Combined antifungal therapy against systemic murine infections by rare Cryptococcus species.

Cryptococcus albidus and Cryptococcus laurentii are uncommon species of this genus that in recent decades have increasingly caused opportunistic infections in humans, mainly in immunocompromised patients; the best therapy for such infection being unknown. Using a murine model of systemic infection by these fungi, we have evaluated the efficacy of amphotericin B (AMB) at 0.8 mg/kg, administered intravenously, fluconazole (FLC) or voriconazole (VRC), both administered orally, at 25 mg/kg and the combination of AMB plus VRC against three C. albidus and two C. laurentii strains. All the treatments significantly reduced the fungal burden in all the organs studied. The combination showed a synergistic effect in the reduction in fungal load, working better than both monotherapies. The histopathological study confirmed the efficacy of the treatments.

Therapies against murine Candida guilliermondii infection, relationship between in vitro antifungal pharmacodynamics and outcome / Terapia antifúngica frente a la infección por Candida guilliermondii en ratones, correlación entre los parámetros farmacodinámicos in vitro y la eficacia in vivo

Background. Candida guilliermondii has been recognized as an emerging pathogen showing a decreased susceptibility to fluconazole and considerably high echinocandin MICs. Aims. Evaluate the in vitro activity of anidulafungin in comparison to amphotericin B and fluconazole against different isolates of C. guilliermondii, and their efficacy in an immunosuppressed murine model of disseminated infection. Methods. The in vitro susceptibility of four strains against amphotericin B, fluconazole and anidulafungin was performed by using a reference broth microdilution method and time-kill curves. The in vivo efficacy was evaluated by determination of fungal load reduction in kidneys of infected animals receiving deoxycholate AMB at 0,8 mg/kg i.v., liposomal amphotericin B at 10 mg/kg i.v., fluconazole at 50 mg/kg, or anidulafungin at 10 mg/kg. Results. Amphotericin B and anidulafungin showed fungicidal activity, while fluconazole was fungistatic for all the strains. In the murine model, liposomal amphotericin B at 10 mg/kg/day was effective in reducing the tissue burden in kidneys of mice infected with any of the tested strains. However, amphotericin B, anidulafungin and fluconazole were only effective against those strains showing low MIC values. Conclusions. Liposomal amphotericin B showed the higher activity and efficacy against the two strains of C. guilliermondii, in contrast to the poor effect of fluconazole and anidulafungin. Further studies with more isolates of C. guilliermondii representing a wider range of MICs should be carried out to assess whether there is any relationship between MIC values and anidulafungin efficacy (AU)

Antecedentes. Candida guilliermondii es un patógeno emergente, con reducida sensibilidad al fluconazol y a las equinocandinas. Objetivos. Evaluar la actividad in vitro de la anidulafungina, en comparación con la de la anfotericina B y el fluconazol, frente a C. guilliermondii y su eficacia en un modelo animal de infección diseminada. Métodos. La sensibilidad in vitro se valoró mediante microdilución en caldo y curvas de mortalidad. La eficacia in vivo se evaluó mediante la determinación de la carga fúngica en riñón de ratones inmunosuprimidos con infección diseminada por C. guilliermondii tratados con anfotericina B desoxicolato (0.8 mg/kg i.v.), anfotericina B liposomal (10 mg/kg i.v.), fluconazol (50 mg/kg) o anidulafungina (10 mg/kg). Resultados. La anfotericina B y la anidulafungina mostraron actividad fungicida, mientras que el fluconazol fue fungistático frente a todas las cepas. En el modelo murino, la anfotericina B liposomal redujo para todas las cepas la carga fúngica en riñones, mientras que la anfotericina B desoxicolato, la anidulafungina y el fluconazol fueron efectivas solo en aquellos animales infectados con las cepas de menor valor de concentración mínima inhibitoria (CMI). Conclusiones. La anfotericina B liposomal mostró la mayor actividad y eficacia frente a C. guilliermondii, en contraste con el limitado efecto del fluconazol y de la anidulafungina. Se necesitan estudios que incluyan cepas con un rango más amplio de CMI que permitan determinar la relación entre la actividad in vitro y la eficacia de la anidulafungina (AU)

Therapies against murine Candida guilliermondii infection, relationship between in vitro antifungal pharmacodynamics and outcome.

