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We treated a case of laryngeal Cryptococcus neoformans infection in a glucocorticosteroid-dependent patient with chronic obstructive pulmonary disease. To our knowledge, this is the first report of successful treatment of laryngeal cryptococcus using oral fluconazole as a single agent.
Sexual reproduction in basidiomycete fungi is governed by MAT loci (P/R and HD), which exhibit remarkable evolutionary plasticity, characterized by expansions, rearrangements, and gene losses often associated with mating system transitions. The sister genera Cryptococcus and Kwoniella provide a powerful framework for studying MAT loci evolution owing to their diverse reproductive strategies and distinct architectures, spanning bipolar and tetrapolar systems with either linked or unlinked MAT loci. Building on recent comparative genomic analyses, we generated additional chromosome-level assemblies, uncovering distinct trajectories shaping MAT loci organization. Contrasting with the small-scale expansions and gene acquisitions observed in Kwoniella, our analyses revealed independent expansions of the P/R locus in tetrapolar Cryptococcus, possibly driven by pheromone gene duplications. Notably, these expansions coincided with a pronounced GC-content reduction best explained by reduced GC-biased gene conversion following recombination suppression, rather than relaxed codon usage selection. Diverse modes of MAT locus linkage were also identified, including three previously unrecognized transitions: one resulting in a pseudobipolar arrangement and two leading to bipolarity. All three transitions involved translocations. In the pseudobipolar configuration, the P/R and HD loci remained on the same chromosome but genetically unlinked, whereas the bipolar transitions additionally featured rearrangements that fused the two loci into a nonrecombining region. Mating assays confirmed a sexual cycle in C. decagattii, demonstrating its ability to undergo mating and sporulation. Progeny analysis in K. mangrovensis revealed substantial ploidy variation and aneuploidy, likely stemming from haploid-diploid mating, yet evidence of recombination and loss of heterozygosity indicates that meiotic exchange occurs despite irregular chromosome segregation. Our findings underscore the importance of continued diversity sampling and provide further evidence for convergent evolution of fused MAT loci in basidiomycetes, offering new insights into the genetic and chromosomal changes driving reproductive transitions.
Since 1923, eight cases of cryptococcosis have been described in Norway, all with meningeal affection. A distinct, solitary infiltration in the upper lobe of the right lung was discovered in a 68 year-old woman. The lobe was extirpated. Microbiological and histological investigations showed infection with Cryptococcus neoformans. No antimycotic treatment was given. Four months later the patient developed osteomyelitis of the skull due to cryptococcosis, and was treated successfully with amphotericin B and flucytosin. Further investigation of the patient revealed a defect in the T-lymphocyte immune system, but it is uncertain whether this was of any significance for the development of the cryptococcus infection.
CSF studies of 14 cases of cryptococcus meningitis revealed: 1. Direct discerning of yeast cells in the blood cell counting chamber, by Indian ink stain, and by cytological examination based on Sayk's technic, all were highly positive in repeated examinations. 2. Morphology of cryptococcus and inflammatory cellular reactions in CSF were investigated, and were quite characteristic. 3. Suppression and destruction of yeast cells were attainable only when doses of amphotericin B were sufficient.
The in vitro interaction between yeast cells of Cryptococcus neoformans and Lewis rat alveolar macrophages (AM phi) was studied in the absence of serum. AM phi were harvested by lung lavage, and monolayers of adherent cells were established in wells of microtiter plates. Radiolabeled yeast cells were added to fresh AM phi monolayers, the plates were incubated at 37 degrees C under 5% CO2, nonadherent yeasts were removed, and phagocytosis (i.e., attachment or ingestion) was determined by measuring adherent radioactivity. AM phi were able to bind or ingest, and kill, encapsulated strains of C. neoformans in the absence of serum. Serum-free phagocytosis was suboptimal by comparison with phagocytosis in the presence of serum. The mechanism of serum-free phagocytosis involves a receptor on the AM phi with affinity for mannose-rich determinants present on the yeast cell walls and unrelated to the capsular polysaccharide. Opsonin-independent phagocytosis was only detected with nonencapsulated, small, and medium encapsulated strains of C. neoformans. Large encapsulated strains were not taken up without serum. Serum-free phagocytosis could be of critical importance in the alveolar spaces, where only marginal concentrations of serum opsonins are initially present.
