Cryptococcus neoformans var. neoformanspresently includes isolates which have been determined by the immunologic reactivity of their capsular polysaccharides to be serotype A and those which have been determined to be serotype D. However, recent analyses of the URA5 sequences and DNA fingerprinting patterns suggest significant genetic differences between the two serotypes. Therefore, we propose to recognize these genotypic distinctions, as well as previously reported phenotypic differences, by restricting C. neoformans var. neoformans to isolates which are serotype D and describing a new variety, C. neoformans var. grubii, for serotype A isolates.

High-frequency reversible changes in colony morphology were observed in three strains of Cryptococcus neoformans. For one strain (SB4, serotype A), this process produced three colony types: smooth (S), wrinkled (W), and serrated (C). The frequency of switching between colony types varied for the individual colony transitions and was as high as 10 ؊3 . Mice infected with colony type W died faster than those infected with other colony types. The rat inf lammatory response to infection with colony types S, W, and C was C > S > W and ranged from intense granulomatous inf lammation with caseous necrosis for infection with type C to minimal inf lammation for infection with type W. Infection with the various colony types was associated with different antibody responses to cryptococcal proteins in rats. Analysis of cellular characteristics revealed differences between the three colony types. High-frequency changes in colony morphology were also observed in two additional strains of C. neoformans. For one strain (24067A, serotype D) the switching occurred between smooth and wrinkled colonies. For the other strain (J32A, serotype A), the switching occurred between mucoid and nonmucoid colonies. The findings indicate that C. neoformans undergoes phenotypic switching and that this process can affect virulence and host inf lammatory and immune responses. Phenotypic switching may play a role in the ability of this fungus to escape host defenses and establish chronic infections.

Cryptococcus neoformans is a major fungal pathogen for patients with debilitated immune systems. However, no information is available on the stability of virulence or of phenotypes associated with virulence for C. neoformans laboratory strains. A serendipitous observation in our laboratory that one isolate of C. neoformans ATCC 24067 (strain 52D) became attenuated after continuous in vitro culture prompted us to perform a comparative study of nine strain 24067 isolates obtained from six different research laboratories. Each isolate was characterized by DNA typing, virulence for mice, proteinase production, extracellular protein synthesis, melanin synthesis, carbon assimilation pattern, antifungal drug susceptibility, colony morphology, growth rate, agglutination titers, phagocytosis by murine macrophages, capsule size, and capsular polysaccharide structure. All isolates had similar DNA typing patterns consistent with their assignment to the same strain, although minor chromosome size polymorphisms were observed in the electrophoretic karyotypes of two isolates. Several isolates had major differences in phenotypes that may be associated with virulence, including growth rate, capsule size, proteinase production, and melanization. These findings imply that C. neoformans is able to undergo rapid changes in vitro, probably as a result of adaptation to laboratory conditions, and suggest the need for careful attention to storage and maintenance conditions. In summary, our results indicate thatC. neoformans (i) can become attenuated by in vitro culture and (ii) is capable of microevolution in vitro with the emergence of variants exhibiting new genotypic and phenotypic characteristics.

Little is known about the global molecular epidemiology of the human pathogenic fungus Cryptococcus neoformans. We studied 51 clinical and environmental (pigeon excreta) isolates from two cities in Brazil (Belo Horizonte and Rio de Janeiro) by analyzing their carbon assimilation patterns, electrophoretic karyotypes, restriction fragment length polymorphisms (RFLPs) with the C. neoformans repetitive element-1 (CNRE-1), and URA5 sequences. Results were compared to those previously obtained for isolates from New York City by the same DNA typing methods. Computer-assisted analysis of RFLPs and contour-clamped homogeneous electrophoresis (CHEF) patterns and URA5 sequences was performed to generate dendrograms. Some environmental and clinical isolates were found to be indistinguishable by CHEF, CNRE-1 RFLP, and URA5 sequence analyses. Similarly, some isolates from Rio de Janeiro and Belo Horizonte were indistinguishable by the three DNA typing techniques. Overall, Brazilian isolates appeared to be less heterogeneous by DNA analysis than isolates from other regions. Several Brazilian isolates were highly related to New York City isolates. Phylogenetic analysis of the sequences obtained for the Brazilian isolates and those obtained for New York City isolates was congruent with the dendrogram generated from the CNRE-1 RFLP data. In summary our results indicate (i) that the discriminatory power of the DNA typing method differs for Brazilian and New York City strains, with the order being CNRE-1 RFLP analysis > URA5 sequence analysis > CHEF analysis and CHEF analysis > URA5 sequence analysis > CNRE-1 RFLP analysis, respectively; (ii) that there are differences in local genetic diversity for Brazilian and New York City isolates; (iii) that there is additional evidence linking clinical isolates to those in pigeon excreta; and (iv) that some isolates from Brazil and New York City are closely related, consistent with the global dispersal of certain pathogenic strains. Cryptococcus neoformans is an encapsulated yeast which causes life-threatening infections in approximately 5 to 10% of patients with AIDS (10, 44). In Brazil, 4.3% of AIDS-related infections are caused by C. neoformans (26), but this number is likely to be an underestimate because cryptococcosis is not a reportable disease. In AIDS patients, meningoencephalitis is the most common clinical manifestation of C. neoformans infection, and it is usually incurable, despite antifungal therapy (44). AIDS patients with cryptococcosis who survive the initial presentation are treated with lifelong suppressive antifungal therapy to reduce the likelihood of recurrent infection. DNA typing analysis of initial and relapse isolates obtained from patients with recurrent cryptococcal meningoencephalitis consistently reveals persistence of the same strain, despite antifungal therapy in the majority of patients (4, 37). Genetic differences among C. neoformans strains have been detected by several typing methods, including restriction fragment length polymorphism (RFLP) analysis (11, 1...

