Molecular typing of environmental Cryptococcus neoformans/C. gattii species complex isolates from Manaus, Amazonas, Brazil.

Cryptococcus neoformans and Cryptococcus gattii are the main causative agents of cryptococcosis, a systemic fungal disease that affects internal organs and skin, and which is acquired by inhalation of spores or encapsulated yeasts. It is currently known that the C. neoformans/C. gattii species complex has a worldwide distribution, however, some molecular types seem to prevail in certain regions. Few environmental studies of Cryptococcus have been conducted in the Brazilian Amazon. This is the first ecological study of the pathogenic fungi C. neoformans/C. gattii species complex in the urban area of Manaus, Amazonas, Brazil. A total of 506 samples from pigeon droppings (n = 191), captive bird droppings (n = 60) and tree hollows (n = 255) were collected from June 2012 to January 2014 at schools and public buildings, squares, pet shops, households, the zoo and the bus station. Samples were plated on niger seed agar (NSA) medium supplemented with chloramphenicol and incubated at 25°C for 5 days. Dark-brown colonies were isolated and tested for thermotolerance at 37°C, cycloheximide resistance and growth on canavanine-glycine-bromothymol blue agar. Molecular typing was done by PCR-RFLP. Susceptibility to the antifungal drugs amphotericin B, fluconazole, itraconazole and ketoconazole was tested using Etest(®) strips. In total, 13 positive samples were obtained: one tree hollow (C. gattiiVGII), nine pigeon droppings (C. neoformansVNI) and three captive bird droppings (C. neoformansVNI). The environmental cryptococcal isolates found in this study were of the same molecular types as those responsible for infections in Manaus.

Molecular characterisation of the causative agents of Cryptococcosis in patients of a tertiary healthcare facility in the state of Amazonas-Brazil.

As there are four major molecular types of Cryptococcus neoformans (VNI, VNII, VNIII and VNIV) and four molecular types of Cryptococcus gattii (VGI, VGII, VGIII and VGIV), it is important to identify the specific groups causing cryptococcosis in different geographical regions. Here, we investigated the molecular types of 57 cryptococcal isolates from patients in a tertiary care hospital in the state of Amazonas, Brazil, between 2006 and 2010. The isolates were characterised by PCR fingerprinting using the M13 minisatellite and confirmed by URA5-RFLP analysis, and the presence of specific genes from the mating type locus (MATα and MATa) of these species was analysed by PCR. Most of the patients were male (66.7%), between 16 and 30 years of age (51.7%), and HIV-positive (75.0%). Most isolates were collected from cerebrospinal fluid samples (71.7%). Most of the C. neoformans isolates (n=40) were characterised as members of the VNI molecular group (n=39), a unique isolate was characterised as VNII whereas all isolates of C. gattii (n=17) were members of the VGII molecular group. With regard to mating types, 55 isolates were type 'α', and only two were type 'a'. This study revealed the prevalence of the VNI molecular group and provides the first reported observation of the VNII molecular group in the northern region of Brazil.

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.

Selection and optimization of PCR-based methods for the detection of Histoplasma capsulatum var. capsulatum.

BACKGROUND: Current methods for the laboratory diagnosis of histoplasmosis are problematic in terms of their sensitivity, specificity and runtime. OBJECTIVES: Thus, in this study, we sought to select and optimize methods for the detection of Histoplasma capsulatum var. capsulatum by polymerase chain reaction (PCR). METHODS: Three DNA extraction methods and three PCR methods were evaluated. We optimised the concentration of the components of this PCR reaction and determined its sensitivity and specificity using blood samples to which H. capsulatum had been added. RESULTS: The DNA extraction method that yielded the highest-quality DNA used silica membranes (DNeasy Blood & Tissue Kit, Qiagen, Hilden, Germany), and the amplification method with the best detection capacity used a target gene encoding a 100-kDa protein. Our optimisation of the PCR conditions indicated that the reaction works over a significant range of component concentrations; in addition, it was able to detect H. capsulatum better than traditional culture techniques, with a detection limit of only 10 pg of DNA. CONCLUSIONS: In our experimental conditions, the PCR method selected in this work (instead of nested-PCR) is a tool sensitive enough for the diagnosis of histoplasmosis.