Species diversity, antifungal susceptibility and phenotypic and genotypic characterisation of Exophiala spp. infecting patients in different medical centres in Brazil.

The Exophiala genus is responsible for many superficial and invasive infections resulting from black fungi. Identification of Exophiala at the species level is based on morphological observations complemented by molecular tests. The aim of this study was to identify 23 clinical isolates of Exophiala spp. and evaluate the antifungal susceptibility to seven different agents. Molecular identification was based on an analysis of ITS region of rDNA using genomic databases. The micromorphology was evaluated by microculture and scanning electron microscopy. The susceptibility tests were performed using the antifungal agents 5-fluorocytosine (5-FC), amphotericin B (AMB), itraconazole (ITC), voriconazole (VRC), posaconazole (PSC), caspofungin (CFG) and terbinafine (TRB). The ITS analysis identified 100% of the following isolates as: E. dermatitidis (8), E. xenobiotica (6), E. bergeri (4), E. oligosperma (3), E. spinifera (1) and E. mesophila (1). The antifungal susceptibility tests showed that the triazoles compounds were in vitro the most active agents against Exophiala. ITS sequencing enabled the accurate identification of the 23 tested isolates. The triazoles, particularly itraconazole and posaconazole, exhibited MIC values lower than AMB, CAS and 5-FC. Although the guidelines do not indicate AMB for treatment against Exophiala spp., this study showed activity for all of the tested species, except E. mesophila.

Subcutaneous phaeohyphomycosis caused by Exophiala spinifera in a European patient with lymphoma: a rare occurrence case report and literature review.

Exophiala spinifera is a dematiaceous fungus responsible for rare skin infections presenting as phaeohyphomycosis or chromoblastomycosis which has been primarily reported in tropical and subtropical areas (Asia, South and North America). We report the first case of E. spinifera phaeohyphomycosis in a European patient. The phaeohyphomycosis was limited to the skin, involving the finger of an immunocompromised patient presenting with a large B-cell lymphoma treated by R-mini-CHOP regimen. Remission was initially achieved by surgical excision; however, a local subcutaneous relapse required treatment with itraconazole. We performed a literature review of the 32 previously published cases of E. spinifera phaeohyphomycosis highlighting its clinical phenotype: disseminated infection with extracutaneous involvement and poor prognosis were reported in young patients, of whom some were recently associated with CARD9 mutations, whereas cases in older immunocompromised patients were limited to the skin and showed better prognosis. There is currently no standard treatment for E. spinifera phaeohyphomycosis; however, itraconazole, alone or in combination, allowed partial or complete response in 16 out of 20 cases.

Subcellular distribution and chemical forms of cadmium in a dark septate endophyte (DSE), Exophiala pisciphila.

Our objective was to understand the cadmium (Cd) tolerance mechanisms by investigating the subcellular distribution, chemical forms of Cd and adsorptive groups in the mycelia of Exophiala pisciphila. We grew E. pisciphila in the liquid media with increasing Cd concentrations (0, 25, 50, 100, 200, and 400 mg L(-1)). Increased Cd in the media caused a proportional increase in the Cd uptake by E. pisciphila. Subcellular distribution indicated that 81 to 97% of Cd was associated with the cell walls. The largest amount and proportion (45-86%) of Cd was extracted with 2% acetic acid, and a concentration-dependent extraction was observed, both of which suggest that Cd-phosphate complexes were the major chemical form in E. pisciphila. A large distribution of phosphate and Cd on the mycelia surface was observed by scanning electron microscopy-energy dispersive spectrometer (SEM-EDS). The precipitates associated with the mycelia were observed to contain Cd by transmission electron microscopy-energy dispersive X-ray spectroscopy (TEM-EDX). Fourier transform infrared (FTIR) identified that hydroxyl, amine, carboxyl, and phosphate groups were responsible for binding Cd. We conclude that Cd associated with cell walls and integrated with phosphate might be responsible for the tolerance of E. pisciphila to Cd.

[Electron microscopy of spindle pole bodies in pathogenic fungi].

Morphology and dynamics of the spindle pole bodies (SPBs) in Exophiala dermatitidis and Cryptococcus neoformans were examined by freeze-substitution and serial ultrathin sectioning electron microscopy. The SPBs showed double forms and were located on the nuclear envelope in G1 phase, entered the nuclear membrane or the periphery of nucleus in M phase, and appeared to duplicate in early G1 phase in these yeasts. Thus, the SPBs in these yeasts are clearly different from those of Saccharomyces cerevisiae.

Exophiala xenobiotica infection in cultured striped jack, Pseudocaranx dentex (Bloch & Schneider), in Japan.

