Impact of Antimalarial Treatment and Chemoprevention on the Drug Sensitivity of Malaria Parasites Isolated from Ugandan Children

Abstract

Changing treatment practices may be selecting for changes in the drug sensitivity of malaria parasites. We characterized ex vivo drug sensitivity and parasite polymorphisms associated with sensitivity in 459 Plasmodium falciparum samples obtained from subjects enrolled in two clinical trials in Tororo, Uganda, from 2010 to 2013. Sensitivities to chloroquine and monodesethylamodiaquine varied widely; sensitivities to quinine, dihydroartemisinin, lumefantrine, and piperaquine were generally good. Associations between ex vivo drug sensitivity and parasite polymorphisms included decreased chloroquine and monodesethylamodiaquine sensitivity and increased lumefantrine and piperaquine sensitivity with pfcrt 76T, as well as increased lumefantrine sensitivity with pfmdr1 86Y, Y184, and 1246Y. Over time, ex vivo sensitivity decreased for lumefantrine and piperaquine and increased for chloroquine, the prevalences of pfcrt K76 and pfmdr1 N86 and D1246 increased, and the prevalences of pfdhfr and pfdhps polymorphisms associated with antifolate resistance were unchanged. In recurrent infections, recent prior treatment with artemether-lumefantrine was associated with decreased ex vivo lumefantrine sensitivity and increased prevalence of pfcrt K76 and pfmdr1 N86, 184F, and D1246. In children assigned chemoprevention with monthly dihydroartemisinin-piperaquine with documented circulating piperaquine, breakthrough infections had increased the prevalence of pfmdr1 86Y and 1246Y compared to untreated controls. The noted impacts of therapy and chemoprevention on parasite polymorphisms remained significant in multivariate analysis correcting for calendar time. Overall, changes in parasite sensitivity were consistent with altered selective pressures due to changing treatment practices in Uganda. These changes may threaten the antimalarial treatment and preventive efficacies of artemether-lumefantrine and dihydroartemisinin-piperaquine, respectively. M alaria, in particular disease caused by Plasmodium falciparum, remains an overwhelming problem in most of subSaharan Africa (1, 2). Malaria control was greatly limited by resistance to chloroquine (CQ) and sulfadoxine-pyrimethamine (SP), leading to adoption of artemisinin-based combination therapy (ACT) as the standard treatment for uncomplicated falciparum malaria in the last decade (3). ACT consists of a rapid-acting artemisinin derivative plus a longer-acting partner drug that clears parasites not eliminated by the artemisinin component and limits selection of artemisinin resistance (4, 5). In nearly all countries in sub-Saharan Africa, either artemether-lumefantrine (AL) or artesunate-amodiaquine (AS-AQ) is recommended to treat uncomplicated malaria (6). Other ACTs are dihydroartemisinin (DHA)-piperaquine (DP), a first-line therapy in some countries in Asia, with particular promise for malaria prevention due to the extended halflife of piperaquine (7), and artesunate-mefloquine (AS-MQ), which is used in some countries in Asia and South America. In Uganda, AL was named the national ma...

Intermittent Preventive Treatment with Dihydroartemisinin-Piperaquine in Ugandan Schoolchildren Selects for Plasmodium falciparum Transporter Polymorphisms That Modify Drug Sensitivity

“…We tested whether DP selected for parasites with genotypes associated with altered sensitivity to aminoquinolines. Compared to parasites not under drug pressure, those that emerged within 30 days of IPT with DP were more likely to harbor two mutations, pfmdr1 86Y and pfcrt 76T; these mutations are associated with resistance to chloroquine and amodiaquine (36,(43)(44)(45). Thus, the marked preventive effi- cacy of IPT with DP may be accompanied by selection of decreased sensitivity to aminoquinolines.…”

“…Thus, new infections emerging within 2 months of treatment with AL showed selection of wild-type sequences at the pfcrt K76T and pfmdr1 N86Y and D1246Y alleles (26)(27)(28)(29); mutant sequences are selected at these same alleles by aminoquinolines. Of recent concern has been resistance to artemisinins, manifest as delayed parasite clearance after therapy, in Southeast Asia (22,(30)(31)(32), but recent studies utilizing clinical, parasitological, and molecular markers (33,34) suggest that the artemisinin-resistant phenotype is not yet prevalent in Uganda (26,35,36) or other parts of Africa (37,38).…”

Lack of Artemisinin Resistance in Plasmodium falciparum in Uganda Based on Parasitological and Molecular Assays

“…With adoption of artemether-lumefantrine as standard therapy for malaria within the last decade, parasites have demonstrated increasing prevalence of pfcrt and pfmdr1 alleles that mediate decreased lumefantrine sensitivity (20) and, in ex vivo assays, decreasing sensitivity to lumefantrine (17). Thus, even without the artemisinin resistance phenotype, changes in parasite drug sensitivity may threaten the antimalarial efficacies of ACTs, in particular artemether-lumefantrine, in Uganda.…”

“…However, the recent spread of resistance in Asia suggests that the introduction or de novo emergence of resistance in Africa, the major world focus of P. falciparum malaria, is likely. Further, altered sensitivity to artemisinin partner drugs already threatens the efficacy of ACTs for the treatment of P. falciparum malaria in Africa (17,20). Thus, regular surveillance for markers of artemisinin resistance is needed across Africa.…”

“…The susceptibility of fresh isolates to DHA and chloroquine was assessed by a standard ex vivo histidine-rich protein 2-based enzyme-linked immunosorbent microplate assay, as previously described (17), except that drugs were freshly diluted from frozen stocks prior to each assay. Results from single assays were fitted to a variable-slope sigmoidal function using GraphPad Prism 6.0f to generate 50% inhibitory concentrations (IC 50 s).…”