Adequate clinical and parasitologic cure by artemisinin combination therapies relies on the artemisinin component and the partner drug. Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes are associated with decreased sensitivity to amodiaquine and lumefantrine, but effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) have not been clearly defined. Individual patient data from 31 clinical trials were harmonized and pooled by using standardized methods from the WorldWide Antimalarial Resistance Network. Data for more than 7,000 patients were analyzed to assess relationships between parasite polymorphisms in pfcrt and pfmdr1 and clinically relevant outcomes after treatment with AL or ASAQ. Presence of the pfmdr1 gene N86 (adjusted hazards ratio = 4.74, 95% confidence interval = 2.29 – 9.78, P < 0.001) and increased pfmdr1 copy number (adjusted hazards ratio = 6.52, 95% confidence interval = 2.36–17.97, P < 0.001) were significant independent risk factors for recrudescence in patients treated with AL. AL and ASAQ exerted opposing selective effects on single-nucleotide polymorphisms in pfcrt and pfmdr1. Monitoring selection and responding to emerging signs of drug resistance are critical tools for preserving efficacy of artemisinin combination therapies; determination of the prevalence of at least pfcrt K76T and pfmdr1 N86Y should now be routine.

Polymorphisms in the Plasmodium falciparum pfmdr1 gene were assayed in pretreatment samples and in samples from patients reinfected following therapy with artemether-lumefantrine. The pfmdr1 alleles 86N, 184F, and 1246D significantly increased in prevalence after treatment. All samples had a single pfmdr1 copy. Treatment with artemether-lumefantrine selects for polymorphisms that may alter antimalarial drug response.Due to widespread resistance of Plasmodium falciparum to drugs such as chloroquine and sulfadoxine-pyrimethamine, artemisinin combination therapy (ACT) is currently advocated in Africa as a means of improving treatment efficacy and slowing the spread of resistance. The rationale behind ACT is to rapidly reduce the parasite burden with a short-acting artemisinin compound, leaving a longer-acting partner drug to eliminate the remaining parasites, thus reducing the chance of selection of drug-resistant parasites. In Southeast Asia, where the risk of reinfection following therapy is low, the combination of artesunate and mefloquine has been effective at slowing the spread of resistance (10). It is unclear whether ACT will be as successful in preventing the selection of resistant parasites in Africa, where parasite transmission rates are generally much higher. In a recent study from Tanzania, treatment with the widely advocated ACT artemether-lumefantrine (AL) was associated with selection of newly infecting parasites containing the pfmdr1 86N allele (16), which has been associated with decreased in vitro sensitivity to artemisinins and lumefantrine (4, 5).We recently completed a clinical trial including AL at a rural site in Uganda with extremely high transmission intensity (1). Briefly, children aged 1 to 10 years with uncomplicated falciparum malaria received directly observed therapy and were followed for 28 days. Molecular genotyping techniques were used to distinguish recrudescent from new infections for all patients failing therapy after day 3. Briefly, filter paper blood samples collected on the day of enrollment and the day of failure (late clinical failure or late parasitological failure) were analyzed for polymorphisms in merozoite surface protein 1 (MSP-1) and MSP-2 using nested PCR as previously described (2). An outcome was defined as recrudescence if all MSP-1 and MSP-2 gene alleles present at the time of failure were present at the time of treatment initiation and as a new infection otherwise. To test the hypothesis that AL selects for polymorphisms in the pfmdr1 gene, we compared the prevalences of key alleles and pfmdr1 copy numbers between pretreatment isolates and new isolates following treatment with AL.Polymorphisms studied were pfmdr1 N86Y, Y184F, N1042D, and D1246Y. Alleles were identified using nested PCR and restriction fragment length polymorphism methods, and copy number was assessed by TaqMan quantitative PCR as previously described (4, 12). Single (3D7)-and three-copy (W2mef) standards were used as positive controls. Reactions were done in quadruplicate and repeated for a cha...

Wellcome Trust and the Bill & Melinda Gates Foundation.

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...

The HIV-1 seroprevalence rate was surprisingly high in adults presenting with malaria. This finding supports the implementation of routine HIV counseling and testing for adults with uncomplicated falciparum malaria. HIV-1 infection increased the susceptibility to new malarial infections but did not increase the risk of recrudescences in adults.