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Survey revealed that the mating type alpha is predominant among natural and clinical isolates of Cryptococcus neoformans regardless of the serotype. The ratio of alpha and a type was about 40:1 among 105 natural isolates and 30:1 in 233 clinical isolates.
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.
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.
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.
Melanogenesis in Cryptococcus neoformans begins with the oxidation of dihydroxyphenylalanine by the enzyme phenol oxidase. The succeeding steps are very rapid. Two intermediates, dopachrome and 5,6-dihydroxyindole, have been isolated and characterized by high performance liquid chromatography. A pathway of melanin formation in C. neoformans is proposed, based on the presence of these intermediates.
The electrokaryotype of the pathogenic yeast Cryptococcus neoformans is described for the first time. Three different patterns were seen: (a) serotypes B and C (variety gattii) are similar and consist of nine chromosome mobility groups of greater than 580 kb; (b) serotype A (variety neoformans) revealed eight chromosome-like groups greater than 700 kb; (c) serotype D (the second serotype of variety neoformans) not only differs from those described above, but each D isolate tested showed a different distribution of bands. The discrepancy, and the importance of electrokaryotyping as a taxonomic tool, are discussed.
The restriction patterns of mitochondrial DNA from 20 isolates of the two varieties of Cryptococcus neoformans were compared. The patterns exhibited extensive heterogeneity among the isolates regardless of their serotype or varietal status. Hybridizations with cloned fragments of the conserved cytochrome oxidase gene from Saccharomyces cerevisiae exhibited at least seven patterns among the 20 isolates. There were, however, similarities in the restriction patterns among isolates within the same serotype that were not shared by isolates of other serotypes. Intra-varietal similarities were observed in the restriction patterns among the isolates of C. neoformans var. neoformans which were not present in the restriction patterns among the isolates of C. neoformans var. gattii. Hybridization of some cloned mitochondrial DNA fragments to total DNA digests of various isolates revealed polymorphic as well as variety-specific patterns of homology. These findings agree with the antigenic heterogeneity among the isolates and support the current taxonomic classification of C. neoformans into two varieties.
Cryptococcus neoformans was found in the crops of four (1.3%) of 319 pigeons in the Charity Bird Hospital and the University Campus, Delhi. It was demonstrated repeatedly at the Bird Hospital in old pigeon excreta as well as in the air. The findings substantiate the current view that the pigeon itself is not a reservoir of C. neoformans although it may serve as a carrier of the fungus.
Pseudomonas aeruginosa was found to produce a factor or factors that inhibited Cryptococcus neoformans and appeared to be extracellular because the anti-C. neoformans activity was readily demonstrable in medium after the removal and killing of Pseudomonas organisms. Production of the inhibitor material was greatest in DST Agar after prolonged incubation and was reduced in the presence of glucose. A part of the inhibitory material was found to be chromatographically distinct from pyocyanin.
We investigated the opsonic activity of the serum factors affecting phagocytosis of Cryptococcus neoformans in vitro to elucidate the role of humoral factors in the host defense mechanisms against cryptococcosis. Two strains of C. neoformans, one heavily and one weakly encapsulated, were used. Guinea pig peripheral blood leukocytes (PBLs) were used for phagocytosis. The viable weakly encapsulated cells were ingested effectively by PBLs, in the presence of guinea pig normal fresh serum, while the heavily encapsulated cells were not ingested. Neither immune serum, its IgG fraction alone, nor heated serum promoted the phagocytosis of either the weakly or heavily encapsulated strain. On the other hand, immune serum promoted adherence of PBLs to viable cells of the heavily encapsulated strain, forming rosettes in the presence of fresh serum. A substantial amount of C3b component was detected on yeast cells when weakly encapsulated cells were incubated with human fresh serum, or heavily encapsulated cells were incubated with rabbit immune serum together with human fresh serum. Serum chelation experiments also indicated that the factors involved in the alternative complement pathway are opsonins for the weakly encapsulated strain. These results suggest that the alternative pathway plays an important normal opsonic role for weakly encapsulated strains and that specific antibody plays an immune opsonic role for heavily encapsulated strains of C. neoformans via the classical pathway of complement activation.
