The genus Malassezia: old facts and new concepts.

Lipophilic yeasts are being considered as major opportunistic pathogens for a very long time. Most of the yeasts show an absolute requirement for long fatty acid chains and specific procedures are required for their isolation, conservation and identification. For that reason, the history of the nomenclature used for the Malassezia genus is quite complex. Before 1996, only 3 species were recognized: Malassezia furfur, M. pachydermatis and M. sympodialis. To date, the genus is composed of one non lipid-dependent species (M. pachydermatis) and 12 lipid-dependent species. No doubt that additional new taxa will be described in close future. Very recently the genome and secretory proteome of two Malassezia species was described. This analysis demonstrated the presence of multiple secreted lipases to aid in harvesting host lipids. It also revealed the presence of mating-type genes, providing an indication that Malassezia yeasts may be capable of sex.

Identification and typing of Malassezia yeasts using amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE).

Three molecular tools, amplified fragment length polymorphism (AFLP), denaturing gradient gel electrophoresis (DGGE) and random amplified polymorphic DNA (RAPD) analysis, were explored for their usefulness to identify isolates of Malassezia yeasts. All seven species could be separated by AFLP and DGGE. Using AFLP, four genotypes could be distinguished within M. furfur. AFLP genotype 4 contained only isolates from deep human sources, and ca. 80% of these isolates were from patients with systemic disease. Most of the systemic isolates belonged to a single RAPD genotype. This suggests that systemic conditions strongly select for a particular genotype. Although the clinical use of DGGE may be limited due to technical demands, it remains a powerful tool for the analysis of complex clinical samples.

A single PCR-restriction endonuclease analysis for rapid identification of Malassezia species.

AIMS: The present study describes a system based on PCR and restriction endonuclease analysis (REA) to distinguish the seven currently recognized Malassezia species. METHODS AND RESULTS: Fifty-five representative yeast isolates were examined. A single primer pair was designed to amplify the large subunit ribosomal RNA (LSU rRNA) gene of the seven Malassezia species, and identification was achieved by digestion of the PCR products with three restriction endonucleases: BanI, HaeII and MspI. A specific restriction endonuclease analysis pattern was determined for each species investigated. Moreover, PCR-REA allowed the detection and characterization of mixtures of several Malassezia species. CONCLUSION: PCR-REA of only the LSU rRNA gene is a reliable and rapid method to distinguish all Malassezia species. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR-REA represents a considerable saving in time over currently available identification procedures. This method should be evaluated on clinical material directly.

Phylogenetic position and physiology of Cerinosterus cyanescens.

Partial 25S rRNA sequencing of Cerinosterus cyanescens showed it to be a close relative of Microstroma juglandis, a member of the basidiomycetous order Microstromatales. It is unrelated to the generic type species, C. luteoalba, which is a member of the order Dacrymycetales. The clinical occurrence of C. cyanescens is possibly explained by its thermotolerance and lipolytic activity. The species' nutritional profile is established. Growth on n-hexadecane is rapid; it grows well on typical plant constituents like gallic, tannic, vanillic, quinic and p-coumaric acids, but not on 3-hydroxybenzoic acid, phenol and hydroquinone. The failure to assimilate D-galactose, L-sorbose and ethylamine, the presence of urease and sensitivity to cycloheximide are diagnostic for the species.

Osteoarthritis caused by Neocosmospora vasinfecta.

We report the case of a patient who developed an ankle osteoarthritis due to the Ascomycete Neocosmospora vasinfecta, following accidental multiple trauma to his legs in whilst in Africa. Antifungal susceptibility testing was performed. Despite a low amphotericin B minimal inhibitory concentration, parenteral antifungal therapy failed and amputation was required to resolve the osteoarthritis. Possible reasons for the failure of this antifungal treatment are examined.

Genetic diversity in the yeast species Malassezia pachydermatis analysed by multilocus enzyme electrophoresis.

Fifty-two strains of the yeast species Malassezia pachydermatis were analysed by multilocus enzyme electrophoresis. M. pachydermatis appeared to be genetically heterogeneous. A total of 27 electrophoretic types were identified that could be divided into five distinct groups with different host specificities. The diversity revealed by this electrophoretic method matched remarkably well the reported genetic variability obtained by comparing large subunit rRNA sequences. This study also suggests that genetic exchanges can occur in the anamorphic species M. pachydermatis.