BACKGROUND: Candida guilliermondii has been recognized as an emerging pathogen showing a decreased susceptibility to fluconazole and considerably high echinocandin MICs. AIMS: Evaluate the in vitro activity of anidulafungin in comparison to amphotericin B and fluconazole against different isolates of C. guilliermondii, and their efficacy in an immunosuppressed murine model of disseminated infection. METHODS: The in vitro susceptibility of four strains against amphotericin B, fluconazole and anidulafungin was performed by using a reference broth microdilution method and time-kill curves. The in vivo efficacy was evaluated by determination of fungal load reduction in kidneys of infected animals receiving deoxycholate AMB at 0,8 mg/kg i.v., liposomal amphotericin B at 10 mg/kg i.v., fluconazole at 50 mg/kg, or anidulafungin at 10 mg/kg. RESULTS: Amphotericin B and anidulafungin showed fungicidal activity, while fluconazole was fungistatic for all the strains. In the murine model, liposomal amphotericin B at 10 mg/kg/day was effective in reducing the tissue burden in kidneys of mice infected with any of the tested strains. However, amphotericin B, anidulafungin and fluconazole were only effective against those strains showing low MIC values. CONCLUSIONS: Liposomal amphotericin B showed the higher activity and efficacy against the two strains of C. guilliermondii, in contrast to the poor effect of fluconazole and anidulafungin. Further studies with more isolates of C. guilliermondii representing a wider range of MICs should be carried out to assess whether there is any relationship between MIC values and anidulafungin efficacy.

In vitro antifungal susceptibility of Candida glabrata to caspofungin and the presence of FKS mutations correlate with treatment response in an immunocompromised murine model of invasive infection.

It has been argued that the in vitro activity of caspofungin (CSP) is not a good predictor of the outcome of echinocandin treatment in vivo. We evaluated the in vitro activity of CSP and the presence of FKS mutations in the hot spot 1 (HS1) region of the FKS1 and FKS2 genes in 17 Candida glabrata strains with a wide range of MICs. The efficacy of CSP against systemic infections from each of the 17 strains was evaluated in a murine model. No HS1 mutations were found in the eight strains showing MICs for CSP of ≤ 0.5 µg/ml, but they were present in eight of the nine strains with MICs of ≥ 1 µg/ml, i.e., three in the FKS1 gene and five in the FKS2 gene. CSP was effective for treating mice infected with strains with MICs of ≤ 0.5 µg/ml, showed variable efficacy in animals challenged with strains with MICs of 1 µg/ml, and did not work in those with strains with MICs of >1 µg/ml. In addition, mutations, including one reported for the first time, were found outside the HS1 region in the FKS2 gene of six strains with different MICs, but their presence did not influence drug efficacy. The in vitro activity of CSP was compared with that of another echinocandin, anidulafungin, suggesting that the MICs of both drugs, as well as mutations in the HS1 regions of the FKS1 and FKS2 genes, are predictive of outcome.

Evaluation of the correlation of caspofungin MICs and treatment outcome in murine infections by wild type strains of Candida parapsilosis.

We have evaluated the in vitro activity of caspofungin against 36 wild-type strains of Candida parapsilosis sensu stricto using 3 techniques: broth microdilution, disk diffusion, and the determination of minimal fungicidal concentration (MFC). The first 2 methods showed a good in vitro activity of caspofungin, but the MFCs were ≥2 dilutions above their corresponding MICs. In a murine model of disseminated infection, we evaluated the efficacy of caspofungin at 5 mg/kg against 8 strains of C. parapsilosis representing different degrees of in vitro susceptibility (0.12-1 µg/mL). All the isolates responded to treatment and (1→3)-ß-D-glucan levels were reduced in all the cases; however, the study revealed differences among isolates, since caspofungin reduced the tissue burden of mice infected with isolates with MICs ≤0.5 µg/mL but was less effective against those with MICs of 1 µg/mL.

MIC values of voriconazole are predictive of treatment results in murine infections by Aspergillus terreus species complex.

We evaluated the efficacy of voriconazole against nine strains of Aspergillus terreus with different MICs (0.12 to 4 µg/ml) by using a murine model. Markers of efficacy included survival, tissue burden, galactomannan antigenemia, and drug serum levels. Voriconazole was especially effective in prolonging survival and reducing the fungal load in infections by strains that showed MICs that were less than or equal to the epidemiological cutoff value (1 µg/ml). In vitro data might be useful for predicting the outcome of A. terreus infections.

Experimental murine model of disseminated infection by Saksenaea vasiformis: successful treatment with posaconazole.

We have determined the in vitro activity of amphotericin B (AMB) and posaconazole (PSC) against Saksenaea vasiformis using broth microdilution and disk diffusion methods and determined the minimal fungicidal concentration (MFC). PSC was found to have the greatest in vitro activity in all cases and was the most efficacious in prolonging survival and reducing the fungal load in an immunocompetent murine model of disseminated infection caused by four strains of the fungus.

