An aberrant variant of Histoplasma capsulatum var. capsulatum.

We studied an aberrant culture of Histoplasma capsulatum var. capsulatum isolated from synovial fluid collected from the right elbow of a patient from Kansas. Colonies on Sabouraud glucose agar and other routine mycological media were glabrous to soft, moist, heaped, deeply folded or convoluted, and orange-brown with a white, irregular margin. Microscopically, hyphae were hyaline, septate, and branched and remained totally devoid of conidiation over a period of 2 years on all mycological media. Conversion to the yeast form was achieved on Pine's medium at 37 degrees C. Colonies at early stages of growth were smooth, moist, pasty, shiny, and orange-brown but soon became wrinkled and slightly raised and produced oval, thin-walled cells measuring 2 to 3 by to 4.5 microns which multiplied by polar budding. The identity of the isolate was further confirmed by utilizing the Accuprobe DNA and the exoantigen test for H. capsulatum var. capsulatum.     

Immunological studies on Histoplasma capsulatum.

Alveolar macrophages freshly harvested from normal and immunized rabbits were parasitized with yeast cells and protoplasts of Histoplasma capsulatum. Macrophages obtained from either normal or sensitized rabbits failed to phagocytize protoplasts, whereas, the yeast cells were actively ingested. There was no detectable intracellular killing by macrophages. A serological similarity was found between the whole yeast cell, the purified isolated cell wall, and the protoplasts of the fungus. Aprecipitin test of the protoplasts of the fungus gave a postive band, whereas immunodiffusion in agar was negative. Addition of immune sera activated phagocytosis, the immune sera against cell walls being the most active.     

Histoplasma capsulatum infection in nude mice.

Congenitally athymic nude (nu/nu) mice, when injected intraperitoneally with Histoplasma capsulatum, developed a rapidly fatal disseminated infection characterized by heavy parasitization of reticuloendothelial tissues. In contrast, their heterozygous (nu/X) littermates, which possessed a functioning thymus, developed only a low-grade infection which was apparently self-limited and rarely fatal. Transplantation of thymic tissue into nu/nu mice diminished greatly the severity of infection and reduced mortality by about 50%. These studies emphasize the importance of cell-mediated immunity in host defense against histoplasmosis and suggest that the nude mouse may be a valuable model for the study of this chronic intracellular infection.     

Morphogenesis and pathogenicity of Histoplasma capsulatum.

The sulfhydryl blocking agent p-chloromercuriphenylsulfonic acid (PCMS) irreversibly inhibited the mycelium-to-yeast transitions of two virulent strains of Histoplasma capsulatum, G184A and G222B, when the temperature of incubation was raised to 37 degrees C, and the block persisted even after the cultures were washed free of PCMS. Instead of transforming to yeast cells, PCMS-treated mycelia continued to grow as mycelia at the elevated temperatures. A less virulent strain (Downs) was more temperature sensitive, but it showed a similar irreversible effect at 34 degrees C. Therefore, the mycelium-to-yeast transition of H. capsulatum is not required for the adaptation of mycelia to elevated temperatures but probably results from the temperature-dependent activation of yeast-specific genes. The transition to yeast is inferred to be obligate for pathogenicity in mice because PCMS-treated mycelia failed to cause infection, and no fungi were seen in tissues after PCMS-treated mycelia were injected into mice.     

Molecular detection of Histoplasma capsulatum var. capsulatum in human clinical samples

We developed a seminested PCR for the diagnosis of histoplasmosis that amplifies a portion of the Histoplasma capsulatum H antigen gene. This assay is highly sensitive and specific, being able to detect genomic material corresponding to less than 10 yeast cells without cross-reaction against other bacterial or fungal pathogens.     

Hydroxamate siderophores of Histoplasma capsulatum

The zoopathogenic fungus Histoplasma capsulatum, like other eukaryotic aerobic microorganisms, requires iron for growth. Under conditions of low iron availability, the fungus secretes hydroxamates that function as siderophores (iron chelators). The experiments to be reported were designed to gather further information on the hydroxamate siderophores of H. capsulatum. The fungus was grown in a synthetic medium deferrated with the cationic exchange resin Chelex 100. Siderophores were detected after 4 days of incubation at 37 degrees C in media containing 0.3 to 1.0 microM iron. The secretion was suppressed by 10 microM iron. The hydroxamates were purified by reverse-phase and size-exclusion chromatography. On the basis of ions observed during electrospray mass spectroscopy, five hydroxamate siderophores were tentatively identified: dimerum acid, acetyl dimerum acid, coprogen B, methyl coprogen B, and fusarinine (monomeric). A polyclonal antibody to dimerum acid was generated. This reagent cross-reacted with coprogen B and fusarinine. Thus, the antibody detects hydroxamates in all three families of siderophores excreted by H. capsulatum.     