Murine natural killer (NK) cell-mediated inhibition of growth of a yeast-like target cell, Cryptococcus neoformans, was completely abrogated by blocking the effector cell secretory process with monensin. Therefore, further studies were performed to determine the ability of various cytoplasmic fractions of NK cells to mediate inhibition of cryptococcal growth. Percoll-fractionated homogenates of rat LGL tumor cells demonstrated that the granule-containing fractions plus three additional sets of less dense cytoplasmic fractions displayed anti-cryptococcal activity; whereas only the cytoplasmic granule-containing fractions had cytotoxic activity against YAC-1 tumor cell and sheep erythrocyte targets. Maximal cryptococcal growth inhibition induced by LGL granules occurred after a 1 h incubation, required the presence of Ca2+ (1.0 mM) or Mg2+ (0.5 mM or 5.0 mM), and was completely abrogated in the presence of rabbit anti-LGL granule IgG. Cytolysin, the granule component which mediates tumor cell and sheep erythrocyte lysis, effectively limited the growth of cryptococci. Since Percoll gradient fractionation of the LGL homogenates demonstrated three separate peaks of anti-cryptococcal activity other than the granule peak, it is possible that the cytolysin-containing granules are not the only subcellular component of NK cells playing a role in inhibition of C. neoformans growth.
Ninety-seven strains of Cryptococcus neoformans and C. bacillisporus were examined for 44 biochemical characters and the results were analyzed numerically. One phenon emerged at the 86% level of similarity when strains were clustered according to their M-similarity values. All strains grew in ten carbon sources (D-glucose, D-galactose, arbutin, maltose, sucrose, D-melezitose, D-xylose, D-mannitol, D-glucitol, and meso-inositol), and also grew at 37 degrees C and produced urease and phenoloxidase. None of them grew in melibiose, lactose, nor valine, and none reduced nitrate to nitrite. Comparison of selected biochemical characters, creatinine utilization, and serotypes of 49 aberrant strains is presented. Forty-eight of the 97 strains produced the Filobasidiella state either alone or when paired with a strain of compatible mating-type. Filobasidiella neoformans serotypes A and D were interfertile with compatible mating-types of F. bacillispora serotypes B and C. The 44 biochemical characters and 4 serotypes did not predict barriers to mating competence. The present study further substantiates that Filobasidiella neoformans and F. bacillispora are one species.
Cryptococcus neoformans cell and culture supernatant extracts were fractionated by ion exchange and gel filtration column chromatography. Various fractions were used to immunize mice, and to assess the release of migration inhibition factor, delayed type hypersensitivity, and protective immunity after challenge with C. neoformans. Results suggest that the C. neoformans fractions, which protect mice, contain a high molecular weight, predominantly carbohydrate antigen that can be distinguished from the capsular polysaccharide.
The effectiveness of the fluorescent viability test (fluorescein diacetate-FDA and ethidium bromide-EB-solution) compared to the plaque counting test (Miles & Misra M & M) was performed on 10 samples of Cryptococcus neoformans cultivated in Sabouraud dextrose agar at 25 degrees C. The optimum incubation period of 50 minutes was determined. Growth curves of the fungal strains studied based on the mean cell number were drawn for both the FDA & EB and M & M methods. The statistical evaluation (Student's T test) of the average sum of the viable cell counts showed that the FDA-EB method is more sensitive than the M & M test for the studied species. The growth curves of the samples usually followed a homogeneous pattern comparable to other non-dimorphic fungi.
Cryptococcus neoformans was isolated from 13 patients (7 females and 6 males) suffering from systemic cryptococcosis. Eight patients were suffering from central nervous system cryptococcosis and 5 were suffering from disseminated cryptococcosis. Systemic lupus erythematosus was found to be the common underlying disease in 3 patients. The results of treatment depended on the underlying diseases (7 improved, 6 died). Also, 13 isolates of C. neoformans were obtained from feces of 30 pet birds. All 26 isolates of C. neoformans were cultured in glycine cycloheximide medium and were found to be of serotypes A and D.
Acapsular mutant Cryptococcus neoformans cap 67 was grown in Pine's citrate broth medium for 3 days and the cells then transferred to a nitrogen-free medium for 6 days. The cells were subjected to a four stage extraction with buffered Triton-X100, cold dilute alkaline borohydride, hot dilute acetic acid, and a second alkaline extraction. Galactoxylomannan antigens were recovered from the culture supernates of both 3 day-old and 9 day-old yeast cells. The alkaline extracts contained water-soluble galactoxylomannan and a water-insoluble glucan. Dilute acid treatment released a minor amount of carbohydrate from the cells. The second alkaline extraction yielded increased amounts of glucan and galactoxylomannan from the 9 day-old cells. Soluble non-dialyzable cell extracts were antigenically identical in immunodiffusion with the culture supernate antigens. After the extraction sequence, all of the galactose, xylose, and mannose were removed from the cells. The walls retained their shape after extraction but their layers were loosened. Cells resuspended in nitrogen-free medium for six days developed thickened walls with alternating electron-dense and electron-lucent layers. The major constituent of the thickened 9 day-old cell walls was glucose, only 5% glucosamine was detected.
