis). The taxonomy of the genus has changed considerably and is likely to change more in the future. Our understanding of the interaction between Malassezia and the host demonstrates that it has the paradoxical ability to both stimulate and suppress the immune response directed against it and there is a fine balance in its existence at the interface between commensalism and pathogenicity.     

Malassezia related diseases]

The genus Malassezia is now divided into eleven species. Different species initiate or aggravate different skin diseases. In seborroheic dermatitis, M. restricta play an important role, while in atopic dermatitis, M. globosa and/or M. restricta are major cutaneous microflora. M. globosa is a causative species of tinea versicolor, and this species is also a causative species of malassezia folliculitis. We should therefore obtain better knowledge of the ecological and pathogenic roles of malassezia.     

Cutaneous immune and inflammatory reactions to Malassezia furfur]

Initiation and aggravation of several inflammatory skin diseases are associated with Malassezia furfur. These are divided into at least two groups. In one group including tinea versicolor and Malassezia folliculitis, the growth of Malassezia furfur directly triggers the development of the cutaneous lesions. In another group including atopic dermatitis, seborrheic dermatitis, and psoriasis, cutaneous lesions already developed by other mechanisms are aggravated by the growth of Malassezia furfur. Recent progress of molecular biology techniques revealed that Malassezia furfur is divided into at least seven species. Since their clinical and histological findings are quite diverse, their differences cannot be explained solely by the difference in antigenicity of each Malassezia. Instead, the cutaneous defense mechanisms against Malassezia furfur must be considered. In this article, we reviewed the mechanisms at three levels: 1) barrier functions of the uppermost layer of the skin, the stratum corneum, 2) cytokine production by epidermal keratinocytes, and 3) immune and inflammatory responses by infiltrating neutrophils and T cells.     

Description of a new yeast species, Malassezia japonica, and its detection in patients with atopic dermatitis and healthy subjects

Lipophilic yeasts of the genus Malassezia are part of the normal cutaneous microflora and are considered one of the factors that trigger atopic dermatitis (AD). We isolated two strains of Malassezia from a healthy Japanese female. Analysis of the D1/D2 26S ribosomal DNA and internal transcribed spacer region sequences of the isolates suggested that they are new members of the genus Malassezia. We propose the name Malassezia japonica sp. nov. for the isolates. M. japonica is easily distinguished from the seven known lipophilic species by its ability to assimilate Tween 40 and Tween 60 and its inability to assimilate Tween 20 and Tween 80 and to grow at 40 degrees C. Furthermore, by applying transparent dressings to the skin lesions of 36 patients with AD and the skin of 22 healthy subjects, M. japonica DNA was detected by a non-culture-based method consisting of nested PCR with M. japonica species-specific primers. M. japonica DNA was detected from 12 of the 36 patients (33.3%) and 3 of the 22 healthy subjects (13.6%). Although it is not known whether M. japonica plays a role in AD, this species was part of the microflora in both patients with AD and healthy subjects.     

New yeast species, Malassezia dermatis, isolated from patients with atopic dermatitis

Malassezia species are considered to be one of the exacerbating factors in atopic dermatitis (AD). During examination of the cutaneous colonization of Malassezia species in AD patients, we found a new species on the surface of the patients' skin. Analysis of ribosomal DNA sequences suggested that the isolates belonged to the genus MALASSEZIA: They did not grow in Sabouraud dextrose agar but utilized specific concentrations of Tween 20, 40, 60, and 80 as a lipid source. Thus, we concluded that our isolates were new members of the genus Malassezia and propose the name Malassezia dermatis sp. nov. for these isolates.     