Molecular reidentification of human pathogenic Trichoderma isolates as Trichoderma longibrachiatum and Trichoderma citrinoviride.

Several species of the well-known saprophytic genus Trichoderma have been identified as the cause of infections in immunosuppressed humans. Because the differentiation and identification of Trichoderma species based on morphological characters only, is very difficult, two molecular approaches were applied for species identification. Six human pathogenic Trichoderma isolates were investigated by PCR-fingerprinting and analysis of ribosomal DNA internal transcribed spacer (ITS) sequences and compared with the corresponding data sets established for described species of the genus. Five of these strains were identified as T. longibrachiatum, whereas one single strain turned out to be T. citrinoviride. Both species are very closely related and belong to Trichoderma section Longibrachiatum. These data indicate that the occurrence of pathogenic Trichoderma strains may be restricted to species of section Longibrachiatum.

Exophiala (Wangiella) dermatitidis keratitis after keratoplasty.

We report the first French case of an intraocular infection due to Exophiala (Wangiella) dermatitidis. Two months after a second corneal transplant for congenital hereditary endothelial dystrophy, the patient presented with ocular pain and corneal infiltrates leading to the graft rejection. Diagnosis was established by positive direct examination and cultures of the same fungus from corneal buttons, iris biopsies and ablated sutures.

[Cutaneous Alternaria infectoria infection after liver transplantation]. / Infection cutanée à Alternaria infectoria après greffe hépatique.

We report a case of cutaneous alternariosis after liver transplantation. This rare phaeohyphomycosis is mainly observed in patients treated by corticosteroids. This case is remarkable for the species isolated: Alternaria infectoria. Clinical and histopathological features are studied and diagnostic problems are discussed.

The role of Malassezia species in the ecology of human skin and as pathogens.

The new taxonomic structure of the lipophilic genus Malassezia was presented with key characteristics for the seven described species. Among techniques used for epidemiological surveys, the pulsed field gel electrophoresis (PFGE) was found to be of little value in contrast to randomly amplified polymorphic DNA (RAPD). Immunological studies still yielded conflicting results but at least the immunomodulatory capacity of Malassezia yeasts appeared to be related to the cell wall lipids. A review of Malassezia infections together with the present consensus for their prevention and treatment was also made.

Molecular typing of Malassezia species with PFGE and RAPD.

The currently recognized seven species of Malassezia all have different karyotypes which do not vary intraspecifically, except in M. furfur which displayed two different karyotypes. In contrast, random amplified polymorphic DNA (RAPD) typing showed the presence of genetic variation in all species. It is concluded that karyotype analysis is useful for species identification, and RAPD typing can be used in epidemiological investigations.

Molecular taxonomy and epidemiology of Blastomyces and Histoplasma species.

Cladistic analysis of partial 26S rRNA sequences was used to estimate evolutionary distances among species and varieties of the dimorphic onygenalean genera Blastomyces, Coccidioides, Emmonsia, Histoplasma and Paracoccidioides. With the exception of Coccidioides, all genera were closely related, with about 5% base differences and even less (1-2%) between Blastomyces and Emmonsia. These data were supported by a teleomorph in the same genus Ajellomyces. In a phylogenic study of a wide range of ascomycete orders and families, Coccidioides immitis was found to be closest to Aphanoascus fulvescens and Chrysosporium keratinophilum, and to have relative distances to the remaining dimorphic genera (family Onygenaceae) similar to those of the dermatophytes (family Arthrodermataceae). The sequencing data were confirmed by genomic comparisons. All dimorphic genera had a nuclear DNA base composition in the same range of 46.6-47.3% G + C. The DNA melting curves of Blastomyces and Histoplasma strains showed irregularities that were ascribed to the presence of AT-rich stretches in satellite DNA rather than in mitochondrial DNA. Derivative profiles proved to be highly reproducible within regional populations and coincided with differences in clinical behaviour of each species. Blastomyces dermatitidis generated two kinds of curves, corresponding to the geographically distinct serotypes 1 and 2. The African type (serotype 2) was characterized by a classical sigmoidal melting curve similar to that for all strains of Coccidioides, Emmonsia and Paracoccidioides. In contrast, the American type (serotype 1) contained satellite DNA (27% G + C). A rRNA base difference of 1.5% was observed between geographical types, a value slightly higher than that noted between Histoplasma capsulatum and its variety farciminosum (0.9%). All three H. capsulatum varieties presented irregularities in their DNA melting curves. The molecular data support the recognition of two of them as agents of blastomycosis and the assignment of more than one species and two varieties to the genus Emmonsia.

