Francisca Jakelyne de Farias Marques

Candida tropicalis isolates obtained from veterinary sources show resistance to azoles and produce virulence factors

Candida tropicalis has been associated with invasive candidiasis, being the first or second most common non-Candida albicans Candida species isolated in humans with candidemia and candiduria, as well as being frequently isolated from healthy animals. This study aimed to characterize C. tropicalis isolates (n = 64) obtained from several animal species regarding antifungal susceptibility and production of virulence factors. The isolates were obtained from the microbiota of healthy animals (goats, n = 25; sheep, n = 6; psittacines, n = 14; rheas, n = 6; horses, n = 2; sirenians, n = 5; shrimp, n = 1), as well as from aquatic mammals found dead in the environment (cetaceans, n = 5). The isolates were subjected to in vitro susceptibility testing by broth microdilution according to the CLSI M27-A3 protocol against amphotericin B, caspofungin, itraconazole, and fluconazole. We also evaluated the virulence attributes, such as proteases and phospholipases, as well as biofilm formation. Resistance to itraconazole (n = 29) and fluconazole (n = 30) was detected among isolates from every source; resistance to both azoles was detected in 24 isolates, but none of them were resistant to amphotericin B and caspofungin. Protease production was detected in the majority of the isolates (n = 59), but phospholipase was produced by only a few of them (n = 6). The isolates showed different patterns in biofilm production, being considered strong producers (n = 41), moderate producers (n = 11), weak producers (n = 9) or non-producers (n = 3). In summary, C. tropicalis isolated from animals showed high rate of resistance to azoles, expressed virulence factors and therefore may represent a potential threat to human and animal health.

Coccidioides posadasii infection in bats, Brazil.

To analyze the eco-epidemiologic aspects of Histoplasma capsulatum in Brazil, we tested 83 bats for this fungus. Although H. capsulatum was not isolated, Coccidioides posadasii was recovered from Carollia perspicillata bat lungs. Immunologic studies detected coccidioidal antibodies and antigens in Glossophaga soricina and Desmodus rotundus bats.

In vitro inhibitory effect of miltefosine against strains of Histoplasma capsulatum var. capsulatum and Sporothrix spp.

Miltefosine (MIL), originally developed for use in cancer chemotherapy, has been shown to have important antifungal activity against several pathogenic fungi. Our aim in this study was to determine the in vitro activity of MIL against the dimorphic fungi Histoplasma capsulatum and Sporothrix spp. This was done using the broth microdilution method. MIL had an in vitro inhibitory effect against all strains of H. capsulatum var. capsulatum and Sporothrix spp. analyzed. The minimal inhibitory concentrations (MIC) varied from 0.25 μg/ml to 2 μg/ml for H. capsulatum var. capsulatum in the filamentous phase and from 0.125 μg/ml to 1 μg/ml in the yeast phase. The MIC interval for Sporothrix spp. in the filamentous phase was 0.25-2 μg/ml. The minimal fungicidal concentrations (MFCs) were ≤4 μg/ml for isolates of both analyzed species. This study demonstrates that MIL has an antifungal effect in vitro against two potentially pathogenic fungi and that more studies should be performed in order to evaluate its applicability in vivo.

Synthesis and Antifungal Activity In Vitro of Isoniazid Derivatives against Histoplasma capsulatum var. capsulatum

ABSTRACT 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.

