Additional studies of histoplasmin formation

Summary Culture filtrates of 20 strains ofHistoplasma capsulatum were studied to determine the effect of certain growth conditions on histoplasmin formation. The presence of histoplasmin was denoted by an antigenic titer of 1:4 or higher with the complement fixation test.The data indicated that, in addition to verifying that the strain used affected histoplasmin formation, the morphological condition of the inoculum was extremely important. It was found that most strains which converted readily to the yeast phase at 37° C produced histoplasmin poorly. Tests with different volumes of media also showed that 500 ml volumes of culture media produced histoplasmin with higher titers than 3 liter volumes when cultured at 25° C for six months.Some additional histoplasmin could be liberated by sonification of the mycelial pad from culture filtrates which contained histoplasmin. A few strains produced high titer histoplasmin by the shake method if incubated for three months, but they had low titers after only six weeks.Complement fixation tests with sera from proven cases of histoplasmosis indicated that histoplasmin from a single strain ofH. capsulatum can give identical results with those obtained with histoplasmin from a pool ofH. capsulatum strains if H and M antigen components are present.     

Two-dimensional immunoelectrophoresis of histoplasmin and a purified polysaccharide-protein antigen ofHistoplasma capsulatum

Crude histoplasmin and a polysaccharide-protein complex (PPC-histo) antigens obtained from culture filtrates ofHistoplasma capsulatum were analyzed by single and tandem two-dimensional immunoelectrophoresis (TD-IEP) using a rabbit hyperimmune anti-histoplasmin polyvalent serum. Single TD-IEP showed 14 arc precipitates for histoplasmin. Continuity of arcs 2, 6, and 7, and 9 and 10 was observed, suggesting a different polymeric configuration of the same antigen. This was also confirmed in tandem TD-IEP of histoplasmin with homologous (PPC-histo) and heterologous PPC's fromBlastomyces dermatitidis, Paracoccidioides brasiliensis andCoccidioides immitis. Tandem TD-IEP of histoplasmin and PPC-histo displayed a similar antigenic pattern to histoplasmin alone, being arcs 1 and 3 more evident and apparently present only in histoplasmin and PPC-histo. Tandem TD-IEP showed common antigens among the other heterologous fungal purified antigens, and seems useful to observe the multiplicity of antigens present in fungal preparations and to identify those precipitates (arcs 1 and 3) that are predominant in the purified preparation.     

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.     

Immunological studies with an m-deficient histoplasmin skin-test antigen

Previous studies have shown that a single skin test to histoplasmin may induce complement-fixing antibodies or M precipitins (or both) to histoplasmin in histoplasmin-sensitive, but serologically negative, individuals. Ideally a skin-test antigen should be one which detects hypersensitivity without stimulating humoral antibodies. Histoplasmin skin-test antigens presently used contain both H and M antigens. The present study was undertaken to evaluate an histoplasmin skin-test antigen deficient in the M component but containing the H antigen. Thirty histoplasmin-hypersensitive subjects were bled prior to administration of the experimental skin-test antigen and at various time intervals thereafter. Only six of the thirty hypersensitive subjects showed serological responses. The sera of the six, however, only showed weak precipitin reactions, five showed M bands, and only one showed an M and an H band. None showed complement-fixation titers with either the yeast or mycelial antigens of Histoplasma capsulatum. Our data suggest that the use of a skin-test antigen purified to contain only H component would detect histoplasmin hypersensitivity without inducing antibodies and would eliminate false-positive serological reactions caused by the M component.     

A survey of histoplasmosis and blastomycosis in U.A.R. (Egyptian sector) a preliminary report

Summary A selected group of 525 individuals with pulmonary diseases, granulomas and other medical conditions was tested for histoplasmin and blastomycin dermal reactions. No positive results were observed. Few doubtful positive reactions were recorded (3 to histoplasmin and 7 to blastomycin). None of the patients with chronic cutaneous granulomas exhibited any reaction.Although the number of subjects studied is small, these preliminry findings suggest the probable absence of histoplasmosis and blastomycosis in Egypt.     