The ultrastructure of Cryptococcus neoformans in the cerebrospinal fluid, which was obtained from a patient suffering from systemic lupus erythematosus and cryptococcal meningitis, before treatment and on the 10th and 20th day after the start of treatment was studied. Numerous cryptococci were detected in their cerebrospinal fluid before treatment. However, in most of them their biological activity was low and their organelles were not clear. A few yeasts preserved well their organelles. Such cells showed a tendency to develop vesicular membrane structures (lomasomes) touching the internal part of the cell wall. In the cerebrospinal fluid on the 20th day all of the yeasts were dead, even though numerous yeasts were observed by an India-ink method. At this time no colonies were recovered from the fluid.
A simple melanin assay using DL.DOPA as the substrate was developed to aid in the identification of Cryptococcus neoformans. The DL-DOPA drop test was simple and efficient. The best results (100% of the C. neoformans isolates were positive) occurred when C. neoformans was grown for two days at room temperature on Sabouraud agar modified. One to three loopfuls of yeast cells were then transferred to a starvation medium for 18-24 hours. Two of three drops of 0.3% DL-DOPA solution was applied to the transferred yeast cells. Only C. neoformans produced a brown or blackgrey pigment within 24 hrs, with 85% of the isolates becoming brown or black-grey within thirty minutes.
Forty-one strains of Cryptococcus neoformans were examined after 3 days growth on a fresh and aged medium at pH 5 & pH 7 for comparison of capsule formation. Over one-half of the strains did not form visible capsules on aged medium at pH 5. Serotypes and source of isolation did not correlate with ability or inability to form capsules. Growth of C. neoformans in the nonencapsulated state makes it possible to culture many strains of C. neoformans in the form that more closely simulates the true infectious particles.
Cryptococcus neoformans was instilled intranasally into mice which were periodically sacrificed to determine the course of infection. Cryptococci persisted within the nasal passages throughout the 90 day study. Extranasal dissemination began 14-28 days after instillation and was still demonstrable 90 days post-exposure. Ten percent mortality was observed in mice receiving 10(6) cryptococci, while no mortality was observed in mice exposed to 10(3) or 10(4) cryptococci. Our research suggests that nasal colonization with C. neoformans can precede pulmonary and systemic cryptococcosis by weeks or months.
A strain of Cryptococcus neoformans that was isolated from the cerebrospinal fluid of a human diagnosed as having acquired immunodeficiency syndrome (AIDS), and that produced cutaneous lesions in experimentally infected, normal mice is described. Although no unusual cutaneous manifestations were noted in the patient's records, this isolate of C. neoformans proved to be dermotropic when injected intravenously into CD-1 mice. The LD50 at 28 days post infection ranged from 3.6-7.5 X 10(5) cells per mouse, and in vitro growth rate studies demonstrated that this isolate grew well at 35 degrees C and at 37 degrees C, but did not grow at 40 degrees C and higher. This isolate was rhinotropic producing large granulomatous lesions in the nasal tissues. Other cutaneous tissues affected were the periocular tissues, ears, feet and tail, although the granulomas were nodular in structure and less necrotic than the nasal lesions. The brain, lungs, liver, kidneys and spleen also were culture positive for C. neoformans. Histopathologically, each affected tissue examined had large densities of yeast cells and a chronic, granulomatous host response. Animals surviving the infection appeared to develop a commensal-type relationship with the infective yeast. This is the first report of an isolate of C. neoformans from an AIDS patient that has caused cutaneous manifestations in an animal model. The model described in this report may be useful for elucidating pathogenic mechanisms of cryptococcosis, particularly cutaneous manifestations of the disease.
Mice were vaccinated with formalin-killed Cryptococcus neoformans either intranasally, intravenously, or intraperitoneally. Resistance to lethal challenge and in vitro phagocytic activity of alveolar and peritoneal macrophages was studied. Increased resistance to lethal challenge was seen following vaccination by each of the three routes but increased phagocytic activity was seen only in alveolar macrophages from mice vaccinated intranasally.
Three pseudohyphal isolates of Cryptococcus neoformans were inoculated intracranially into mice. Four weeks post-inoculation the animals showed no symptoms of disease and the number of viable cells per brain decreased to zero. Possible roles of pseudohyphal forms of C. neoformans in the immunology and pathogenesis of cryptococcosis are discussed.