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En el presente trabajo se describen 5 pacientes con infección por el virus de inmunodeficiencia humana e hitosplasmosis diseminada progresiva con formas clínicas inusuales. Tres de ellos tenían diseminación cutánea, uno diseminación gastrointestinal, y uno ambas presentaciones. Además se observó que el paciente sobrevivió 18 meses presentaba anticuerpos contra el Hitoplasma capsulatum.

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Sao apresentados 25 casos de histoplasmose disseminada em pacientes com SIDA. O envolvimento mucocutaneo estabeleceu o diagnostico de histoplasmose disseminada em 68 por cento dos casos. Destacamos a importancia da histoplasmose como infeccao oportunistica em paciente HIV positivo.

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Histoplasma capsulatum var. capsulatum is a dimorphic fungus that, under special conditions, converts from its more common mycelial form to a yeast-like form. Achieving this conversion, however, has been problematical for researchers. The present study tested conversion rates in ten Histoplasma capsulatum var. capsulatum strains using seven culture media, four of wich were conventional and three novel. One of our novel media, MLGema, induced complete conversion, of two strains within five days of incubation at 35 degrees centigrades, and of all strains that eventually converted by the time of the second subculturing transfer, under defined experimental conditions. MLGema is also inexpensive and easy to produce

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These patients demonstrate the difficulty in arriving at the diagnosis of disseminated histoplasmosis. The diagnosis in two of the three patients also served as the initial AIDS case-defining opportunistic infection. In each of these patients, the clinical presentations were atypical and in only one patient was a positive exposure history elicited. Recurrent bowel obstruction was the presenting complaint in the first patient and the diagnosis was made only on pathologic exam of the resected small bowel. The second patient's diagnosis was made on biopsy of the colon via colonoscopy. The third patient's diagnosis also eluded an extensive FUO workup; he was diagnosed by bone marrow culture and silver stain of a mediastinal lymph node biopsy, despite serial negative serologic tests for histoplasmosis. The first two patients had significant gastrointestinal disease which is a relatively unusual manifestation for disseminated histoplasmosis. The third patient illustrates the limited diagnostic usefulness of serologic testing in AIDS patients and the continued usefulness of bone marrow analysis in an FUO evaluation. In conclusion, these case presentations demonstrate that disseminated histoplasmosis in patients with HIV infection can present with unusual manifestations, outside of the typical endemic arca, without a positive exposure history or positive serologic test, and may be the initial AIDS case-defining opportunistic infection in these patients. Consequently, a disseminated histoplasmosis should be considered in all AIDS patients with perplexing clinical presentations.

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Se estudiaron 50 muestras de tierra de cuatro cuevas de la region de Rio Claro (Antioquia, Colombia), con el fin de aislar Histoplasma capsulatum en su forma natural; se encontraron dos muestras positivas (4%) tomadas en sitios diferentes de la Cueva del Condor. Se describen los metodos empleados y se discuten algunas implicaciones de la presencia del hongo para las personas que visitan las cuevas.

Fifty soil specimens from 4 caves located in the Río Claro region of the Department of Antioquia, Colombia, were studied In order to recover Histoplasma capstilatum in its natural form. Two of the specimens, obtained from different sites of one of the caves, were positive. The methods employed for cultivation are described and the implications of the presence of the fungus for tourists visiting the caves are discussed

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Pneumocystis carinii pneumonia is a major cause of morbidity and mortality in immunocompromised patients. An indirect fluorescent antibody (IFA) test has been developed using monoclonal antibodies specific for antigens on the surface of P carinii. We tested the sensitivity and specificity of this IFA test for detecting P carinii in respiratory specimens of immunocompromised patients with pulmonary symptoms undergoing bronchoscopy. Both the bronchial wash and bronchoalveolar lavage specimens of patients with and without P carinii pneumonia were studied. The bronchoalveolar lavage and bronchial wash specimens were examined using modified Wright-Giemsa and methenamine silver stains. In addition, aliquots of the specimen were fixed and stained with IFA and read with a fluorescent microscope. Fifty-nine patients were found to have P carinii organisms. The bronchial wash specimen has been shown to be less sensitive than the bronchoalveolar lavage specimen for detecting the presence of P carinii. In the bronchial wash specimen from these 59 patients, only 60% had positive modified Wright-Giemsa stains, and 70% had positive methenamine silver stains. The IFA stain was positive in 93% of the specimens tested (significantly higher than the other two stains). There was only one false-positive IFA test result among the 54 patients tested with negative results. We found the IFA stain to be superior to conventional stains when examining less-than-adequate specimens, such as those from bronchial washes.

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Immunogold electron microscopy using LR Gold as a resin was undertaken to determine the distribution of actin and tubulin within the hyphae of Histoplasma capsulatum capsulatum and H. capsulatum duboisii. Both of these proteins were found throughout the cytoplasm when probed with the appropriate monoclonal antibodies.

