Guerreiro Machado reaction. Chromatographic analysis of Trypanosoma cruzi antigens

The aqueous extract of Trypanosoma cruzi, previously treated by benzene, was filtered through a column of Sephadex G-200. The eluted fractions were tested by complement-fixation with the chagasic reference serum. It was found that the CF antigens were eluted in the first fractions and the contaminants in the last ones. The aqueous antigen, when dried and extracted with methanol, showed a very high titer in the complement-fixation quantitative test. This antigen is free of contaminants and can be recommended for use in the serological test for the diagnosis of Chagas' disease.     

Agar-Gel Precipitin-Inhibition Technique for Histoplasmosis Antibody Determinations

The agar-gel precipitin-inhibition technique has been modified to detect antibodies of Histoplasma capsulatum in sera from human clinical cases and experimental animals infected with this organism. By use of this modified technique, the histoplasmosis can be detected more consistently and reliably than with the direct agar-gel diffusion test, and titers are comparable to those attained by the complement-fixation serological technique.     

Application of agar-gel precipitin test for the detection of Sendai virus antibody in rat sera.

A simplified agar-gel precipitin test was performed for the detection of Sendai virus antibody in rat sera. A close correlation was observed between detection of antibodies by complement-fixation test and agar-gel precipitin test. No correlation was found between results obtained by hemagglutination-inhibition test and agar-gel precipitin test in sera with HI titer of less than 1:8.     

Serological Response of Rhesus Monkeys to Histoplasma, Blastomyces, and Coccidioides Antigens

Fifteen adult rhesus monkeys were inoculated with nonviable Histoplasma capsulatum, Blastomyces dermatitidis, or Coccidioides immitis. Antibody assays were made periodically during a 2-year period by use of a complement-fixation (CF) test employing four antigens and a latex-agglutination test. Selected sera were also studied in an immunodiffusion test and a coccidioidin-precipitin test. The serological patterns obtained with the anti-Histoplasma, anti-Blastomyces, and anti-Coccidioides monkey sera were comparable to those of sera from patients with diseases caused by the respective organisms. Rhesus monkeys should provide a good laboratory model for additional studies, including the influence of multiple antigenic stimuli on serologic response and patterns. Monkeys could also be used for the production of antisera required for studies to improve the specificity of the currently available CF antigens.     

Effect of Histoplasmosis on Antibody Response to an Erythrocyte Antigen

Infection of mice with Histoplasma capsulatum depressed their ability to form agglutinins against foreign erythrocytes. Animals previously inoculated with 10(8) yeast cells of H. capsulatum showed the most significant depression, occurring when erythrocytes were injected 8 days after infection. The average log(2) hemagglutinin titer was 2.7 compared to 8.0 for the control (noninfected) group. In general, depression of hemagglutinin response in all infected mice was greatest 8 days after infection, but response was back to near normal after 16 days and stayed at that level for the remaining time tested (24 days).     

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.     

Influence of pH on the Growth Characteristics of Neisseria gonorrhoeae in Continuous Culture

A series of three continuous cultures of Neisseria gonorrhoeae was performed at pH values ranging from 6.4 to 7.2, with an average duration of 3 weeks. The maximal yield obtained was 1.12 mg/ml at a pH of 6.75 and a dilution rate of 0.26 per hr. Some morphological variations were observed at lower dilution rates, but complement-fixation titers remained stable for the duration of the cultures.     

Development and Use of a Polyvalent Conjugate to Differentiate Histoplasma capsulatum and Histoplasma duboisii from Other Pathogens

Previous investigations have demonstrated the existence of five Histoplasma capsulatum serotypes. Available specific fluorescent-antibody reagents stain only four of the five serotypes. Antibodies produced against the most complete H. capsulatum serotype were labeled with fluorescein isothiocyanate to develop a reagent specific for H. capsulatum that was reactive with all the known serotypes. The unadsorbed reagent not only stained all the H. capsulatum serotypes, but it also stained cultures of Blastomyces dermatitidis, H. duboisii, several Candida species, and a variety of other fungi. Adsorption of the conjugate with antigens of C. albicans produced a reagent that intensely stained only H. capsulatum, H. duboisii, and B. dermatitidis. Differentiation of B. dermatitidis from the Histoplasma species was accomplished by application of a B. dermatitidis specific fluorescent antibody to antigens positive with the H. capsulatum reagent. At present, differentiation of H. capsulatum from H. duboisii may be accomplished only by animal inoculation. Our data substantiate the antigenic relationships hypothesized earlier, and they indicate that H. capsulatum shares at least two antigens with the other fungi that were studied.     

