Blastomycosis in man after kinkajou bite.

We report transmission of Blastomyces dermatitidis fungal infection from a pet kinkajou to a man. When treating a patient with a recalcitrant infection and a history of an animal bite, early and complete animal necropsy and consideration of nonbacterial etiologies are needed.

Cutaneous blastomycosis.

We report on a patient with cutaneous blastomycosis and no evidence of systemic involvement. This diagnosis was made on the basis of clinical findings and confirmed by histologic examination and results of culture. The primary lesion in blastomycosis is almost always pulmonary. However, occasionally, as in our patient, the pulmonic focus resolves spontaneously before the patient presents. Disseminated lesions occur most often in the skin, followed by bone, genitourinary tract, and central nervous system. Our patient had an excellent response to ketoconazole without adverse effects.

A basis for resistance of Blastomyces dermatitidis killing by human neutrophils: inefficient generation of myeloperoxidase system products.

The mechanism by which the yeast form of Blastomyces dermatitidis resists killing by human peripheral blood polymorphonuclear neutrophils (PMN) was investigated. The metabolic products of the oxidative burst generated during the interaction of PMN and B. dermatitidis or Candida albicans were detected by lucigenin- or luminol-enhanced chemiluminescence (CL). Interaction of PMN and C. albicans resulted in luminol-enhanced CL 100-fold greater than that generated by PMN and B. dermatitidis. This correlated with killing of C. albicans and resistance of B. dermatitidis. Since B. dermatitidis and PMN interactions resulted in significant lucigenin-enhanced CL, deficient luminol CL was not due to a lack of products from the NADPH oxidase system. Killed B. dermatitidis cells at 37 degrees C were more efficient than live cells in stimulating PMN for luminol-enhanced CL; however, only fragmented B. dermatitidis cells elicited luminol-enhanced CL equivalent to that of C. albicans. Since lysates of PMN were active in a cell-free hydrogen peroxide-peroxidase-halide system, resistance of B. dermatitidis to PMN was not due to a defect in PMN peroxidase. Taken together, these findings indicate that resistance of B. dermatitidis to killing by PMN results from inefficient generation of products from the peroxidase-dependent PMN microbicidal system.

Isolation and partial characterization of a plasma membrane-enriched fraction of Blastomyces dermatitidis.

The isolation and characterization of a plasma membrane-enriched fraction is reported from the pathogenic fungus Blastomyces dermatitidis employing a simplified and inexpensive procedure. The yeast cells, suspended in an osmotic stabilizer buffer, were subjected to gentle mechanical disruption followed by a three-step differential centrifugation to obtain a 40,000 x g sediment. This fraction showed 2.7- and 3.3-fold relative enrichment of the plasma membrane marker enzymes Mg2(+)-ATPase and 5'-nucleotidase, respectively, with modest cross-contamination of other intracellular membranes. The fraction comprised approximately 49% protein, 31% carbohydrate, 11% phospholipids and 9% sterol with sterol to phospholipid molar ratio of 1.1. Transmission electron microscopy of the plasma membrane-enriched fraction showed homogeneous vesicles with a tripartite unit membrane of 115 A thickness.

Isolation, purification, and radiolabeling of a novel 120-kD surface protein on Blastomyces dermatitidis yeasts to detect antibody in infected patients.

