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Cryptococcus neoformans in the crops of pigeons following its experimental administration.

Cryptococcus neoformans (5 X 10(6) yeast cells) was given per os to 10 pigeons (Columba livia) proved to be free (crops and excreta) of C. neoformans prior to experimentation. The yeast was recovered from the droppings of 9 pigeons the day after ingestion but was still present in the droppings of 1 pigeon on the 22nd day after ingestion. The crop was much more constantly positive than the droppings and for a much longer time since positive in 9 pigeons on the first day it was still positive in 2 pigeons on the 86th day at the end of the observation period. The results of the experiment presented here and the results of previous work, indicate that C. neoformans can survive and could so be carried in the crop of pigeons.

Animals

Study of the role of pigeons in the dissemination of Cryptococcus neoformans in nature.

Cryptococcus neoformans was recovered from droppings collected within the first 24 h from pigeons experimentally fed with a dose of 5 X 10(6) cells. The fungus proved to multiply well though differently in the sterilized pigeon and chicken excreta seeded with the organism. In both unsterile types of droppings no viable cells of C. neoformans were detected after 4 weeks incubation. Isolated bacterial flora from the intestinal contents of apparently healthy pigeons showed a complete inhibitory effect on the growth of C. neoforms in vitro. It has been concluded that pigeons do not favor multiplication of the fungus in their gut and consequently they do not seem to play an active biological role in dissemination of C. neoformans in nature.

Animals

Cryptococcus neoformans: gastronomic delight of a soil ameba.

During 7 days of incubation in vitro the trophozoite stage of the free-living soil amoeba, Acanthamoeba polyphaga, phagocytized and killed 78-97% of the cells of three strains of Cryptococcus neoformans. With one strain, incubation time was increased to nine days and 99% of the yeast cells were killed. It was calculated that during 4-9 days of incubation a single trophozoite phagocytized and killed a daily average of 84 yeast cells. The lethal effect of A. polyphaga on C. neoformans may represent a biological control mechanism in nature. Some of the surviving cells of C. neoformans developed into colonies containing pseudohyphae; these pseudolhyphal forms may be a biological 'escape hatch'.

Amoeba

Comparative morphological and biological studies on the itraconazole- and ketoconazole-resistant mutants of Cryptococcus neoformans.

Studies were carried out on the resistance in vitro of Cryptococcus neoformans to the oral antifungal drugs itraconazole and ketoconazole. None of the six sensitive strains tested developed resistance to itraconazole or ketoconazole by serial transfer on Sabouraud's glucose agar plates containing increasing concentrations of either drug. One mutant resistant to itraconazole, and one mutant resistant to ketoconazole, were isolated from the progenies of yeast cells surviving after treatment with a mutagenic substance, N-methyl-N'-nitro-N-nitroso-guanidine. These mutants were capable of growing in the presence of high concentrations of the drugs to which they were resistant. The itraconazole- and ketoconazole-resistant mutants obtained by mutagenesis were compared morphologically and biologically. The itraconazole-resistant mutant was characterized by the formation of very rough colonies which varied in size and shape, production of a large number of cell clusters, complete loss of capsule formation, and major degenerative changes in the cells, while in the ketoconazole-resistant mutant these changes were less pronounced and no cell clusters were formed. The acquisition of resistance was more stable in the itraconazole-resistant mutant than in the ketoconazole-resistant mutant. Both mutants showed partial cross-resistance and complete loss of virulence for mice.

Animals

Poorly encapsulated Cryptococcus neoformans from patients with AIDS. II. Correlation of capsule size observed directly in cerebrospinal fluid with that after animal passage.

Cryptococcus neoformans recovered from the cerebrospinal fluid (CSF) of eight patients, seven with the acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC), were studied to assess the relationship between degree of encapsulation noted in fresh CSF with that observed after animal passage. We further correlated encapsulation with extent of immunodeficiency in these patients. Results of these studies showed poor encapsulation (mean capsule plus cell diameter less than 10 micron) in six patients, intermediate in one (mean 15.5 micron), and full encapsulation in one (mean 24.4 micron). The last isolate was observed in the CSF from the only patient without convincing clinical evidence for AIDS. Mouse passage of cryptococci from 5 AIDS patients and one with ARC resulted in a statistically significant (P less than 0.05) increase in capsule size over that observed directly in fresh cerebrospinal fluid. Cryptococci derived from the non-AIDS patient did not show an increase in encapsulation after mouse passage. These studies suggest that the immune deficiency state associated with AIDS exerts little selective pressure on inhaled poorly encapsulated C. neoformans.

AIDS-Related Complex

Recovery of Cryptococcus neoformans from sputum using new technics for the isolation of fungi from sputum.

Three sputum-digesting agents, N-acetyl-l-cytseine, dithiothreitol, and pancreatin-trypsin, were shown to be equally effective in allowing for the isolation of Cryptococcus neoformans from sputum samples in quantitative comparisons. By quantitative plating on bird-seed medium it was also shown the centrifugation after digestion concentrated C. neoformans into a platable sediment and, further, that the organisms, when present in concentrations as low as 10 yeasts per ml. of sputum, could be isolated with much higher frequencies than when no digestion-centrifugation procedure was used.