Terpinen-4-ol, tyrosol, and β-lapachone as potential antifungals against dimorphic fungi

This study aimed to evaluate the in vitro antifungal activity of terpinen-4-ol, tyrosol, and β-lapachone against strains of Coccidioides posadasii in filamentous phase (n = 22) and Histoplasma capsulatum in both filamentous (n = 40) and yeast phases (n = 13), using the broth dilution methods as described by the Clinical and Laboratory Standards Institute, to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of these compounds. The mechanisms of action of these compounds were also investigated by analyzing their effect on cell membrane permeability and ergosterol synthesis. The MIC and MFCf these compounds against C. posadasii, mycelial H. capsulatum, and yeast-like H. capsulatum, were in the following ranges: 350–5720 μg/mL, 20–2860 μg/mL, and 40–1420 μg/mL, respectively for terpinen-4-ol; 250–4000 μg/mL, 30–2000 μg/mL, and 10–1000 μg/mL, respectively, for tyrosol; and 0.48–7.8 μg/mL, 0.25–16 μg/mL, and 0.125–4 μg/mL, respectively for β-lapachone. These compounds showed a decrease in MIC when the samples were subjected to osmotic stress, suggesting that the compounds acted on the fungal membrane. All the compounds were able to reduce the ergosterol content of the fungal strains. Finally, tyrosol was able to cause a leakage of intracellular molecules.

Synergistic effect of antituberculosis drugs and azoles in vitro against Histoplasma capsulatum var. capsulatum

ABSTRACT This study evaluated in vitro interactions of antituberculosis drugs and triazoles against Histoplasma capsulatum. Nine drug combinations, each including an antituberculosis drug (isoniazid, pyrazinamide, or ethambutol) plus a triazole (itraconazole, fluconazole, or voriconazole), were tested against both growth forms of H. capsulatum. Stronger synergistic interactions were seen in isoniazid or pyrazinamide plus triazoles for the mold form and ethambutol plus voriconazole for the yeast-like form. Further studies should evaluate these combinations in vivo.

Cotrimoxazole enhances the in vitro susceptibility of Coccidioides posadasii to antifungals

The aim of the present study was to evaluate the effect of cotrimoxazole on the in vitro susceptibility of Coccidioides posadasii strains to antifungals. A total of 18 strains of C. posadasii isolated in Brazil were evaluated in this study. The assays were performed in accordance with the Clinical and Laboratory Standards Institute guidelines and the combinations were tested using the checkerboard method. The minimum inhibitory concentrations were reduced by 11, 2.4, 4.3 and 3.5 times for amphotericin B, itraconazole, fluconazole and voriconazole, respectively. Moreover, it was seen that cotrimoxazole itself inhibited C. posadasii strains in vitro. The impairment of folic acid synthesis may be a potential antifungal target for C. posadasii.

In vitro inhibitory activity of terpenic derivatives against clinical and environmental strains of the Sporothrix schenkii complex

Sporotrichosis is a subacute or chronic subcutaneous infection, caused by the fungus Sporothrix schenkii complex, occurring in human and animal tissues. Potassium iodide and itraconazole have been used as effective therapy for first-choice treatment, while amphotericin B may be indicated for disseminated infection. However, the adverse effects of potassium iodide and amphotericin B or the long duration of therapy with itraconazole often weigh against their use, leading to the search for alternatives for the treatment of severe infections. Terpinen-4-ol and farnesol are components of essential oils present in many plant species and have been described to have antifungal activity against microorganisms. In this study, 40 strains of Sporothrix spp. were tested for the susceptibility to terpinen-4-ol and farnesol. Changes in cytoplasmic membrane permeability were also investigated. Terpenes inhibited all Sporothrix strains with MIC values ranging from 87.9 to 1,429.8 μg/ml for terpinen-4-ol and from 0.003 to 0.222 μg/ml for farnesol. The MFC values ranged from 177.8 to 5,722.6 μg/ml and from 0.027 to 0.88 μg/ml, respectively, for terpinen-4-ol and farnesol. Farnesol was the most active compound for the Sporothrix strains. Significant loss of 260 and 280 nm-absorbing material did not occur after treatment with concentrations equivalent to the MIC and sub-MIC of the tested terpenes, when compared to corresponding untreated samples. The failure of terpenes to lyse Sporothrix cells suggests that their primary mechanism of action is not by causing irreversible cell membrane damage. Thus, new studies are needed to better understand the mechanisms involved in the antifungal activity.

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.