Efficacy of cell-free antigens in evaluating cell immunity and inducing protection in a murine model of histoplasmosis.

Histoplasma capsulatum is a dimorphic pathogenic fungus that causes a wide spectrum of disease when mycelial fragments are inhaled. Resistance to H. capsulatum is dependent on cellular immunity mediated by T cells and macrophages. Here we standardized the production of extracts containing cell-free antigens (CFAgs) and observed their efficacy in evaluating cellular immunity during murine histoplasmosis. CFAgs induced a more potent delayed-type hypersensitivity (DTH) response in H. capsulatum-infected mice than did histoplasmin-a classical antigen. This DTH response to CFAgs is able to determine the immune status of infected mice and to predict their death. Moreover, CFAgs stimulated spleen cells from immune mice to produce higher amounts of gamma interferon (IFN-gamma) in vitro. Finally, immunization with CFAgs protected against a lethal inoculum of H. capsulatum. These results demonstrate that CFAgs may be useful for the evaluation of cellular immune response and as a potential source for the development of a vaccine against histoplasmosis.

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.

ELISA for early diagnosis of histoplasmosis.

An ELISA was developed and evaluated as a method for detecting antibodies against glycosylated and deglycosylated histoplasmin (HMIN). Sera from patients with histoplasmosis, paracoccidioidomycosis, sporotrichosis, coccidioidomycosis, aspergillosis, cryptococcosis and healthy donors were tested by ELISA against purified, deglycosylated histoplasmin (ptHMIN) and compared with purified, native (i.e. glycosylated) histoplasmin (pHMIN). Although cross-reactivity was not abolished when ptHMIN was used in the test, it was reduced (pHMIN ELISA 93 % versus ptHMIN ELISA 96 %). However, there were statistically significant differences between the sensitivities of these two methods for the detection of antibodies (pHMIN ELISA 57 % versus ptHMIN ELISA 92 %; P < 0.001) and between the efficiency of the methods (pHMIN ELISA 83 % versus ptHMIN ELISA 95 %; P < 0.001). These parameters compare better than previously published data relating to the use of treated HMIN in diagnostic ELISAs. Some of the reactivities of serum samples were compared by immunoblotting using deglycosylated HMIN and by immunodiffusion using the crude antigen. The results demonstrated that cross-reactions with heterologous sera in both ELISAs could also be observed in immunoblotting and arose from shared protein epitopes. These data suggest that ELISA using deglycosylated HMIN is a very sensitive diagnostic method and, by using commercially available antigen, it can be easily standardized and performed faster than previous Western blot-based tests using the same antigen. It provides a useful adjunct to existing methods of diagnosis that could be applied even in situations where laboratory facilities were relatively limited.

[A primary investigation on disseminated histoplasmosis in Hubei].