Acetylcysteine

Differentiation of Cryptococcus neoformans serotypes by isoenzyme electrophoresis.

Cryptococcus neoformans has been divided into four serotypes by specific agglutination in immune rabbit sera. Based on mating characteristics of the perfect state and epidemiologic and biochemical differences, the serotypes have been divided into two major pairs. In an attempt to characterize the serotypes further, the authors studied 22 strains of C. neoformans by the technic of horizontal starch-gel isoenzyme electrophoresis. The glucose-phosphate isomerase and phosphoglucomutase of serotypes A, C, D, and a subset of the serotype B strains migrated to distinguishable locations in this system. The activities of the remainder of the serotype B strains co-migrated with the serotype C strains. Thus, this technic distinguishes all the serotypes of C. neoformans except for a subset of serotype B and should be a useful adjunct for further elucidation of the epidemiologic and biochemical differences among serotypes.

Cryptococcus

Role of the capsule in phagocytosis of Cryptococcus neoformans.

The capsule is closely associated with the virulence of Cryptococcus neoformans. The capsule inhibits phagocytosis by macrophages, monocytes, and neutrophils. Studies in our laboratory have shown that incubation of encapsulated cryptococci in normal human serum leads to deposition of large amounts of C3 fragments at the surface of the yeast and lesser amounts of IgG within the capsule. Thus, the capsule mediates two biologic activities with opposing effects. It is our current view that phagocytosis of the yeast is dependent on a balance between the antiphagocytic action of cryptococcal polysaccharide and the ability of the yeast to focus opsonically active complement fragments and the IgG at the capsular surface.

Animals

Capsular polysaccharides of Cryptococcus neoformans.

Polysaccharides of Cryptococcus neoformans are considered to have a role in the virulence of this encapsulated fungus. The structure has been determined for the most abundant polysaccharide, a glucuronoxylomannan of varying xylose and ester content. The structural complexity of the capsular material increases from serotype D to A to B to C, but even for the simplest capsule (type D), the immunodeterminants seem to occur only on the side chains. The interactions of some of these groups with antibodies is discussed.

Antibodies, Fungal

Susceptibility of coccidioides immitis, Candida albicans, and Cryptococcus neoformans to amphotericin B, flucytosine, and clotrimazole.

Toxicity and failure of treatment with amphotericin B are stimuli for researchers to evaluate alternative antifungal antimicrobics. Also, data from susceptibility tests of Coccidioides immitis are sparse. With use of a defined, synthetic culture medium, C. immitis (25 strains). Candida albicans (21 strains), and Cryptococcus neoformans (21 strains) were tested against flucytosine, clotrimazole, and amphotericin B. Molecule for molecule, the sequency of activity was: clotrimazole greater than amphotericin B greater than flucytosine (totally inactive) C. immitis; and clotrimazole greater than amphotericin B greater than flucytosine with C. albicans and C. neoformans. With four strains of C. immitis, the minimal inhibitory concentration (of amphotericin B) was the same when inocula of arthrospores were tested as when corresponding spherules/endospores were tested simultaneously and identically. The clinical outcome of coccidioidomycosis in 17 patients treated with amphotericin B correlated best with minimal inhibitory concentration after incubation of cultures for 48 hr; a favorable response was associated with minimal inhibitory concentrations of less than or equal 1.0 mug/ml. Because clinical isolates of fungi appear to vary in susceptibility, in vitro tests may have clinical utility.

Adult

Amphotericin B and amphotericin B methyl ester ascorbate. I. Chemotherapeutic activity against Candida albicans, Cryptococcus neoformans, and Blastomyces dermatitidis in mice.

Amphotericin B methyl ester (AME) has been reported to possess in vitro antifungal activity similar to that of amphotericin B and to have less intrinsic toxicity in mice and dogs. For these reasons AME has been porposed as an alternative to amphotericin B in the therapy of deep mycoses. For comparison of the therapeutic efficacy of the two polyenes in laboratory animals before initiation of studies in humans, groups of mice were infected with Candida albicans, Cryptococcus neoformans, and Blastomyces dermatitidis. Treatment consisted of two or more doses of each drug given by the intravenous route. Concurrently, studies of subacute toxicity were conducted in the same species to permit calculation of therapeutic indices. These studies have shown that AME, as the ascorbate salt, is substantially less efficacious than amphotericin B (in colloidal dispersion with sodium deoxycholate) for treatment of the fungal infections and that amphotericin B had a higher therapeutic ratio for all infections studied than did AME.

Amphotericin B

Fungicidal components of mammalian granulocytes active against Cryptococcus neoformans.