Differentiation of Malassezia species: selectivity of cremophor EL, castor oil and ricinoleic acid for M. furfur.

In recent years, the genus Malassezia has been reclassified based on molecular data. In addition to M. furfur, M. pachydermatis and M. sympodialis, four new species, M. globosa, M. obtusa, M. restricta and M. slooffiiae, have been described. However, apart from their lipid dependence, little is known about the metabolism and nutritional requirements of all the seven species. Further to recent studies, 10 hydrophilic emulsifiers (HLB > 10) were examined in an agar diffusion test to determine their growth-promoting effect on reference strains of the different Malassezia species. Polyethylene glycol (PEG) 7 glyceryl monoalcanoate (Cetiol HE). PEG-glyceryl stearate (Tagat S2) and macrogol-50 stearate (Myrj 53) were metabolized by all strains, while PEG-35 castor oil (Cremophor EL) was metabolized only by M. furfur. The latter observation is due to a different metabolism of castor oil and its main component, ricinoleic acid (12-hydroxy oleic acid), which may also give an insight into the pathogenesis of diseases that are associated with Malassezia spp. As hydroxy fatty acids are important in maintaining the epidermal structure and function, their metabolism specifically by M. furfur might clarify some clinical aspects of pityriasis versicolor. Apart from this speculation, use of Cremophor EL, with splitting of esculin as an additional key character, improves the distinction of the species M. furfur, M. slooffiae and M. sympodialis.

Epidemiological analysis of Malassezia pachydermatis isolates by partial sequencing of the large subunit ribosomal RNA.

The opportunistic yeast Malassezia pachydermatis is commonly recovered from both normal and diseased skin of warm-blooded animals. The diversity of M pachydermatis isolates obtained from a wide range of hosts was investigated by the partial sequencing of the large subunit (LSU) ribosomal RNA. Among 100 isolates examined, seven types (Ia-Ig) were discriminated on the basis of nucleotide sequence diversity. The seven types differed by one to five mutations, all of them corresponding to transitions. The predominant sequence, type Ia, appeared to be ubiquitous since it was observed in isolates recovered from domestic and wild carnivora, from a monkey and from man. In contrast the sequence types Ic, Id and Ig seemed to be more host-specific; they included isolates recovered exclusively from rhinoceros, dogs and ferrets, respectively. None of the seven sequence types correlated with isolation from healthy skin or a particular lesion (otitis externa or other dermatitis). The study indicated that the skin of an animal may be colonised by more than one type of M pachydermatis.

Assignment and serotyping of Trichosporon species: the causative agents of summer-type hypersensitivity pneumonitis.

We re-valued three antigenic types within the genus Trichosporon as the causative agents of summer-type hypersensitivity pneumonitis (SHP). Corresponding standard strains, TIMM 1573 (serotype I), TIMM 1318 (serotype II), and M9456 (serotype III) were assigned to species T. mucoides, T. asahii and T. montevideense, respectively, based on 95% or more DNA/DNA relatedness with each type of culture and other genetical, physiological and morphological characteristics. To confirm the significance of these serotypes, 98 other strains of Trichosporon isolated from patients' environments and 24 CBS strains, including type cultures of species described within the genus, were serotyped. Serum antibody analysis of 220 SHP patients against Trichosporon spp. were also examined to estimate the antigenic profile of SHP. The present results indicate that T. asahii and T. mucoides, which are the most common causes of trichosporonosis, appear to be the major causative agents of SHP. These observations suggest a common pathogenesis of Trichosporon inducing hypersensitivity and infection, depending on the immunological status of the host.