Studies on the catalase of Histoplasma capsulatum.

Factors which control the levels of catalase within yeast cells of Histoplasma capsulatum were studied. Only a small fraction of the total catalase activity could be detected in whole cells. The bulk of the activity was revealed in cell-free extracts or in cells permeabilized with acetone. The formation of the enzyme was regulated by glucose and by oxygen. There were large, consistent differences in the levels of catalase among strains of H. capsulatum. The sensitivity of the strains to H2O2 toxicity also varied remarkably. Peroxidase activity could not be detected in cell-free extracts of the strains. Resistance to H2O2 did not correspond to levels of catalase. There was no obvious correlation of H2O2 sensitivity and virulence among the strains.     

A serendipitous isolate of Histoplasma capsulatum

This is a report of an unexpected laboratory diagnosis of Histoplasma capsulatum. The fungus was isolated from an acute cellulitic lesion on the forearm of an elderly male patient with a functioning renal transplant. The patient resides within the environs of Brisbane and has not travelled outside Australia. We consider the isolation of H. capsulatum from a rare site in a patient resident in a non-endemic area indicative of a latent opportunistic infection in an immunocompromised patient.     

Recovery of Histoplasma capsulatum from BACTEC TB media.

From 1990 through 1994, we fortuitously isolated Histoplasma capsulatum from six patients with AIDS whose specimens of blood were processed by the BACTEC system using Middlebrook broth selective for acid-fast bacilli (13A medium). Growth indices became positive after an average of 17 days of incubation (range, 11 to 20 days). No acid-fast bacilli were seen, but small budding yeasts characteristic of H. capsulatum were present.     

Evaluation of cross-reactions in Histoplasma capsulatum serologic tests.

Cross-reactivity in Histoplasma serologic tests was evaluated by using sera from patients with histoplasmosis and other infections. Serum samples from 127 of 134 (95%) patients with histoplasmosis were judged positive by complement fixation tests, and 121 (90%) showed H bands, M bands, or both by immunodiffusion. Of these 134 patients, cross-reactions were seen to Blastomyces dermatitidis in 53 patients (40%), to Coccidioides immitis in 20 patients (16%), and to Aspergillus fumigatus in 3 patients (2%) by complement fixation. Serum samples from 5 of 99 patients (5%) with other fungal infections and from 5 of 46 patients (11%) with tuberculosis had M precipitin bands by the Histoplasma immunodiffusion test, whereas none of the 123 sera from patients with other bacterial, Mycoplasma, or viral infections showed H or M precipitin bands. In the complement fixation test, positive reactions were observed in 16 of 90 patients (18%) with other fungal infections, in 14 of 41 patients (34%) with tuberculosis, and in 18 of 105 patients (17%) with other bacterial, Mycoplasma, or viral infections. Positive reactions were seen by radioimmunoassay in 54 of 110 patients (49%) with other fungal infections, in 23 of 46 patients (50%) with tuberculosis, and in 35 of 123 patients (28%) with with other bacterial, Mycoplasma, or viral infections. These results demonstrate a wider range of cross-reactions in Histoplasma serology than has been previously recognized, and the cross-reactivity was greatest when observed by radioimmunoassay. Caution should be exercised in the interpretation of serologic data from patients with suspected fungal infections.     

Evaluation of Gen-Probe's Histoplasma capsulatum and Cryptococcus neoformans AccuProbes.

Gen-Probe's DNA probes were evaluated for use in the identification of clinical isolates of Histoplasma capsulatum var. capsulatum and Cryptococcus neoformans. Ninety-five mould-phase fungi were probed, including 41 isolates of H. capsulatum var. capsulatum. Similarly, 98 yeasts, including 42 C. neoformans isolates, were examined by using the C. neoformans DNA probe. In the study, both probes demonstrated 100% specificity and 100% sensitivity. Their use in the clinical laboratory may significantly reduce the time required for definitive identification of fungi.     

Histoplasma capsulatum yeast cells attach and agglutinate human erythrocytes.