OBJECTIVE: To investigate the epidemiologic features of disseminated histoplasmosis (PDH) in Hubei province. METHODS: Bone marrow smears of 12 patients diagnosed as Kala-azer in Hubei province including 4 patients in Jingsan, 2 patients in Shashi and each 1 in Yichang, Jinmen, Zhongxiang, Luotian, Xianning and Guanghua respectively were re-examed under microscope. Peripheral blood and bone marrow smears of several patients were detected. After inoculated the bone marrow, peripheral blood, liver and spleen tissue of patients in MLI, the single colony was trans-inoculated in BHIB, SDA and CMA and incubated at 25 degrees C and 35 degrees C. Bone marrow, peripheral blood and bacterial fluid of yeast-phase Histoplasma capsulatum (H.cap) were injected into the abdominal cavity of Kunming mice and nude mice. When symptoms and signs developed, the spleen tissue was separated, then observed under microscope and cultured. Mycelium-phase and Yeast-phase H.cap were inoculated in urase and gelatin medium, then incubated at 25 degrees C and 35 degrees C. Histoplasmin was injected subcutaneously into patients, and then followed for 48 - 72 hours. Amphotericin B was selected to treat the PDH patients. RESULTS: Moriform cell cluster and sausage-shaped cell were not observed in mononuclear-macrophages in the bone marrow smears from 12 patients. Leishman-Donovan body was found only in one patient. There wasn't kinetoplast in the cellular plasm of spores in 11 patients and no transeptae was found. The reaction of H.cap to urease was positive and H.cap did not liquefy the gelatin. It appeared to be mycelium-phase at 25 degrees C but no penicillus and catenulate conidia was found. The characteristic denticle macroconidia was observed but produced red coloring matter. It also appeared to be yeast-phase at 35 degrees C. Yeast-phase spores were observed under microscope. No sausage-shaped spore and transeptae were identified. H.cap could be acquired in the spleen tissue in Kunming mice and nude mice. Bacterium forms, characteristics under microscope and biochemical reaction of mycelium-phase and yeast-phase H.cap were different from some other kinds of dimorphic fungi such as Penicillium marneffei and Histoplasm duboisii etc. CONCLUSION: There were scattered epidemics of PDH in Hubei province. The detection rate of PDH was higher in the southeast area then in the northwest area. The golden standards of clinic diagnosis were mycological culture and inoculation to animals. Amphotericin B was necommended as the first choice for treatment.

Cellular immune response in HIV-infected patients with histoplasmosis.

The relationship of immunity to Histoplasma capsulatum and CD4 count in HIV-1-infected patients is unknown. Samples of blood from people with HIV infection and from HIV-negative volunteers were assessed for immune responsiveness to the histoplasmin antigen using proliferation and interferon-gamma production as indicators of immunity. Results of histoplasmin skin tests, lymphoproliferative responses (LPR), and interferon-gamma production were positive in 9 of 20 (45%) HIV-negative controls, and in vitro measurements agreed highly with skin test reactivity. Among HIV-1-infected patients with recent histoplasmosis, skin test results were positive in none, LPR results were positive in 14%, and interferon-gamma production in 18%. Among HIV-1-infected patients with CD4 counts between 200 and 500 cells/mm(3), LPR was positive in 8% and interferon-gamma production in 33%, and among those with CD4 counts >500 cells/mm(3), LPR was positive in 31% and interferon-gamma production in 46%. In conclusion, immune responsiveness to H. capsulatum was depressed in HIV-1-infected persons with CD4 counts between 200 and 500 cells/mm(3), but approached normal in those with CD4 counts >500 cells/mm(3).

Incidence of histoplasmin hypersensitivity in the Philippines.

Long-term residents of the Philippines were skin tested with histoplasmin skin test material. This study was conducted with 143 electric company (MERALCO) employees from Manila, Philippines. We found that 37 (26%) of the subjects were skin test positive. Characteristics of the positive group were: average age of 37 years; all except one were lifelong inhabitants of Metro Manila; 25 were male and 12 were female; one-half of the subjects reported extended contact with chickens. Despite these findings, histoplasmosis is considered to be a very rare disease in the Philippines. This survey indicates that Histoplasma capsulatum is sufficiently present in the Philippines to come in contact with one-fourth of the test population. This reinforces the hypothesis that histoplasmosis is present in the Philippines and is probably being misdiagnosed as granulomatous-inducing diseases such as tuberculosis, e.g., so-called "drug resistant" tuberculosis. We recommend larger surveys of this type and attempts to culture the etiologic agent from natural sources such as chicken and bat droppings.

[Paracoccidioides brasiliensis infection in a subtropical region with important environmental changes]. / Infection par Paracoccidioides brasiliensis dans une région subtropicale avec changements importants de l'environnement.

