Evaluation of Mycograb®, amphotericin B, caspofungin, and fluconazole in combination against Cryptococcus neoformans by checkerboard and time-kill methodologies

Abstract

This article reported the identification of heat shock protein 90 (hsp90) homologues by immunoblot in Cryptococcus neoformans. Mycograb®, a genetically recombinant antibody against hsp90, was evaluated against 8 clinical isolates and the National External Quality Assessment Service for Microbiology strain of C. neoformans alone and in combination with amphotericin B, caspofungin, and fluconazole by checkerboard assay. At the end point of an optically clear well, the minimum inhibitory concentration (MIC) 0's ranged from 256 to 1024 μg/mL for Mycograb®, from 0.5 to 1 μg/mL for amphotericin B, and from 16 to 32 μg/mL for caspofungin. The combination of Mycograb® and amphotericin B produced a fractional inhibitory concentration index from 0.27 to 0.56, indicating a mainly synergistic effect, whereas for caspofungin, it varied from 0.5 to 2. At an end point of ≥50% inhibition, the MIC-2s varied from 16 to 128 μg/mL for Mycograb® and from 0.125 to 16 μg/mL for fluconazole. The fractional inhibitory concentration index classified the combination as indifferent for 5 isolates, additive for 3 more isolates, and synergistic in a single isolate. Time-kill analysis on 2 isolates (F/7844 and F/10156), which had synergistic and additive results with amphotericin B, respectively, on checkerboard was performed with 4–16 μg/mL of Mycograb®, 2–8 μg/mL of fluconazole, and 0.0625–2 μg/mL of amphotericin B. This demonstrated an increasingly static effect with augmenting concentrations of fluconazole and an initial static effect with amphotericin B at lower concentrations, which became fungicidal as the level of drug increased. The addition of either 4 or 8 μg/mL of Mycograb® to 0.5 μg/mL of amphotericin B with C. neoformans F/7844 changed a static effect to a fungicidal effect at 8 h with an increased killing of 1.2 logs at 48 h. With C. neoformans F/10156, the addition of 16 μg/mL of Mycograb® to 0.25 μg/mL of amphotericin B produced a difference in killing from 1 logarithm after 4 h to 1.5 logarithms after 48 h. These data suggest that the combination of amphotericin B and Mycograb® would be worth exploring in the treatment of infection due to C. neoformans.