The ability of yeast cells of Histoplasma capsulatum to attach and agglutinate human erythrocytes has been described. This is the first report involving these yeasts in the hemagglutination phenomenon. Results revealed that the yeast cells were able to bind to erythrocytes irrespective of blood groups and to agglutinate them when a high density of yeast cells was used. Assays on the inhibition of yeast attachment to erythrocytes were also performed, using sugar-treated yeast cells. Results indicate that galactose (Gal), mainly the beta-anomer, specially inhibited yeast attachment. Disaccharides (Gal-derivatives) and glycosaminoglycans containing Gal residues, mainly chondroitin sulfate C, promote this type of inhibition. In addition, preliminary data of inhibition assays also involved a probable ionic strength driven mechanism mediated by sialic acid and heparan sulfate, suggesting that yeast binding to erythrocytes could be associated with negative charges of both molecules.     

Comparison of radioimmunoassay and enzyme-linked immunoassay methods for detection of Histoplasma capsulatum var. capsulatum antigen.

The purpose of this study was to compare the conventional radioimmunoassay (RIA) to an enzyme-linked immunoassay (EIA) for the measurement of Histoplasma antigen in banked urine specimens. A correlation between the two methods would allow the EIA to be used as a nonradioactive alternative to the established 125I RIA. The study used stored urine from patients diagnosed with histoplasmosis during an outbreak in Indianapolis which began in 1988. Control specimens from healthy adults, patients with other fungal infections, urinary tract infections, or nonfungal pneumonia were also tested. Both the RIA and EIA were run concurrently. The RIA system measured antigen levels of 0.4 to 27.0 RIA units, while the EIA measured antigen levels of 0.6 to 20.1 units. Both the EIA and RIA detected measurable antigen levels in urine from 50 of 56 patients (89%) with disseminated disease and 11 of 30 patients (37%) with self-limiting disease. One of 96 control specimens, from a patient with paracoccidioidomycosis, was positive with both systems. Antigen levels measured by EIA correlated well with those measured by the established RIA method (correlation coefficient, 0.974). The EIA is an acceptable alternative to the RIA for measuring Histoplasma antigen levels in urine specimens.     

Calcium dependence and binding in cultures of Histoplasma capsulatum.

Histoplasma capsulatum is a pathogenic fungus with two distinct morphologies and lifestyles. The saprophytic form of this organism, a mold, thrives in soil and is especially abundant in the Ohio and Mississippi River valleys. Its parasitic counterpart, a yeast, colonizes phagolysosomes of mammalian macrophages. We have observed a major difference in the calcium requirements of the two forms of Histoplasma, potentially implicating the phagolysosome as a calcium-limiting compartment. Deprivation of calcium by the addition of EGTA to culture media inhibited the growth of mycelial H. capsulatum but had no effect on yeast growth in vitro. In addition, yeasts released a calcium-binding protein (CBP) detectable by a 45CaCl2 blotting technique. CBP was a major component of yeast culture supernatant and was also detectable by ruthenium red staining, another assay for calcium-binding activity. Conversely, mycelial H. capsulatum did not produce CBP, a finding that correlates with the dependence of mycelia on calcium for growth. We also describe here the purification of CBP from yeast culture supernatant by reversed-phase high-pressure liquid chromatography.     

Activation of the alternative complement pathway by Histoplasma capsulatum.

The present study was performed to determine whether Histoplasma capsulatum (yeast phase) is able to activate the alternative complement pathway. H. capsulatum yeasts were shown to consume C3 in C4-deficient guinea pig serum. Immunoelectrophoretic conversion of alternative pathway component factor B confirmed that C3 consumption was mediated by activation of the alternative pathway. These results demonstrate that H. capsulatum is able to activate the alternative complement pathway.     

Disseminated infection by Histoplasma capsulatum with AIDS

Histoplasmosis is an illness which occurs very rarely in Europe and it is especially rare in Germany. A generalised infection with Histoplasma capsulatum, a systemic mycosis of the mononuclear phagocyte system (MPS), occurs only in individuals with weakened immune systems. Within the framework of diagnostics, a pathologist can be confronted with histoplasmosis since there has been an increase in travel to and from endemic regions, as well as an increase in the number of diseases of the immune system. The presented case reports the histological intravital and post-mortem diagnostics of disseminated histoplasmosis in existing HIV-infection in the stage of manifest AIDS.