Paracoccidioidomycosis is a systemic mycoses caused by Paracoccidioides brasiliensis, a dimorphic fungus that infects man through respiratory ducts. It has been accepted that its ecological niche is located in the soil and plants of subtropical forests of Latin America. The Province of Corrientes is located at the northeastern border of Argentina, in a subtropical area where important environmental modifications have been introduced in the last decade as consequence of damming the Paranà river at Yacyretà, one of the biggest hydroelectric dams in the world. Since there are no data on human infection provoqued by this fungal agent in Corrientes, the purpose of this study was to obtain information at present time about infection indexes and to assess if environmental changes introduced in the area could impact on the epidemiology of the disease. Skin tests with paracoccidioidin and histoplasmin were performed on 455 persons of both sexes, from 1 to 73 years of age, who live permanently in the area and voluntarily accepted to be included in the study. Both antigens were employed at the same time in order to evaluate crossed type reactions. Of the 455 persons, 52 (39 males-13 females) were reactive to paracoccidioidin (11.4%), with an increasing prevalence with age. According to previous data, these results would indicate an increase in the index of human infection by P. brasiliensis, and this may be related to the important changes in climatic and environmental conditions introduced in the area in the last years.

[Cutaneous sensitivity to histoplasmin in a Martinique hospitalized population]. / Sensibilité cutanée à l'histoplasmine dans une population hospitalière martiniquaise.

OBJECTIVE: Histoplasma capsulatum infection is considered to be endemic in the West Indies. Nevertheless, few epidemiologic studies have been conducted in this area. The histoplasmin skin test reflects the frequency of asymptomatic forms of histoplasmosis. We studied the prevalence of positive skin tests in a population of the French West Indies (Martinique). MATERIALS AND METHODS: Forty one patients (24 females and 17 males), age range 29 to 90 years, were tested for histoplasmin skin sensitivity between August and October 1997, in the department of dermatology of Fort de France (French West Indies). Patients with immunosuppression or personal history of histoplasmosis were excluded. RESULTS: Five patients had a positive skin test (12 p. 100). No significative association was found between a positive skin test and diabetes, rural occupations or exposure to bats. DISCUSSION: Despite the small number of cases, related with difficulties in obtaining histoplasmin, our study showed a sensitivity level similar to medium endemic areas of the USA. The positive skin test rate is much higher than the rate reported before in West Indies, in a sample of the population under 25 years of age.

Outbreak of histoplasmosis among cavers attending the National Speleological Society Annual Convention, Texas, 1994.

In June 1994, 18 people developed serologically confirmed histoplasmosis following cave exploration associated with the annual National Speleological Society Convention in Bracketville, Texas. Six others had an undiagnosed illness suspected to be histoplasmosis. Two persons were hospitalized. We conducted a survey of convention attendees and a nested case-control study of those entering caves. We also conducted a histoplasmin skin test survey of a subgroup of the society, the Texas Cavers Association, who were attending a reunion in October 1994. Among the national convention attendees, exposure to two caves was identified as responsible for 22 (92%) of the 24 cases; 12 (75%) of 16 people exploring one cave (Cave A) and 10 (77%) of 13 exploring a separate cave (Cave B) developed acute histoplasmosis. Additional risk-factors included fewer years of caving experience, longer time spent in the caves, and entering a confined crawl space in Cave A. Of 113 participants in the separate skin test survey, 68 (60%) were found to be skin test positive, indicating previous exposure to Histoplasma capsulatum. A positive skin test was significantly associated with male sex and more years of caving experience. Those less experienced in caving associations should be taught about histoplasmosis, and health care providers should pursue histories of cave exposure for patients with bronchitis or pneumonia that does not respond to initial antibiotic therapy.

Histoplasmosis and Paracoccidioidomycosis in northwestern Argentina III. Epidemiological survey in Vipos, La Toma, and Choromoro - Trancas, Tucumán, Argentina.