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Seven chromosome-sized DNA molecules in the Downs strain of Histoplasma capsulatum were resolved by using chromosome-specific DNA probes in blot hybridizations of contour-clamped homogeneous electric field (CHEF) and field-inversion gel electrophoresis (FIGE) agarose gels. The sizes of the chromosomal DNA bands extended from that of the largest Saccharomyces cerevisiae chromosome to beyond that of the Schizosaccharomyces pombe chromosomes. Under our experimental conditions, the order of the five largest DNA bands was inverted in the FIGE gel relative to the CHEF gel, demonstrating a characteristic of FIGE whereby large DNA molecules may have greater rather than lesser mobility with increasing size. Comparison of the Downs strain with other H. capsulatum strains by CHEF and FIGE analysis revealed considerable variability in band mobility. The resolution of seven chromosome-sized DNA molecules in the Downs strain provides a minimum estimate of the chromosome number.

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Se estudió la cinética de fagocitosis de levaduras de Histoplasma capsulatum en una línea celular de macrófagos (J774.2), sincronizando el inóculo y la ingestión (internalización) de levaduras a 4-C por 1h, a fin de permitir un mejor contacto de los macrófagos y las levaduras durante la fase de adherencia. La fagocitosis se llevó a cabo a 37-C con diferentes períodos de incubación 0,5,15,30,60min, 3 y 24 h, para determinar el tiempo de internalización de las células levaduriformes a los macrófagos. El seguimineto de la internalización fue realizado por microscopía electrónica de transmisión y de barrido. Se utilizó Mycobacterium tuberculosis como microorganismo de referencia. La fagocitosis de Histoplasma fue retardada en comparación con la del bacilo tuberculoso y los macrófagos (J774.2) internalizaron levaduras en un tiempo aproximado de 15-30 minutos. Mientras H. capsulatum sólo fue observado en el interior del macrófago a partir de los 15 min de fagocitosis, M. tuberculosis fue encontrado desde los 0-5 minutos. Además, a los 60 min aún se observaron levauras adheridas, siendo que los macrófagos infectados con M. tuberculosis presentaron daño y muerte celular. Estos resultados apoyan las diferencias en la internalización de ambos microorganismos

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Cell wall composition of isogenic virulent-avirulent strain pairs of Histoplasma capsulatum varied markedly with respect to alpha-(1,3)-glucan content. When yeast cell walls were fractionated by standard techniques, the avirulent strains contained up to 1,000-fold less alpha-(1,3)-glucan than did their virulent parents. No alpha-(1,3)-glucan could be detected on the surface of the avirulent strain yeast cells if we used a mouse monoclonal antibody that recognized this polymer. A similar relationship between virulence and alpha-(1,3)-glucan has been described for Paracoccidioides brasiliensis. alpha-(1,3)-Glucan is also found in several other pathogenic fungi and may thus be an important common virulence determinant.

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Although antigen-reactive T lymphocytes play a central role in the host response to Histoplasma capsulatum, little is known of the nature of Histoplasma antigens recognized by these cells in vitro. Employing a murine T-cell line and two clones that are reactive with histoplasmin, we examined whether activation of T cells by histoplasmin required the presence of carbohydrate or protein moieties. The approach taken was to modify carbohydrate or protein molecules in histoplasmin by chemical or enzymatic digestion or by lectin adsorption. In parallel, antigen was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis to correlate alterations in functional activity with changes in the electrophoretic appearance of histoplasmin. Treatment of histoplasmin with periodate (0.1 M, 0.05 M, and 0.01 M) or with the endoglycosidases N-glycanase and endoglycosidase H sharply diminished the capacity of histoplasmin to trigger responses by T cells. Reactivity of T cells to histoplasmin that had been adsorbed with lectins binding mannose, glucose, or galactose was reduced by greater than 70%; conversely, the responses by T cells to antigen that had been adsorbed with lectins specific for fucose, N-acetylgalactosamine, or N-acetylglucosamine ranged from 82 to 91% of that to control antigen. Proliferative responses by T cells to histoplasmin that had been digested with chymotrypsin, protease, or trypsin were 2 to 43% of control values. The electrophoretic appearance of histoplasmin was modified by some but not all of the treatments. Partially purified H and M antigens triggered proliferation of T cells. Thus, both carbohydrates and proteins must be present to induce optimal responses by T cells. A portion of the carbohydrates is N linked to proteins, and alpha-D-mannose (or alpha-D-glucose) and beta-D-galactose are the sugar ligands of carbohydrate-containing antigens.

