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. © 2010 Revista Iberoamericana de Micología

Production of Trametes versicolor laccase by solid state fermentation using a fixed-bed bioreactor

The aim of this work was to investigate the production of Trametes versicolor laccase by SSF (solid state fermentation) using a fixed-bed bioreactor. The SSF optimization assays were carried out in Erlenmeyer flasks (250 mL) containing 12.5 g of dried wheat bran. The medium moisture (50, 60, 70, 80 and 90% w/w), inoculum (78, 240, 400, 560 and 720 mycelium plugs/kg D.M.- dried matter) and the contents of glucose (0, 10, 30, 50 and 70 g/kg D.M.), yeast extract (24 and 48 g/kg D.M.) and CuSO4 (2.4 and 4.8 g/kg D.M.) were evaluated for laccase production. After this optimization an experiment in a fixed-bed bioreactor (250 mL) with 25g D.M. of wheat bran was accomplished, and this reactor was aerated (1.5 L/min) with humidified air. The laccase activity was quantified using ABTS as substrate. The results of the experiments demonstrated that the moisture of 70% w/w and the inoculum of 240 mycelium plus/kg D.M. were optimized conditions producing, on the 20th day of the experiment, a laccase activity of 19,341 UI/kg D.M. Glucose, yeast extract and CuSO4, in the experimental conditions, decreased the laccase production. The experiment carried out in the fluidized bed reactor, with the optimized conditions, resulted on the 16th day of experiment in a laccase activity of 18,917 UI/kg D.M. These experiments demonstrated that it was possible to produce high laccase levels using the wheat bran as substrate without any supplementation and that the fluidized bed reactor showed to be adequate for the enzyme production, as well as the Erlenmeyer flasks

Characterization of clinical isolates of the Cryptococcus neoformans-Cryptococcus gattii species complex from the Amazonas State in Brazil

Background: The differentiation and classification of pathogenic Cryptococcus species provides useful data for epidemiological studies and for the clinical diagnosis and treatment of patients. Aims: The aim of this study was to characterise 40 clinical Cryptococcus isolates obtained from patients at the Tropical Medicine Foundation of Amazonas (FMTAM) from 2006 to 2008. Methods: It was used phenotypic (i.e., enzyme production and antifungal resistance) and molecular biological (URA5-RFLP) experiments. Results: Patients with HIV/AIDS were most affected with cryptococcosis. Thirty-one (75.5%) of the clinical isolates were classified as Cryptococcus neoformans and 9 (22.5%) as Cryptococcus gattii. High amounts of protease and phospholipase enzymes were produced by most of the isolates. Using the disk diffusion test (CLSI M44-A), 81, 35 and 100% of the C. neoformans isolates were characterized as susceptible to fluconazole, itraconazole and amphotericin B, respectively, whereas 78, 56 and 100% of the C. gattii isolates were susceptible to these antimicrobial agents. The average of Minimal Inhibitory Concentration (MIC) for C. neoformans and C. gattii isolates was 0.26 and 0.58 μg/mL, respectively. The 9 isolates of C. gattii had a fingerprint pattern comparable with the VGII molecular type, while all 31 isolates of C. neoformans presented with a pattern consistent with the VNI type. Conclusions: This study confirms the importance of HIV/AIDS for the cryptococcosis epidemiology, the susceptibility of the isolates to amphotericin B and the high prevalence of the molecular genotypes VNI and VGII in the north of Brazil. © 2010 Revista Iberoamericana de Micología

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 30years 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. © 2012 Blackwell Verlag GmbH

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. © 2019, Sociedade Brasileira de Microbiologia