Piperaquine resistance is associated with a copy number variation on chromosome 5 in drug-pressured Plasmodium falciparum parasites

Abstract

The combination of piperaquine and dihydroartemisinin has recently become the official first-line therapy in several Southeast Asian countries. The pharmacokinetic mismatching of these drugs, whose plasma half-lives are ~20 days and ~1 h, respectively, implies that recrudescent or new infections emerging shortly after treatment cessation will encounter piperaquine as a monotherapy agent. This creates substantial selection pressure for the emergence of resistance. To elucidate potential resistance determinants, we subjected cloned Plasmodium falciparum Dd2 parasites to continuous piperaquine pressure in vitro (47 nM; ~2-fold higher than the Dd2 50% inhibitory concentration [IC(50)]). The phenotype of outgrowth parasites was assayed in two clones, revealing an IC(50) against piperaquine of 2.1 μM and 1.7 μM, over 100-fold greater than that of the parent. To identify the genetic determinant of resistance, we employed comparative whole-genome hybridization analysis. Compared to the Dd2 parent, this analysis found (in both resistant clones) a novel single-nucleotide polymorphism in P. falciparum crt (pfcrt), deamplification of an 82-kb region of chromosome 5 (that includes pfmdr1), and amplification of an adjacent 63-kb region of chromosome 5. Continued propagation without piperaquine selection pressure resulted in "revertant" piperaquine-sensitive parasites. These retained the pfcrt polymorphism and further deamplified the chromosome 5 segment that encompasses pfmdr1; however, these two independently generated revertants both lost the neighboring 63-kb amplification. These results suggest that a copy number variation event on chromosome 5 (825600 to 888300) is associated with piperaquine resistance. Transgene expression studies are underway with individual genes in this segment to evaluate their contribution to piperaquine resistance.

Fig. 1.

Flow diagram illustrating the selection of piperaquine-resistant P. falciparum lines and derivation of the revertant clones following prolonged culture in the absence of drug pressure. Piperaquine-resistant clones 1 and 2 were cultured under 47 nM continuous piperaquine pressure and cloned by limiting dilution. All genetic and phenotypic analyses were conducted with cloned lines, other than when we assessed the stability of the piperaquine-resistant phenotype, which was measured with lines maintained without piperaquine pressure for 70 days. The parental Dd2 1pa clone, the piperaquine-resistant clones 1 and 2, and the piperaquine-revertant clone 1 were further analyzed by whole-genome hybridization.

Fig. 2.

In vitro antimalarial response of the piperaquine-resistant and revertant clones. In vitro [³H]hypoxanthine incorporation assays (72 h) were performed with the piperaquine-resistant clones, revertant clones, and the parental Dd2 line, which were tested in duplicate against each antimalarial drug on 4 to 15 separate occasions. IC₅₀s (shown as means ± SEMs) were derived by nonlinear regression analysis. Numerical values are listed in Table S2 in the supplemental material. For statistical comparisons, Mann-Whitney U tests were performed (P values of <0.05 [*], <0.01 [**], and <0.001 [***], unless indicated comparison is with the Dd2 parental line). CQ, chloroquine; DHA, dihydroartemisinin; LMF, lumefantrine; mdAQ, monodesethylamodiaquine; MFQ, mefloquine; PQP, piperaquine.

Fig. 3.

Time course of piperaquine accumulation in P. falciparum. Cell suspensions of either Dd2 (PQP sensitive [black circles]) or PQP clone 1 (PQP resistant [orange squares]) were incubated in 5 nM [³H]piperaquine at 37°C, and the amounts of internalized piperaquine were determined at the time points indicated. Results represent the means ± SEMs of three independent experiments.

Fig. 4.

Genetic analysis of piperaquine-resistant parasites. (A) Genomic tiling arrays identified shared-copy-number variations in the piperaquine-resistant clones compared to those of the parental Dd2 line. Log₂ ratios of probe intensities, plotted along chromosome 5 for the hybridization of genomic DNA from PQP clone 1 relative to the Dd2 parental line, demonstrated an increased copy number of ch5 with the approximate breakpoints 825600 and 888300 (probe intensity reflects the comparative abundance of genomic DNA from the two lines hybridized to the microarray; orange indicates no difference or similar genomic abundance; white/yellow indicates higher probe intensity for PQP clone 1 compared to that for Dd2 1pa; red/brown indicates lower probe intensity for PQP clone 1 or rev 1 compared to that for Dd2 1pa). In addition, there was a reduction in the copy number of the region surrounding pfmdr1 (888300 to 970100). Comparison of the PQP clone 1 and revertant clone 1 found deamplification of the region that was amplified in PQP clone 1 (825600 to 888300) and further deamplification of the region surrounding pfmdr1 (888300 to 970100). Locations of PFE1010w, PFE1085w, and pfmdr1 are noted. (B) Tiling array analysis also identified a polymorphism in pfcrt. PCR amplification and sequencing revealed this polymorphism to be a nonsynonymous change in the coding region, encoding the predicted point mutation C101F. Copy number variations identified by genomic tiling array were confirmed by quantitative PCR for PFE1010w (ch5, 831614 to 834340) (C), PFE1085w (ch5, 882373 to 884898) (D), and pfmdr1 (PFE1150w; ch5, 957885 to 962144) (E). P. falciparum 3D7 and FCB were included as controls. 3D7 has been demonstrated previously to have 1 copy of pfmdr1, and FCB has 2 copies.