This report describes Exophiala infection in cultured striped jack, Pseudocaranx dentex, in Japan in 2005. One hundred out of 35,000 fish died per day and mortalities continued for 1 month. Diseased fish showed swelling of the abdomen and kidney distension. Numerous septate hyphae, pale brown in colour, were seen in kidney in squash preparations. Histology revealed abundant fungal hyphae and conidia in gill, heart and kidney. Fungal hyphae were accompanied by cell necrosis and influx of inflammatory, mainly mononuclear cells. The fungus isolated from the diseased fish had septate hyphae, pale brown in colour and 1.8-3.0 microm in diameter. Conidiogenous cells were conspicuous annellides, short or cylindrical or fusiform in shape. Conidia were one-celled, ellipsoidal with smooth walls, accumulated in balls at the apices of annellides that tended to slide down, 1.5-2.0 microm in width and 3.0-5.0 microm in length. The fungus was classified into the genus Exophiala based on its morphology and as Exophiala xenobiotica based on the sequences of the ITS 1-5.8S-ITS 2 regions of rDNA. This is the first record of this fungus in a marine fish.

Histopathology of the mangrove land crab Ucides cordatus (Ocypodidae) affected by lethargic crab disease.

Lethargic crab disease (LCD) has caused extensive epizootic mortality of the mangrove land crab Ucides cordatus (Linnaeus 1763) (Brachyura: Ocypodidae) along the Brazilian coast. Direct culture of tissue samples from sick crabs and subsequent isolation and purification identified the causative agent as an Exophiala species of fungus. The histopathology of crabs with variable signs of LCD indicates that the most affected tissues are the epidermis, connective tissue, heart, hepatopancreas, nervous system, and gills. Gonads, somatic muscles, and digestive system are less affected by the fungus. The observed pathology is compatible with the clinical signs of LCD. Necrosis, tissue degeneration, and congestion of hemal sinuses and vessels are present in heavily infected organs. Nerve fibers may be compressed by accumulations of yeast-like cells. In heavy infections the tissue of gill lamellae is destroyed with subsequent dilation or compression. Cellular immune responses include hemocytic infiltration, agglutination and encapsulation, and phagocytosis. Phagocytosis of yeast-like cells is abundant in the connective tissue associated with the exoskeleton. These results indicate that LCD is the result of a systemic phaeohyphomycosis caused by a species of Exophiala. The present study also suggests that dispersal of the fungus within the crab occurs through the hemal system.

Effects of disrupting the polyketide synthase gene WdPKS1 in Wangiella [Exophiala] dermatitidis on melanin production and resistance to killing by antifungal compounds, enzymatic degradation, and extremes in temperature.

BACKGROUND: Wangiella dermatitidis is a human pathogenic fungus that is an etiologic agent of phaeohyphomycosis. W. dermatitidis produces a black pigment that has been identified as a dihydroxynaphthalene melanin and the production of this pigment is associated with its virulence. Cell wall pigmentation in W. dermatitidis depends on the WdPKS1 gene, which encodes a polyketide synthase required for generating the key precursor for dihydroxynaphthalene melanin biosynthesis. RESULTS: We analyzed the effects of disrupting WdPKS1 on dihydroxynaphthalene melanin production and resistance to antifungal compounds. Transmission electron microscopy revealed that wdpks1Delta-1 yeast had thinner cell walls that lacked an electron-opaque layer compared to wild-type cells. However, digestion of the wdpks1Delta-1 yeast revealed small black particles that were consistent with a melanin-like compound, because they were acid-resistant, reacted with melanin-binding antibody, and demonstrated a free radical signature by electron spin resonance analysis. Despite lacking the WdPKS1 gene, the mutant yeast were capable of catalyzing the formation of melanin from L-3,4-dihyroxyphenylalanine. The wdpks1Delta-1 cells were significantly more susceptible to killing by voriconazole, amphotericin B, NP-1 [a microbicidal peptide], heat and cold, and lysing enzymes than the heavily melanized parental or complemented strains. CONCLUSION: In summary, W. dermatitidis makes WdPKS-dependent and -independent melanins, and the WdPKS1-dependent deposition of melanin in the cell wall confers protection against antifungal agents and environmental stresses. The biological role of the WdPKS-independent melanin remains unclear.

Quantitative three-dimensional structural analysis of Exophiala dermatitidis yeast cells by freeze-substitution and serial ultrathin sectioning.

