Prior subclinical histoplasmosis revealed in Nigeria using histoplasmin skin testing.

OBJECTIVES: Disseminated histoplasmosis is an AIDS-defining illness. Histoplasmosis is commonly misdiagnosed as tuberculosis. Nigeria has the second highest number of people living with HIV/AIDS in Africa. The present study was carried out to investigate the prevalence of skin sensitivity amongst Nigerians to histoplasmin. DESIGN: A cross-sectional study was conducted in six centres across five geopolitical zones of Nigeria. METHODS: We recruited both healthy non-HIV and HIV-positive adults with CD4 count ≥ 350 cells/mm3 regardless of their ART status from March to May 2017. Skin tests were performed intradermally; induration ≥5 mm were considered to be histoplasmin positive. RESULTS: 750 participants were recruited from Lagos (n = 52), Yola (n = 156), Ilorin (n = 125), Calabar (n = 120), Ibadan (n = 202) and Benin (n = 95). 467 (62.3%) were HIV negative, 247 (32.9%) were HIV positive and 36 (4.8%) did not know their HIV status. A total of 32/735 (4.4%) participants had a positive skin test. Study centre (p<0.001), education (p = 0.002) and age (p = 0.005) appeared to be significantly associated with positive skin reactivity at the 0.5% significance level, while sex (p = 0.031) and occupation (p = 0.031) would have been significant at the 5% significance level. Males had a higher rate of reactivity than females (p = 0.031, 7% vs 3%). The highest positive rates were recorded from Benin City (13/86 (15%)) and Calabar (7/120 (6%)) and no positives were recorded in Lagos (p<0.001). HIV status was not statistically significant (p = 0.70). CONCLUSION: Histoplasmosis diagnostics should be included in the Nigerian HIV guidelines. Epidemiological vigilance of progressive disseminated histoplasmosis should be considered by local health authorities.

Histoplasmosis infection in Spanish travelers to Latin America.

The aim of the study presented here was to assess the incidence of histoplasma infection in a cohort of 342 individuals in Spain who had traveled to Latin America for the first time. The histoplasmin skin test was positive in 20% of the travelers, and Central America posed a higher risk for infection than South America (p=0.013). Sleeping outdoors (p=0.031) and the duration of travel (p=0.016) were also identified as significant risk factors. Serological testing demonstrated poor overall sensitivity for detecting infection in the travelers, but for the symptomatic acute cases the results were improved. Histoplasmosis must be considered in patients presenting with fever (odds ratio=3.51 [1.52-8.12]) or cough (odds ratio=4.24 [1.32-13.58]) after visiting Latin America. The results of this study have public health implications and indicate the risks of acquiring histoplasmosis should be included in pre-travel counseling.

Paracoccidioidomycosis: an epidemiologic survey in a pediatric population from the Brazilian Amazon using skin tests.

Since Paracoccidioides brasiliensis and Histoplasma capsulatum are known to be present in similar environments, there have been many epidemiologic investigations regarding the prevalences of these two organisms. However, cross-reactivity can occur in paracoccidioidin and histoplasmin skin tests, and this usually results in the overestimation of the prevalence of P. brasiliensis. The prevalence of infection with P. brasiliensis was evaluated in a cross-sectional study of 298 asymptomatic school children in the Brazilian Amazon region (Mato Grosso State). In this investigation, the reactivity of children to two different P. brasiliensis antigen preparations, paracoccidioidin and a purified 43-kD glycoprotein (gp43), was compared with or without the co-administration of histoplasmin. In the group of individuals receiving paracoccidioidin who had a positive histoplasmin skin test result, the prevalence of exposure to P. brasiliensis was 44% (16 of 36). This reactivity to P. brasiliensis was significantly higher than that observed in other groups, which ranged from 4% to 6% (P < 5 x 10(-4) for each). Overall prevalence was 4.6% (95% confidence interval = 2.5-7.7%). These data suggest that gp43 provides a better estimate of exposure to P. brasiliensis when the co-administration of histoplasmin is desired.

