Emerging Southeast Asian PfCRT mutations confer Plasmodium falciparum resistance to the first-line antimalarial piperaquine

Abstract

The widely used antimalarial combination therapy dihydroartemisinin + piperaquine (DHA + PPQ) has failed in Cambodia. Here, we perform a genomic analysis that reveals a rapid increase in the prevalence of novel mutations in the Plasmodium falciparum chloroquine resistance transporter PfCRT following DHA + PPQ implementation. These mutations occur in parasites harboring the K13 C580Y artemisinin resistance marker. By introducing PfCRT mutations into sensitive Dd2 parasites or removing them from resistant Cambodian isolates, we show that the H97Y, F145I, M343L, or G353V mutations each confer resistance to PPQ, albeit with fitness costs for all but M343L. These mutations sensitize Dd2 parasites to chloroquine, amodiaquine, and quinine. In Dd2 parasites, multicopy plasmepsin 2, a candidate molecular marker, is not necessary for PPQ resistance. Distended digestive vacuoles were observed in pfcrt-edited Dd2 parasites but not in Cambodian isolates. Our findings provide compelling evidence that emerging mutations in PfCRT can serve as a molecular marker and mediator of PPQ resistance.

An open dataset of Plasmodium falciparum genome variation in 7,000 worldwide samples

“…Antimalarial drug resistance represents a major focus of research for many partner studies within the Pf Community Project, and this dataset therefore contains a significant body of data that have appeared in previous reports on drug resistance. Readers are referred to these publications for more detailed analyses of local patterns of resistance [9][10][11][12][13][14][16][17][18][19][20][21][22] and of resistance to specific drugs including chloroquine 16,21 , sulfadoxine-pyrimethamine 16,19,21 and artemisinin combination therapy [9][10][11][13][14][15]17,18,21,22 .…”

“…The crt locus is also relevant to other types of drug resistance, e.g. crt variants that are relatively specific to SE Asia form the genetic background of artemisinin resistance, and newly emerging crt alleles have been associated with the spread of ACT failure due to piperaquine resistance 13,14,22,68 .…”

“…9 Data produced by the Pf Community Project have been used to address a broad range of research questions, both by the groups that generated samples and data and by the wider research community, and have generated over 50 previous publications (refs . These data have become a key resource for the epidemiology and population genetics of antimalarial drug resistance [9][10][11][12][13][14][15][16][17][18][19][20][21][22] and an important platform for the discovery of new genetic markers and mechanisms of resistance through genome-wide association studies [23][24][25][26][27] and combined genome-transcriptome analysis. 28 The data have also been used to study gene deletions that cause failure of rapid diagnostic tests 29 ; to characterise genetic variation in malaria vaccine antigens 30,31 ; to screen for new vaccine candidates 32 ; to investigate specific host-parasite interactions 33,34 ; and to describe the evolutionary adaptation and diversification of local parasite populations.…”

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Evolution and expansion of multidrug resistant malaria in Southeast Asia: a genomic epidemiology study

“…The recent, rapid international expansion of some KEL1/PLA1 subgroups after several years of confinement in Cambodia raises the question of whether new genetic changes have produced advantageous phenotypic effects in these subgroups. One candidate set of driver mutations are substitutions in the crt gene (PF3D7_0709000) that were recently associated with piperaquine resistance 22 . To investigate this possibility, we calculated the changes in allele frequency in ESEA for all non-synonymous crt SNPs from earlier sampling years (2007-2011) to later years (2016-2018) (Supplementary Table 3).…”