Efficacy of posaconazole in murine experimental sporotrichosis.

We developed a murine model of systemic sporotrichosis by using three strains of each of the two commonest species causing sporotrichosis, i.e., Sporothrix schenckii sensu stricto and Sporothrix brasiliensis, in order to evaluate the efficacy of posaconazole (PSC). The drug was administered at a dose of 2.5 or 5 mg/kg of body weight twice a day by gavage, and one group was treated with amphotericin B (AMB) as a control treatment. Posaconazole, especially at 5 mg/kg, showed good efficacy against all the strains tested, regardless of their MICs, as measured by prolonged survival, tissue burden reduction, and histopathology.

In vitro and in vivo activities of posaconazole and amphotericin B in a murine invasive infection by Mucor circinelloides: poor efficacy of posaconazole.

The in vitro susceptibility of 17 strains of Mucor circinelloides to amphotericin B and posaconazole was ascertained by using broth microdilution and disk diffusion methods and by determining the minimal fungicidal concentration (MFC). We evaluated the efficacy of posaconazole at 40 mg/kg of body weight/day and amphotericin B at 0.8 mg/kg/day in a neutropenic murine model of disseminated infection by M. circinelloides by using 6 different strains tested previously in vitro. In general, most of the posaconazole MICs were within the range of susceptibility or intermediate susceptibility, while the small inhibition zone diameters (IZDs) were indicative of nonsusceptibility for all isolates tested. The MFCs were ≥ 3 dilutions higher than the corresponding MICs. In contrast, amphotericin B showed good activity against all of the strains tested regardless of the method used. The in vivo studies demonstrated that amphotericin B was effective in prolonging survival and reducing the fungal load. Posaconazole showed poor in vivo efficacy with no correlation with the MIC values. The results suggested that posaconazole should be used with caution in the treatment of infections caused by Mucor circinelloides or by strains of Mucor not identified to the species level.

Efficacy of intrathecal administration of liposomal amphotericin B combined with voriconazole in a murine model of cryptococcal meningitis.

Meningitis is one of the most fatal manifestations of cryptococcosis, even with specific treatment. Combination of a prompt diagnosis and appropriate therapy are critical to reduce the fungal load and the inflammatory response effects of the proliferation of yeast into the central nervous system (CNS). Mice with experimental acute meningitis caused by Cryptococcus neoformans were treated with liposomal amphotericin B (L-AmB) administered intrathecally (i.t.c.) at 0.006 mg/kg weekly or intravenously (i.v.) at 10 mg/kg daily or with voriconazole (VCZ) administered orally at 30 mg/kg per dose twice daily or with combinations of both drugs, i.e. L-AmB i.t.c.+VCZ or L-AmB i.v.+VCZ at the same doses as used in the monotherapies. All treatments significantly increased the survival of animals in comparison with the control group, with VCZ being less effective in comparison with all other treatments (P ≤ 0.012). All treatments, with the exception of VCZ (P=0.533), reduced fungal burdens in the brain in comparison with controls. The combination of L-AmB i.t.c.+VCZ showed a synergistic effect in the reduction of fungal load that was significantly superior to any tested therapy (P ≤ 0.039). Histologically, untreated animals showed a marked inflammatory response with massive fungal cells in the meninges, whilst treated animals showed a variable number of fungal cells in the CNS, with the exception of animals receiving L-AmB i.t.c.+VCZ in which neither yeasts nor inflammation were observed.

Anidulafungin in treatment of experimental invasive infection by Candida parapsilosis: in vitro activity, (1-->3)-beta-D-glucan and mannan serum levels, histopathological findings, and in vivo efficacy.

We have evaluated the in vitro activity of anidulafungin (AFG) against 31 strains of Candida parapsilosis sensu stricto by using broth microdilution, disk diffusion, and minimal fungicidal concentration (MFC) determination procedures. The two first methods showed a high level of activity of the drug, while MFCs were 1 to 5 dilutions higher than their corresponding MICs. To assess if MICs were predictive of in vivo outcomes, six strains representing different AFG MICs (0.12 to 2 µg/ml) were tested in a murine model of disseminated infection treated with different doses of the drug (1, 5, or 10 mg/kg of body weight). AFG was able to prolong the survival of mice infected with all the strains tested but was able to reduce the tissue burden of those mice infected only with the strains that showed the lowest MIC (0.12 µg/ml).

Efficacy of anidulafungin against Aspergillus niger in vitro and in vivo.