Relatedness analyses of Histoplasma capsulatum isolates from Mexican patients with AIDS-associated histoplasmosis by using histoplasmin electrophoretic profiles and randomly amplified polymorphic DNA patterns.

The present paper analyzes the histoplasmin electrophoretic profiles and the randomly amplified polymorphic DNA (RAPD) patterns of the fungus Histoplasma capsulatum isolated from Mexican patients with AIDS-associated histoplasmosis. Clinical isolates from Guatemala, Colombia, and Panama, as well as H. capsulatum isolates from different sources in nature, were also processed. All histoplasmin samples shared four antigenic fractions of 200, 49, 10.5, and 8.5 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). According to their percentage of relatedness, based on SDS-PAGE histoplasmin electrophoretic image analysis, H. capsulatum isolates were divided in two groups: group A contained all AIDS-associated isolates studied and two human reference strains from Mexican histoplasmosis patients without AIDS; group B included bat guano, infected bat, and cock excreta isolates from the State of Guerrero, Mexico, plus three human histoplasmosis strains from Guatemala, Panama, and Colombia. Polymorphic DNA patterns evaluated by RAPD-PCR showed three major bands of 4.4, 3.2, and 2.3 kb in most H. capsulatum isolates studied. Four groups were related by DNA polymorphisms: group I was formed by most of the AIDS-associated H. capsulatum isolates studied, one human histoplasmosis strain from Colombia, two human reference strains from Mexican patients without AIDS, and one human histoplasmosis strain from Guatemala. Group II consisted of only a single strain from Panama. Group III included three strains: one from a Mexican patient with AIDS and two isolated from nature in Guerrero (cock excreta and bat guano). The last, group IV, consisted of only one strain isolated from an infected bat, captured in Guerrero. A tight relationship between phenotypic and genotypic characterization was observed, and both analyses could be useful tools for typing H. capsulatum from different sources and geographic origins.

In situ localization of antigens of Histoplasma capsulatum using colloidal gold immune electron microscopy.

Histoplasma capsulatum contains multiple antigens, among them the H antigen and M antigen, which are useful in serologic testing for histoplasmosis. We prepared 7 mouse monoclonal antibodies (5 IgG, 2 IgM) to histoplasmin, and compared these with polyclonal histoplasmin antibodies raised in rabbits and mice. Both monoclonal and polyclonal antibodies were high titered by ELISA. Colloidal gold immune electron microscopy (CGIEM) showed that polyclonal antibodies to histoplasmin or H antigen bound at multiple sites in the cell wall, cytoplasm, and nucleus of Histoplasma yeast cells. In contrast, antibodies to M antigen selectively label the cell membrane and antibodies to alkali soluble cell wall antigen label only the cell wall. Polyclonal antibodies cross reacted extensively with other fungi, both by ELISA and CGIEM. Monoclonal antibodies stained only cytoplasmic epitopes, but also cross reacted with other fungi by electron microscopy. Only periodate treated H antigen elicited polyclonal antibodies which were more specific than those of untreated H antigen or histoplasmin.

Characterization of antigenic determinants in histoplasmin that stimulate Histoplasma capsulatum-reactive T cells in vitro.

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.

Monoclonal antibodies against the M-protein and carbohydrate antigens of histoplasmin characterized by the enzyme-linked immunoelectrotransfer blot method.

Monoclonal antibodies (MAbs) of two different specificities were produced by immunizing mice with the semipurified M antigen of histoplasmin. One type, from clone CB4, was an immunoglobulin M that precipitated a polysaccharide present in histoplasmin and also formed immunoprecipitates with a cross-reactive polysaccharide present in extracts of Blastomyces dermatitidis and Coccidioides immitis. The second type of MAb, from clone EC2, was an immunoglobulin G that reacted in the enzyme-linked immunoelectrotransfer blot (EITB) assay with a doublet of proteins with an apparent molecular size of 70 to 75 kilodaltons. This molecule is proposed as the authentic M protein antigen that is recognized by M antibodies in sera from mice and rabbits immunized with Histoplasma capsulatum and from persons with histoplasmosis. The M factor also occurs in an abundant disulfide-bridged dimer which has a molecular size of 150 kilodaltons and is nonimmunoreactive under the conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Lymphocyte transformation in presumed ocular histoplasmosis.

