Treatment of Aspergillus terreus infections: a clinical problem not yet resolved.

Despite the use of recommended therapies, invasive infections by Aspergillus terreus show a poor response. For years, investigative studies on the failure of therapy of fungal infections have focused on in vitro susceptibility data. However, it is well known that low minimum inhibitory concentrations (MICs) are not always predictive of response to therapy despite a correct dosage schedule. Many experimental and clinical studies have tried to establish a relationship between MICs and outcome in serious fungal infections but have come to contradictory and even surprising conclusions. The success or failure of treatment is determined by many factors, including the in vitro susceptibility of the causative fungal isolate, the pharmacokinetics/pharmacodynamics of the drug used for treatment, pharmacokinetic variability in the population, and the underlying disease that patients suffer. To try to understand this poor response to treatment, available data on the in vitro susceptibility of A. terreus, the experimental and clinical response to amphotericin B, triazoles and echinocandins, and the pharmacokinetics/pharmacodynamics of these antifungals have been reviewed. Of special interest are the fungistatic activites of these drugs against A. terreus and the high interpatient variability of serum drug levels observed in therapy based on triazoles, which make monitoring of infected patients necessary.

Crystallization and preliminary X-ray diffraction analysis of Xyn30D from Paenibacillus barcinonensis.

Xyn30D, a new member of a recently identified group of xylanases, has been purified and crystallized. Xyn30D is a bimodular enzyme composed of an N-terminal catalytic domain belonging to glycoside hydrolase family 30 (GH30) and a C-terminal family 35 carbohydrate-binding domain (CBM35) able to bind xylans and glucuronic acid. Xyn30D shares the characteristic endo mode of action described for GH30 xylanases, with the hydrolysis of the ß-(1,4) bonds of xylan being directed by α-1,2-linked glucuronate moieties, which have to be placed at the -2 subsite of the xylanase active site. Crystals of the complete enzyme were obtained and a full data set to 2.3 Šresolution was collected using a synchrotron X-ray source. This represents the first bimodular enzyme with the domain architecture GH30-CBM35. This study will contribute to the understanding of the role that the different xylanases play in the depolymerization of glucuronoxylan.

In vitro pharmacodynamics and in vivo efficacy of fluconazole, amphotericin B and caspofungin in a murine infection by Candida lusitaniae.

The in vitro activities of fluconazole (FLC), amphotericin B (AmB) and caspofungin (CSP) were evaluated against three isolates of Candida lusitaniae using time-kill curves. AmB showed in vitro fungicidal activity, whilst FLC and CSP exerted mainly strain-dependent fungistatic activity. The in vivo efficacies of the three drugs were evaluated in a murine model of disseminated infection. The doses administered were FLC 50 mg/kg/day, AmB 0.8 mg/kg/day and CSP 5 mg/kg/day. All three drugs were able to reduce the fungal burden in the kidneys of infected mice, with AmB showing the highest efficacy, followed by CSP. At least in this model, FLC, AmB and CSP are good candidates for treating invasive infections by C. lusitaniae.

Efficacy of Amphotericin B at Suboptimal Dose Combined with Voriconazole in a Murine Model of Aspergillus fumigatus Infection with Poor In Vivo Response to the Azole.

The combination of amphotericin B at a suboptimal dose (0.3 mg/kg) with voriconazole has shown efficacy in prolonging survival and reducing tissue burden in a murine model of disseminated infection by an isolate of Aspergillus fumigatus that had showed a poor in vivo response to the azole. The efficacy of the combined treatment was higher than that obtained with amphotericin B at 0.8 mg/kg.

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.

Evaluation of the in vitro activity of voriconazole as predictive of in vivo outcome in a murine Aspergillus fumigatus infection model.

We have evaluated the in vitro activity of voriconazole against 61 strains of Aspergillus fumigatus by using broth microdilution, disk diffusion, and minimal fungicidal concentration procedures. We observed an excellent correlation between the results obtained with the three methods. Five percent of the strains showed MICs greater than or equal to the epidemiological cutoff value (ECV; 1 µg/ml). To assess whether MICs were predictive of in vivo outcome, we tested the efficacy of voriconazole at 25 mg/kg of body weight daily in an immunosuppressed murine model of disseminated infection using 10 strains representing various patterns of susceptibility to the drug as determined by the in vitro study. Voriconazole prolonged survival and reduced fungal load in the kidneys and brain in those mice infected with strains with MICs of ≤0.25 µg/ml, while in mice infected with strains with MICs of 0.5 to 2 µg/ml, the efficacy was varied and strain dependent and in mice infected with the strain with a MIC of 4 µg/ml, the antifungal did not show efficacy. Voriconazole reduced galactomannan antigenemia against practically all strains with a MIC of <4 µg/ml. Our results demonstrate that some relationship exists between voriconazole MICs and in vivo efficacy; however, further studies testing additional strains are needed to better ascertain which MIC values can predict clinical outcome.

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.

Are epidemiologic cut-off values predictors of the in vivo efficacy of azoles in experimental aspergillosis?

We tried to correlate the in vitro activity and the in vivo efficacy of voriconazole (VRC) and posaconazole (POS) against Aspergillus flavus and Aspergillus niger. The in vitro susceptibility of a large set of isolates was determined by broth microdilution and disk diffusion methods, while the in vivo efficacy was assessed in a murine model of disseminated infection. For A. flavus, VRC showed efficacy even for strains with MICs above the epidemiologic cutoff value (ECV) (1 µg/mL). For A. niger, VRC was ineffective against those strains for which the MIC was 1 dilution below the corresponding ECV (2 µg/mL). POS showed efficacy against all the strains of both species included in the study, although isolates with MICs > ECV were not tested.