Agar-Gel Precipitin-Inhibition Test for Coccidioidomycosis: I. Preliminary Evaluation of the Complement-Fixation and Agar-Gel Precipitin Tests in the Serodiagnosis of Human Coccidioidomycosis

The agar-gel precipitin-inhibition serological test for coccidioidomycosis was a more sensitive indicator of Coccidioides immitis antibodies than the tube precipitin, the agar-gel immunodiffusion, in the complement-fixation tests in assaying monkey sera, whether these sera were from prechallenge-vaccinated or postchallenged animals. When applying this technique to the assay of human sera, an analogous finding generally persisted. However, some human sera were positive by the complement-fixation test and negative by the agar-gel precipitin-inhibition test. These sera were diffused in agar-gel against various coccidioidin complement-fixation, tube precipitin, and agar-gel precipitin-inhibition test antigens with essentially negative results.     

Effects of a Single Histoplasmin Skin Test on the Serological Diagnosis of Histoplasmosis

Numerous reports have indicated that a single histoplasmin skin test may stimulate humoral antibodies to Histoplasma capsulatum antigens in histoplasmin-hypersensitive individuals. Although these investigations concur that antibody elevations are evoked, they vary in the reported degree of incidence and response induced, and they cast doubt on the interpretation of serological tests in the diagnosis of histoplasmosis. Histoplasmin-hypersensitive subjects (114) were bled prior to administration of the skin test, 2 days later, at the time this test was read, and 15 and 30 days after testing. No significant antibody titers were observed at 2 days. At 15- and 30-day intervals, only 17 (15%) of the subjects demonstrated circulating antibodies. All 17 showed agar gel bands; 5 demonstrated no complement-fixation (CF) titers, 10 produced CF antibodies ranging from 1:8 to 1:16, and 2 demonstrated titers of 1:32. The data suggest that skin testing does not interfere significantly with antibody levels in sera drawn approximately 2 days after administration of antigen. However, since titers as high as 1:32 were obtained at later intervals, such reactions should be evaluated cautiously and only after consideration of clinical findings.     

Estudio comparativo de las reacciones serologicas cuantitativas con un antigeno metabolico de Aspergillus fumigatus

Summary The following quantitative serologic reactions: agar-gel immunodiffusion, complement-fixation, opposite electrophoresis and latex particle agglutination tests have been performed in 38 sera from mycologically proved pulmonary aspergillosis cases. A metabolic antigen from a strain ofAspergillus fumigatus according toAjello et al technic modified by us, has been employed. Sera from 120 subjects suffering from non-mycotic lung conditions, as well as 10 sera from histoplasmosis cases, 10 sera from S. A. blastomycosis and 2 sera from patients with lung aspergillosis produced byA. niger, gave negative results with the above mentioned seroligic reactions.One hundred per cent of positive results were obtained with the complement-fixation test (titre ranging from 1/20 to 1/1280), agar-gel immunodiffusion test (titre up to 1/64) and the opposite immunoelectrophoresis (titre ranging from 1/2 to 1/256). Twenty five per cent negative and 4 non-specific results were registered with the latex particle agglutination test.A correlation of the number of serum precipition bands obtained by the electrophoresis technic with the titre of the quantitative serologic reactions, as well as a correlation of the titre of the circulating antibodies with the severity of the clinical form of aspergillosis seems to be present.Electrophoretic motility of the specific antibody performed in 10 sera showed results like the IgM in 1 instance and an intermediate position between IgA and IgG in 9 samples.     