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From the yeast phase of the human pathogen Histoplasma capsulatum, three novel glycolipids were isolated, shown to react with sera from histoplasmosis patients, and partially characterized: compound V, ceramide-P-inositol-[mannose2]; compound VI, ceramide-P-inositol-[mannose2, galactose]; compound VIII, an isomer of compound VI [Barr, K., & Lester, R.L. (1984) Biochemistry (preceding paper in this issue)]. Ammonolysis of these lipids has yielded all the carbohydrate (oligosaccharides V, VI, and VIII) as novel, intact oligosaccharides suitable for characterization. Anomeric configurations were determined by specific glycosidase digestion and by the stability of peracetylated saccharides to CrO3 oxidation. Linkages were established by methylation analysis. These experiments yielded the following structural assignments: (formula; see text) The occurrence of galactofuranose is novel for glycosphingolipids, and it is noteworthy that compound VI is immunoreactive.

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Iron added to a chemically defined liquid medium suppressed hydroxamic acid production at 37 degrees C by yeast cells of Histoplasma capsulatum. Four hydroxamic acids, HA-I, HA-II, HA-III, and HA-IV, present in the low-iron fluid after the culture of H. capsulatum were isolated by extraction and cation-exchange chromatography through cellulose phosphate (0.35% formic acid). Visible spectra of prepared ferrihydroxamates indicated that HA-II and HA-III were monohydroxamates, whereas HA-I and HA-IV were identified as di- and trihydroxamates, respectively. Reductive hydrolysis of HA-I (the major hydroxamic acid isolated) yielded ornithine. Hydrolysis of HA-IV in water or in 0.1 N NaOH resulted in the formation of HA-I (dihydroxamic acid) and HA-II (monohydroxamic acid). Based on their charge at pH 5.2 and 2 determined by paper electrophoresis, Rf values on thin-layer chromatography, infrared spectra, and reactivity to ninhydrin, three of the isolated hydroxamic acids were identified as deferricoprogen B (HA-IV) and its breakdown products, dimerumic acid (HA-I) and trans fusarinine (HA-II). HA-I and HA-IV exhibited growth factor activity for both yeast and mycelial forms of growth of H. capsulatum.

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Electron cytochemical determinations of periodic acid-thiocarbohydrazide-silver proteinate (PATAg)-reactive sites in the vegetative and reproductive cells of the pathogenic dimorphic fungus Histoplasma capsulatum, are described an illustrated by electron micrographs. Ultrastructural sites of PATAg-reactive substances were detected at the plasma membrane, along various cytomembranes of the cytoplasm, and in aggregates of storage material believed to be partly composed of glycogen or a glycogen-like polymer. Cell walls were either minimally or negatively stained. The positive staining of PATAg-reactive sites was eliminated by use of the aldehyde-blocking agent sodium borohydride. The limiting membrane and matrix of Woronin bodies seen at the hyphal cell septum were devoid of PATAg reactivity.

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The electron microprobe analyzer was used to study the chemical compositions of some pathogenic fungi for humans. Elementary analysis was based on the characteristic X-ray spectra generated by the electrons passing through the specimens. This is a non-destructive method allowing to perform multiple elementary analysis in the same tissue. In order to increase our understanding of pathogenic fungi, small pieces of fungi tissue were analyzed. The species examined were Histoplasma capsulatum, Blastomyces dermatitidis and Fonsecaea pedrosoi.

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Four strains of Histoplasma capsulatum were analyzed to observe any variations in their cell wall composition. Strain G-184B produced the same amount of sugars reported by Domer (2) although qualitative analyses agreed with Kanetsuna's report (4) as far as the presence of glucose, galactose, and mannose was concerned. Our results agree with previous reports in classifying H. capsulatum into chemotypes I and II, the latter having more alpha-glucan in its cell wall. A decrease in the amount of fraction 2 (alpha-glucan) of strain G-184B was observed when the strain was subcultured in vitro for many years, an effect similar to that reported for Paracoccidioides brasiliensis (12, 13).

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A quantitative technique is presented for differentiating live and dead yeast cells grown in culture through the use of the fluorescent dye acridine orange. The method gives results that correlate well with those of other commonly used vital staining techniques and is free of certain interpretative errors inherent in them. Vital staining of yeasts with acridine orange also allows for more precise assessment of the physiological state of individual cells and the culture as a whole. The progressive senescence of yeast cells in culture can be monitored by the changing staining characteristics of several subcellular organelles. The method is simple and reliable.

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An inhibitor of ribonucleic acid polymerases has been obtained from the mycelial phase of Histoplasma capsulatum and partially characterized. The inhibitor, called histin, was purified 200-fold by heat treatment at 100 C and electrophoresis on polyacrylamide gels. Histin moved in electrophoresis as if negatively charged; it was insensitive to treatment with ribonuclease of deoxyribonuclease but was completely digested by Pronase. Sucrose gradient centrifugation suggests a molecular weight of 24,000. The possibility of a regulatory role for histin in the life cycle of H. capsulatum is discussed.

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