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Isolation of representatives of the Cryptococcus neoformans/Cryptococcus gattii species complex can be made using dopamine containing media, such as Niger seed agar and l-DOPA agar. Here, we describe an alternative medium that uses banana flowers. Banana is a dopamine containing fruit and is widely available in tropical and subtropical countries that have high numbers of cryptococcosis patients. This banana blossom-based agar is useful for the enrichment of isolates of the C. neoformans/C. gattii species complex from environmental and clinical materials. The banana blossom agar (BABA) with and without creatinine can differentiate between the melanin forming isolates of the C. neoformans/C. gattii species complex from other yeasts that do not form melanin., (© 2018 Blackwell Verlag GmbH.)

Melanin is an important virulence factor for several microorganisms, including Cryptococcus neoformans sensu lato and Cryptococcus gattii sensu lato, thus, the assessment of melanin production and its quantification may contribute to the understanding of microbial pathogenesis. The objective of this study was to standardise an alternative method for the production and indirect quantification of melanin in C. neoformans sensu lato and C. gattii sensu lato. Eight C. neoformans sensu lato and three C. gattii sensu lato, identified through URA5 methodology, Candida parapsilosis ATCC 22019 (negative control) and one Hortaea werneckii (positive control) were inoculated on minimal medium agar with or without L-DOPA, in duplicate, and incubated at 35°C, for 7 days. Pictures were taken from the third to the seventh day, under standardised conditions in a photographic chamber. Then, photographs were analysed using grayscale images. All Cryptococcus spp. strains produced melanin after growth on minimal medium agar containing L-DOPA. C. parapsilosis ATCC 22019 did not produce melanin on medium containing L-DOPA, while H. werneckii presented the strongest pigmentation. This new method allows the indirect analysis of melanin production through pixel quantification in grayscale images, enabling the study of substances that can modulate melanin production., (© 2017 Blackwell Verlag GmbH.)

Pathogenesis of cryptococcosis in the central nervous system (CNS) is a topic of ongoing research, including the mechanisms by which this fungus invades and infects the brain. Astrocytes, the most common CNS cells, play a fundamental role in the local immune response. Astrocytes might participate in cryptococcosis either as a host or by responding to fungal antigens. To determine the infectivity of Cryptococcus neoformans var. grubii and Cryptococcus gattii in a human astrocytoma cell line and the induction of major histocompatibility complex (MHC) molecules. A glioblastoma cell line was infected with C. neoformans var. grubii and C. gattii blastoconidia labelled with FUN-1 fluorescent stain. The percentage of infection and expression of HLA class I and II molecules were determined by flow cytometry. The interactions between the fungi and cells were observed by fluorescence microscopy. There was no difference between C. neoformans var. grubii and C. gattii in the percentage infection, but C. neoformans var. grubii induced higher expression of HLA class II than C. gattii. More blastoconidia were recovered from C. neoformans-infected cells than from C. gattii infected cells. Cryptococcus neoformans var. grubii may have different virulence mechanisms that allow its survival in human glia-derived cells., (© 2017 Blackwell Verlag GmbH.)

Ambisome (L-Amb) was used to treat nine cases of meningitis or menigoencephalitis by Cryptococcus neoformans and 28 cases of other deep fungal infections. A retrospective study on conventional amphotericin B (C-Amb) was performed as the control. A series of indices was observed including curative effect, fungal clearance rate, course of treatment, daily dose, cumulative dose and adverse effects. Nine cases of cryptococcal meningitis or menigoencephalitis treated with Ambisome were clinically cured with an effective rate of 100%, within a mean course of 50 days, which was shorter than that of C-Amb, by a mean cumulative dose of 1807.2 mg, which was not statistically significant in comparison with C-Amb. Fungal clearance rate on the second month of treatment was 89% with Ambisome, which was higher than that of C-Amb. Twenty-eight cases of other deep fungal infections treated with Ambisome were clinically cured with an effective rate of 92%, within a mean course of 19.3 days, by a mean cumulative dose of 907.5 mg, and fungal clearance rate on the second and third month was 75 and 92%, respectively. The adverse effects by Ambisome decreased evidently compared with those by C-Amb.