Phylogenetic relationship of the genus Malassezia based on mitochondrial cytochrome B gene]

Mitochondrial cytochrome b genes of pathogenic yeasts and fungi were analyzed for identification and phylogenetic relationship. The species of genus Malassezia also were analyzed and each sequence was specific in the same domain of cytochrome b gene. Some species represented intraspecies variation. The structure and function of cytochrome b protein was retained and its substitution rates may be in proportion to the evolutionary period. The deduced amino acid sequence was encoded by each nucleotide sequence of cytochrome b gene, and the phylogenetic tree of eukaryote and basidiomycetous yeast was obtained using this sequence. The species of genus Malassezia formed one cluster in this tree, meaning that the concepts of this genus reflect its evolution. The mitochondrial cytochrome b gene analysis was valuable for the identification and phylogenetic analysis of the genus.     

A study of culture-based easy identification system for Malassezia

Most species of this genus are lipid-dependent yeasts, which colonize the seborrheic part of the skin, and they have been reported to be associated with pityriasis versicolor, Malassezia folliculitis, seborrheic dermatitis, and atopic dermatitis. Malassezia have been re-classified into 7 species based on molecular biological analysis of nuclear ribosomal DNA/RNA and new Malassezia species were reported. As members of the genus Malassezia share similar morphological and biochemical characteristics, it was thought to be difficult to differentiate between them based on phenotypic features. While molecular biological techniques are the most reliable methods for identification of Malassezia, they are not available in most clinical laboratories. We studied ( i ) development of an efficient isolation media and culture based easy identification system, ( ii ) the incidence of atypical biochemical features in Malassezia species and propose a culture-based easy identification system for clinically important Malassezia species, M. globosa, M. restricta, and M. furfur.     

Human infections due to Malassezia spp.

The genus Malassezia contains three member species: Malassezia furfur and Malassezia sympodialis, both obligatory lipophilic, skin flora yeasts of humans, and Malassezia pachydermatis, a nonobligatory lipophilic, skin flora yeast of other warm-blooded animals. Several characteristics suggest the basidiomycetous nature of these yeasts, although a perfect stage has not been identified. Classically, these organisms are associated with superficial infections of the skin and associated structures, including pityriasis versicolor and folliculitis. Recently, however, they have been reported as agents of more invasive human diseases including deep-line catheter-associated sepsis. The latter infection occurs in patients, primarily infants, receiving parenteral nutrition (including lipid emulsions) through the catheter. The lipids presumably provide growth factors required for replication of the organisms. It is unclear how deep-line catheters become colonized with Malassezia spp. Skin colonization with M. furfur is common in infants hospitalized in neonatal intensive care units, whereas colonization of newborns hospitalized in well-baby nurseries and of older infants is rarely observed. Catheter colonization, which may occur without overt clinical symptoms, probably occurs secondary to skin colonization, with the organism gaining access either via the catheter insertion site on the skin or through the external catheter hub (connecting port). There is little information on the colonization of hospitalized patients by M. sympodialis or M. pachydermatis. Diagnosis of superficial infections is best made by microscopic examination of skin scrapings following KOH, calcofluor white, or histologic staining. Treatment of these infections involves the use of topical or oral antifungal agents, and it may be prolonged. Diagnosis of Malassezia catheter-associated sepsis requires detection of the organism in whole blood smears or in buffy coat smears of blood drawn through the infected catheter or isolation of the organism from catheter or peripheral blood or the catheter tip. Culture of M. furfur from blood is best achieved with Isolator tubes and plating onto a solid medium supplemented with a lipid source. Appropriate treatment of patients requires removal of the infected catheter with or without temporary stoppage of lipid emulsions; administration of antifungal therapeutic agents does not appear to be necessary. Because many patients who develop Malassezia catheter-associated sepsis have severe underlying illnesses, caution must be exercised in attributing all clinical deterioration to Malassezia infection. Our better understanding of how these organisms cause disease awaits the development of a useful typing scheme for epidemiologic studies and further studies on microbial virulence factors and the role of the immune response in pathogenesis.     