Lymphocytes from individuals with inactive macular disciform lesions of presumed ocular histoplasmosis challenged with three histoplasmin antigens incorporated tritiated thymidine at a significantly higher rate than histoplasmin-stimulated lymphocytes of matched control and peripheral scar groups. This finding is consistent with the etiologic association of the disciform ocular syndrome and previous systemic infection with Histoplasma capsulatum. The disciform group had a higher mean response than the other two groups to pokeweed mitogen but not to phytohemagglutinin and had higher mean counts per minute to the specific antigens Toxoplasma gondii, Blastomyces dermatitidis, Cryptococcus neoformans, Mycobacterium tuberculosis, M battery, and M gaus, but not to Candida albicans. These data would suggest that individuals with the disciform lesion of presumed ocular histoplasmosis have a hyperreactive cellular immune response; this response may play an important role in the development of the disciform.

Evaluation of purified H and M antigens of histoplasmin as reagents in the complement fixation test.

Complement-fixation (CF) tests were performed with purified H and M antigens, histoplasmin, and Histoplasma capsulatum whole cell yeast phase antigen using sera of 126 patients with proven or suspected histoplasmosis. Specific titers for either H or for M antibody were obtained with the individual purified antigens; the highest titers were comparable to those obtained with histoplasmin. However, in sera containing only anti-M antibody, the titers obtained with the purified M antigen were 2 to 16 times those obtained with the histoplasmin or yeast phase antigens. The CF test for either H or M antibody was 4 to 32 times as reactive as the agar-gel microimmunodiffusion test; in general precipitin lines were obtained with either H or M antigens from sera with CF titers greater than or equal to 8. With sera containing H antibody, there was an excellent correlation between the CF titers obtained with purified M antigen and histoplasmin. The correlations of CF titers with H antigen and either histoplasmin or yeast phase antigen were very low.

Determination of molecular weight, isoelectric point, and glycoprotein moiety for the principal skin test-reactive component of histoplasmin.

A purified component designated HPD (histoplasmin-purified derivative) dII was isolated from two different cru-e histoplasmin lots by a combination of gel filtration and polyacrylamide disc electrophoresis (Sprouse, 1969). A 0.05-mug portion of HPD dII was reactive and specific in detection of delayed hypersensitivity in guinea pigs experimentally infected with Histoplasma capsulatum. The objective of this study was to characterize this skin test-reactive component for (i) homogeneity, (ii) molecular weight, (iii) isoelectric point, and (iv) composition. Sephadex chromatography, polyacrylamide disc electrophoresis, sucrose density gradient ultracentrifugation, acid and heat denaturation, and immunoelectrophoresis indicate that HPD dII is (i) homogeneous, (ii) of approximately 12,000 molecular weight, and (iii) a glycopeptide of approximately 60% carbohydrate and 40% proteinaceous composition. The marked acid and heat stability exhibited by the compound probably is attributable to the prominent carbohydrate moiety in the molecule. Isoelectric focusing indicated an isoelectric point of 5.68. This would suggest that dII is an acidic compound with either predominance of acidic amino acid residues in the molecule or, more probably, an abundance of electron donors in the carbohydrate moiety. In summary, HPD dII appears to be a glycopeptide of approximately 12,000 molecular weight, reactive in elicitation of delayed hypersensitivity of histoplasmosis.

Specific immunological test for the rapid identification of members of the genus Histoplasma.

