The efficacies of common dyes in primary isolation media for recovery of pathogenic fungi.

Dyes incorporated into a basal medium of brain heart infusion, Sabhi, tryptic soy, or yeast extract--pepton--glucose (YxPG) agar for selective isolation of fungi were investigated. Dilutions of 1:500, 1:750, 1:1,000, 1:5,000, and 1:10,000 of 33 common dyes were tested against 11 gram-positive and 16 gram-negative bacteria. In addition, these dyes were tested against Cryptococcus neoformans, Candida albicans, and the dimorphic phases of Histoplasma capsulatum and Blastomyces dermatitidis. Twenty-one of the dyes did not inhibit any of the organisms tested. Brilliant green, gentian violet, and malachite green (at three dilutions) inhibited all the organisms tested. Methyl red was found to be the best dye in selecting for fungi. Several dyes were also found to inhibit selectively C. neoformans or C. albicans and the dimorphic fungi H. capsulatum or B. dermatitidis.

Accidental inoculation blastomycosis.

The pathogenesis of blastomycosis as a primary pulmonary infection has been well established and widely accepted. The rate cases of primary cutaneous inoculation are in all documented instances related to "laboratory" accidents. A case of inoculation with a culture suspension of Blastomyces Dermatitidis is herein reported. A slow healing chancre remained active for about eight months.

Identification of the active precipitin components in a purified preparation of the A antigen of Blastomyces dermatitidis.

A purified A-antigen preparation of Blastomyces dermatitidis was determined to be composed of five major glycoprotein bands, visible with Coomassie blue and periodic acid-Schiff staining of polyacrylamide gels. At least 20 additional protein bands were detected by using a silver stain, which was 100 times more sensitive than the Coomassie method. Two components of this mixture were determined to be associated with the A-antigenic activity of B. dermatitidis. Of several antigen preparations examined in Ouchterlony precipitation tests, those reactive with a reference anti-A antiserum contained the slowest moving of the Coomassie blue bands. The antigen preparations without precipitin reactivity lacked this protein band. Two protein bands were shown to disappear from an antigen preparation after incubation with an affinity gel linked to the reference anti-A serum. One of the bands was the slowest Coomassie blue band, and the other was a fast-migrating protein detectable only with the silver stain. Characterization of the components responsible for the A-antigenic activity has important applications in the production and standardization of serological reagents for the diagnosis of blastomycosis.

Production and characterization of a hybridoma-derived antibody to Blastomyces dermatitidis.

A hybridoma cell line was isolated which produced monoclonal antibody to one protein component of a yeast-phase cytoplasmic antigenic complex of Blastomyces dermatitidis. The immunoglobulin M antibody product was characterized by immunodiffusion, autoradiography of polyacrylamide gels, and cellulose acetate electrophoresis. By attaching the antibody to an affinity gel, one major protein band was identified by polyacrylamide gel electrophoresis as the antigen for which the antibody was specific.

Comparative activities of glycolytic enzymes in yeast and mycelial forms of Candida albicans.

Through use of a synthetic defined medium which allows for the exclusive growth of yeast or mycelial forms of Candida albicans the activity of several major glycolytic enzymes in these forms were examined and compared. The results indicate vast metabolic differences between the forms. These data are discussed in relationship to the phenomenon of morphogenesis in C. albicans which in turn relates to problems in immunology and pathogenics of this important opportunistic organism.

Crossed immunoelectrophoretic analysis of two antigen extracts of Thermoactinomyces candidus.

A pyridine extract antigen and a double-dialysis antigen (DDA) obtained from Thermoactinomyces candidus were analyzed by crossed immunoelectrophoresis. In addition, the heat lability, pronase sensitivity, and isolectric points of the components of the DDA were determined. By using antisera raised against crude pyridine extract antigen, two immunogenic components were resolved by crossed immunoelectrophoresis. A similar analysis of DDA using antisera raised against crude DDA revealed 15 immunogens. All but six components were heat labile, whereas pronase had little effect on the number of resolvable components. Intermediate gel crossed immunoelectrophoresis using antiserum raised to whole spores detected six immunogenic components, four of which were also detected by the anti-DDA serum. A total of 19 bands were obtained when the DDA was subjected to flatbed isoelectric focusing on polyacrylamide gels. The isoelectric points for the various components were found to range from 3.5 to 5.7. Crossed immunoelectrophoresis using isoelectric focusing in the first dimension yielded at least 16 immunogenic components. Six components with isoelectric points falling in the range of 4.5 to 6.4 were found to be resistant to heat. A comparison with antigens obtained from other thermophilic actinomycetes is presented.