The morphologies, numbers, sizes and volumes of all organelles and cell components identified on ultrathin sections of aerobically grown exponential phase yeast cells of Exophiala dermatitidis in G1 phase were examined by freeze-substitution fixation and serial ultrathin sectioning. The cell wall consisted of three layers and occupied approximately 22% of the cell volume. The nucleus was approximately 1.8 microm in diameter and occupied approximately 7% of the cell volume. There was only one nucleolus in the nucleus and it occupied approximately 16% of the nuclear volume. There were 17-52 mitochondria per cell, occupying 7-12% of the cell volume. Five to ten endoplasmic reticula were present per cell; these occupied only 0.2% of the cell volume and did not form a network. There were 1-4 vacuoles per cell and they occupied 4-10% of the cell volume. Storage material was round and electron transparent and occupied 4-11% of the cell volume. The cytosol occupied 43-53% of the cell volume. The Golgi apparatus, spindle pole body, autophagosomes, multivesicular bodies, lipid bodies, microtubules and microfilaments occupied approximately 1% of the cell volume in total. About 200,000 ribosome particles, 1000 glycogen granules and several tens of microtubules (average length 0.78 microm) were present per yeast cell. The membranes of this yeast could be classified into three groups by their appearance and thickness. This is the first report, to our knowledge, that analysed all the components in the yeast cell quantitatively and in three dimensions, and provides fundamental information for understanding various aspects of cell biology.

Three-dimensional reconstruction of a pathogenic yeast Exophiala dermatitidis cell by freeze-substitution and serial sectioning electron microscopy.

The structure of a budding cell of the pathogenic yeast Exophiala dermatitidis was observed in three dimensions after freeze-substitution, serial ultrathin sectioning and computer reconstruction. The nucleus occupied about 10% of the cell volume. The spindle pole body was composed of two disk elements connected by an intervening midpiece, and occupied about 0.01% of the cell volume. The cell wall consisted of an inner transparent layer, a middle electron-opaque layer, and an outer fibrous layer. The mitochondria occupied about 10% of the cell volume. There were numerous mitochondria in the mother cell and the bud, but no 'giant mitochondrion' was seen. The ratio of mitochondrial volume within the bud to the mitochondrial volume of the cell was close to the ratio of bud:cell cytoplasmic volume. The results emphasize the importance of good cryofixation for 'perfect' preservation of yeast cell structure.

The spindle pole body duplicates in early G1 phase in the pathogenic yeast Exophiala dermatitidis: an ultrastructural study.

The spindle pole body of the pathogenic yeast Exophiala dermatitidis was observed during the cell cycle using freeze-substitution and serial ultrathin sectioning electron microscopy. The spindle pole body was located on the outer membrane of the nuclear envelope and consisted of two disk elements connected by an intervening midpiece in G1 through G2 phases. Each disk element was composed of filamentous materials and measured 150 nm in diameter and 100 nm in thickness. The midpiece had higher electron density and measured 60 nm in length and 40 nm in thickness. At the beginning of prophase, each disk element of the spindle pole body enlarged to more than double in size. They were separated on the nuclear envelope, and associated with numerous cytoplasmic microtubules. At mitosis, the spindle pole body entered the nuclear envelope, associated with numerous nuclear microtubules, and was located at the spindle poles. At the end of telophase, it was extruded back into the cytoplasm from the nuclear envelope. Three-dimensional analysis of cells in different cell cycles suggested that duplication of the spindle pole body took place in early G1 phase. Thus, the location, structure, and duplication cycle of the E. dermatitidis spindle pole body were different from those of Saccharomyces cerevisiae.

Electron microscopy of pathogenic yeasts Cryptococcus neoformans and Exophiala dermatitidis by high-pressure freezing.

A high-pressure freezing method was used to observe the ultrastructure of pathogenic yeasts, Cryptococcus neoformans and Exophiala dermatitidis, after freeze-substitution and ultrathin sectioning. The method well preserved the cell structure in its natural state, since the capsule, cell wall, plasma membrane, nucleus, outer and inner nuclear membranes, nuclear pores, nucleolus, mitochondria, mitochondrial membrane and cristae, vacuoles, endoplasmic reticulum, Golgi apparatus, spindle pole body, ribosomes, lipid droplets, microtubules, actin filaments, and glycogen granules were clearly visible. The method was shown to freeze cells as deep as 0.1 mm by sectioning the sample perpendicular to specimen surface. The quality of the cell image was similar to that obtained by a rapid freezing method when compared using the same materials. Thus, high-pressure freezing would be useful for making serial ultrathin sections for three-dimensional analysis of cells, which should give basic information of structure and function of pathogenic yeast cells necessary for finding an effective therapy for mycoses.

The fine structure of Hortaea werneckii.