We examined seven strains, comprising five serotypes, of Cryptococcus neoformans to determine what constituents of the organisms are responsible for pathogenicity and virulence in BALB/c mice. C. neoformans strains were divided into three virulence classes by survival rates after intravenous inoculation of 1 X 10(5) or 1 X 10(7) viable cells, and virulence was found not to be correlated with serotype or capsular size. C. neoformans cells resisted phagocytosis in different degrees in the presence of normal serum. Sensitivity of the C. neoformans strains to singlet oxygen ranged from resistance to susceptibility. Histological examination revealed that a weakly encapsulated virulent strain induced inflammatory responses with granuloma formation in the liver, lung, and kidney in addition to formation of cystic foci in the brain. In contrast, although the heavily encapsulated virulent strain produced granulomatous lesions in the liver, this strain preferably produced mucinous cystic foci in the lung, kidney, and brain. Correlation between virulence, and biological, histopathological and physiological evidence suggests that C. neoformans strains are endowed with the implicated multiple pathogenic constituents in various degrees and proportions. The following are suggested as the most important pathogenic constituents: a polysaccharide capsule responsible for resistance to phagocytosis and formation of cystic foci; a cell surface structure for responsible for resistance to intra- or extracellular killing and induction of the granulomatous lesion; a growth rate suitable for interacting with phagocytic elimination.
During a study of serotyping of Cryptococcus neoformans, we found that the type strain of C. neoformans (CBS 132) was serotype A-D. This strain agglutinated with both factor 7 serum (specific for serotype A) and factor 8 serum (specific for serotype D) in our serotyping system. Therefore, we investigated the chemical structure of the antigenic capsular polysaccharide of this strain. The soluble capsular polysaccharide was obtained from the culture supernatant fluid by precipitation with ethanol. Column chromatography of the polysaccharide on DEAE-cellulose yielded three fractions (F-1 to F-3). The major antigenic activity was found in the F-3 fraction. The results obtained by methylation analysis, controlled Smith degradation-methylation analysis, partial acid hydrolysis, and other structural studies of F-3 polysaccharide indicated that the polysaccharide contains mannose, xylose, and glucuronic acid at a ratio of 7:2:2, and has a backbone of alpha (1-3)-linked D-mannopyranoside residues with a single branch of beta (1-2)-xylose and glucuronic acid. The ratio of mannose residues with or without a branch in the F-3 polysaccharide was 4:3 and its molecular weight calculated from the average of the degree of polymerization was 46,500 daltons. These results indicate that the chemical structure of the capsular polysaccharide of serotype A-D is very similar to those from serotypes A and D, suggesting that small differences in the molar ratio and pattern of linkage of monosaccharides in the branch of the polysaccharides of the three serotypes may be responsible for their different specificities.
Mitochondrial kinetics during mitosis in Cryptococcus neoformans was examined with ultrathin serial sections using a computer-aided three-dimensional reconstruction technique. The number of mitochondria varied during mitosis: there was an increase in prophase to telophase cells and a decrease in interphase cells. No appreciable differences in the form and number of mitochondria were found between the cells in the logarithmic growth phase and stationary phase. Fluctuations in the ratio of mitochondrial volume/total cytoplasmic volume were minimum during mitosis. However, the ratio was affected by the growth condition of the cells; that is, the ratio in the logarithmic growth cells was significantly higher than that in stationary cells. A giant mitochondrion, which is composed of a coalescence of all the mitochondria in a cell, was not found in this study.
The polysaccharide capsule is a characteristic virulence factor in the yeast-pathogen, Cryptococcus neoformans. Growth in hypertonic growth media results in yeast cells with visibly smaller capsules. We investigated this suppression quantitatively, using a chemical assay for cell-bound and dissolved capsular polysaccharide. Molar NaCl suppressed production of cell-bound polysaccharide by a factor of 2.5- to 5-fold. The possibility of salt-induced physico-chemical contraction of capsular gel was tested by dialysis of fixed cells from hypotonic medium against medium containing 1 M NaCl and against the original medium again, while capsular thickness, packed cell volume and cell-bound polysaccharide were followed. We detected a physical contraction of gel following dialysis against medium containing 1 M NaCl. Mutants which gave mucoid colonies on hypertonic agar were isolated. One of these gave twice as much polysaccharide as the wild type when cultivated in medium containing 1 M NaCl. The hypercapsular trait was passed through serial outcrosses to the wild type and segregated as a chromosomal gene. This mutant may represent a gene which regulates production of capsular polysaccharide.