An effective medium for the selective growth of yeast or mycelial forms of Candida albicans: biochemical aspects of the two forms.

A new synthetic medium, based on a modification of a commercially available tissue culture medium, allows Candida albicans to be grown in the yeast or mycelial form. Salient features of the system are described and comparisons with previous physiological investigations are discussed. A concise biochemical profile of these two forms of C. albicans is also presented. The results indicate vast metabolic differences between the two forms.

Laboratory examination of organic fungicides against zoopathogenic fungi in soil.

Seven commercial and five experimental organic fungicides were tested against Histoplasma capsulatum, Cryptococcus neoformans, Allescheria boydii, and Sporotrichum schenckii in a series of laboratory studies. Preliminary agar dilution plate tests indicated that Lanstan, DAC 649, DAC 469, DAC 2787, He 3944, maneb, and nabam, in order of decreasing activity, inhibited all test fungi. Terraclor, Dexon, and He 13274 were active only in high concentration. The best fungicidal properties were exhibited by Lanstan, Vapam, and DAC 649; maneb, nabam, DAC 469, and DAC 2787 were fungicidal but to a lesser degree than the former compounds. In subsequent tests against H. capsulatum and C. neoformans in artificially infested soil or by use of the buried plug technique, Lanstan, Vapam, and DAC 649 showed good activity and merit further study.

Yeast- and mycelial-phase antigens of Blastomyces dermatitidis: comparison using disc gel electrophoresis.

The disc gel electrophoretic patterns obtained with skin test-active (mycelial and yeast) antigens of Blastomyces dermatitidis were compared. As would be expected, the blastomycins (mycelial-phase) and the cytoplasmic ultrafiltrates (yeast-phase) were heterogeneous mixtures containing proteins, glycoproteins, lipoproteins, and carbohydrates. The skin test-active cytoplasmic ultrafiltrates and the blastomycins contained glycoproteins that had similar R(f) values which allows the possibility that one or more of these components is responsible for the skin-test reactivity of these antigens. The electrophoretic migration of the alkali-soluble, water-soluble cell wall antigen differed from those of the cytoplasmic antigens and the two blastomycins. Electrophoresis, Sephadex chromatography, and ultrafiltration studies showed that the alkali-soluble, water-soluble cell wall antigen is comprised of lipid, polysaccharide, and protein and has a molecular weight range of 30,000 to 50,000. The increased number and mobility of both the protein and carbohydrate bands after denaturation and electrophoresis of this antigen in sodium dodecyl sulfate indicate that there are several cross-linkages between the polysaccharide and/or protein moieties, possibly via lipid or disulfide bridges.

Isolation of skin test-active preparations from yeast-phase cells of Blastomyces dermatitidis.

Cell wall and cytoplasmic fractions were isolated from mechanically disrupted yeast-phase cells of Blastomyces dermatitidis in an effort to obtain a reliable skin-test antigen. The biological activities of these fractions were compared with those of two blastomycin preparations. The cytoplasmic antigens exhibited a low index of specificity yet exceeded the specificities of the blastomycins. The skin test-active component(s) of the cytoplasmic material had a molecular weight between 10,000 and 30,000 and could easily be concentrated by ultrafiltration on a PM-10 membrane. Unlike the cytoplasmic antigens and blastomycins, an alkali-soluble, water-soluble cell wall antigen effectively distinguished guinea pigs that were sensitized to B. dermatitidis from those sensitized to Histoplasma capsulatum. The biological activity of the yeast-phase antigen could be quantitated, on a weight basis, after purification by ultrafiltration (PM-10 membrane).

Chitin synthetase from the yeast and mycelial phases of Blastomyces dermatitidis.

Chitin synthetase (E.C.2.4.1.16) from mixed membrane fractions of the yeast and mycelial phases of Blastomyces dermatitidis were compared. The behavior of the enzyme from both phases was very similar: N-acetylglucosamine was stimulatory (Km 8.5 mM for yeast and 3.9 mM for mycelium); substrate Michaelis-Menten kinetics were sigmoidal; substrate Km of enzyme from yeast decreased from 3.0 mM at low N-acetylglucosamine (5 mM) levels to 1.4 mM at high (100 mM) levels; substrate Km of enzyme from mycelium was essentially unchanged at 1.4 mM; temperature optimum was 28 degrees C; pH optimum was 7-7.5; Mg+2 optimum was 5-10 mM. The greatest difference was that enzyme from yeast was extracted in a mostly latent form that required trypsin treatment for maximal in vitro activity while enzyme from mycelium was extracted in an active form which was rapidly deactivated by trypsin treatment.