Molecular analysis of Malassezia microflora on the skin of atopic dermatitis patients and healthy subjects

Members of the genus Malassezia, lipophilic yeasts, are considered to be one of the exacerbating factors in atopic dermatitis (AD). We examined variation in cutaneous colonization by Malassezia species in AD patients and compared it with variation in healthy subjects. Samples were collected by applying transparent dressings to the skin lesions of AD patients. DNA was extracted directly from the dressings and amplified in a specific nested PCR assay. Malassezia-specific DNA was detected in all samples obtained from 32 AD patients. In particular, Malassezia globosa and M. restricta were detected in approximately 90% of the AD patients and M. furfur and M. sympodialis were detected in approximately 40% of the cases. The detection rate was not dependent on the type of skin lesion. In healthy subjects, Malassezia DNA was detected in 78% of the samples, among which M. globosa, M. restricta, and M. sympodialis were detected at frequencies ranging from 44 to 61%, with M. furfur at 11%. The diversity of Malassezia species found in AD patients was greater (2.7 species detected in each individual) than that found in healthy subjects (1.8 species per individual). Our results suggest that M. furfur, M. globosa, M. restricta, and M. sympodialis are common inhabitants of the skin of both AD patients and healthy subjects, while the skin microflora of AD patients shows more diversity than that of healthy subjects. To our knowledge, this is the first report of the use of a nested PCR as an alternative to fungal culture for analysis of the distribution of cutaneous Malassezia spp.     

Epidemiology of Malassezia yeasts associated with pityriasis versicolor in Ontario, Canada.

The genus Malassezia was recently revised to include seven species, but the clinical significance of each of these species is not clearly understood. To obtain a better understanding of the contribution of individual Malassezia species to the epidemiology of pityriasis (tinea) versicolor, we used Leeming-Notman medium to culture patient skin specimens showing positive evidence of Malassezia infection in direct microscopy. Isolates were identified on the basis of recently published morphological and physiological tests for distinction of the new species. Identification using recently developed molecular criteria was also carried out for the ambiguous cases. Malassezia species were cultured from 111 cases of pityriasis versicolor in the Canadian province of Ontario. The most frequently isolated species were Malassezia sympodialis, M. globosa and M. furfur which respectively made up 59.4%, 25.2% and 10.8% of the isolated etiological agents. M. globosa was commonly isolated from a small number of pityriasis versicolor specimens obtained from investigators outside Canada. A large number of additional Ontario specimens with positive direct microscopy failed to yield a culture; however, it is suggested that this is consistent with the standard sampling practice of scraping the older portions of pityriasis lesions rather than the margins, where viable fungal cells are most likely to occur.     

Taxonomy of genus Malassezia: progress and problems]

The genus Malassezia is composed of lipophilic basidiomycetous yeasts which were recently shown to consist of seven species, one lipid-independent species, M. pachydermatis and six lipid-dependent species, M. furfur, M. sympodialis, M. globosa, M. obtusa, M. restricta and M. slooffiae. Based on this classification, we will be able to analyze pathogenicity or relationship between Malassezia-related diseases and each species.     

Factors associated with the prevalence of Malassezia species in the external ears of cattle from the state of Minas Gerais, Brazil.

Swabs collected from the external ears of 942 cattle, including both European and zebu breeds, were used to evaluate the epidemiology of Malassezia species. High numbers of Malassezia isolates were significantly associated with maturity of the animals and with diagnosed otitis. In healthy animals, a particularly low prevalence of the genus was found in Holstein cows, especially in the summer months, a finding perhaps correlating with the open, air-exposed ears of this breed. Pendutlous-eared zebu breeds and hybrids had higher levels of colonization, although this effect was more pronounced in humid regions, and was least in the dry north and west of the state. The most common species seen was M. sympodialis, which accounted for 47.1% of isolates from healthy cattle and 39.1% from cattle with otitis. In cattle with otitis sampled in summer, we found that the relatively thermotolerant M. sympodialis made up 80.8% of isolates. By contrast, during the winter the less thermotolerant M. globosa was the most common species from otitis, making up 56% of the isolates. Humidity and temperature may be the main factors explaining the epidemiology of Malassezia diseases and colonizations in both animals and humans.     

