Activity of antifungal agents alone and in combination against echinocandin-susceptible and -resistant Candida parapsilosis strains / Actividad de antifúngicos solos y en combinación frente a cepas de Candida parapsilosis resistentes y sensibles a equinocandinas

Background: Candida parapsilosis may acquire resistance to echinocandins, a fact that prompts the search for new therapeutic options. Aims: The present study aimed to evaluate the in vitro activity of antifungal agents, alone and in combination, against four groups of C. parapsilosis strains: (1) echinocandin-susceptible (ES) clinical isolates (MIC ≤ 2 μg/ml), (2) anidulafungin-resistant strains (MIC ≥ 8 μg/ml), (3) caspofungin-resistant strains (MIC ≥ 8 μg/ml), and (4) micafungin-resistant strains (MIC ≥ 8 μg/ml). Methods: Antifungal interactions were evaluated by a checkerboard micro-dilution method. The determination of the MIC to each drug for every isolate according to the Clinical and Laboratory Standards Institute documents M27 (2017) and M60 (2017) was also done. Results: The echinocandins-resistant (ER) strains showed higher MICs to the tested antifungals than the ES strains, except for amphotericin B, for which the ER groups remained susceptible. Conclusions: Most combinations showed indifferent interactions. The use of monotherapy still seems to be the best option. As resistance to echinocandins is an emergent phenomenon, further studies are required to provide clearer information on the susceptibility differences between strains to these antifungal agents

Antecedentes: Candida parapsilosis puede volverse resistente a las equinocandinas, lo que requiere la búsqueda de nuevas opciones terapéuticas. Objetivos: El presente estudio tenía como objetivo evaluar la actividad in vitro de algunos antifúngicos, solos y en combinación, frente a cuatro grupos de cepas de C. parapsilosis: 1) cepas clínicas sensibles a las equinocandinas (SE) (CIM ≤ 2 μg/ml), 2) cepas resistentes a la anidulafungina (CIM ≥ 8 μg/ml), 3) cepas resistentes a la caspofungina (CIM ≥ 8 μg/ml) y 4) cepas resistentes a la micafungina (CIM ≥ 8 μg/ml). Métodos: Se evaluaron las interacciones de los antifúngicos con el método de microdilución en damero. También se determinó el valor de la CIM de cada cepa en cada antifúngico de acuerdo con los documentos Clinical and Laboratory Standards Institute M27 (2017) y M60 (2017). Resultados: Las cepas resistentes a las equinocandinas (RE) presentaron los valores de CIM más altos a los antifúngicos probados que las cepas SE a excepción de la anfotericina B, frente a la cual los grupos RE se mantuvieron sensibles. Conclusiones: La mayoría de las combinaciones evidenciaron interacciones indiferentes. El uso de monoterapias aún parece la mejor opción. Puesto que la resistencia a las equinocandinas es un fenómeno emergente, se requieren estudios adicionales con el fin de proporcionar una información más clara acerca de las diferencias de sensibilidad de diferentes cepas a estos antifúngicos

In vitro activity of diphenyl diselenide and ebselen alone and in combination with antifungal agents against Trichosporon asahii.

This study evaluated the in vitro susceptibility of Trichosporon asahii strains to diphenyl diselenide (DPDS) and ebselen (EBS) alone and in combination with amphotericin B (AMB), fluconazole (FCZ), itraconazole (ITZ) and caspofungin (CAS) using the microdilution method. EBS showed in vitro activity against T asahii strains with minimal inhibitory concentration (MIC) ranged from 0.25 to 8.0 µg/mL. For DPDS, the MIC ranged from 8.0 to 64 µg/mL. The combinations demonstrating the greatest synergism rate against fluconazole-resistant T asahii strains were the following: CAS + DPDS (96.67%), AMB + DPDS (93.33%), FCZ + DPDS (86.67%) and ITZ + DPDS (83.33%). The combinations AMB + DPDS and AMB + EBS exhibited the highest synergism rate against the fluconazole-susceptible (FS) T asahii strains (90%). Antagonism was observed in the following combinations: FCZ + EBS (80%) and FCZ + DPDS (13.33%) against the FS strains, and ITZ + EBS (20%) against the FR strains. Our findings suggest that the antimicrobial activity of DPDS and EBS against T. asahii and its use as an adjuvant therapy with antifungal agents warrant in vivo experimental investigation.

Evaluation of the efficacy of a posaconazole and anidulafungin combination in a murine model of pulmonary aspergillosis due to infection with Aspergillus fumigatus.