Attention is drawn to the observation that the type strain Cryptococcus laurentii CBS 139, producing killer toxins (mycocins) directed at Cr. neoformans var. gattii, causes a green colour effect (GCE) on Staib agar (Guizotia abyssinica creatinine agar) in combination with an intense assimilation of creatinine. Five (9.6%) out of 52 strains of Cr. laurentii of various origin, showed a GCE and intense creatinine assimilation. Further research must show if all Cr. laurentii strains, characterized by a GCE similar to that of the strain CBS 139, are also capable of producing killer toxins. For further ecological and epidemiological research on strains producing killer toxins directed against species of the genus Cryptococcus, it is proposed to use Staib agar as differential culture medium indicating both colour effects, i.e. the GCE and the brown colour effect (BCE).

Yeast isolates that share the same habitats as Cryptococcus neoformans var. neoformans serotype A in a restricted Mediterranean area were assayed in order to verify their killer activity against Cr. neoformans strains isolated from clinical and environmental sources. Many of the environmental yeast isolates expressed the killer phenomenon against the assayed strains of Cr. neoformans. Two species of Candida: Candida parapsilosis and Candida famata, and Pichia carsonii, were the most active killers at pH 4.6, 5.0 and 5.6 levels encountered in pigeon and canary guanos. Killer activity by C. parapsilosis is reported for the first time. The authors hypothesized that the killer phenomenon exerted by yeast species with heavy killer activity against Cr. neoformans would lend themselves for use as biological control agents against sensitive strains of Cr. neoformans when directly inoculated into the habitats of Cr. neoformans.

Two isolates of Cryptococcus neoformans serotype A and one isolate of serotype D from pigeon droppings were found to grow on canavanine-glycine-bromthymol blue (CGB) medium, when the Japanese isolates of Cr. neoformans were examined for their serotype and biochemical characteristics. The susceptibility to canavanine and the activity in assimilation of glycine were analysed on these three isolates. They were resistant to canavanine at the high concentration of 3.6 mmol l-1 and developed by assimilating the glycine even at a concentration of 7 mmol l-1. These isolates were proved to develop well on CGB medium, which contains 0.1 mmol l-1 of canavanine and 133 mmol l-1 of glycine. Three isolates of Cr. neoformans developed on CGB medium were also confirmed to be serotype A or D by the molecular analysis.

The effect of four media or media combinations on the growth and the in vitro susceptibility to amphotericin B, 5-fluorocytosine, fluconazole and itraconazole of 51 isolates of Cr. neoformans was studied. The drug concentration producing a 50% decrease in OD405 (IC50) was determined in microtiter format. The following MOPS buffered media or media combinations (antifungal drug diluent/inoculum diluent) were used: RPMI 1640 (RPMI)/RPMI, Yeast nitrogen base (YNB)/YNB, RPMI/YNB and RPMI/High resolution broth. Amphotericin B was further tested in M3 broth in the microdilution format and on RPMI agar with the E-test. Growth rates (OD405) were higher in YNB, RPMI/YNB, RPMI/HR and M3 as compared to RPMI alone. After 48 h of incubation, IC50 values obtained with RPMI alone and with RPMI/YNB correlated within two dilutions for 98, 95, 95 and 100% of test strains with amphotericin B, 5-fluorocytosine,fluconazole and itraconazole respectively. The E-test provided the widest range of IC values with amphotericin B. Growth of Cr. neoformans can be enhanced in susceptibility tests using microtiter plates with drugs diluted in RPMI by preparing the inoculum in YNB. However, when using spectrophotometric endpoint determinations, sufficient growth is obtainable with most isolates for test interpretation at 48 h in RPMI alone.