Commercially available histoplasmin sensitized latex particles in an agglutination test for histoplasmosis

Summary A commercial preparation of histoplasmin sensitized latex particles was tested in an agglutination test with sera from 50 culturally confirmed cases of histoplasmosis in varying stages of the infection. The reactions were superior to those obtained with collodion agglutination and complement fixation tests in which the antigen histoplasmin was also used. The latex agglutination test with the commercially available antigen is easy to do, and can be done in any laboratory equipped to carry out agglutination tests with the common bacterial antigens. It warrants more extensive trial in the general hospital laboratory as a screening test for histoplasmosis, especially the primary, pulmonary type.     

A preliminary note on inter-reaction of skin test sensitivity between Histoplasmin and Chrysosporin in experimental animals

Summary Species ofChrysosporium have been isolated from soil in Iran. Guinea pigs inoculated withChrysosporium keratinophilum gave a positive skin test to histoplasmin and vice versa. This would suggest thatChrysosporium species might be reponsible for positive skin tests to histoplasmin in areas known not to be endemic for histoplasmosis.     

Results of skin test surveys for systemic Mycoses in Iran

Several investigators had carried out histoplasmin skin test surveys in 3 different areas of Iran before 1960 (5). To detect skin sensitivity to both histoplasmin and coccidioidin a study was carried out by the Institute of Public Health Research in parts of 7 of the country's 13 provinces on both apparently healthy persons and on clinic patients from February 1962–September 1966. These studies uncovered a small focus with a rather high level of positive reaction to histoplasmin (about 17 %). Positive cultures ofHistoplasma capsulatum were not obtained from either sputum of the positive reactors or from collected soils. In spite of a previously reported case of coccidioidomycosis, very low percentages of positive reaction to coccidioidin were noted.These studies were supported in part by the Institute of Public Health Research Teheran University, and funds of the Ministry of Health and Plan Organization for Project No. 631101 and N.I.H. Grant No. TW00170.Paper read at the Eighth International Congresses for Tropical Medicine and Malaria, September 1968, Teheran (Iran).     

Serological and skin test cross-reactivity between Histoplasma capsulatum and Oidiodendron kalrai

Guinea pigs were infected with yeast cells ofHistoplasma capsulatum andOidiodendrom kalrai. They were skin tested 6 and 12 weeks post-infection with both homologous and heterologous antigens. There was almost complete skin test cross-reactivity between histoplasmin and oidiodendrin after 12 but not after 6 weeks. There were also strong serologic cross-reactions among 6 different anti-H. capsulatum hyperimmune sera and oidiodendrin. However, cross-reactions among anti-O. kalrai sera and histoplasmin could not be demonstrated.     

Epidemiologic skin test survey of sensitivity to paracoccidioidin,histoplasmin and sporotrichin among gold mine workers of Morro Velho Mining,Brazil

Skin tests with paracoccidioidin, histoplasmin and sporotrichin were applied to 417 workers of Morro Velho Mining in the State of Minas Gerais, Brazil, with the main purpose of detecting the prevalence of paracoccidioidomycosis-infection, histoplasmosis capsulate-infection and sporotrichosis-infection. The rates of positivity to the skin tests were 13.43% for paracoccidioidin, 17.50% for histoplasmin and 13.67% for sporotrichin. Several epidemiological factors were investigated for a better interpretation of the results. Paracoccidioides brasiliensis, Histoplasma capsulatum var. capsulatum and Sporothrix schenkii were not isolated from the soil samples from the mines investigated.     

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.     

Immunomodulation by Histoplasma capsulatum products; polyclonal activation and mitogenic effects

The presence of reactive spleen cells to sheep red blood cells (SRBC) in nonimmunized BALB/c mice injected with histoplasmin, the culture filtrate of Histoplasma capsulatum, was monitored for 21 days following inoculation. Polyclonal activation, as evidenced by a sharp increase in the number of anti-SRBC rosetteforming cells (RFC), as well as an enhanced response to heterologous non-cross-reactive erythrocytes from other species, was found in the spleens of these rodents on Days 11 to 13. Elimination of B-cell-derived RFC by the addition of complement indicated that the erythrocyte-binding cells consisted of both T- and B-lymphocytes. An immunosuppressive effect was detected if histoplasmin was injected 2 days before the antigen (SRBC), but could be reversed by injecting the filtrate 30 min prior to the antigen, as is found with polyclonal activators displaying immunosuppressive activity. Histoplasmin also had a mitogenic effect on lymphocyte obtained from the spleen, bone marrow, and thymus similar in magnitude to that produced by lipopolysaccharide (LPS) and concanaval in A. The biological significance of these findings is discussed.     