Cryptococcus neoformans infections in patients with AIDS are often incurable, despite aggressive antifungal therapy. Combination regimens with additive or synergistic drugs could provide additional options for treating cryptococcal meningitis. We evaluated the efficacy of combination therapies using L-743,872, a pneumocandin antifungal drug, and amphotericin B or fluconazole against 18 strains of C. neoformans, including 11 C. neoformans var. neoformans, 3 C. neoformans var. gattii, and 4 fluconazole-resistant isolates. The combination of subinhibitory concentrations of L-743,872 with amphotericin B significantly enhanced amphotericin B activity against C. neoformans as measured by turbidity (antifungal susceptibility studies using the National Committee of Clinical and Laboratory Standards method), quantitative CFU, and tetrazolium salt reduction assays. Similarly, the addition of subinhibitory concentrations of L-743,872 to fluconazole enhanced fluconazole activity, but the effect was less dramatic than for the pneumocandin-amphotericin B combination. A marked synergism was observed in all combinations of amphotericin B and L-743, 872 (fractional inhibitory concentration index [FIC] of < or = 0.5). Fluconazole-resistant strains showed a susceptibility to amphotericin B and L-743,872 which was comparable to that of susceptible isolates. Combinations of pneumocandin with fluconazole revealed different activities for the various strains, including synergism (FIC < 1.0), additivity (FIC = 1.0), and autonomy (FIC between 1.0 and 2.0). Combination studies with fluconazole and L-743,872 showed additive and autonomous activities against fluconazole-resistant isolates. No antagonistic interactions (FIC < 2.0) were observed for any combination of L-743,872 with either amphotericin B or fluconazole. The results of this study suggest that L-743,872 can enhance the efficacy of fluconazole or amphotericin B in vitro and indicate a potential role for L-743,872 in combination therapy against C. neoformans.

Amphotericin B (AmB) and fluconazole (FLU) are the major antifungal drugs used in the treatment of cryptococcosis. Both drugs are believed to exert their antifungal effects through actions on cell membrane sterols. In this study we investigated whether AmB and FLU had other, more subtle effects on C. neoformans that could contribute to their therapeutic efficacy. C. neoformans cells were grown in media with subinhibitory concentrations of either AmB or FLU and analyzed for cellular charge, phagocytosis by macrophages with antibody and complement opsonins, appearance by scanning electron and light microscopies, and release of the capsular polysaccharide glucuronoxylomannan into the culture medium. Growth in the presence of either AmB or FLU resulted in major reductions in cellular charge, as measured by determination of the zeta potential. Phagocytosis studies demonstrated that exposure of C. neoformans to subinhibitory concentrations of AmB or FLU enhanced phagocytosis by macrophages. Scanning electron microscopy revealed that a large proportion of cells had an altered capsular appearance. Cells grown in medium with either AmB or FLU were smaller and released more glucuronoxylomannan into the culture medium than cells grown without antibiotics. The results suggest additional mechanisms of action for AmB and FLU that may be operative in body compartments where drug levels do not achieve the MICs. Furthermore, the results suggest mechanisms by which AmB and FLU can cooperate with humoral and cellular immune defense systems in controlling C. neoformansinfections.

Cryptococcus neoformans serotypes A and D are responsible for the overwhelming majority of infections in patients with AIDS. The genetic relationship between the serotypes is poorly understood, but there are significant differences in the epidemiology and clinical presentation of serotype A and D infections. We evaluated the genetic relationship between reference C. neoformansstrains belonging to serotypes A and D by analyzing theirURA5 sequences and restriction fragment length polymorphisms (RFLPs) with the C. neoformans repetitive element 1 (CNRE-1) probe. The results were compared to those previously obtained for isolates from Brazil and New York City by the same typing methods, and dendrograms were generated. Serotype A and D strains produced distinct RFLP patterns consistent with their separation into two major clusters in the dendrogram generated on the basis of RFLP data. Similarly, serotype A and D strains clustered independently on the basis of the nucleotide sequences of their URA5 genes. Pairwise comparisons revealed average numbers of nucleotide differences within serotypes A and D of 3.0 ± 1.7 and 7.2 ± 3.4, respectively (P < 0.0001), and between serotypes A and D of 41.9 ± 2.7. In summary, our results indicate phylogenetic differences between the two serotypes of C. neoformans var. neoformans and suggest that these serotypes could probably be considered different varieties of C. neoformans.

A total of 53 Cryptococcus neoformans strains, including clinical and environmental Brazilian isolates, were tested for their susceptibilities to amphotericin B, 5-flucytosine, ketoconazole, fluconazole, and itraconazole. The tests were performed according to the National Committee of Clinical Laboratory Standards recommendations (document M27-P). In general, there was a remarkable homogeneity of results for all strains, and comparable MICs were found for environmental and clinical isolates. This paper represents the first contribution in which susceptibility data for Brazilian C. neoformans isolates are provided.