Hortaea werneckii (strain CBS 107.67) was examined by light and transmission electron microscopy (TEM). Special attention was paid to the wall architecture, the septum with a simple pore apparatus, the annellidic type of collar and the nuclei. Two-celled organisms showed signs of distoseptation; nuclear events appeared to be rather synchronous in both cells. The fine structural results provided evidence of endogenous conidia development.

Successful treatment of systemic Exophiala dermatitidis infection in a patient with chronic granulomatous disease.

Exophiala dermatitidis, one of the saprophytic dematiaceous fungi, is a rare cause of human infection that, when invasive, is nearly always fatal. Besides the more common subcutaneous infection usually caused by traumatic inoculation, infection can also spread hematogenously, in which case the organism has a distinct neurotropism. A patient with autosomal recessive chronic granulomatous disease of childhood who was found to have a progressive pulmonary and central nervous system infection with E. dermatitidis responded to an aggressive, multifaceted therapeutic approach. Scanning electron microscopy of the cultured conidiogenous cells confirmed that the manner of conidiogenesis is typical of the genus Exophiala. We report the first successful treatment of an infection involving the lungs and central nervous system by a combination of surgical resection of the pulmonary source and medical therapy with amphotericin B, flucytosine or ketoconazole, and transfused white cells, followed by a prolonged course of fluconazole.

Karyology and hyphal characters as taxonomic criteria in ascomycetous black yeasts and related fungi.

Mycelial development of seventy-three strains of black yeasts and related fungi were studied, and numbers of nuclei per hyphal cell were counted. Two main patterns were apparent in expanding hyphae, viz. (1) uninucleate expanding hyphal cells, septum formation strictly following mitosis, and (2) multinucleate, branched, aseptate hyphal tips, septa being formed in a later stage, leading to oligo- or uninucleate mature cells. Characteristic genera in the two groups are Exophiala and Aureobasidium, respectively. In Zasmidium and in some Ramichloridium species all mycelial cells are oligonucleate. The character is indicative for relationships at the family level in black yeasts.

Corneal phaeohyphomycosis caused by Wangiella dermatitidis.

A case is described of a corneal abscess of the left eye of a patient, male, 35 years, from which there was found in pure culture a mycotic micro-organism, Exophiala (Wangiella) dermatitidis, determined also in the native preparation from the pus. The patient is feeble-minded, suffers from von Recklinghausen's disease and has an impairment of the immune system (above all the reduction of T lymphocytes).

A previously undescribed synanamorph of Wangiella dermatitidis.

A black yeast isolated from a Korean patient with subcutaneous phaeohyphomycosis was studied and identified as Wangiella dermatitidis (Kano) McGinnis on the basis of its distinctive morphology, and physiologic and serologic characteristics. In addition to the characteristic diagnostic features of W. dermatitidis, we encountered unusual multicellular structures. These consisted of globose endoconidia contained within thick-walled, dematiaceous bodies. Mycologic observation indicated that a cell increased in size, developed a thickened outer cell wall, and subsequently developed internal septa that split or lysed down their central portions to separate the developing endoconidia. To our knowledge, this reproductive form has not been previously associated with W. dermatitidis. We describe this new synanamorph of the highly polymorphic black fungus W. dermatitidis.

Exophiala pisciphila. A study of its development.

Exophiala pisciphila is a dematiaceous fungus that belongs to a group of fungi known as the 'black yeasts'. It was isolated from the skin lesions of a smooth dogfish, Mustelus canis Mitchill, that had been born in the shark exhibit tank of the New York Aquarium. The different stages of development of this fungus were studied by light microscopy and scanning electron microscopy to illustrate the morphology and surface structures of conidia and mycelium. The list of marine and fresh water fish, which have been infected by Exophiala spp. and Exophiala-like fungi has been up-dated. Potato Dextrose Agar and Malt Agar proved to be the best growth media, while Corn Meal Agar proved to be the best medium for studying the morphological features of the conidia and mycelial development of E. pisciphila, which exhibited polymorphic conidiogenesis.

Further studies on the phylogenesis of the genus Exophiala and Hortaea.

The conidial ontogenesis of the pathogenic black yeasts is studied at an ultrastructural level and their phylogenesis is discussed. Five cultures of Exophiala dermatitidis, four of E. jeanselmei, one of E. moniliae, one of E. spinifera and six of H. werneckii were observed using a scanning electron microscope. The conidial ontogenesis of the Exophiala species is not pleomorphic but only annellidic. There are definite differences in morphology of annellated tips among the Exophiala species. The ontogenesis of Hortaea werneckii consists of a combination of sympodial and annellidic conidiogenesis. Its sympodial anamorph is unique and the annellidic anamorph is considered to be a homology of the sympodial one.