Citric acid extracts of granule-rich fractions, prepared from rabbit and guinea pig heterophils or human neutrophis, killed Cryptococcus neoformans in vitro. These extracts sere fractionated by micropreparative electrophoresis on polyacrylamide gels. In preparation of rabbit and guinea pig heterophils, cryptococcidal activity was associated predominantely with lysosomal cationic protein complex. Human neutrophils lacked strictly comparable cationic proteins but contained other components that killed C. neoformans. These components appeared to be identical to previously described proteins of the human neutrophil active against Candida parapsilosis.

Animals

Functional versus phenotypic analysis of T cells in subjects seropositive for the human immunodeficiency virus: a prospective study of in vitro responses to Cryptococcus neoformans.

We performed a prospective study of 50 subjects at high risk for human immunodeficiency virus (HIV) infection to determine if assays of antigen-specific T cell function provide an earlier indication of future progression to AIDS or a better assessment of immune function than do current methods of evaluation. We measured in vitro T cell responses to Cryptococcus neoformans and tetanus toxoid, response to mitogens, HIV p24 antigenemia, and clinical parameters. Progression to AIDS was significantly associated with loss of T cell response to cryptococci (P = .015), HIV antigenemia (P = .001), and low CD4+ cell numbers (P = .001). Most importantly, we found that loss of antigen-specific responses to cryptococci and tetanus can occur before changes in CD4 cell number. Abnormal response to mitogens and marked depletion of CD4+ cells were late signs of progressive HIV infection. Measurement of antigen-specific T cell function may be useful for assessing the efficacy of antiviral therapy in HIV infection before onset of symptoms.

Acquired Immunodeficiency Syndrome

Epidemiologic differences among serotypes of Cryptococcus neoformans.

In the USA, the most prevalent serotype of the fungus, Cryptococcus neoformans, was serotype A. The serotype constituted 203 of 272 isolates from infections and 85 of 89 isolates from the environment. Serotype B or C isolates were infrequent causes of infection, except in Southern California, and were infrequent causes of infecand were not isolated at all from environmental sources. In Southern California, the absence of serotypes B and C in 67 soil and pigeon dropping isolates was striking, considering that 25 of 49 isolates from infections were serotypes B or C. The site in nature where serotypes B and C exist is currently unknown but differs from that of serotypes A and D. Serotype D may be unusually prevalent in both environmental and patient isolates from Denmark and Italy. Of 24 isolates from those countries, 21 were serotype D.

Animals

Epidemiologic differences between the two varieties of Cryptococcus neoformans.

This report of the worldwide distribution of two varieties of Cryptococcus neoformans was drawn from data on 628 clinical isolates and from data on 97 additional isolates from other laboratories. Tests showed that 100% of the cultures from Austria, Belgium, Denmark, France, Germany, Holland, Italy, Switzerland, and Japan belonged to C. neoformans var. neoformans. More than 85% of the isolates from Argentina, Canada, the United Kingdom, and the United States (except southern California) were of C. neoformans var. neoformans, the remainder being of C. neoformans var. gattii . There was an unusually high prevalence (35-100%) of C. neoformans var. gattii in Australia, Brazil, Cambodia, Hawaii, southern California, Mexico, Paraguay, Thailand, Vietnam, Nepal, and countries in central Africa. These findings indicated that C. neoformans var. gattii is prevalent only in tropical and subtropical regions. Seventy per cent of the total isolates studied were of serotype A of C. neoformans var. neoformans. Serotype D (9% of the total) was common in Europe, but was found infrequently in other regions. Among the two serotypes of C. neoformans var. gattii , serotype B was 4.5 times more prevalent than serotype C. The majority (88%) of type C isolates in our collection were from southern California.

Africa

Postitional specificity of fatty acids in pyrophosphatidic acid from Cryptococcus neoformans.

Pyrophosphatidic acid isolated from Cryptococcus neoformans was degraded to phosphatidic acid in aqueous pyridine. The phosphatidic acid was hydrolyzed by phospholipase A (EC 3.1.1.4) of Crotalus adamanteus to lysophosphatidic acid and 2-positioned fatty acids. From the analyses of the fatty acid composition of pyrophosphatidic acid and its degraded products (phosphatidid acid, lysophosphatidic acid, and fatty acid), it was concluded that most of the saturated fatty acids of pyrophosphatidic acid were at the 1,1'-positions while the unsaturated fatty acids were largely confined to the 2,2'-positions. The positional specificity of the fatty acids in pyrophosphatidic acid coincided with that of ordinary glycerophosphatides.

Chromatography

Isolation of Cryptococcus neoformans from houses of AIDS-associated cryptococcosis patients in Bujumbura (Burundi).

Cryptococcus neoformans var. neoformans, which is responsible for AIDS-associated cryptococcosis in Bujumbura, was isolated in the domestic environment of seven out of 20 patients with AIDS-associated cryptococcosis. The findings prove that in his own domestic environment, the HIV-positive patient in central Africa is frequently exposed to the yeast and these observations lead us to insist on the suitability of carrying out a systematic survey by means of soluble antigens-sensitized latex in every HIV-positive patient. This also proves the importance of a follow-up of the 'cured' patients who easily can be recontaminated after their return home.

Acquired Immunodeficiency Syndrome