Plasmodium falciparum resistance to piperaquine driven by PfCRT

The Articles by William Hamilton and colleagues¹ and Rob van der Pluijm and colleagues² illustrate the plummeting clinical efficacy of dihydroartemisinin–piperaquine as a first-line treatment for Plasmodium falciparum malaria in southeast Asia. These authors also report a rapid regional spread of clonal parasite lineages harbouring novel variants of the P falciparum chloroquine resistance transporter PfCRT (emerging on the chloroquine-resistant Dd2 isoform). These lineages exclusively harboured the Cys580Tyr mutation in the K13 gene that is associated with decreased artemisinin efficacy. These studies raise important questions about whether these novel PfCRT variants cause piperaquine resistance, how they effect other antimalarials, and whether changes in prevalence over time reflect differences in parasite fitness.

Here, we show that the now predominant PfCRT Thr93Ser and Ile218Phe mutations, which of all mutations have expanded the most rapidly in the past 5 years,^1,2 confer piperaquine resistance when individually edited into the pfcrt locus of Dd2 parasites. Assays with cultured intra-erythrocytic parasites show 8–13% survival at piperaquine concentrations ranging from 200 nM to 1600 nM (figure; appendix p 5). The Phe145Ile mutation was highly piperaquine-resistant, with 57–69% survival at these elevated concentrations, consistent with previous findings.³ Parental or control Dd2 parasites with an edited pfcrt showed less than 1% survival, reflecting background rates. Quantitative PCR assays showed a single copy of plasmepsin II in all edited parasites (data not shown), indicating that amplification of this initial resistance marker^4,5 was not required for piperaquine resistance.

Figure: Piperaquine survival and fitness of pfcrt-edited lines.

(A) Survival of pfcrt-modified parasite lines cultured with various concentrations of piperaquine (0–6 h rings, treated for 48 h). The 10% cutoff represents a standard working threshold for piperaquine resistance at 200 nM piperaquine. Data are mean with SEM for three to four independent assays done in duplicate. (B) In-vitro growth of the indicated parasite lines in competition with a GFP-positive reporter line. Data are mean with SEM for two independent experiments done in triplicate. Methods are provided in the appendix. GFP=green fluorescent protein.

Whole-genome sequence data of 84 Cambodian isolates support the expansion of Thr93Ser and Ile218Phe between 2013 and 2016, overtaking the prevalence of Phe145Ile (appendix p 4). Recent years showed a reduced percentage of parasites harbouring four or more copies of plasmepsins II and III, although parasites with two to three copies remained the majority (appendix p 4). pfmdr1 amplification, a marker of reduced susceptibility to lumefantrine and mefloquine, became less common over time (appendix p 4). Survival rates of piperaquine-treated cultured parasites increased over the years (appendix p 4), mirroring increasing dihydroartemisinin–piperaquine clinical failure rates. Emerging PfCRT mutations also increased P falciparum susceptibility to chloroquine, amodiaquine, quinine, pyronaridine, and ferroquine, with the Phe145Ile mutation causing the greatest sensitisation (appendix pp 6, 7). Dihydroartemisinin, lumefantrine, and mefloquine were unchanged. These data highlight the broad effect of PfCRT mutations on multiple antimalarials.

To test for differences in parasite fitness between mutants, we used a competitive growth rate assay in which each parasite line was individually cocultured with an isogenic green fluorescent protein (GFP)-positive Dd2 line. Parasites expressing the Thr93Ser allele showed a negligible fitness cost compared with control Dd2 pfcrt-edited parasites, with both lines out-proliferating GFP-positive Dd2 parasites (figure). The Ile218Phe mutation showed a mild growth attenuation. Phe145Ile parasites showed a substantial fitness cost, potentially explaining why this allele is ceding ground to the less resistant but fitter Thr93Ser and Ile218Phe mutations.

The data support a key role for PfCRT mutations in driving the recent expansion of highly piperaquine-resistant parasites in southeast Asia and highlight the need for vigilance in screening for novel PfCRT mutations in other malaria-endemic regions, notably in Africa or South America where piperaquine use has been increasing.