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Adaptive evolution of malaria parasites in French Guiana: Reversal of chloroquine resistance by acquisition of a mutation in pfcrt

Significance This study addresses the evolutionary dynamics of antimalarial drug resistance after changes in drug use. We show that chloroquine resistance in Plasmodium falciparum from French Guiana was lost after sustained drug removal, whereas the resistance marker PfCRT K76T remained fixed in the parasite population. This phenotypic reversion was caused by the acquisition of a single additional C350R substitution in PfCRT. This genetic change also impaired susceptibility to piperaquine, suggesting that piperaquine pressure drove the expansion of this allele. These findings have important implications for understanding drug resistance evolution when standard resistance alleles reach fixation and can lose their utility as markers because of adaptive changes at other amino acid positions or loci in the genome. In regions with high malaria endemicity, the withdrawal of chloroquine (CQ) as first-line treatment of Plasmodium falciparum infections has typically led to the restoration of CQ susceptibility through the reexpansion of the wild-type (WT) allele K76 of the chloroquine resistance transporter gene (pfcrt) at the expense of less fit mutant alleles carrying the CQ resistance (CQR) marker K76T. In low-transmission settings, such as South America, drug resistance mutations can attain 100% prevalence, thereby precluding the return of WT parasites after the complete removal of drug pressure. In French Guiana, despite the fixation of the K76T allele, the prevalence of CQR isolates progressively dropped from >90% to <30% during 17 y after CQ withdrawal in 1995. Using a genome-wide association study with CQ-sensitive (CQS) and CQR isolates, we have identified a single mutation in pfcrt encoding a C350R substitution that is associated with the restoration of CQ susceptibility. Genome editing of the CQR reference strain 7G8 to incorporate PfCRT C350R caused a complete loss of CQR. A retrospective molecular survey on 580 isolates collected from 1997 to 2012 identified all C350R mutant parasites as being CQS. This mutation emerged in 2002 and rapidly spread throughout the P. falciparum population. The C350R allele is also associated with a significant decrease in piperaquine susceptibility in vitro, suggesting that piperaquine pressure in addition to potential fitness costs associated with the 7G8-type CQR pfcrt allele may have selected for this mutation. These findings have important implications for understanding the evolutionary dynamics of antimalarial drug resistance.