Identification of a Mutant PfCRT-Mediated Chloroquine Tolerance Phenotype in Plasmodium falciparum

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Abstract
Mutant forms of the Plasmodium falciparum transporter PfCRT constitute the key determinant of parasite resistance to chloroquine (CQ), the former first-line antimalarial, and are ubiquitous to infections that fail CQ treatment. However, treatment can often be successful in individuals harboring mutant pfcrt alleles, raising questions about the role of host immunity or pharmacokinetics vs. the parasite genetic background in contributing to treatment outcomes. To examine whether the parasite genetic background dictates the degree of mutant pfcrt-mediated CQ resistance, we replaced the wild type pfcrt allele in three CQ-sensitive strains with mutant pfcrt of the 7G8 allelic type prevalent in South America, the Oceanic region and India. Recombinant clones exhibited strain-dependent CQ responses that ranged from high-level resistance to an incremental shift that did not meet CQ resistance criteria. Nonetheless, even in the most susceptible clones, 7G8 mutant pfcrt enabled parasites to tolerate CQ pressure and recrudesce in vitro after treatment with high concentrations of CQ. 7G8 mutant pfcrt was found to significantly impact parasite responses to other antimalarials used in artemisinin-based combination therapies, in a strain-dependent manner. We also report clinical isolates from French Guiana that harbor mutant pfcrt, identical or related to the 7G8 haplotype, and manifest a CQ tolerance phenotype. One isolate, H209, harbored a novel PfCRT C350R mutation and demonstrated reduced quinine and artemisinin susceptibility. Our data: 1) suggest that high-level CQR is a complex biological process dependent on the presence of mutant pfcrt; 2) implicate a role for variant pfcrt alleles in modulating parasite susceptibility to other clinically important antimalarials; and 3) uncover the existence of a phenotype of CQ tolerance in some strains harboring mutant pfcrt. Plasmodium falciparum resistance to the antimalarial drug chloroquine has been found to result primarily from point mutations in PfCRT, which provide a highly sensitive marker of in vivo treatment failure and in vitro resistance. Debate has nonetheless continued about the singular role of mutant PfCRT and the contribution of the parasite genetic background. To address this, we have generated recombinant P. falciparum lines expressing a mutant pfcrt allele, or the reference wild type allele, in three distinct chloroquine-sensitive strains. Their analysis reveals a spectrum of responses ranging from high-level resistance to a previously unrecognized tolerance phenotype. The latter is characterized by virtually unchanged chloroquine IC50 values, significantly elevated IC90 values, and the ability to recrudesce after exposure to drug concentrations that are lethal to chloroquine-sensitive parasites. This tolerance phenotype was also observed in an isolate from French Guiana, confirming its presence in malaria-endemic regions. Mutant PfCRT significantly affected parasite responses to other antimalarials, including ones used in artemisinin-based combination therapies, in a strain-dependent manner. Our data suggest that successful CQ treatment of drug-resistant parasites is dependent on both host immunity and the strain-dependent extent to which mutant pfcrt imparts resistance.