Experimental Evolution of Reduced Susceptibility to Artemether-Lumefantrine in Plasmodium berghei Following Serial Drug Pressure.

Abstract

Malaria remains a major global health problem, with uneven progress and the greatest disease burden clustered in sub-Saharan Africa. Antimalarial drug resistance continues to undermine malaria control efforts, with widespread resistance reported in Plasmodium falciparum, including reduced susceptibility to artemisinin-based combination therapies (ACTs). However, despite increasing concerns over the long-term effectiveness of ACTs, the processes through which resistance to artemether-lumefantrine (AL) partner drugs evolves and contributes to resistance against individual drug components remain poorly understood. Consequently, we investigated AL resistance in Plasmodium berghei ANKA towards understanding resistance development to full ACT regimens rather than individual drugs. Firstly, drug efficacy was evaluated using the standard 4-day suppressive test (4-DT), with baseline effective doses (ED₅₀ and ED₉₀) for the parental line determined as 1.45 and 2.80 mg/kg, respectively. Following 20 stepwise in vivo drug selection pressure passages, from the parental (0th) to the 20th passage in mice over 26 weeks, resistance increased by 2.85-fold in ED₅₀ and 2.65-fold increase in ED₉₀, indicating reduced drug susceptibility and stepwise resistance selection. After five drug-free passages and recovery following 45 days of cryopreservation at -80°C, the parasite retained slight resistance across all assays, indicating a stable resistance phenotype. In conclusion, continuous drug exposure successfully led to the development of P. berghei parasites with reduced sensitivity to AL, as evidenced by significantly lower chemo-suppression in resistant lines compared with sensitive controls (p < 0.05). These findings demonstrate that AL resistance can be progressively selected over a period of time and stability maintained in P. berghei, providing insights into the durability and evolutionary vulnerability of ACTs under sustained drug pressure. Future studies should enhance the level of resistance, evaluate its stability, and investigate the molecular basis of AL resistance to identify reliable genetic markers for monitoring resistance to ACTs.