Drug selection pressure and fitness cost for artemether-resistant Plasmodium berghei ANKA parasites in vivo

Abstract

The clinical use of artemisinin-based combination therapies is threatened by increasing failure rates due to the emergence and spread of multiple drug resistance genes in most human Plasmodium strains. The aim of this study was to generate artemether-resistant (AMR) parasites from Plasmodium berghei ANKA (AMS), and determine their fitness cost. Artemether resistance was generated by increasing drug pressure doses gradually for 9 months. Effective doses (ED50 and ED90) were determined using the 4-day suppressive test, and the indices of resistance (I) at 50% and 90% (I50 and I90) were determined using the ratio of either ED50 or ED90 of AMR to AMS, respectively. The stability of the AMR parasites was evaluated by: five drug-free passages (5DFPs), 3 months of cryopreservation (CP), and drug-free serial passages (DFSPs) for 4 months. Analysis of variance was used to compare differences in growth rates between AMR and AMS with 95% confidence intervals. ED50 and ED90 of AMS were 0.61 and 3.43 mg/kg/day respectively. I50 and I90 after 20 cycles of artemether selection pressure were 19.67 and 21.45, respectively; 5DFP values were 39.16 and 15.27, respectively; 3-month CP values were 29.36 and 10.79, respectively; and DFSP values were 31.34 and 12.29, respectively. The mean parasitaemia value of AMR (24.70% ± 3.60) relative to AMS (37.66% ± 3.68) at Day 7 post infection after DFSPs revealed a fitness cost of 34.41%. A moderately stable AMRP. berghei line was generated. Known and unknown mutations may be involved in modulating artemether resistance, and therefore molecular investigations are recommended.