In this study, anidulafungin (AFG) showed high in vitro activity against 10 isolates of Aspergillus niger by broth microdilution and disk diffusion methods. The efficacy of AFG at 1, 5 and 10 mg/kg was tested against six of the isolates in a murine model of disseminated infection. AFG was able to reduce mortality, showing survival rates of 70-100%, 60-100% and 30-60% in mice treated with AFG at 10, 5 and 1 mg/kg, respectively. AFG also showed a dose-response efficacy in reducing tissue burden in kidneys and spleen. A parallel experiment demonstrated that administration of AFG did not reduce serum concentrations of galactomannan in mice. Histopathological studies confirmed the efficacy of AFG.

In vitro activity and in vivo efficacy of posaconazole in treatment of murine infections by different isolates of the Aspergillus terreus complex.

Posaconazole (PSC) is an antifungal drug recommended as an alternative for the treatment of invasive aspergillosis in patients who are refractory or intolerant to primary antifungal therapy. We have evaluated the in vitro activity of PSC against 21 strains of the Aspergillus terreus complex using both broth microdilution and disk diffusion (Neo Sensitabs) methods. PSC showed the same high level of activity against all the strains with the two in vitro methods used. We developed a murine model of disseminated infection to evaluate the efficacy of PSC at 5, 10, or 20 mg/kg of body weight twice a day by using 6 different strains chosen randomly. PSC showed good efficacy, especially at 20 mg/kg, as measured by prolonged survival, tissue burden reduction, histopathology, and lowered galactomannan levels. The PSC levels in serum on the fourth day of treatment were higher than the MICs for the strains tested.

In vitro and in vivo antifungal susceptibilities of the Mucoralean fungus Cunninghamella.

We have determined the in vitro activities of amphotericin B (AMB), voriconazole, posaconazole (PSC), itraconazole (ITC), ravuconazole, terbinafine, and caspofungin against five strains of Cunninghamella bertholletiae and four of Cunninghamella echinulata. The best activity was shown by terbinafine against both species (MIC range = 0.3 to 0.6 µg/ml) and PSC against Cunninghamella bertholletiae (MIC = 0.5 µg/ml). We have also evaluated the efficacies of PSC, ITC, and AMB in neutropenic and diabetic murine models of disseminated infection by Cunninghamella bertholletiae. PSC at 40, 60, or 80 mg/kg of body weight/day was as effective as AMB at 0.8 mg/kg/day in prolonging survival and reducing the fungal tissue burden in neutropenic mice. PSC at 80 mg/kg/day was more effective than AMB at 0.8 mg/kg/day in reducing the fungal load in brain and lung of diabetic mice. Histological studies revealed an absence of fungal elements in organs of mice treated with either AMB at 0.8 mg/kg/day or PSC at 60 or 80 mg/kg/day in both models. ITC showed limited efficacy in both models. PSC could be a therapeutic option for the treatment of systemic infections caused by Cunninghamella bertholletiae.

Iron-radiofrequency synergism in lymphomagenesis.

The parenteral iron administration effects on the acceleration of lymphomagenesis by radiofrequency exposure were investigated using an animal model that develops spontaneous lymphomas with ageing. Complementary studies of the in vivo uptake of 59Fe-labeled ferric gluconate and ferric-ATP complex showed differences ob absorption and excretion between both iron compounds. In vitro assays of their effects on calcium cellular uptake using a cell model and tissues homogenates showed a molecular structure-dependence. The current results (mortality, clinical and histopathological examinations) demonstrated a synergism between radiofrequency and ferric gluconate, and the increased risk of radiofrequency exposure when it is simultaneous to parenteral iron administration.

Combined action of uranium and stress in the rat. II. Effects on male reproduction.

The effects of stress on the potential reproductive toxicity of long-term exposure to uranyl acetate dihydrate (UAD) were assessed in adult male rats. Six groups of animals were given UAD at 10, 20, and 40 mg/kg/day in the drinking water during 3 months. Animals in three of these groups were also subjected to restraint for 2 h/day during the same period. Control groups included restrained and unrestrained male rats not exposed to UAD. To evaluate the fertility, male rats were mated with untreated females for 2 weeks. Although body weight was not affected by uranium at any dose, there was a significant (not dose-related) decrease in the pregnancy rate. Moreover, spermatid number/testis was significantly decreased by uranium administration. Histopathological examination of the testes in rats killed after 3 months of treatment revealed few differences in the tubule and interstitial alterations (focal atrophy, binucleated cells) between control and uranium-exposed animals. The results of this investigation show that at the current UAD doses, restraint stress did not enhance the uranium-induced adverse effects on reproduction in male rats.