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 a murine model of disseminated infection caused by Apophysomyces variabilis.

OBJECTIVES: We evaluated the in vitro activity of posaconazole and amphotericin B against several clinical strains of the mucoralean fungus Apophysomyces variabilis, and their efficacy in a murine model of disseminated infection caused by that fungus. METHODS: The in vitro susceptibility of seven strains of A. variabilis to posaconazole and amphotericin B was determined by using a broth microdilution method. The in vivo efficacy of both drugs, posaconazole at 20 mg/kg twice daily orally by gavage and amphotericin B at 0.8 mg/kg once daily intravenously, was evaluated against six of the strains previously tested in vitro using immunocompetent mice. RESULTS: In general, MICs of both drugs were within the range of susceptibility or intermediate susceptibility. Posaconazole and amphotericin B were able to significantly reduce the percentages of positive cultures in the affected tissues. However, in general, posaconazole significantly improved survival (median, 23 days; range, 7-30 days) compared with untreated controls (median, 6 days; range, 4-7 days) and, in some cases, with respect to the animals treated with amphotericin B (median, 15 days; range, 5-30 days). CONCLUSIONS: Our results demonstrate the efficacy of posaconazole in the treatment of a disseminated murine infection caused by A. variabilis. However, further clinical studies are required to ascertain the potential use in human infections caused by this fungus.

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.

Histopathology and antifungal treatment of experimental murine chromoblastomycosis caused by Cladophialophora carrionii.

OBJECTIVES: A murine model of chromoblastomycosis caused by Cladophialophora carrionii was used to compare the efficacy of posaconazole and voriconazole with that of terbinafine and itraconazole, the currently used drugs in the management of chromoblastomycosis. METHODS: Athymic nude mice were infected with 2 × 10(7) cfu of a clinical isolate of C. carrionii. When typical lesions were established, treatments with posaconazole at 20 mg/kg/day, voriconazole at 20 mg/kg/day, itraconazole at 50 mg/kg/day or terbinafine at 250 mg/kg/day were initiated. Treatment efficacy was evaluated for 4 months by measuring the size of the lesions, observing any histopathological changes and culturing the excised tissue. RESULTS: Posaconazole was the only drug that reduced the initial lesion size, while voriconazole and terbinafine reduced growth relative to controls. CONCLUSIONS: This study suggests that the newer triazoles have potential in the treatment of chromoblastomycosis caused by C. carrionii.

Combined therapy of voriconazole and anidulafungin in murine infections by Aspergillus flavus.

The efficacy of the combination of anidulafungin (AFG) at 1 mg/kg plus voriconazole (VRC) at 25 mg/kg was evaluated in a murine model of disseminated infection by Aspergillus flavus using three isolates previously tested in vitro. All the combinations showed indifferent in vitro interaction with the exception of one, which showed synergy. In general, the combined treatment prolonged the survival and reduced the fungal load in comparison with AFG alone, and only in a few cases, it improved the results of the VRC monotherapy. The combination of the two drugs and VRC alone reduced the galactomannan levels in serum in comparison with the control group.

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.

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).

Antifungal therapy in an athymic murine model of chromoblastomycosis by Fonsecaea pedrosoi.

We have compared the efficacy of terbinafine (TRB) and itraconazole (ITZ), the recommended drugs for the treatment of chromoblastomycosis, with that of posaconazole (PSC) and voriconazole (VRC) in athymic mice infected with the fungus Fonsecaea pedrosoi. Three weeks after challenge, mice were treated for 4 months with PSC at 10 or 20 mg/kg of body weight/day, with VRC at 10 or 20 mg/kg/day, with ITZ at 25 or 50 mg/kg/day, or with TRB at 150 or 250 mg/kg/day. The progress of the infection was evaluated by measuring the size of the lesions, by histopathological studies, and by cultures of the excised tissue. The two doses of PSC followed by the highest doses of ITZ and TRB showed the best results while VRC did not show efficacy. PSC could be an alternative in the treatment of chromoblastomycosis.

In vitro activity and in vivo efficacy of anidulafungin in murine infections by Aspergillus flavus.

Anidulafungin (AFG) showed high activity against 27 strains of Aspergillus flavus by use of broth microdilution and disk diffusion methods. This drug was effective in vivo in a murine model of disseminated infection with five isolates tested. AFG was able to prolong survival and reduce tissue burden of infected mice but not able to reduce galactomannan serum concentrations. The AFG serum levels were above the corresponding minimum effective concentrations (MEC) for all of the strains tested.

Treatment of murine Fusarium verticillioides infection with liposomal amphotericin B plus terbinafine.

Using a murine model of disseminated infection by Fusarium verticillioides, the efficacy of liposomal amphotericin (L-AmB) B at 10mg/kg body weight once daily and terbinafine (TRB) at 150 mg/kg body weight twice daily, alone and in combination, was evaluated. The combination of L-AmB with TRB was the only treatment able to prolong survival and to reduce fungal loads in the spleen and kidneys of mice infected with either strain of F. verticillioides used. The results suggest that this combination may have a role in the treatment of F. verticillioides infection.

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.