Background: The incidence of invasive mycoses is increasing worldwide. PCR-RFLP was applied to the identification of 10 reference strains and 90 cultures of agents of invasive mycoses. In addition, the new approach was applied to detect fungal agents in 120 biological samples (blood, cerebrospinal fluid and bone marrow). PCR-RFLP results were compared with the ones obtained with conventional methods (culture, microscopy, and biochemical testing). Results: The assays carried out with the reference strains (Candida albicans, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida guilliermondii, Cryptococcus neoformans, Cryptococcus gattii and Histoplasma capsulatum), demonstrated that the RFLP profiles were correctly predicted by the in silico investigation and allowed unequivocal identification of all chosen reference strains. The PCR-RFLP also identified 90 cultures of agents of invasive mycoses correctly, 2.5 times faster than the conventional assays. Evaluating PCR-RFLP with biological samples it was observed that the PCR was found to be 100% accurate and the RFLP profiles allowed the identification of the etiological agents: C. neoformans (n = 3) and C. gattii (n = 1) in CSF samples, H. capsulatum (n = 1) in bone marrow and C. albicans (n = 2) in blood cultures. The detection and identification by PCR-RFLP were found to be between two to ten times faster than the conventional assays. Conclusion: The results showed that PCR-RFLP is a valuable tool for the identification of invasive mycoses that can be implemented in hospital laboratories, allowing for a high number of clinical analyses per day.

In recent years, many antimicrobial peptides have been found in the venoms of animals from different sources and have been intensively studied to elucidate their ability to inhibit the growth of potential pathogenic microorganisms. The aim of this study was to characterize and evaluate the in vitro antifungal activity of crude venom from two amazonian snakes: Bothrops atrox and Crotalus durissus ruruima. The molecular profile of representative proteins from the venom samples was obtained by reversed-phase high-performance liquid chromatography (RP-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Fungal inhibition was investigated by microdilution assays against two Candida albicans strains. Based on the chromatography and electrophoresis analyses, the venom from B. atrox and C. durissus ruruima were characterized. In addition, the venoms (400 µg/mL) were not able to cause significant inhibition (> 50%) of the growth of C. albicans KL-07, at only 9.09% (200 µg/mL) and 7.88% (400 µg/mL), respectively, and neither presented any influence on the growth of strain C. albicans ATCC 36232.

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Glucose oxidase (GOx) has several industrial applications. It is believed that there are several species of fungi that have the ability to produce this enzyme, most of which are unexplored. This work aimed to investigate the production of glucose oxidase (EC 1.1.3.4) by fungi isolated from soil samples of the Amazonian forest. Filamentous fungi were isolated from soil samples from the Adolpho Ducke Forest Reserve, located in Manaus, Amazonas. Strains were subjected to submerged bioprocessing to select for the best GOx producers. Those selected for the production of isolated enzymes were subjected to kinetic tests that evaluated production of the enzyme and consumption of the biomass substrate by the isolates. In addition, experiments to evaluate the optimal carbon, nitrogen and phosphorus sources as well as the influence of the bioprocess factors were carried out. Finally, GOx production was investigated in a semi-continuous system for 7 days. The most frequent isolates isolated from soil samples belonged to the genera Aspergillus, Penicillium and Trichoderma. Aspergillus niger LMM01 was the best GOx producer. Glucose, peptone and KH 2 PO 4 were demonstrated to be the optimal carbon, nitrogen and phosphorus sources, respectively. Multivariate experiments demonstrated that the parameters with the greatest effect on GOx production were pH and agitation. Stable expression results for GOx (7.74 U/ml) were obtained over 7 days in a semi-continuous process. In this context, the new Amazonian source of this enzyme (A. niger LMM01), and enzyme production in a semi-continuous process, demonstrates the importance of the present work.