Cryptococcus gattii is an emergent human pathogen. Fluconazole is commonly used for treatment of cryptococcosis, but the emergence of less susceptible strains to this azole is a global problem and also the data regarding fluconazole-resistant cryptococcosis are scarce. We evaluate the influence of fluconazole on murine cryptococcosis and whether this azole alters the polysaccharide (PS) from cryptococcal cells. L27/01 strain of C. gattii was cultivated in high fluconazole concentrations and developed decreased drug susceptibility. This phenotype was named L27/01F, that was less virulent than L27/01 in mice. The physical, structural and electrophoretic properties of the PS capsule of L27/01F were altered by fluconazole. L27/01F presented lower antiphagocytic properties and reduced survival inside macrophages. The L27/01F did not affect the central nervous system, while the effect in brain caused by L27/01 strain began after only 12 hours. Mice infected with L27/01F presented lower production of the pro-inflammatory cytokines, with increased cellular recruitment in the lungs and severe pulmonary disease. The behavioral alterations were affected by L27/01, but no effects were detected after infection with L27/01F. Our results suggest that stress to fluconazole alters the capsule of C. gattii and influences the clinical manifestations of cryptococcosis.

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Acanthamoeba is a genus of free-living amoebas distributed worldwide. Few studies have explored the interactions between these protozoa and their infecting giant virus, Acanthamoeba polyphaga mimivirus (APMV). Here we show that, once the amoebal encystment is triggered, trophozoites become significantly resistant to APMV. Otherwise, upon infection, APMV is able to interfere with the expression of a serine proteinase related to amoebal encystment and the encystment can no longer be triggered. Acanthamoeba is a genus of free-living amoebas found in a variety of environments and distributed worldwide (1-3). The life cycle of the amoebas involves two cellular forms: one that is metabolically active, known as the trophozoite form, and a dormant form, called the cyst, that is also responsible for promoting resistance in adverse environments (3). In natural environments, members of Acanthamoeba spp. are hosts and reservoirs for many microorganisms, including pathogenic bacteria and yeasts (4-7). Recently, the study of members of the Acanthamoeba genus has gained increased attention since the description of them as natural hosts for viruses of the Mimiviridae family, which are among the largest and most complex viruses described to date (8). Despite the recent interest in both organisms, there are few studies focusing on the interactions between Acanthamoeba spp. and the infecting mimivirus (9, 10). Thus, the objective of this study was to analyze the interactions between Acanthamoeba spp. and Acanthamoeba polyphaga mimivirus (APMV) in response to encystment stimulation that may commonly happen in the natural environment.First, we performed a one-step growth curve analysis to compare the levels of replication of APMV at the amoebal trophozoite and cyst stages. Acanthamoeba castellanii cells (ATCC 30234) were seeded on 24-well microplates (Corning Incorporated, Corning, NY) in phosphate-amoeba saline (PAS) at 10 5 cells per well. The cells (cysts or trophozoites) were then infected with APMV at a multiplicity of infection (MOI) of 10, collected at different time points, and titrated as described before (8). All experiments were performed three times in triplicate. APMV was unable to infect cysts of A. castellanii, given that final viral titers remained close to the initial inoculum titer, revealing no viral replication (Fig. 1A). On the other hand, when trophozoites were infected, the viral progeny titer increased about 2.5 logs (500-fold) 24 h postinfection and evident cytopathic effect was observed (Fig. 1A). These results were confirmed by electron microscopy (12 h postinfection). APMV morphogenesis, including the presence of mature virions, was observed in trophozoites (Fig. 1B), while no virions could be seen inside the cysts (Fig. 1C).Next, we investigated if the stimulation of encystment affects APMV infection of amoebas. Amoebal trophozoites were infected with APMV after being incubated in Neff saline solution (Neff) to trigger encystment. Trophozoites were transferred to 24-well microplates, at 10 5 cells per we...

The genotype 677 CT was associated with increased risk of developing ALL (OR=1.6, 95% CI=1.1-2.7). Further epidemiological study is needed to unravel the role of complex multiple gene-environment interactions in leukemogenesis.

The Cryptococcus gattii species complex harbors the main etiological agents of cryptococcosis in immunocompetent patients. C. gattii molecular type VGII predominates in the north and northeastern regions of Brazil, leading to high morbidity and mortality rates. C. gattii VGII isolates have a strong clinical relevance and phenotypic variations. These phenotypic variations among C. gattii species complex isolates suggest that some strains are more virulent than others, but little information is available related to the pathogenic properties of those strains. In this study, we analyzed some virulence determinants of C. gattii VGII strains (CG01, CG02, and CG03) isolated from patients in the state of Piauí, Brazil. The C. gattii R265 VGIIa strain, which was isolated from the Vancouver outbreak, differed from C. gattii CG01, CG02 and CG03 isolates (also classified as VGII) when analyzed the capsular dimensions, melanin production, urease activity, as well as the glucuronoxylomannan (GXM) secretion. Those differences directly reflected in their virulence potential. In addition, CG02 displayed higher virulence compared to R265 (VGIIa) strain in a cryptococcal murine model of infection. Lastly, we examined the genotypic diversity of these strains through Multilocus Sequence Type (MLST) and one new subtype was described for the CG02 isolate. This study confirms the presence and the phenotypic and genotypic diversity of highly virulent strains in the Northeast region of Brazil.

Cryptococcosis is an invasive infection caused by yeast-like fungus of the genera Cryptococcus spp. The antifungal therapy for this disease provides some toxicity and the incidence of infections caused by resistant strains increased. Thus, we aimed to assess the consequences of fluconazole subdoses during the treatment of cryptococcosis in the murine inflammatory response and in the virulence factors of Cryptococcus gattii. Mice infected with Cryptococcus gattii were treated with subdoses of fluconazole. We determined the behavior of mice and type 1 interferon expression during the treatment; we also studied the virulence factors and susceptibility to fluconazole for the colonies recovered from the animals. A subdose of fluconazole prolonged the survival of mice, but the morbidity of cryptococcosis was higher in treated animals. These data were linked to the increase in: (i) fluconazole minimum inhibitory concentration, (ii) capsule size and (iii) melanization of C. gattii, which probably led to the increased expression of type I interferons in the brains of mice but not in the lungs. In conclusion, a subdose of fluconazole altered fungal virulence factors and susceptibility to this azole, leading to an altered inflammatory host response and increased morbidity.