Assessment of Efficacy of Antifungals against Aspergillus fumigatus: Value of Real-Time Bioluminescence Imaging

Abstract

c Aspergillus fumigatus causes life-threatening infections, especially in immunocompromised patients. Common drugs for therapy of aspergillosis are polyenes, azoles, and echinocandins. However, despite in vitro efficacy of these antifungals, treatment failure is frequently observed. In this study, we established bioluminescence imaging to monitor drug efficacy under in vitro and in vivo conditions. In vitro assays confirmed the effectiveness of liposomal amphotericin B, voriconazole, and anidulafungin. Liposomal amphotericin B and voriconazole were fungicidal, whereas anidulafungin allowed initial germination of conidia that stopped elongation but allowed the conidia to remain viable. In vivo studies were performed with a leukopenic murine model. Mice were challenged by intranasal instillation with a bioluminescent reporter strain (5 ؋ 10 5 and 2.5 ؋ 10 5 conidia), and therapy efficacies of liposomal amphotericin B, voriconazole, and anidulafungin were monitored. For monotherapy, the highest treatment efficacy was observed with liposomal amphotericin B, whereas the efficacies of voriconazole and anidulafungin were strongly dependent on the infectious dose. When therapy efficacy was studied with different drug combinations, all combinations improved the rate of treatment success compared to that with monotherapy. One hundred percent survival was obtained for treatment with a combination of liposomal amphotericin B and anidulafungin, which prevented not only pulmonary infections but also infections of the sinus. In conclusion, combination therapy increases treatment success, at least in the murine infection model. In addition, our novel approach based on real-time imaging enables in vivo monitoring of drug efficacy in different organs during therapy of invasive aspergillosis.

Longitudinal, in vivo assessment of invasive pulmonary aspergillosis in mice by computed tomography and magnetic resonance imaging

“…[16][17][18][19] BLI appeared to be suitable to investigate the effectiveness of antifungal compounds in vitro but proved to be very challenging to detect infection in vivo, possibly owing to problems with substrate availability. 20 Nuclear imaging techniques, such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT), have also been used for detecting pulmonary aspergillosis in rodents by using radioactive probes.…”

Administration of Zinc Chelators Improves Survival of Mice Infected with Aspergillus fumigatus both in Monotherapy and in Combination with Caspofungin

“…The in vitro susceptibility of the A. fumigatus 2/7/1 strain in liquid cultures against chelators was determined by seeding 5 ϫ 10 4 conidia in a 24-well plate. Each well contained 500 l of RPMI 1640 cell culture medium (Invitrogen 22409-015; Gibco, France) supplemented with 10% fetal calf serum (FCS) (complete RPMI) (34). For experiments with other sera, 10% human, rat, mouse, or rabbit serum was used to ensure that the serum environment specific to each species does not interfere individually with the properties of the chelators.…”

“…These were added at different concentrations and at the time points indicated for the specific experiments. Plates were incubated for 10 h at 37°C, 5 l phosphatebuffered saline (PBS) containing 0.16 mg of D-luciferin was then added to each well, and plates were incubated for 10 min prior to luminescence acquisition on an IVIS 100 system (PerkinElmer, Boston, MA) as previously reported (34). Photons were collected for 1 and 3 min on the highsensitivity setting.…”

“…Plates were incubated for an additional 5 h at 37°C, and luminescence measurements were repeated as described above. Experiments were repeated twice for each concentration, and cultures were made in triplicate (34). The length of hyphae was measured by taking photographs on an EVOS Core microscope (Thermo Fisher Scientific, Waltham, MA) at a magnification of ϫ20, followed by measuring the lengths of 100 hyphae for each sample using the ImageJ software.…”

“…Conidia were produced from A. fumigatus strain 2/7/1, a modified version of the bioluminescent C3 strain containing a codon-optimized version of the Photinus pyralis luciferase gene. This strain behaved and caused virulence similarly to the wild-type strain (34). Cultures were grown for 7 days on 2% malt agar slants and recovered by vortexing with 0.01% aqueous Tween 20 solution.…”