Cryptococcus gattii VGII isolated from native forest and river in Northern Brazil.

BACKGROUND: Cryptococcosis is a global invasive mycosis associated with significant morbidity and mortality. In the northern region of Brazil, this disease is caused by Cryptococcus neoformans genotype VNI and Cryptococcus gattii genotype VGII. However, few environmental studies have been conducted in this large tropical area. AIMS: This study was performed to isolate, genotype, and determine the frequency of cryptococcal agents in environmental samples near Manaus, Amazonas, Brazil. METHODS: A total of 970 environmental samples (290 from soil, 290 from decaying plants, 5 from insects, 280 from the Negro river, and 105 from small streams within the city of Manaus) were collected and plated on Niger seed agar. In addition, 20 sub-cultures obtained from each positive sample were analyzed by PCR-RFLP (URA5) and PCR for genotyping and determination of mating type. RESULTS: Six samples were positive for isolates from the C. gattii species complex. Of those, three samples were from Adolpho Ducke Forest Reserve and three were from the Negro river. All isolates were C. gattii genotype VGII (mating type MATα). CONCLUSION: Genotype VGII proved to be the most important genotype found in the environmental samples. The genotype VGII has been described as one of the most virulent and less susceptible to antifungals and responsible for important outbreaks. This is the first study to demonstrate isolation of C. gattii (VGII) from the Negro river.

MLST reveals a clonal population structure for Cryptococcus neoformans molecular type VNI isolates from clinical sources in Amazonas, Northern-Brazil.

Cryptococcosis is considered endemic in Amazonas state, occurring more frequently in individuals with AIDS, who are predominantly infected by Cryptococcus neoformans molecular type VNI. Infections by Cryptococcus gattii VGII predominate in immunocompetent hosts from the American continent and are associated with outbreaks in North America, particularly the subtypes VGIIa and VGIIb, which are also present in the Brazilian Amazon region. Despite few environmental studies, several aspects of the molecular epidemiology of this disease in Amazonas remain unclear, including the limited use of multilocus sequence typing (MLST) to evaluate the genetic population structure of clinical isolates, mainly C. neoformans. Therefore, we used MLST to identify the sequence types of 38 clinical isolates of C. neoformans VNI and C. gattii VGII and used phylogenetic analysis to evaluate their genetic relationship to global isolates. Records of 30 patients were analyzed to describe the current scenario of cryptococcosis in the region and their associations with the different subtypes. Broth microdilution was also performed to determine the susceptibility profile to the antifungals amphotericin B, fluconazole and itraconazole. MLST identified that patients with HIV (n = 26) were exclusively affected by VNI strains with ST93, and among the VGII strains (n = 4), three STs (ST5, ST172 and the new ST445) were identified. An in-hospital lethality of 54% was observed in the HIV group, and there were no significant differences in the clinical aspects of the disease between the HIV and non-HIV groups of patients. In addition, all isolates were susceptible to the antifungals tested. Therefore, in Amazonas state, VNI isolates are a genetically monotypic group, with ST93 being highly important in HIV individuals.

Production of Biosurfactants by Soil Fungi Isolated from the Amazon Forest.

Biosurfactants are surface-active compounds that have sparked interest in recent years because of their environmental advantages over conventional surfactants. The aim of this study was to investigate the production of biosurfactants by soil fungi isolated from the Amazon forest. Fungi colonies were isolated from soil samples and screened for biosurfactant production in submerged fermentation. In addition, the influences of bioprocess factors (carbon source, nitrogen source, pH, and fermentation time) were investigated. Finally, the biosurfactant produced was semipurified and submitted to stability tests. One hundred fungal cultures were obtained from the soil samples, identified by micromorphology, and submitted to screening for biosurfactant production. Sixty-one strains produced biosurfactants. The strain Penicillium 8CC2 showed the highest emulsification index (54.2%). The optimized bioprocess conditions for biosurfactant production by Penicillium 8CC2 were as follows: soybean oil, 20 g/L; yeast extract, 30 g/L; pH 9; duration of 9 days. The semipurified biosurfactant showed stability after heating at 100°C for 60 min and after the addition of 30% NaCl (w/v). Tween 80 (0.2% w/v), a conventional surfactant, was used as the control.