Many studies have evaluated the toxicity of mycotoxins to mammals, but there is little information on their action against fungal cells, even although mycotoxins are frequently active against fungi in nature. A crude extract of Aspergillus sulphureus was tested for its growth-inhibitory effect on Cryptococcus neoformans. The reduction in cell growth of Cr. neoformans caused by the extract was dose dependent. Using a liquid medium containing 2% A. sulphureus extract, the RNA content of Cryptococcus amounted to about 60% of that of non-treated cells. Capsule thickening, demonstrated biochemically and with cytological stains, occurred at doses that had minimal effect on cell growth and RNA content. Our results suggest that the virulence of Cr. neoformans may increase in cases of coenobiosis with A. sulphureus, which is theoretically possible in places where corn-fed pigeons are numerous.

Tenacity studies of Cryptococcus neoformans in bird droppings originated from different ornamental birds and chickens showed that there is less chance for this fungus species to survive in non-sterile or bacteria-free droppings of large parakeets and chickens in comparison with droppings of small parakeets. Survival rates of Cr. neoformans in buffer solutions with pH-values ranging from 8.5-9.5 allow to conclude that this species is not alkali-sensitive. Therefore, the increase of pH is not regarded responsible for the survival of Cr. neoformans in bird droppings. Possibly fungistatic substances present in droppings are involved.

A 5-year-old female Siamese cat was presented to the veterinary teaching hospital with a history of bronchopneumonia for 20 days. The cat had not responded to antibacterial chemotherapy and had developed a pronounced submandibular lymphadenopathy. Characteristically encapsulated yeast cells with narrow-necked buds were clearly seen in a fine-needle aspirate of the lymph node with an India ink preparation. Cryptococcus neoformans var. neoformans was identified. Susceptibility tests on the isolated strain were performed using antifungal tablets. The strain was sensitive to amphotericin B, fluconazole, itraconazole and ketoconazole and was resistant to 5-fluorocytosine. The cat was positive for feline immunodeficiency virus. Nevertheless, the cat was treated with ketoconazole for 3 months and apparently recovered. Three months later the animal was presented in a precomatose state. The owners refused to treat the animal and the cat was destroyed.

The interaction between yeasts and bacteria may have clinical implications in polymicrobial septicaemia. The in vitro effect of Pseudomonas aeruginosa on five pathogenic yeast species, Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata and Cryptococcus neoformans, was investigated. Yeast inhibition assays were performed in an aerobic blood culture medium, inoculated with yeast cells (inoculum 1-10 CFU ml-1) and bacterial cells (inoculum 10-10(7) CFU ml-1). Interactions between P. aeruginosa and yeasts were determined after incubation by enumeration of pure and mixed cultures. Growth of all isolates tested was completely or partially inhibited by P. aeruginosa in blood culture medium, the phenomenon depending on the yeast genus and bacterial inoculum. Suppression of fungal growth was also observed in bacterial culture filtrate. This in vitro antifungal activity may preclude yeast recovery from blood cultures in mixed infections.

Cryptococcus neoformans with a rare morphology of hand-mirror appearance was demonstrated by direct microscopic preparation of both cerebrospinal fluid (CSF) and sputum of a patient with AIDS. In addition, one to six blastoconidia were seen at the tip of a germ-tube like structure. Cr. neoformans was isolated in pure culture and the identification was confirmed by biochemical and serological tests as well as by animal pathogenicity.

The open questions of the persistence of Cryptococcus neoformans in the urogenital tract under antimycotic treatment can be examined under optimal mycological-diagnostic conditions only. The example of a case of cryptococcosis in an AIDS patient diagnosed and treated with itraconazole in the early secondary stage of cryptococcosis is used to discuss the problems of the persistence of Cr. neoformans involvement in the urogenital tract (prostate). Data from a ten-week follow-up study are presented and discussed. The observations made have shown that itraconazole is effective in all regions of the body, with the exception of the urogenital tract. In addition to clinical examinations, cases treated with itraconazole should be finally subjected to cultural examination of prostatic secretion and/or seminal fluid, to exclude the possible presence of a symptom-free involvement of the prostate by Cr. neoformans.

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.

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.

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.