The present work was undertaken to obtain epidemiological data on the extent and distribution of Histoplasma capsulatum var. capsulatum and Paracoccidiodes brasiliensis diseases in the Vipos, La Toma and Choromoro areas. Skin test surveys with histoplasmin and paracoccidioidin were carried out in the permanent human population of those localities. Mycological sputum studies and serological tests were performed to skin test reactors to determine if there were signs or symptoms of active mycotic disease. La Toma and Choromoro are highly prevalent areas of histoplasmosis (>30% the histoplasmin positive individuals) whereas Vipos can be relatively considered a highly prevalent area (between 20-30% the histoplasmin reactors) according to the normally accepted range used to define an endemic disease [2]. Early Histoplasma capsulatum infection (<10 years old) is reported for Vipos and Choromoro. La Toma has the highest rate of previous exposure to P. brasiliensis detected in the studied area (10.2%). Vipos residents are not infected with P. brasiliensis.

Inmunolocalización del complejo polisacárido-proteína en ultraestructuras de la fase micelial y levaduriforme de Histoplasma capsulatum / Immunolocalization of the polysaccharide-protein complex (PPCD-Histo) in the Histoplasma capsulatum mycelium and yeast phase ultrastructures

Introducción: El empleo de un antígeno parcialmente purificado denominado complejo polisacárido-proteína desproinizado de Histoplasma capsulatum (CPPD-Histo), utilizado para discriminar la histoplasmosis de diversas micosis pulmonares y otras enfermedades respiratorias en métodos inmunodiagnósticos de alta sensibilidad, ha sido motivo de estudio desde hace años por nuestro grupo de investigación: Objetivo: En este trabajo se planteó conocer la ubicación celular preferencial del antígeno CPPD-Histo, en las diferentes formas y estructuras de las fases micelial y levaduriforme del hongo. Material y métodos: El estudio se desarrolló mediante inmunolocalización con marcaje de oro coloidal para microscopia electrónica, usando anticuerpos primarios CPPD-Histo específicos. Resultados y discusión: La localización en microscopia electrónica mostró mayor concentración del CPPD-Histo en las zonas de mayor grosor de la capa externa de la pared celular de las proyecciones digitiformes de macroconidios, poco marcaje en pared celular de hifas, y una distribución dispersa de la marca en las levaduras. Conclusión: La pared celular de los macroconidios de la fase micelial del hongo es la estructura fúngica con mayor concentración del antígeno CPPD-Histo

[Investigation on the epidemiology of Histoplasma capsulatum infection in Nanjing district].

Two hundred and ninety-two permanent or temporary residents in Nanjing district were tested with histoplasmin (Histolyn-CYL, ALK/Berkeley Laboratories, USA) and PPD. Forty-nine (16.78%) subjects reacted to histoplasmin with 5.0-23.0 (9.5 +/- 4.2) mm induration. Positive reaction rate among people without pulmonary diseases (normal group) was 15.10% comparing to 17.74% to patients with pulmonary diseases. Positive reaction to PPD with 5-50 (14.2 +/- 4.7) mm induration was 56.16%, with 59.43% in normal group and 54.30% in patients with pulmonary diseases while 8.90% subjects reacted to both histoplasmin and PPD, 7.88% of the research subjects reacted to histoplasmin but not to PPD. Result suggested that there was herd infection of Histoplasma capsulatum in Nanjing district.

Histoplasmin reaction: comparison of a polysaccharide antigen to the filtrate antigen

O estudo envolve a comparaçäo entre o antigeno polissacaridico de Histoplasma capsulatum com a histoplasmina classica em inquerito epidemiologico, atraves de provas intradermicas de hipersensibilidade do tipo tardio, realizado em 115 individuos da regiäo de Santo Amaro. Os resultados revelaram 46 por cento de provas positivas com a histoplasmina classica e 51,30 por cento de resultados positivos com o antigeno polissacaridico em sua maior concentraçäo. A principal conclusao da pesquisa: e possível utilizar o antigeno polissacaridico como histoplasmina, em substituiçäo ao antigeno filtrado

Immunologic, genetic and social human risk factors associated to histoplasmosis: studies in the State of Guerrero, Mexico.