Atypical lipid-dependent Malassezia species isolated from dogs with otitis externa

During a survey of the occurrence of Malassezia species in the external ear canals of dogs with chronic otitis externa, lipid-dependent Malassezia species were isolated in three dogs. These species were identified as Malassezia furfur and M. obtusa but showed atypical assimilation patterns. To our knowledge, this is the first report of the isolation of lipid-dependent species of the genus Malassezia in association with canine otitis.     

The effects of lipid extraction on the immunomodulatory activity of Malassezia species in vitro.

Malassezia spp. are believed to be the causative agents of pityriasis versicolor and are strongly implicated in seborrhoeic dermatitis. The yeast also forms part of the normal human cutaneous microflora. We have previously shown that when Malassezia yeast cells are incubated with human peripheral blood mononuclear cells (PBMCs), they are capable of reducing the levels of pro-inflammatory cytokines produced. In order to test the hypothesis that this immunoevasive phenomenon may be related to the unusually high level of lipid in the Malassezia yeast cell wall, we have compared the immunomodulatory capacity of normal and lipid-depleted yeast cells. Stationary phase yeast cells of Malassezia sympodialis, M. globosa and M. restricta were treated with chloroform/methanol to extract the surface lipids. The lipid-depleted and non-depleted yeast cells were then co-cultured with human PBMCs from three different human donors at a ratio of 20 yeasts per leukocyte for 24 h. The levels of interleukin (IL)-1beta, IL-6 and tumour necrosis factor (TNF)-alpha were then determined by enzyme-linked immunosorbent assay (ELISA). The results demonstrated that extraction of lipid reversed the yeast cell capacity to reduce the levels of pro-inflammatory cytokines. The levels of IL-1beta, IL-6 and TNF-alpha produced in response to lipid-extracted Malassezia of all three species were either no different from or significantly greater (P < 0.05; ANOVA) than the constitutive control levels. These results suggest that the lipid microfibrillar layer of Malassezia may prevent the yeast cells from inducing inflammation and provide an explanation for the normal commensal status of the organism on human skin. The hypothesis that the lipid layer is absent or altered in seborrhoeic dermatitis may provide an explanation for the inflammatory nature of this dermatosis.     

Otitis externa associated with Malassezia sympodialis in two cats

The lipid-dependent species Malassezia sympodialis was isolated from two cats with otitis externa. To our knowledge, this is the first report of the isolation of lipid-dependent species of the genus Malassezia associated with skin disease in domestic animals.     

Immunity and resistance to astrovirus infection

Astroviruses are one of the leading causes of acute viral enteritis in infants, and are recognized as a clinically important pathogen in the elderly and the immunocompromised. In spite of this, we still know very little about the immune response to astrovirus infection. Clinical observations and human volunteer studies have indicated a role for the humoral response and suggest neutralizing antibodies are important in limiting infection. Studies of human intestinal biopsies have suggested that cellular immunity; specifically CD4(+) T-cells may also be involved in the anti-astrovirus response. Additionally, various animal models have indicated potential roles for the innate immune system in controlling infections. How these various effector arms of the immune system collaborate to result in immunity and resistance to astrovirus infection is still unknown. This review summarizes our current understanding of the immune response to this pathogen and highlights the key concepts that still need to be addressed. Until we understand the role of the immune system in astrovirus infection or other enteric viruses, we will continue to be limited in our ability to treat and control gastrointestinal diseases.     

