Synthesis and antifungal activity in vitro of isoniazid derivatives against histoplasma capsulatum var. capsulatum.

Histoplasmosis is a severe infection that affects millions of patients worldwide and is endemic in the Americas. Amphotericin B (AMB) and itraconazole are highly effective for the treatment of severe and milder forms of the disease, but AMB is toxic, and the bioavailability of itraconazole is erratic. Therefore, it is important to investigate new classes of drugs for histoplasmosis treatment. In this study, a series of nine isoniazid hydrazone derivatives were synthesized and evaluated for their antifungal activities in vitro against the dimorphic fungus Histoplasma capsulatum var. capsulatum. The drugs were tested by microdilution in accordance with CLSI guidelines. The compound N'-(1-phenylethylidene)isonicotinohydrazide had the lowest MIC range of all the compounds for the yeast and filamentous forms of H. capsulatum. The in vitro synergy of this compound with AMB against the planktonic and biofilm forms of H. capsulatum cells was assessed by the checkerboard method. The effects of this hydrazone on cellular ergosterol content and membrane integrity were also investigated. The study showed that the compound alone is able to reduce the ergosterol content of planktonic cells and can alter the membrane permeability of the fungus. Furthermore, the compound alone or in combination with AMB showed inhibitory effects against mature biofilms of H. capsulatum. N'-(1-Phenylethylidene)isonicotinohydrazide alone or combined with AMB might be of interest in the management of histoplasmosis.

Antigens of Coccidioides posadasii as an important tool for the immunodiagnosis of coccidioidomycosis.

Serologic diagnosis has been presented as a safe alternative for coccidioidomycosis. However, commercial kits based on coccidioidal antibodies available in the USA are considered too expensive for laboratories outside that country. In this study, we describe the preparation of antigens for detection of human coccidioidal antibodies by the immunodiffusion test (ID) and enzyme immunoassay (EIA). Antigens were tested against serum samples from patients with coccidioidomycosis, histoplasmosis and paracoccidioidomycosis, as well as healthy individuals. The highest reactivity in the ID tests was seen in the F0-90 antigen. In the EIAs, the best results were obtained with the F60-90 antigen. None of the serum samples from healthy individuals were recognized by any of the antigen extracts tested by ID or EIA. In conclusion, the F0-90 and F60-90 antigens have the potential to be commercially employed in presumptive diagnosis of coccidioidomycosis by ID or EIA, respectively. The tests could improve serological diagnosis of coccidioidomycosis in South America.

Trichosporon inkin biofilms produce extracellular proteases and exhibit resistance to antifungals.

The aim of this study was to determine experimental conditions for in vitro biofilm formation of clinical isolates of Trichosporon inkin, an important opportunistic pathogen in immunocompromised patients. Biofilms were formed in microtitre plates in three different media (RPMI, Sabouraud and CLED), with inocula of 104, 105 or 106 cells ml- 1, at pH 5.5 and 7.0, and at 35 and 28 °C, under static and shaking conditions for 72 h. Growth kinetics of biofilms were evaluated at 6, 24, 48 and 72 h. Biofilm milieu analysis were assessed by counting viable cells and quantification of nucleic acids released into biofilm supernatants. Biofilms were also analysed for proteolytic activity and antifungal resistance against amphotericin B, caspofungin, fluconazole, itraconazole and voriconazole. Finally, ultrastructural characterization of biofilms formed in microtitre plates and catheter disks was performed by scanning electron microscopy. Greater biofilm formation was observed with a starter inoculum of 106 cells ml- 1, at pH 7.0 at 35 °C and 80 r.p.m., in both RPMI and Sabouraud media. Growth kinetics showed an increase in both viable cells and biomass with increasing incubation time, with maximum production at 48 h. Biofilms were able to disperse viable cells and nucleic acids into the supernatant throughout the developmental cycle. T. inkin biofilms produced more protease than planktonic cells and showed high tolerance to amphotericin B, caspofungin and azole derivatives. Mature biofilms were formed by different morphotypes, such as blastoconidia, arthroconidia and hyphae, in a strain-specific manner. The present article details the multicellular lifestyle of T. inkin and provides perspectives for further research.