No well-defined Blastomyces-specific antigens are currently available. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting to identify immunologically active molecules in the cell wall of B. dermatitidis. A major immunoreactive 120-kD protein (WI-1) was present in all five strains studied and comprised 5% of the protein in the cell wall extract obtained after freezing and thawing yeast cells. WI-1 was recognized by serum from all 10 patients with blastomycosis but by none of those from 5 patients with histoplasmosis. It was purified by electroelution, radiolabeled with 125I, and incorporated into a radioimmunoassay (RIA) for serodiagnosis of blastomycosis. Antibody to WI-1 was detected in 58 (85%) of 68 patients with blastomycosis (geometric mean titer, 1:2,981), in two (3%) of 73 patients with histoplasmosis, coccidioidomycosis, sporotrichosis, or candidiasis (titers, 1:86 and 1:91) and in none of 44 healthy persons. WI-1 was shown to be a surface molecule abundant on B. dermatitidis yeasts that were indirectly stained with serum from a rabbit immunized with WI-1. Approximately 0.93 pg of WI-1 or 4.7 x 10(6) WI-1 molecules were found on the surface of an individual yeast using an antigen-inhibition RIA; none was found on Histoplasma capsulatum or Candida albicans yeasts. We conclude that WI-1 is a novel, immunologically active surface molecule on the invasive form of B. dermatitidis and that WI-1 can be used to reliably detect antibody and study the immunopathogenesis of blastomycosis.

Subcutaneous blastomycosis: diagnosis by fine-needle aspiration cytology.

Blastomycosis is a rare disease. Fine-needle aspiration of nodules and neck masses is generally well accepted for head and neck cancer. This method has recently been used to make a rapid diagnosis of blastomycosis so that therapy could be instituted before the usual 4 to 6 week delay that is necessary for fungal cultures to mature. We believe this technique is reliable, provided the services of an experienced cytopathologist are available. The current treatment for blastomycosis is amphotericin B or ketoconazole, though newer antifungal agents hold promise for the treatment of this disease in the future.

Ultrastructural changes in Blastomyces dermatitidis after in vitro exposure to lidocaine.

Electron microscopic examination of yeasts of Blastomyces dermatitidis, exposed in vitro to concentrations of lidocaine that occur when the drug is used for topical anesthesia, showed that lidocaine rapidly damaged intracellular structures. The extent of damage was dependent on the concentration of drug and length of exposure. The observed ultrastructural changes were very similar to those reported for other drugs that directly damage membranes. This relationship suggests that the antifungal effect of lidocaine is the result of direct membrane damage.

Cerebral blastomycosis: an immunodiagnostic study.

Cerebral blastomycosis may simulate a brain tumor. Its diagnosis is sometimes very difficult. The morphologic identification of the fungus may be misleading because it shares some common features with many other dimorphic fungi. Culturing and conversion of the organism from mycelial phase to yeast phase are not always successful. Immunofluorescent staining of the biopsy tissue is useful in confirming the diagnosis. However, a combination of double immunodiffusion (DID) test and complement fixation (CF) test makes the diagnosis more accurate and reliable. The direct role of macrophages in defending the host against blastomycosis is illustrated by electron microscopy.

Ultrastructural aspects of cytoplasmic ribosomes from Histoplasma capsulatum and Blastomyces dermatitidis as revealed by heavy metal staining.

Cytoplasmic ribosomes were isolated and purified from sonicates of the mycelial and yeastlike growth forms of the pathogenic dimorphic fungi, Histoplasma capsulatum and Blastomyces dermatitidis. Similar ribosomal fractions were prepared from Neurospora crassa and Saccharomyces cerevisiae. These latter organisms were selected as typical filamentous and yeastlike monophasic fungi, and their ribosomes were used as reference standards. High resolution electron microscopy permitted a comparison of both positively and negatively-stained ribosomes to those dehydrated without heavy metal salt. Such studies revealed statistically significant differences in physical dimensions. Cautious interpretations of substructural detail of the various ribosomal preparations suggested both interphasic and interspecies differences.

The effect of growth media upon the ultrastructure of Blastomyces dermatitidis.