Nocardia infections: clinical and biological aspects]

The nocardiosis is an infection caused by a bacterial pathogen agent, Nocardia, belonging to the Actinomycetales order. They are Gram-positive, strictly aerobic bacteria. Members of the genus Nocardia are ubiquitous. They are frequently isolated from soil, water, air dusts. The mode of contamination occurs by inhalation or by cutaneous or ocular traumatic lesion. Clinically, nocardiosis is essentially characterized by pulmonary diseases. Others secondary localizations are described, such as in the central nervous system. Nocardia can be responsible for important cutaneous, subcutaneous and lymphocutaneous manifestations. In the same way, some extrapulmonary diseases and spread nocardiosis are more rarely observed. Several factors seem to favour the development of Nocardia. The immunocompromised patients, particularly those with organ transplant and the patients treated with immunosuppressor treatments, offer strong predispositions to this opportunistic disease. The nocardiosis is nevertheless observed in healthy persons. In front of polymorphic and specific-less clinical manifestations, large phenotypic heterogeneity, and resistance profiles to specific antibiotics, a correct diagnosis for Nocardia species is necessary to apply an adequate treatment. The techniques of identification based on the chemotaxonomic analysis and the susceptibility to different inhibitors are efficient for the identification of genus and species. However, because of the slow growth rate of Nocardia, the reading of these tests can require several weeks of incubation. With the intention of the rapid identification of genus and species, the molecular techniques (PCR-RFLP) seem to be efficient. The technique of RAPD allows an efficient molecular typing, which will give a better knowledge concerning transmission, ecological niches and epidemic reservoirs.     

Ichthyophthirius multifiliis: a model of cutaneous infection and immunity in fishes

Summary: The parasitic ciliate Ichthyophthirius multifilus offers a useful system for the study of cutaneous immunity against an infectious microorganism. Naive fish usually die following infection, but animals surviving sublethal parasite exposure become resistant to subsequent challenge. This resistance correlates with the presence of humoral antibodies in the sera and cutaneous mucus of immune fish. A mechanism of immunity has recently been elucidated that involves and body binding to surface proteins (referred to as immobilization antigens or i-antigens) located on the parasite cell and ciliary membranes. Antibody-mediated cross-linking of i-antigens triggers a response by the parasite resulting in its exit from the host. These effects can be observed directly on the surface of live fish. In addition to allowing the observation of effector responses in vivo, Ichthyophthirius also provides a means to study the ontogeny of the mucosal immune response. The sites of antigen capture and presentation, and the sites of antibody production, are unknown with regard to cutaneous immunity. Because the external epithelial surfaces of fish are often the points of pathogen entry, a basic understanding of the inductive immune mechanisms and immune cell interactions in the skin and gills is extremely important with regard to vaccine development. The development of Ichthyophthirius as an experimental system and how it might be used to address these issues are discussed in this review.     

Immunopathogenesis of infection with the visceralizing Leishmania species

Human leishmaniasis is a spectral disease that includes asymptomatic self-resolving infection, localized skin lesions, and progressive visceral leishmaniasis. With some overlap, visceral and cutaneous leishmaniasis are usually caused by different species of Leishmania. This review focuses on host responses to infection with the species that cause visceral leishmaniasis, as they contrast with species causing localized cutaneous leishmaniasis. Data from experimental models document significant differences between host responses to organisms causing these diverse syndromes. The visceralizing Leishmania spp. cause localized organ-specific immune responses that are important determinants of disease outcome. Both the Leishmania species causing cutaneous and those causing visceral leishmaniasis require a Type 1 immune response to undergo cure in mouse models. However, during progressive murine infection with the visceralizing Leishmania sp., the Type 1 response is suppressed at least in part by TGF-beta and IL-10 without type 2 cytokine production. This contrasts with the cutaneous species L. major, in which a Type 2 response suppresses type 1 cytokines and leads to murine disease progression. Population and family studies are beginning to elucidate human genetic determinants predisposing to different outcomes of Leishmania infection. These studies should eventually result in a better understanding of the immunopathogenesis and the spectrum of human leishmaniasis.