Inquérito epidemiológico com paracoccidioidina e histoplasmina em área agrícola de café em Ibiá, Minas Gerais, Brasil.

An epidemiological survey in an endemic area for paracoccidioidomycosis in Ibiá town, Minas Gerais state, Brazil was carried out. For this, we used a standard form for each household and intradermal antigens of histoplasmin and paracoccidioidin. Out of 194 individuals, 109(56%) were engaged in the survey showing reactivity of 44% and 49.5% for these antigens respectively. The reactivity to paracoccidioidin was observed in 50% of children below ten years. Individuals who revealed reactivity to paracoccidiodin with prior history of coffee growing activities, and those reporting the grain collection as the main work, showed statistically significant association p = 0.0092, p = 0.0007 and p = 0,01 respectively. These results could suggest that activities related to coffee crops, may favour the transmission of Paracoccidioides brasiliensis, specially among the grain collectors. In addition, the reactivity for histoplasmin antigen, suggest that this region is endemic for both fungi.     

Heated histoplasmin as a control for non-specificity in the complement fixation test for histoplasmosis

We present data in support of the use of a heated histoplasmin control for the complement fixation (CF) test for histoplasmosis to assist in the detection of the presence of h or m antibody.     

Immunodiffusion studies on the antigens of Histoplasma capsulatum: comparison of mycelial histoplasmin with the yeast phase reagent Histolyn-CYL

Immunodiffusion assays were performed to determine if H and M antigens associated with mycelial histoplasmin were present in the yeast phase reagent, Histolyn-CYL (H-CYL). Neither H nor M antigens could be detected in H-CYL. However, when H-CYL was reacted against human histoplasmal sera a positive reaction was evidenced in all instances in which a response was elicited with a reference mycelial immunodiffusion reagent.     

THE DISTRIBUTION OF COCCIDIOIDOMYCOSIS IN SOUTHERN CALIFORNIA

In coccidioidin skin test surveys among persons of high school age in Saugus, Canoga Park, Banning and Palm Springs areas the average incidence of positive reaction was 15 per cent. Although considerably less than the 68 per cent incidence reported among high school students of Kern County, it is high enough to indicate pockets of relatively high endemicity in Southern California below the San Joaquin Valley.Histoplasmin tests were performed on most of the persons tested with coccidioidin in this survey. The over-all incidence of positive reaction in the group was 7.6 per cent. Most of the subjects with positive reaction to histoplasmin gave a history of having previously lived in some area in the central United States where histoplasmosis is known to be endemic.A few subjects who had positive reaction to coccidioidin tests and who had lived in areas known to be endemic for coccidioidomycosis but not for histoplasmosis, also had positive reaction to histoplasmin. However, the induration produced was always smaller than that caused by the coccidioidin reaction, and there was minimal confusion in interpreting the tests.     

Problemas clínicos en micología médica: problema número 52

The case of a 60 year old woman with hemoptysis and a thin-walled cavitary lesion at the upper lobe of the right lung is presented. The woman presented at the Mycology Unit of the Muñiz Hospital in Buenos Aires City 3 months after the beginning of her clinical manifestations. A hyaline micelial fungus with chlamido-arthroconidias was isolated from the bronchoalveolar lavage. Immunodiffusion and counter-immnunoelectrophoresis with coccidioidin and histoplasmin rendered positive results against both antigents, and skin tests with coccidioidin and histoplasmin were also positive with strong reactions. The isolated fungus was identified as Coccidioides posadasii at the National Microbiology Institute Carlos Malbrán, by means of a molecular technique. The patient was treated with itraconazole by oral route at a daily dose of 200 mg with good clinical response, but due to the persistence of the lung cavity, a surgical removal of the upper lobe of the right lung had to be scheduled.     

