Joanne Peter

Experimental pulmonary aspergillosis due to Aspergillus terreus : pathogenesis and treatment of an emerging fungal pathogen resistant to amphotericin B

Aspergillus terreus is an uncommon but emerging fungal pathogen, which causes lethal infections that are often refractory to amphotericin B (AmB). In comparison to Aspergillus fumigatus, A. terreus was resistant to the in vitro fungicidal effects of safely achievable concentrations of AmB. These in vitro findings correlated directly with resistance of A. terreus to AmB in experimental invasive pulmonary aspergillosis. Residual fungal pulmonary burden and galactomannan antigenemia demonstrated persistent infection, despite therapy with deoxycholate AmB or liposomal AmB. By comparison, posaconazole and itraconazole resolved GM antigenemia, reduced residual fungal burden, and improved survival. There were no differences in phagocytic host response to A. terreus versus A. fumigatus; however, the rate of conidial germination of A. terreus was slower. The strain of A. terreus with the highest minimum inhibitory and minimum lethal concentration of AmB also had the lowest membrane ergosterol content. The hyphae of A. terreus in vivo displayed distinctive aleurioconidia, which may be a practical microscopic feature for rapid preliminary diagnosis.

Activities of amphotericin B and antifungal azoles alone and in combination against Pseudallescheria boydii.

In order to develop new approaches to treatment of infections due to Pseudallescheria boydii, the in vitro antifungal activity of amphotericin B alone and in combination with miconazole, itraconazole, and fluconazole was studied. Combinations of amphotericin B and antifungal azoles were synergistic, additive, or indifferent in their interaction against P. boydii. Antagonism was not observed.

Pathogenesis of Aspergillus fumigatus and the Kinetics of Galactomannan in an In Vitro Model of Early Invasive Pulmonary Aspergillosis: Implications for Antifungal Therapy

BACKGROUND Little is known about the pathogenesis of invasive pulmonary aspergillosis and the relationship between the kinetics of diagnostic markers and the outcome of antifungal therapy. METHODS An in vitro model of the human alveolus, consisting of a bilayer of human alveolar epithelial and endothelial cells, was developed. An Aspergillus fumigatus strain expressing green fluorescent protein was used. Invasion of the cell bilayer was studied using confocal and electron microscopy. The kinetics of culture, polymerase chain reaction, and galactomannan were determined. Galactomannan was used to measure the antifungal effect of macrophages and amphotericin B. A mathematical model was developed, and results were bridged to humans. RESULTS A. fumigatus penetrated the cellular bilayer 14-16 h after inoculation. Galactomannan levels were inextricably tied to fungal invasion and were a robust measure of the antifungal effect of macrophages and amphotericin B. Neither amphotericin nor macrophages alone was able to suppress the growth of A. fumigatus; rather, the combination was required. Monte Carlo simulations showed that human dosages of amphotericin B of at least 0.6 mg/kg were required to achieve adequate drug exposure. CONCLUSIONS This model provides a strategy by which relationships among pathogenesis, immunological effectors, and antifungal drug therapy for invasive pulmonary aspergillosis may be further understood.

Concentration-Dependent Synergy and Antagonism within a Triple Antifungal Drug Combination against Aspergillus Species: Analysis by a New Response Surface Model

ABSTRACT Triple antifungal combinations are used against refractory invasive aspergillosis without an adequate understanding of their pharmacodynamic interactions. We initially studied the in vitro triple combination of voriconazole, amphotericin B, and caspofungin against Aspergillus fumigatus, A. flavus, and A. terreus by a spectrophotometric microdilution broth method after 48 h of incubation. We then analyzed these results with a recently described nonlinear mixture response surface Emax-based model modified to assess pharmacodynamic interactions at various growth levels. The new model allows flexibility in all four parameters of the Emax model and is able to describe complex pharmacodynamic interactions. Concentration-dependent pharmacodynamic interactions were found within the triple antifungal combination. At the 50% growth level, synergy (median interaction indices of 0.43 to 0.82) was observed at low concentrations of voriconazole (<0.03 mg/liter) and amphotericin B (≤0.20 mg/liter) and at intermediate concentrations of caspofungin (0.95 to 14.88 mg/liter), whereas antagonism (median interaction indices of 1.17 to 1.80) was found at higher concentrations of voriconazole and amphotericin B. Ternary plot and interaction surface analysis further revealed the complexity of these concentration-dependent interactions. With increasing concentrations of amphotericin B, the synergistic interactions of voriconazole-caspofungin double combination decreased while the antagonistic interactions increased. A similar effect was observed when voriconazole was added to the double combination of amphotericin B and caspofungin. In conclusion, the new nonlinear mixture-amount response surface modeling of the triple antifungal combination demonstrated a net antagonism or synergy against Aspergillus species depending upon drug concentrations and species.