Immunologic and occupational aspects of the susceptible population exposed to Histoplasma capsulatum, the causative agent of histoplasmosis were analyzed in the Mexican State of Guerrero. Three areas were studied, Juxtlahuaca, Olinala, and Coyuca; in the first two, their populations refer contact with bat guano and/or avian excreta, which contain nutrients for fungal growth, while the Coyuca population referred no contact with the above mentioned excreta. Previous infection with H. capsulatum was determined by histoplasmin-skin test, and the response was higher in men than in women (93.87, 85.71, and 6.6% for men, and 78.94, 66.6, and 0% for women) in Juxtlahuaca, Olinala, and Coyuca, respectively. Labor activities related to a persistent contact with the fungus were considered as an occupational risk factor, histoplasmin-skin test reached 88.57 and 36.36% of positive response in individuals with high and low risk activities. A high percentage of histoplasmin responses was observed in subjects with constant contact with H. capsulatum, such as, cave-tourist guides, peasants, and game-cock handlers, and generally they developed the largest diameter of skin reactions. Genetic risk factor was determined by studying the gene frequency of the Major Histocompatibility Complex antigens in a sample of individuals and their degree relatives in Juxtlahuaca, Olinala, and Coyuca. Significant differences were found for HLA-B22 and B17 antigens in Juxtlahuaca, and for HLA-B22 in Olinala, in comparison to the usual gene frequency observed in the normal Mexican population. HLA results were important, considering that HLA-B22 was previously found to be possibly related to pulmonary histoplasmosis in Guerrero.

Histoplasmin reaction. Comparison of a polysaccharide antigen to the filtrate antigen.

This work was planned by taking into account all the knowledge accumulated from the immunological study of paracoccidioidomycosis. It aimed at comparing a polysaccharide antigen from Histoplasma capsulatum to a classic histoplasmin with the help of intradermal tests of delayed type of hypersensitivity. Tests were applied to 115 individuals in Santo Amaro, a town in the State of São Paulo. Positive results using classic histoplasmin were obtained in 46.0% cases whereas positive results using the polysaccharide antigen at its highest concentration were obtained in 51.30% cases. The major conclusion in this investigation is that it is possible to use the polysaccharide antigen as histoplasmin instead of the filtrate antigen.

Skin and serum reactivity among humans to histoplasmin in the vicinity of a natural focus of Histoplasma capsulatum var. duboisii.

The epidemiology of histoplasmosis duboisii (African histoplasmosis) is not well understood. The present study was carried out to investigate the prevalence of skin sensitivity and to determine by immunodiffusion the presence of antibodies among humans to histoplasmin around a recently discovered natural focus of Histoplasma capsulatum var. duboisii in a bat cave in Ogbunike in the Anambra State of Nigeria. Out of the 40 subjects, all young adults aged 18-30 years, comprising cave guides, traders and farmers examined in the immediate vicinity of the cave, 14 (35.0%) gave a positive skin test. In another population of the same age group, comprising 620 persons, viz. traders, farmers, palm oil workers and some patients attending rural clinics, examined in other nearby areas in Anambra State, 55 (8.8%) reacted positively to histoplasmin. In the immunodiffusion tests, 2 (2.08%) of the 96 school children and 17 (9.4%) of the 181 young adults, including farmers, palm oil workers and traders tested amongst the population around the cave, demonstrated precipitating antibodies to histoplasmin in their sera. Only 5 (0.79%) of the 630 adults of the same age group with similar occupations examined from other areas in Anambra State had precipitating antibodies. Out of another 50 subjects examined, viz.; wood workers, traders, farmers, and school teachers in Nsukka in the Enugu State, two (4.0%) demonstrated antibodies. It is suggested that asymptomatic infections due to the duboisii variety of H. capsulatum may be common in the human population around the cave. A diligent search with the help of local hospitals and public health officials may reveal clinical cases of histoplasmosis duboisii with cutaneous and systemic lesions.