African histoplasmosis: a review

African histoplasmosis caused by Histoplasma capsulatum var. duboisii is an important deep mycosis endemic in Central and West Africa and in the island of Madagascar. The disease is characterized by presence of granulomatous lesions in the skin, subcutaneous tissues and bones. Lungs and other internal organs are rarely involved. The natural reservoir of the etiological agent has only been recently discovered in a bat cave in Nigeria. The status of asymptomatic infection is not certain. Investigations on skin and serum reactivity have suggested frequent prevalence of asymptomatic infections due to H. capsulatum var. duboisii among the residents in the vicinity of the cave microfocus of the fungus. The exact portal of entry into the body is not known, but inhalation into the lungs and direct inoculation in the skin have been incriminated. Laboratory diagnosis is confirmed by in vitro conversion into large yeast forms (8-15 mum in diameter) and by the demonstration of these forms within giant cells of tissues of experimentally infected animals There are no major clean-cut physiological differences between the two varieties, viz. capsulatum and duboisii. The cell wall of H. capsulatum var duboisii contains a glucan with beta 1-4 linkages in addition to a galactomannan shared with H. capsulatum var. capsulatum. Like the var. capsulatum var. duboisii has marked proteinase and collagenase activities in both mycelial and yeast forms, suggesting a possible pathogenic role for these enzymes. Both varieties have a common exoantigen. The yeast form of H. capsulatum var. duboisii contains the antigen found in the serotype 1,4 of var. capsulatum. A monoclonal antibody test has been developed that can recognize some epitopes in H. capsulatum var. capsulatum but not in the var. duboisii. There is need to develop specific serological diagnosis for the disease. Also there should be greater international awareness about African histoplasmosis. Amphotericin B and several antimycotic azoles like ketoconazole, itraconazole and fluconazole have been successfully employed for treatment.     

Immunological Studies on Dermatophytes I. Serological Reactivities of Neutral Polysaccharides with Rabbit Antiserum to Microsporum quinckeanum

Antiserum produced in the rabbit to autoclaved mycelial suspensions of Microsporum quinckeanum reacted with three neutral polysaccharides isolated from each of five species of dermatophytes, M. quinckeanum, Trichophyton granulosum, T. interdigitale, T. rubrum, and T. sch?nleinii. The serological reactivities of these polysaccharides, grouped as galactomannans I, galactomannans II, and glucans, were compared by qualitative precipitation analyses in gel and quantitative complement-fixation analyses. Significant differences were found among the glucans and galactomannans II but not among the galactomannans I of these species.     

Monosaccharide and Chitin Content of Cell Walls of Histoplasma capsulatum and Blastomyces dermatitidis

Cell walls of Histoplasma capsulatum and Blastomyces dermatitidis, obtained by mechanical breakage of yeast- and mycelial-phase cultures, were lipid-extracted and then fractionated with ethylenediamine. Unextracted cell walls, lipid-extracted cell walls, and the three fractions resulting from ethylenediamine treatment were examined for monosaccharide and chitin content. The yeast-phase cell walls of five strains of H. capsulatum fell into two categories, designated chemotypes I and II, one of which, chemotype II, was similar to yeast-phase cell walls derived from three strains of B. dermatitidis. H. capsulatum chemotype I cell walls were characterized by lower content of material soluble in ethylenediamine, higher chitin content, and lower monosaccharide content than H. capsulatum chemotype II or B. dermatitidis cell walls. Approximately 80% of the monosaccharides of chemotype I cell walls was combined in forms susceptible to attack by mild acid hydrolysis, compared with about 50% of the monosaccharides of chemotype II and B. dermatitidis. H. capsulatum and B. dermatitidis yeast-phase cell walls could be distinguished, however, by their susceptibility to attack by a crude enzyme system derived from a Streptomyces sp. incubated with chitin as the only carbon source. Both glucose and acetylglucosamine were released from H. capsulatum cell walls, regardless of chemotype, during enzymatic hydrolysis, whereas only acetylglucosamine was released from B. dermatitidis yeast-phase cell walls. Mycelial-phase cell walls of H. capsulatum and B. dermatitidis were characterized by lower content of material soluble in ethylenediamine, higher proportions of mannose, and lower chitin content than their respective yeast phases. Glucose and acetylglucosamine were both released from all mycelial-phase cell walls, whether H. capsulatum or B. dermatitidis, by the crude enzyme system.     

Toxoplasmosis II. Studies of Animal Sera Using Complement-Fixation Tests

Serological studies were performed in guinea pigs, a sheep, calf, goat and two pigs experimentally infected with toxoplasmosis. The direct complement-fixation method was effective in detecting antibodies in guinea-pig, goat and sheep sera. The modified complement-fixation technique supplementing complement with normal bovine serum fraction, was required when testing bovine serum. With swine sera best reactions occurred in the indirect complement-fixation test and definite but low grade reactions were produced in the direct test after pro-complementary activity was removed by pH treatment of the sera. Allergic skin reactions were produced in the experimental animals but improvement in the antigen is necessary before the test could be used generally in the field as a diagnostic method for animal toxoplasmosis.     

