Pre-incubation with efungumab increases the sensitivity of Cryptococcus neoformans to amphotericin B and 5-flucytosine combination therapy in vitro
Abstract number: P1365
Al-Akeel R., Eyes T., Nooney L., Matthews R., Burnie J.
Objectives: Cryptococcal meningitis is associated with poor outcome: mortality ranges from 88% to 100% in African countries. Conventional treatment is with amphotericin B (AMB) + 5-flucytosine (5-FC). 5-FC has intrinsic toxicity and the emergence of resistance is common during treatment. Efungumab (EFU; Mycograb®) is a recombinant antibody derived against the LKVIRKNIV epitope of Candida heat shock protein 90 (Hsp90). This study devised an assay to evaluate sensitivity of EFU pre-treated Cryptococcus neoformans to AMB and 5-FC in vitro.
Methods: Six isolates of C. neoformans were pre-incubated for 2 hours with EFU 4, 8, 16 or 32 ug/mL. Cells were washed and used to inoculate single (AMB or 5-FC) or combination (AMB+5-FC) antifungals. The MIC was determined by microtitre assay at MIC-0 and MIC-2 endpoints and Fractional Inhibitory Concentration Index (FICI) calculated. Biacore analysis was performed on a sensor chip surface covalently coated with 2 ug/ml EFU to assess binding of EFU to cryptococcal Hsp90.
Results: Before EFU pre-treatment, AMB MIC-0 ranged from 0.25 to 1 ug/mL and 5-FC MIC-2 ranged from 0.25 to 4 ug/mL. Pre-treatment with 4 ug/mL EFU reduced AMB MIC-0 range to 0.250.5 ug/mL, but the range of 5-FC MIC-2 was unchanged. At 8 ug/mL EFU pre-treatment, AMB MIC-0 was 0.125 ug/mL for 5 out of 6 isolates, and 5-FC MIC-2- ranged from 0.1252 ug/mL for 4 out of 6 isolates. At 16 or 32 ug/mL EFU pre-treatment, MICs were reduced by 26 dilutions for each antifungal (0.06250.25 ug/mL for AMB (MIC-0) and 0.1251 ug/mL for 5-FC (MIC-2)). Combination of AMB+5-FC gave lower FICIs when cells were pre-treated with EFU vs not pre-treated. FICI values decreased as EFU concentration increased (Table). Biacore analysis demonstrated the high affinity of EFU for C. neoformans Hsp90: Ka=1.38×105 M-1/S-1, Kd=5.53×10-4 S-1 and KD=4.19×10-9 M.
Conclusion: The tight-binding affinity of EFU to cryptococcal Hsp90 enabled EFU+AMB+5-FC triple therapy to be assayed in C. neoformans. EFU pre-treatment increased the sensitivity of C. neoformans to AMB, 5-FC and AMB+5-FC in vitro, suggesting potential synergy of these combinations.
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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