In vitro and in vivo activity of 2,4-diamino-6-(2-(3,4-dichlorophenyl)acetamido) quinazoline against Cryptococcus neoformans.

The minimum inhibitory concentration of quinazoline derivative was determined by the tube dilution method for Cryptococcus neoformans, strain 184. The effect of this chemical agent on macromolecular metabolism indicated an inhibition of incorporations of labeled precursors into RNA and protein of C. neoformans. A mouse model infection with C. neoformans was established. Following this, the animals were given ip or oral doses of different concentrations of the experimental drug. Infected mice responded to ip administration of the drug in that the percentage of surviving mice increased progressively with increasing drug dosage. The curing dose 50 (CD50) was determined, based on the isolation of C. neoformans from organs of animals during or at the termination of the experiments.

Immunocytoadherence during experimental histoplasmosis.

Immunocytoadherence was investigated as a method of detecting active histoplasmosis in two groups of infected New Zealand white rabbits. Yeast-phase Histoplasma capsulatum was given intravenously to one group and intratracheally to the other. There was a striking correlation between the clinical condition of the animals and the number of rosettes seen. This number increased as the infection progressed and decreased as it regressed. The immunocytoadherence technique might be of value in detecting active histoplasmosis, and investigation in humans is warranted.

Non-specific incorporation of nucleic acid precursors in Blastomyces dermatitidis and Histoplasma capsulatum.

Incorporation of thymidine, thymidine monophosphate (TMP), thymidine triphosphate (TTP), uridine and orotic acid into DNA, RNA and protein in Blastomyces dermatitidis and Histoplasma capsulatum was studied utilizing a specific acid hydrolysis technique developed for these fungi. Thymidine was incorporated to the greatest extent (approximately 0.5% of added label) followed by uridine, orotic acid, TMP and TTP. In Blastomyces, uridine and orotic acid labeled primarily RNA. TMP and TTP labeled RNA, DNA and protein at nearly the same level. In Histoplasma RNA was labeled poorly by any of these precursors. TMP and TTP labeled DNA predominately and protein to a slightly lower level. Deoxyadenosine or uridine media supplements of 250 micrograms/ml did not enhance incorporation. All precursors tested were found to be nonspecific in that RNA, DNA and protein were labeled. All data indicate that neither RNA nor DNA synthesis can be specifically measured in whole cells or acid precipitates by any of these precursors. Specific radiometric monitoring with these isotopes therefore requires the separation of these macromolecules.

The nutritional status of patients with chronic obstructive pulmonary disease.

The nutritional status of 38 patients with chronic obstructive pulmonary disease (COPD) was assessed by dietary intake, anthropometric measurements biochemical analysis, and immunologic testing. The mean intakes for 9 nutrients were significantly greater than the 1974 Recommended Dietary Allowances of the National Academy of Sciences. Results of the anthropometric measurements for usual weight for height, fat stores, and muscle mass were significantly less than standard. Of the 32 subjects evaluated for immunocompetence, 9 were anergic (induration, 0) on all 3 skin tests. The results of this study indicated that the marasmic type of protein calorie malnutrition is a common finding among patients with COPD, and that patients with COPD who are immunoincompetent may be more susceptible to mixed protein calorie malnutrition of the kwashiorkor-marasmus type.

In Vitro and In Vivo Activity of a Synthetic Halogenated Quinoline Against Cryptococcus neoformans.

The minimum inhibitory concentrations of a halogenated quinoline, 3-amino-7-chloro-3,4-dihydro-1-hydroxycarbostyril (CBS), against nine clinical strains of Cryptococcus neoformans were determined by in vitro testing. The CBS was fungistatic at a minimum concentration of 0.2 mug/ml at 48 h for several strains. In vivo toxicity studies were carried out in mice. Mice were also infected with C. neoformans strain Price and injected with various concentrations of CBS. Mean life expectancy of treated groups of animals was increased over infected untreated controls.