Variation in Lipid Content of Strains of Histoplasma capsulatum Exhibiting Different Virulence Properties for Mice

Nielsen, H. S., Jr. (Duke University Medical Center, Durham, N.C.). Variation in lipid content of strains of Histoplasma capsulatum exhibiting different virulence properties for mice. J. Bacteriol. 91:273-277. 1966.-Lipid content and virulence were studied in six isolates of Histoplasma capsulatum in an attempt to determine whether or not the two factors could be correlated in this fungus. Virulence was evaluated by injecting dba line 1 male mice intracerebrally with 2.8 x 10(4) infective yeast-phase units and recording organ involvement and spontaneous deaths occurring in a 20-day period. Yeast cells were extracted with mixtures of ethyl alcohol-diethyl ether (3:1, v/v), and the total extractable lipid, as determined by solubility in petroleum ether, was separated into acetone-soluble and phospholipid fractions by acetone precipitation. Neutral lipids were measured directly by weighing, whereas total phospholipids were calculated after the colorimetric determination of phosphorus. The mixed phosphatides of two isolates, differing in virulence, were separated into five fractions by use of a column of silicic acid and Hyflo Super-Cel. In the six isolates studied, neither total extractable lipid, acetone-soluble lipid, nor phospholipid showed a quantitative correlation with virulence. Phosphatidylserine, cephalin, phosphoinositides, and sphingolipids were present in essentially the same amounts in the two strains investigated; however, a lecithin fraction was absent in the less virulent form. These data suggest that the quantity of phosphatidylcholine demonstrated for a given isolate of H. capsulatum may provide some insight as to its virulence, although such a relationship is lacking for total lipid, the acetone-soluble fraction, and the combined phospholipids of yeast-phase growth.     

Genetic diversity of Histoplasma capsulatum strains in Brazil

This study establishes the genetic relatedness among Brazilian Histoplasma capsulatum samples obtained from different sources. A PCR-based random amplified polymorphic DNA (RAPD) assay was used to delineate polymorphisms among isolates in geographically diverse regions in Brazil. RAPD fingerprints revealed distinct DNA profiles and provided a high level of discrimination among H. capsulatum strains from different locations. Cluster I was composed of H. capsulatum isolates from the northeast region. The majority of strains from southeast and south were categorized as major cluster II. The strain 84564 from Rio de Janeiro State showed no genetic correlation to any of the isolates from the same state. The RAPD patterns of H. capsulatum isolates from Goias (Cluster III) were unrelated to DNA fingerprints observed among the other H. capsulatum strains (48% similarity). This study is the first report that stratifies the clusters of H. capsulatum strains from Brazil by molecular typing and associates them with the geographical origin.     

Two viruses isolated from rodents (Clethrionomys gapperi and Microtus pennsvlvanicus) trapped in St. Lawrence County, New York

Four strains of C. gapperi virus were isolated from 3 Clethrionomys gapperi and 47 strains of Microtus virus from 15 Microtus pennsylvanicus and 1 Mus musculus. One of the Microtus strains was isolated from a pool of 20 mites while the others were from rodent tissues. These agehts were insensitive to ether and sodium desoxycholate, withstood freezing at -70 C for 3 years and lyophilization without loss of titer, and were not killed when heated at 60 C for 1 hour. Their size as determined by filtration was less than 50 mg and greater than 20-35 mmicro. The strains within each group appear to be similar. The illness induced in suckling mice by the C. gapperi agents had a 5-day incubation period followed by prostration and death with a histologic picture of extensive encephalomalacia. The incubation period in mice for the Microtus agents was 9 to 11 days followed by convulsions and death. Histopathology showed meningeal infiltration and necrosis of the molecular layer. No antigenic similarity was detected between the C. gapperi and Microtus viruses by cross complement-fixation test.     

Antígenos del paracoccidioides brasiliensis para las Reacciones serológicas

Summary The sera from normal subjects gave negative results with the following antigens used in the complement-fixation tests: 1) polysaccharide prepared according toFava Netto's technic; 2) a filtrate of shaked cultures followingAjello et al.'s technic; 3) an aqueous extract of mechanically disrupted yeast cells ofP. brasiliensis.The sera from patients of S.A. blastomycosis gave positive c.f. tests with the three antigens with titer ranging from 1/20 to 1/5, 160. Antigen No 2 gave in 11/18 cases higher titers than the other antigens.Immunodiffusion tests gave positive results with the antigen no 2. The sera from 10 cases of histoplasmosis gave cross reactions with the antigen No 3, in 5/10 cases with the antigen No 2 and in not any case with the antigen No 1.