Chronic Cavitary Histoplasmosis of the Lung

Four main clinical types of disease result from infection by the fungus Histoplasma capsulatum: (1) the primary complex; (2) acute pulmonary histoplasmosis; (3) chronic cavitary pulmonary histoplasmosis; (4) disseminated systemic histoplasmosis.Chronic cavitary histoplasmosis of the lung is indistinguishable clinically and radiographically from pulmonary tuberculosis. In this disease, however, the histoplasmin skin test and the histoplasmin complement fixation test are positive in more than 80% of cases and sputum cultures are usually positive for Histoplasma capsulatum. The mortality rate of the chronic cavitary type is greater than 30%. The antibiotic, amphotericin B, is the most effective drug in treatment. If surgical resection of the cavitary lesion is feasible, under amphotericin coverage, this is the treatment of choice.During the past year, two patients with chronic cavitary histoplasmosis illustrating the aforementioned features were diagnosed at the Toronto Hospital for Tuberculosis.     

Preparation and study of an exoantigen of Histoplasma capsulatum for serological reactions.

The aim of this study was to determine the usefulness of a yeast-phase exo-antigen of Histoplasma capsulatum in standard serologic reactions. Three native strains of H.capsulatum which belong to Mycology Center collection were employed. They were maintained in their yeast-phase by weekly subcultures in 2% dextrose broth agar at 37 degrees C. After one week incubation yeast cells were suspended in distilled water containing thimerosal and phenylmethyl sulfonyl fluoride at a concentration of 1:5000. This suspension was left at room temperature for 72 h, then the supernatant was separated by centrifugation and it was lyophilized. Proteins and polysaccharides concentrations were determined. Immunodiffusion (ID) tests were carried out with an antigenic dilution containing 1.4 mg/ml of proteins. This exo-antigen was submitted to SDS-PAGE. Seven protein fractions were detected but only two of them showed antigenic activity against a pool of positive human sera; the molecular weights of these two proteins were 97 kDa and 66 kDa respectively. A metabolic antigen from the mycelial phase of H. capsulatum was used as control. A rabbit gammaglobulin anti-H. capsulatum was prepared and employed as positive control in serologic reactions. The antigenic capacity of ten batches of this exo-antigen was studied by ID and counterimmunoelectrophoresis (CIE) tests using serum samples of 20 hamsters experimentally infected by intracardiac inoculation of the yeast-phase of H. capsulatum. All tests presented positive results after three weeks of the infection. Fifty sera from patients suffering progressive histopasmosis were analyzed: ID, CIE and complement fixation (CF) tests were performed in all cases. HIV negative patients presented 7/7 (100%) positive reactions with the yeast-phase exoantigen and 5/7 (71.4%) with histoplasmin. In HIV positive patients CIE and CF were the most sensitive serologic tests, they gave positive results in 15/43 cases (34.8%) with the yeast-phase exo-antigen and in 7/43 cases (13.9%) with histoplasmin. Sera from 10 patients with paracoccidioidomycosis, aspergillosis and candidiasis respectively were studied by ID with the aim of detecting serologic cross reactions. No cross reaction was detected in these serum samples. This yeast-phase exo-antigen of H. capsulatum is more sensitive than and equally as specific as control histoplasmin.     

Interspecies transfer by "immune" RNA of lymphocyte proliferative response to specific antigen

Ribonucleic acid (RNA) extracted from the lymph nodes of BCG sensitized cattle transferred tuberculin sensitivity to normal guinea pig lymphocytes as indicated by increased incorporation in vitro of ³H-thymidine in response to Purified Protein Derivative (PPD). The RNA treated lymphocytes were unresponsive to a nonspecific antigen, histoplasmin. Ribonuclease treatment of the RNA abolished its ability to transfer tuberculin reactivity and RNA extracted from the lymph nodes of normal cattle was also ineffective.