Efficacy, Safety, and Plasma Pharmacokinetics of Escalating Dosages of Intravenously Administered Ravuconazole Lysine Phosphoester for Treatment of Experimental Pulmonary Aspergillosis in Persistently Neutropenic Rabbits

ABSTRACT Ravuconazole is a new antifungal triazole with broad-spectrum activity and a long half-life in plasma. We studied the antifungal efficacy, safety, and pharmacokinetics of ravuconazole lysine phosphoester in escalating dosages for the treatment of invasive pulmonary aspergillosis due to Aspergillus fumigatus in persistently neutropenic rabbits. Treatment groups consisted of rabbits treated with ravuconazole at 2.5 (RVC2.5), 5 (RVC5), and 10 (RVC10) mg/kg of body weight/day, rabbits treated with amphotericin B (AMB) at 1 mg/kg/day, or untreated controls. There was a dose-dependent reduction of pulmonary residual fungal burden (CFU per gram) in RVC5-, RVC10-, and AMB-treated rabbits in comparison to untreated controls (P < 0.01, P < 0.001, and P < 0.01, respectively). These findings correlated with progressive galactomannan antigenemia in untreated controls and the RVC2.5-treated rabbits, a lower galactomannan index (GMI) in RVC5- and RVC10-treated rabbits, and a similarly low GMI in AMB-treated rabbits (P < 0.01). Rabbits treated with RVC5, RVC10, and AMB also showed a reduction of organism-mediated pulmonary injury, as measured by infarct scores and lung weights, in comparison to untreated controls (P < 0.001). These results were supported by decreased pulmonary infiltrates detected by computed tomography in RVC5- and RVC10-treated rabbits in comparison to untreated controls (P < 0.05). Survival throughout the entire study was achieved in 95% of RVC5-treated rabbits (P < 0.001), 85% of RVC10-treated rabbits (P < 0.001), and 50% of AMB-treated rabbits (P < 0.05) in comparison to none of the untreated controls. Ravuconazole showed linear plasma pharmacokinetics and a large volume of distribution while maintaining concentrations in plasma above the MIC throughout the dosing interval. There was no evidence of hepatotoxicity or nephrotoxicity among ravuconazole-treated animals. Intravenously administered ravuconazole lysine phosphoester showed dose-dependent efficacy and an excellent safety profile for the treatment of invasive pulmonary aspergillosis in persistently neutropenic rabbits.

SCH-39304 in prevention and treatment of disseminated candidiasis in persistently granulocytopenic rabbits.

To investigate the potential use of SCH-39304 for the prevention and treatment of disseminated candidiasis in granulocytopenic patients, we studied its in vivo antifungal activity as preventive, early, and late treatments in three models (acute, subacute, and chronic) of disseminated candidiasis in persistently granulocytopenic rabbits. SCH-39304 was an effective as amphotericin B alone and fluconazole alone for the prevention of disseminated candidiasis. SCH-39304 alone and fluconazole alone were as effective as amphotericin B plus flucytosine for early treatment of subacute disseminated candidiasis. When treatment was delayed for 5 days to establish chronic disseminated candidiasis, SCH-39304 was less effective than amphotericin B plus flucytosine. In comparison with different treatment regimens, SCH-39304 was more effective in early and preventive treatment. Thus, SCH-39304 was comparable to treatment control regimens in prevention and early treatment of subacute disseminated candidiasis. SCH-39304 also was most effective in granulocytopenic rabbits with disseminated candidiasis when used for prevention or early treatment.

Use of Fluorescent Probes To Determine MICs of Amphotericin B and Caspofungin against Candida spp. and Aspergillus spp.

ABSTRACT We investigated the utility of mechanism-based fluorescent probes for determination of MICs (FMICs) of amphotericin B and caspofungin against Candida spp. and Aspergillus spp. Amphotericin B was selected as a membrane-active antifungal agent, and caspofungin was selected as a cell wall-active agent. FMICs were also compared to the MIC determined by CLSI (formerly NCCLS) methods. Five isolates per species of Candida albicans, Candida glabrata, Candida parapsilosis, Aspergillus fumigatus, and Aspergillus terreus were studied with either amphotericin B or caspofungin. The fluorescent probes, carboxyfluorescein diacetate (CFDA) for cytoplasmic esterase activity and dihexyloxacarbocyanine iodide (DiOC6) for cell membrane potential, were each added to their respective plates. MICs and FMICs were determined in at least three separate experiments (in duplicate). Fluorescence was measured using a 96-well plate fluorometer. For amphotericin B and caspofungin, the FMIC end point was the lowest concentration of drug at which the percent growth inhibition from treated organisms versus control organisms displayed 80% inhibition for amphotericin B and 50% inhibition for caspofungin as measured by a fluorescent signal. The MIC for amphotericin B was defined as the lowest concentration of antifungal displaying no visible growth for both Aspergillus and Candida spp. The MIC for caspofungin was the lowest concentration of drug that displayed a minimum effective concentration for Aspergillus spp. For Candida spp., the MIC for caspofungin was defined as the concentration at which the antifungal agent significantly inhibits the organism. The FMICs of both antifungals, as measured by the DiOC6 membrane probe, showed good agreement (83% to 100%), within one well dilution, with the MICs against amphotericin B and caspofungin for all species. Also, the FMICs measured by the CFDA cytoplasmic esterase probe reflecting damage due to cell wall or cell membrane showed strong agreement (79 to 100%) with the MICs of both amphotericin B and caspofungin for all species. There was no significant difference in comparisons of MIC and FMIC values (P ≥ 0.05). The use of fluorescent probes provides a mechanism-based method of determination of MICs of amphotericin B and caspofungin against Candida spp. and Aspergillus spp. that correlates well with standard methods.