The ultrastructure of yeast, intermediate transitional forms, and mycelia of Blastomyces dermatitidis cultured in brain heart infusion (BHI) was compared with that of like forms grown in leucine-glucose (LG) medium. Blastomyces dermatitidis yeasts were also grown in yeast extract - peptone (YP) medium and in glucose - yeast extract - peptone (GYP) medium, and their ultrastructures were compared with BHI-grown yeasts. Yeast, intermediate, and mycelial cells cultured in LG had more granular cytoplasm and better defined membranes than analogous BHI-grown cells. Yeasts grown in LG contained more glycogen and fewer lipid bodies and had no visible intracytoplasmic membrane systems. Some yeasts from LG cultures had bizarre shapes and structure. Intermediate and mycelial forms from LG cultures contained more glycogen and lipid bodies than analogous cells from BHI. Yeasts grown in YP had a more highly defined internal structure than BHI-grown yeasts. Some cells were doubly budded. and some were elongated. Similar elongated yeasts were found growing in GYP. Yeasts grown in GYP lacked a well-defined internal structure and had less glycogen than BHI-grown yeasts. They had no visible lipid bodies, but large vacuoles were present. Intrayeast hyphae were observed only in BHI-grown intermediate cells.

Ultrastructural studies on the dimorphism of Paracoccidioides brasiliensis, Blastomyces dermatitidis and Histoplasma capsulatum.

Whole cells or cell walls of the yeastlike and mycelial forms of Paracoccidioides brasiliensis, Blastomyces dermatitidis and Histoplasma capsulatum were treated successively with sodium hydroxide, beta-1,3-glucanase and pronase. The microfibrils in the insoluble residues, probably composed of chitin, were examined in the electron microscope. In the yeastlike form, tightly interwoven, randomly oriented microfibrils were seen. On the other hand, in the mycelial form, a large portion of microfibrils tended to lie in a more or less longitudinal orientation. A role of chitin in the conversion from the yeastlike form to the mycelial form is discussed.

Problems of differential diagnosis of blastomyces and Russell bodies.

The European dermato-histologist is only rarely confronted with the diagnosis of granulomatous and systemic mycosis. The proper identification in sections and smears of blastomycetes of systemic importance is rather problematic, as Russell bodies may simulate fungi in certain stains. An own observation is reported with particular emphasis on differential diagnosis.

Light and electron microscopic observations of blastomyces dermatitidis in sputum.

Freshly expectorated sputum from a 52-year-old man with extensive pulmonary blastomycosis was examined by light and electron microscopy. Wet preparations of the sputum, stained with 1% aqueous cresyl violet, revealed numerous thick-walled, spherical blastospores and occasional budding forms. Ultrastructurally, the blastospores revealed a double-layered cell wall, the peripheral portion of which consisted of a thick, densely staining lamellar material of variable thickness, suggestive of the glycocalyx of bacterial cells. Mitochrondria and other organelles were easily recognized, but distinct nuclei were not observed. Retraction of the cell membrane and cytoplasmic mass from the cell wall was a common finding. Results of this case study suggest that electron microscopy of sputum may serve as an important adjunct to conventional staining techniques for the presumptive diagnosis of blastomycosis. Similar studies of other fungi in respiratory tract secretions may be helpful in providing more precise morphologic identification.

Role of the conidium in dimorphism of Blastomyces dermatitidis.

Fine details of yeastlike cell development of Blastomyces dermatitidis from its conidium are described and illustrated by electron micrographs. When cultured in an enriched medium at 37C, conidia of two strains of B. dermatitidis readily underwent ultrastructural changes consistent with mycelial to yeast dimorphism. Although hyphal cells contained in the conversion cultures were observed consistently to undergo profound degenerative changes, the conidia rapidly germinated to give rise to short germ tubes which subsequently enlarged to form intermediate yeast mother cells (YMC). The wall of the germ tube arose from the innermost layer of the wall of the germinant. During the transition globoid osmiophilic inclusions of unknown origin and function were observed in vacuolated areas of the germ tube and YMC cytoplasm. Yeastlike daughter cells then budded from the intermediate YMC. Since transformation was readily accomplished under in vitro conditions favoring mycelial to yeast dimorphism, it is suggested that the conidium of B. dermatitidis represents the primary infective unit of this pathogenic fungus.