A sensitive and specific immunological method was developed for rapid identification of the mycelial forms of Histoplasma capsulatum var. capsulatum, H. capsulatum var. duboisii, and H. farciminosum and for separation of these pathogenic fungi from morphologically similar hyphomycetes and other fungal pathogens. This method is based on the fact that all of the Histoplasma spp. produce H and M histoplasmin antigens, whereas the other fungi do not. Inocula consisting of heavy mycelial growth from a pure, full-grown culture were transferred into flasks containing small volumes of brain heart infusion broth. These cultures were placed on a shaker and grown at 25 C. Using the micro-immunodiffusion technique and antisera containing antibodies to H and M precipitinogens, we detected exoantigens in 3-day-old brain heart infusion culture supernatants concentrated 25 and 50 times. The ability of the procedure to identify Histoplasma spp. was evaluated by testing 96 unknown mycelial cultures that grossly or microscopically resembled Histoplasma spp. Three- and six-day-old concentrated culture supernatants prepared from each unknown were tested against rabbit anti-Arthroderma tuberculatum, Chrysosporium keratinophilum, H. capsulatum var. duboisii, and Corynascus (Thielavia) sepedonium sera and human histoplasmosis case serum. Each unknown was also identified by conventional laboratory procedures involving cultural and, where necessary, in vivo studies. In the comparative evaluation the immunological test was observed to be 100% sensitive. It permitted the accurate generic identification of the Histoplasma spp. within 5 days, in contrast to the average of 33 days required by the routine mycological procedure.

Retention of albino and brown phenotypes of Histoplasma capsulatum by liquid nitrogren refrigeration.

Pathogenic differences between albino (A) and brown (B) phenotypes of Histoplasma capsulatum for experimental animals, and serological differences between A- and B- derived histoplasmins or yeast-phase antigens, necessitate the retention of these phenotypes in culture for diagnoses and epidemiologic studies. Selected strains of A and B types were frozen in liquid nitrogen (LN) and stored for 6-9 months in the vapor phase of a LN refrigerator (-165 degrees C). The strains were tested by using Berliner's method for differentiation of these two phenotypes. The results have proved that the LN refrigeration can be used for long-term conservation to prevent the conversion of B type into the A type in vitro.

Purification, composition, and serological characterization of histoplasmin-H and M antigens.

To obtain purified H and M antigens suitable as primary standards in the serological diagnosis of histoplasmosis by agar gel double-diffusion tests, H and M reactive components of histoplasmin were fractionated by column chromatography by using Sephadex G-100, Sephadex G-200, and diethylaminoethyl cellulose. Six fractions from diethylaminoethyl cellulose were reactive in agar gel double-diffusion, complement fixation, and capillary precipitin tests. When examined by electrophoresis on acrylamide gel, one M antigen (fraction 2, molecular weight greater than 200,000) and two H antigens (fractions 5 and 6, molecular weight greater than 200,000) each gave essentially a single protein band. In agar gel double-diffusion and complement fixation tests with sera from patients with proven cases of histoplasmosis, blastomycosis, or coccidioidomycosis, these two fractions of H antigen and the one of M antigen reacted only with sera from proven or suspect cases of histoplasmosis and showed reactivity with those sera known to contain only the anti-H or anti-M antibody, respectively. Fraction 2 (M antigen) and fractions 5 and 6 (H antigens) had carbohydrate-to-protein ratios of 1.60, 0.77 and 0.78, respectively. Both antigens contained galactose, glucose, mannose, and hexosamine, with mannose being the predominant sugar. Fraction 2 was characterized by a high proline and glucose content, whereas fractions 5 and 6 contained higher concentrations of galactose, mannose, glycine, and alanine. Each of these products appeared to separate into two active fractions, one of a molecular weight greater than 200,000 and one of a molecular weight less than 35,000. The M antigen component of fraction 2 was still characterized by a high proline content, whereas the H antigen components of fractions 5 and 6 had a high content of glutamic acid, serine, glycine, and proline.

Reporte Preliminar de dos brotes epidémicos de histoplasmosis pulmonar aguda / Two epidemic outbreaks of acute pulmonary histoplasmosis. A preliminary report

Se presenta dos brotes epidémicos de histoplasmosis pulmonar aguda, surgidos en la relación con la entrada y permanencia en los interiores de una cueva y de un refugio, estando ambos lugares infestados por gran cantidad de murciélagos. Se exponen los resultados de los trabajos de investigación diagnóstica, sobre los pacientes hospitalizados con el diagnóstico presuntivo de histoplasmosis, las encuestas realizadas sobre el personal expuesto a riesgo y los resultados de las investigaciones sobre el medio ambiente en búsqueda del Histoplasma capsulatum(AU)