Comparative Impacts Over 5 Years of Artemisinin-Based Combination Therapies on Plasmodium falciparum Polymorphisms That Modulate Drug Sensitivity in Ugandan Children

(See the editorial commentary by Taylor and Juliano on pages 335–7.) Artemisinin-based combination therapies (ACTs) have shown excellent efficacy and are now recommended to treat falciparum malaria in nearly all countries [1]. ACTs include potent, short-acting artemisinins that rapidly reduce parasite biomass and alleviate malaria symptoms and longer-acting partner drugs that improve antimalarial efficacy and reduce the risk of selection for artemisinin resistance [2]. However, as partner drugs circulate well after artemisinins have been cleared, there is concern that subsequent infections will be exposed to subtherapeutic concentrations, facilitating the selection of parasites with reduced sensitivity to the partner drugs. Artemether-lumefantrine (AL) is the most widely recommended ACT in Africa and the national malaria treatment regimen in Uganda [1, 3]. It has shown outstanding efficacy [4, 5], but treatment selects in recurrent Plasmodium falciparum infections for polymorphisms in pfcrt and pfmdr1 [6–11]—genes encoding 2 putative drug transporters—that reduce sensitivity to artemether, lumefantrine, and other antimalarial drugs [12–15]. AL exerts an opposite selective pressure compared to that of the aminoquinolines chloroquine and amodiaquine. Specifically, the aminoquinolines select for the mutant pfcrt 76T, pfmdr1 86Y, and pfmdr1 1246Y alleles, which decrease sensitivity to these drugs, whereas AL selects for the wild-type alleles [6–11, 16]. AL also selected for the I876 polymorphism in pfmrp1, which encodes another putative drug transporter, in Tanzania [17]. Three additional pfmdr1 polymorphisms (S1034C, N1042D, and increased gene copy number) are associated with altered sensitivity to some drugs, but are primarily seen outside of Africa [12, 15, 18–20]. Ex vivo sensitivities of field isolates to lumefantrine have varied widely, but clinically relevant resistance does not appear to be a problem [14, 21, 22]. Analysis of parasites selected in vitro for high-level lumefantrine resistance demonstrated multiple differentially expressed genes, including pfmdr1, but the phenotype was unstable [23]. Dihydroartemisinin-piperaquine (DP) has shown excellent efficacy in clinical trials in Africa [16, 24–27], but has only been adopted as a first-line therapy in Southeast Asia [1]. Particularly in areas with high malaria transmission intensity, DP benefits from the pharmacokinetics of piperaquine, which has a much longer half-life (3–4 weeks) than that of lumefantrine (3–5 days) and other ACT partner drugs [28], yielding a long posttreatment prophylactic effect [5, 24, 27]. Piperaquine was used extensively to prevent and treat malaria decades ago in China [29, 30], but reported resistance led to reduced use by the 1980s. More recently, with implementation of DP as a standard therapy, piperaquine resistance does not appear to be a major problem, although ex vivo sensitivities of field isolates to piperaquine have varied [14, 22, 31, 32]. Mechanisms of resistance to piperaquine are poorly understood. Parasites selected in vitro for resistance acquired a number of genetic changes, including a novel mutation in pfcrt and deamplification of pfmdr1, but the phenotype was unstable [33]. AL replaced chloroquine plus sulfadoxine-pyrimethamine as the first-line regimen for uncomplicated malaria in Uganda in 2004, although implementation did not begin until 2006 and was initially slow [34, 35]. With improved utilization of AL in recent years, it was of interest to determine the prevalence over time of parasite polymorphisms that alter sensitivity to ACT components and to determine how use of AL impacts upon these polymorphisms. Therefore, we analyzed the prevalence of polymorphisms of interest in samples from a 5-year longitudinal trial comparing the antimalarial efficacies of AL and DP in Ugandan children. Polymorphisms associated with reduced sensitivity to AL increased markedly in prevalence over the course of the study, and this increase was greater in children treated with AL compared to those treated with DP, consistent with our demonstration of opposite selective pressures of the 2 regimens.