Evidence for the early emergence of piperaquine-resistant Plasmodium falciparum malaria and modeling strategies to mitigate resistance

Abstract

Multidrug-resistant Plasmodium falciparum parasites have emerged in Cambodia and neighboring countries in Southeast Asia, compromising the efficacy of first-line antimalarial combinations. Dihydroartemisinin + piperaquine (PPQ) treatment failure rates have risen to as high as 50% in some areas in this region. For PPQ, resistance is driven primarily by a series of mutant alleles of the P. falciparum chloroquine resistance transporter (PfCRT). PPQ resistance was reported in China three decades earlier, but the molecular driver remained unknown. Herein, we identify a PPQ-resistant pfcrt allele (China C) from Yunnan Province, China, whose genotypic lineage is distinct from the PPQ-resistant pfcrt alleles currently observed in Cambodia. Combining gene editing and competitive growth assays, we report that PfCRT China C confers moderate PPQ resistance while re-sensitizing parasites to chloroquine (CQ) and incurring a fitness cost that manifests as a reduced rate of parasite growth. PPQ transport assays using purified PfCRT isoforms, combined with molecular dynamics simulations, highlight differences in drug transport kinetics and in this transporter’s central cavity conformation between China C and the current Southeast Asian PPQ-resistant isoforms. We also report a novel computational model that incorporates empirically determined fitness landscapes at varying drug concentrations, combined with antimalarial susceptibility profiles, mutation rates, and drug pharmacokinetics. Our simulations with PPQ-resistant or -sensitive parasite lines predict that a three-day regimen of PPQ combined with CQ can effectively clear infections and prevent the evolution of PfCRT variants. This work suggests that including CQ in combination therapies could be effective in suppressing the evolution of PfCRT-mediated multidrug resistance in regions where PPQ has lost efficacy.

Mutant PfCRT Can Mediate Piperaquine Resistance in African Plasmodium falciparum With Reduced Fitness and Increased Susceptibility to Other Antimalarials

“…It is important to note the essentiality of the PSA for determining and quantifying PPQ resistance, since both GB4 GB4 + F145I and GB4 GB4 + G353V had lower PPQ IC 50 values in this standard concentration-response assay when compared with the isogenic control line GB4 GB4 . These data are consistent with earlier observations that PPQ resistance is only apparent at high drug concentrations, a phenomenon that we suspect is related to concentration-dependent engagement of drug efflux via mutant PfCRT [ 16 , 23 , 29 ].…”

“…This gain of resistance was accompanied by a very substantial loss of parasite fitness, which we posit could preclude this mutation from being competitive in high-transmission African settings with typically mixed infections. We also find that PPQ resistance results in increased sensitization to md-ADQ, quinine, and CQ, creating a rationale for implementing regimens with different drug combinations that exert opposing selective pressures on parasite populations and thereby hinder the spread of multidrug-resistant P falciparum malaria [ 23 ].…”

“…Mutations in pfcrt (PF3D7_0709000) were edited using customized zinc-finger nucleases, as previously described [ 23 ]. The GB4 donor template was constructed by site-directed mutagenesis of the plasmid pfcrt Dd2 - hdhfr to remove the Dd2-specific N326S and I356T mutations ( Supplementary Figure 1A ).…”

“…GB4 asexual blood stage parasites were cultured in human red blood cells and transfected as described in [ 23 , 24 ] and detailed in the Supplementary Methods .…”

Drug resistance of Plasmodium falciparum and Plasmodium vivax isolates in Indonesia

“…This result demonstrates that DHA used in ACT is still highly productive in suppressing parasite density. Another study described a slight decline in PPQ susceptibility, although it did not appear to have reached clinically significant levels [ 91 ]. During observation periods, no 8 SNPs mutation in pvK12 associated with ART resistance, such as M448, T517, F519, I568, S578, D605, and D691, and L708, has been found in Papua and Jambi.…”