Posaconazole (PSC) in combination with anidulafungin (AFG) was evaluated in a murine model of pulmonary aspergillosis. Immunosuppressed animals were infected via the nasal cavity with 2 different A. fumigatus strains. The animals received PSC (oral, 20mg/kg per day) and/or AFG (i.p., 10mg/kg per day) for 7days. On Day 8, the mice were euthanized and fungal burdens were determined from the lungs. Survival curves were constructed for mortality analysis. Compared to untreated groups, groups singly treated with PSC or AFG showed a reduced fungal burden in the lungs (P=0.0001-0.006) and prevention of mortality (66.66-83.33% of survival). Combination treatment with PSC and AFG significantly reduced the fungal burden (or sterilized the lungs) compared to the findings in the untreated and monotherapy groups and improved the survival rate to 100%. The PSC and AFG combination therapy was highly effective and should be evaluated in larger-scale experiments.

In vitro antifungal susceptibility of clinical and environmental isolates of Aspergillus fumigatus and Aspergillus flavus in Brazil.

The in vitro susceptibility of 105 clinical and environmental strains of Aspergillus fumigatus and Aspergillus flavus to antifungal drugs, such as amphotericin B, azoles, and echinocandins was evaluated by the broth microdilution method proposed by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Following the EUCAST-proposed breakpoints, 20% and 25% of the clinical and environmental isolates of A. fumigatus, respectively, were found to be resistant to itraconazole (Minimal Inhibitory Concentration, MIC>2.0mg/L). Voriconazole showed good activity against A. fumigatus and A. flavus strains, except for one clinical strain of A. fumigatus whose MIC was 4.0mg/L. Posaconazole (≤0.25mg/L) also showed appreciable activity against both species of Aspergillus, except for six A. fumigatus strains with relatively higher MICs (0.5mg/L). The MICs for Amphotericin B ranged from 0.06 to 1.0mg/L for A. fumigatus, but were much higher (0.5-8.0mg/L) for A. flavus. Among the echinocandins, caspofungin showed a geometric mean of 0.078 and 0.113 against the clinical and environmental strains of A. flavus, respectively, but had elevated minimal effective concentrations (MECs) for seven of the A. fumigatus strains. Anidulafungin and micafungin exhibited considerable activity against both A. fumigatus and A. flavus isolates, except for one environmental isolate of A. fumigatus that showed an MEC of 1mg/L to micafungin. Our study proposes that a detailed investigation of the antifungal susceptibility of the genus Aspergillus from different regions of Brazil is necessary for establishing a response profile against the different classes of antifungal agents used in the treatment of aspergillosis.

Antifungal activities of diphenyl diselenide and ebselen against echinocandin-susceptible and -resistant strains of Candida parapsilosis.

We evaluated the in vitro antifungal activity of diphenyl diselenide and ebselen against echinocandin-susceptible and -resistant strains of Candida parapsilosis using the broth microdilution method. Diphenyl diselenide (MIC range =1-8 µg/mL) and ebselen (MIC range =0.25-4 µg/mL) showed in vitro activity against echinocandin-susceptible isolates. However, ebselen also showed the highest antifungal activity against echinocandin-resistant strains (MIC range =0.06-4 µg/mL). This study demonstrated that the antifungal potential of diphenyl diselenide and ebselen deserves further investigation using in vivo experimental protocols.

In vitro synergistic combinations of pentamidine, polymyxin B, tigecycline and tobramycin with antifungal agents against Fusarium spp.

The genus Fusarium is characterized by hyaline filamentous fungi that cause infections predominantly in immunocompromised patients. The remarkable primary resistance to antifungal agents of this genus requires a search for new therapeutic possibilities. This study assessed the in vitro susceptibility of 25 clinical isolates of Fusarium against antifungal agents (amphotericin B, caspofungin, itraconazole and voriconazole) and antimicrobials (pentamidine, polymyxin B, tigecycline and tobramycin) according to the broth microdilution method (M38-A2). The interactions between antifungal and antimicrobial agents were evaluated by the microdilution checkerboard method. Pentamidine and polymyxin B showed MIC values ≥4 µg ml-1 against Fusarium spp. The highest rates of synergism were observed when amphotericin B or voriconazole was combined with tobramycin (80 % and 76 %, respectively), polymyxin B (76 % and 64 %) and pentamidine (72 % and 68 %). The most significant combinations deserve in vivo evaluations in order to verify their potential in the treatment of fusariosis.