For the cultural control of Cryptococcus neoformans (Cr.n.), among the routinely examined standard specimens like CSF, sputum, blood, etc., urine earns special attention. The combination of membrane filtration technique (MFT) and Staib agar for the detection of Cr.n. from body fluids as described by Staib in 1963 was used for the cultural isolation of Cr.n. from urine of AIDS patients. In 3 examplary cases the diagnostic significance of this method could be demonstrated: The brown colour effect (BCE) of Cr.n. of a single CFU, as well as in colonies growing with a high density, was produced on average within 3-5 d/26 degrees C. The method was found to be useful for the evaluation of antimycotic therapy. One example of the survival of a few CFUs of Cr.n. under treatment with fluconazole as compared to the efficacy of therapy with amphotericin B + flucytosine, and one example of a re-emergence of Cr.n. in the urogenital tract after a too short duration of treatment with amphotericin B + flucytosine are shown. For the exclusion of the survival of single CFUs of Cr.n. in the urogenital tract of males, quantities up to 1 l of urine for the combination of MFT and Staib agar are proposed. As a secondary observation, it was found that this diagnostic combination in addition to its primary purpose, can serve to detect the metabolic end products of the human body present in urine which may influence capsule formation of Cr.n. neoformans.
In a 33-year-old HIV-positive homosexual male suffering from unexplained headache, cryptococcosis was diagnosed in a progressive secondary stage. After treatment with the standard combination therapy of amphotericin B + flucytosine for 34 d, the patient was clinically symptom-free and discharged, upon his own request, from the hospital. He remained under ambulatory mycological control. After an interval of 65 d during which the urine had been free from Cryptococcus neoformans (Cr.n.), the fungus could not be isolated from urine but 3 X 10(5) CFUs/ml were found in the seminal fluid. Andrologically, teratospermia and hyposemia were present. There were no clinical signs in the genitourinary tract including the prostate. The significance of ecological niches for Cr.n. colonization of the genitourinary tract after antimycotic therapy is discussed. In such cases, in addition to cultural examination of urine for Cr.n. by the membrane filtration technique (MFT) and Staib agar, an additional cultural examination of seminal fluid is recommended. It is also proposed to pay more attention to Cr.n. in andrological examinations. Special regard should be given to a possible occurrence of Cr.n. in the seminal fluid of AIDS patients. In cytology of the seminal fluid, use of the Giemsa stain is unsuitable for the purpose of Cr.n. detection. For this reason, it should be supplemented by PAS staining.
It was demonstrated that the in vitro growth of a mucoid Escherichia coli strain from the urine of an AIDS patient could disturb the concurrent growth of Cryptococcus neoformans and the development of its brown colour effect (BCE) on Staib agar (syn. Guizotia abyssinica creatinine agar, bird seed agar, niger seed agar etc.) supplemented with penicillin + streptomycin. Owing to the supplementation with the triple antibiotic combination of penicillin + streptomycin + gentamicin and the resulting inhibition of E. coli growth, the formation of an intense BCE of the Cr. neoformans colonies after 3 d at 26 degrees C could be observed. On the same medium supplemented with this triple antibiotic combination 40 Cr. neoformans strains tested showed growth with an intense BCE after 3 d at 26 degrees C; but on Emmons' neutral Sabouraud's dextrose agar (NSDA) supplemented with the same triple antibiotic combination, inhibition of growth was found. For the examination of clinical specimens for Cr. neoformans contaminated with gram-negative rod-like bacteria, Staib agar supplemented with this triple antibiotic combination is proposed. Various antibiotic supplements to primary recovery media for fungi are discussed and ecological interrelations of bacteria and fungi are emphasized.
Trichothecin (T-cin), amphotericin B (AB), and 5-fluorocytosine (FC) were compared singly and in combination for capacities to inhibit growth of Cryptococcus neoformans in culture and to protect mice bearing infections with this yeast. The minimum inhibitory concentrations for T-cin, AB, and FC were found to be 0.5, 0.2, and 5.0 mug/ml, respectively. In vitro viability studies demonstrated a marked reduction in colony counts with the AB-FC combination and additive effects with the AB-T-cin and FC-T-cin combinations for a 3-day period. In mice infected intravenously with C. neoformans, the mean effective dose for AB was 0.38 mg/kg, and for FC it was 100 mg/kg for a 30-day treatment period. No mean effective dose could be ascertained when T-cin was tested at doses of 0.1 to 50 mg/kg. Despite this, marked beneficial effects were noted in vivo with the AB-T-cin combination, whereas additive effects and indifference were observed for AB-FC and FC-T-cin combinations, respectively. High-dose T-cin controls survived despite having received a cumulative dosage of more than twice the reported (LD(50)) mean lethal dose value.