Risk factors for choroidal neovascularization in young patients: a case-control study.

A pair-matched, case-control design was used to study exposure to Histoplasma capsulatum and other environmental factors, and to determine various host characteristics including human leukocyte antigen (HLA) typings in 94 young patients with macular choroidal neovascularization (CNV) and in 94 controls with other eye diseases. Patients with two types of retinal patterns were studied: Type I, or those with CNV with one or no chorioretinal atrophic spots in the posterior pole or periphery (n = 51), and Type II, or those with CNV and 2 or more chorioretinal atrophic spots (n = 43). Our purpose was to explore whether these two variants of idiopathic CNV have different and distinguishable epidemiologies which may or may not be related to prior exposure to Histoplasma. We found that histoplasmin skin tests were negative in all but two Type I cases. The combination of the HLA-B7 and HLA-DR2 markers (but not either marker alone) was significantly increased in Type I cases. Among Type II cases, HLA-B7, HLA-DR2, HLA-DQ1, a positive histoplasmin skin test, myopic refractive error, prior residence in a histoplasmosis endemic area, occupations involving exposure to animals, and hypertension were all significantly increased. Histoplasmin skin test responses were positive in 18 Type II cases (45%). In the multivariate analysis, only DR2 and the combined presence of DQ1 and a positive histoplasmin skin test remained predictive of Type II disease. Our findings suggest that histoplasmin sensitivity is associated with some, but not all, cases of Type II disease. However, histoplasmin sensitivity appears to have no relationship to Type I disease. HLA factors may play a role in both disease types, possibly by producing a modified immune response to Histoplasma and/or other unidentified agents.

Effects of histoplasmin M antigen chemical and enzymatic deglycosylation on cross-reactivity in the enzyme-linked immunoelectrotransfer blot method.

The enzyme-linked immunoelectrotransfer blot (EITB) method was evaluated as a suitable method for detecting antibodies against M antigen of Histoplasma capsulatum by use of both glycosylated and deglycosylated M protein of histoplasmin (HMIN). Sera from patients with histoplasmosis, paracoccidioidomycosis, blastomycosis, coccidioidomycosis, and aspergillosis were tested by the EITB with glycosylated M protein of HMIN. This assay demonstrated 100% sensitivity with histoplasmosis serum samples, all of which reacted with the 94-kDa glycoprotein (M antigen). Although the EITB is highly sensitive, it is not specific for histoplasmosis when glycosylated M protein is used as an antigen. A total of 81% of paracoccidioidomycosis, 25% of blastomycosis, 33% of coccidioidomycosis, 73% of aspergillosis, and 16% of tuberculosis serum samples cross-reacted with M protein of HMIN and yielded patterns indistinguishable from those obtained with histoplasmosis serum samples. The EITB reactions with both untreated M antigen and M antigen altered by periodate oxidation or by deglycosylation with endoglycosidases were compared. Cross-reactions with heterologous sera in the EITB could be attributed to periodate-sensitive carbohydrate epitopes, as reflected by the increase in the test specificity from 46.1 to 91.2% after periodate treatment of M protein. The EITB for the detection of antibodies to M antigen is a potential diagnostic test for histoplasmosis, provided that periodate-treated M protein is used as an antigen.

Histoplasmin sensitivity among a student population in Crema, Po Valley, Italy.

Histoplasmin skin tests were conducted on a student population in Crema, Po Valley, Italy. In this geographical area two autochtonal cases of disseminated histoplasmosis occurred in HIV-negative men. In the last year we tested 776 senior high school students, mean age 18 years, 335 males and 441 females. Histopalsmin skin sensitivity was 1.23%, with a mean area of induration of 5.78 mm. All subjects but one had never been abroad. This preliminary survey confirms the possibility of autochtonal histoplasmal infection in the Po Valley, Italy.