Interference of melanin in the susceptibility profile of Sporothrix species to amphotericin B / Interferencia de la melanina en la sensibilidad de especies del género Sporothrix a la anfotericina B

Background. The presence of melanin in the fungal cell is a major virulence factor of the genus Sporothrix since it protects the fungal cells against the defense systems. Aims. The present study aimed to investigate the interference of melanin in the susceptibility of Sporothrix brasiliensis and Sporothrix schenckii sensu stricto to amphotericin B and itraconazole, drugs recommended as therapy for disseminated and subcutaneous sporotrichosis, respectively. Methods. Yeast cells were cultivated in minimal medium with or without l-DOPA in order to induce the production of melanin. Microdilution and killing assay methods were used to determine the antifungal activity against yeast cells with different mounts of melanin. Results. The killing assay showed that melanization protected isolates within the S. schenckii complex from amphotericin B, particularly in the lower concentrations tested. Conclusions. Studies combining amphotericin B and inhibitors of melanin are required in order to avoid this effect (AU)

Antecedentes. La presencia de melanina en la célula fúngica es un importante factor de virulencia del género Sporothrix en el proceso de infección, pues protege al hongo de la acción del sistema inmunitario. Objetivos. El objetivo del presente estudio fue investigar la interferencia de la melanina en la sensibilidad de Sporothrix brasiliensis y Sporothrix schenckii sensu stricto a la anfotericina B y el itraconazol, antifúngicos recomendados como terapia para la esporotricosis diseminada y la subcutánea, respectivamente. Métodos. Se cultivaron células en la fase levaduriforme en medio mínimo con o sin l-DOPA con el fin de inducir la producción de melanina. La valoración de la actividad antifúngica sobre células de Sporothrix con diferente contenido en melanina se realizó mediante microdilución en caldo y curvas de mortalidad. Resultados. El ensayo mostró que la melanización protege de la acción de la anfotericina B a las especies del complejo S. schenckii, particularmente en las concentraciones más bajas ensayadas. Conclusiones. Se requieren estudios de combinación entre diferentes clases de antifúngicos o de anfotericina B con inhibidores de la melanina, con el fin de disminuir o evitar este efecto (AU)

Lipid peroxidation in cats experimentally infected with Trypanosoma evansi.

The objective of this study was to evaluate the lipid peroxidation and the susceptibility of erythrocytes to in vitro peroxidation as indicators of oxidative damage in erythrocytes and their roles in the pathogenesis of anemia during experimental Trypanosoma evansi infection in cats. Animals were divided into two groups: control and infected with T. evansi. Seven cats were infected with 10(8) trypomastigotes each, and parasitemia was estimated daily for 49 days by microscopic examination of smears. Hematological and biochemical parameters were evaluated for monitoring of the disease. Plasma lipid peroxidation (Thiobarbituric Acid Reactive Substances (TBARS)) and the susceptibility of erythrocytes to in vitro peroxidation were evaluated. Blood samples for analysis were collected at days 21 and 49 post-inoculation. TBARS level, indicated by MDA concentration, was higher in the infected group than in the control group in both analyzed periods, as well as the in vitro erythrocyte peroxidation (P < 0.001). The infected cats had variable degrees of regenerative anemia, which could be explained by the damage in erythrocyte membrane caused by lipid peroxidation.

Differentiation of Candida dubliniensis from Candida albicans on rosemary extract agar and oregano extract agar.

Candida dubliniensis is a recently described pathogenic species which shares many phenotypic features with Candida albicans and therefore, may be misidentified in microbiological laboratories. Because molecular methods can be onerous and unfeasible in routine mycological laboratories with restricted budgets such as those in developing countries, phenotypic techniques have been encouraged in the development of differential media for the presumptive identification of these species. We examined the colony morphology and chlamydospore production of 30 C. dubliniensis isolates and 100 C. albicans isolates on two new proposed media: rosemary (Rosmarinus officinalis) extract agar (REA) and oregano (Origanum vulgare) extract agar (OEA). These substrates are traditionally used as spices and medicinal herbs. In both of these media, all C. dubliniensis isolates (100%) showed rough colonies with peripheral hyphal fringes and abundant chlamydospores after 24 to 48 hr of incubation at 25 degrees C. In contrast, under the same conditions, all isolates of C. albicans (100%) showed smooth colonies without hyphal fringes or chlamydospores. In conclusion, REA and OEA offer a simple, rapid, and inexpensive screening media for the differentiation of C. albicans and C. dubliniensis.