Evolution of Partial Resistance to Artemisinins in Malaria Parasites in Uganda.

BACKGROUND: Partial resistance of Plasmodium falciparum to the artemisinin component of artemisinin-based combination therapies, the most important malaria drugs, emerged in Southeast Asia and now threatens East Africa. Partial resistance, which manifests as delayed clearance after therapy, is mediated principally by mutations in the kelch protein K13 (PfK13). Limited longitudinal data are available on the emergence and spread of artemisinin resistance in Africa. METHODS: We performed annual surveillance among patients who presented with uncomplicated malaria at 10 to 16 sites across Uganda from 2016 through 2022. We sequenced the gene encoding kelch 13 (pfk13) and analyzed relatedness using molecular methods. We assessed malaria metrics longitudinally in eight Ugandan districts from 2014 through 2021. RESULTS: By 2021-2022, the prevalence of parasites with validated or candidate resistance markers reached more than 20% in 11 of the 16 districts where surveillance was conducted. The PfK13 469Y and 675V mutations were seen in far northern Uganda in 2016-2017 and increased and spread thereafter, reaching a combined prevalence of 10 to 54% across much of northern Uganda, with spread to other regions. The 469F mutation reached a prevalence of 38 to 40% in one district in southwestern Uganda in 2021-2022. The 561H mutation, previously described in Rwanda, was first seen in southwestern Uganda in 2021, reaching a prevalence of 23% by 2022. The 441L mutation reached a prevalence of 12 to 23% in three districts in western Uganda in 2022. Genetic analysis indicated local emergence of mutant parasites independent of those in Southeast Asia. The emergence of resistance was observed predominantly in areas where effective malaria control had been discontinued or transmission was unstable. CONCLUSIONS: Data from Uganda showed the emergence of partial resistance to artemisinins in multiple geographic locations, with increasing prevalence and regional spread over time. (Funded by the National Institutes of Health.).

Seasonal Malaria Chemoprevention Drug Levels and Drug Resistance Markers in Children With or Without Malaria in Burkina Faso: A Case-Control Study.

BACKGROUND: Despite scale-up of seasonal malaria chemoprevention (SMC) with sulfadoxine-pyrimethamine and amodiaquine (SP-AQ) in children 3-59 months of age in Burkina Faso, malaria incidence remains high, raising concerns regarding SMC effectiveness and selection of drug resistance. Using a case-control design, we determined associations between SMC drug levels, drug resistance markers, and presentation with malaria. METHODS: We enrolled 310 children presenting at health facilities in Bobo-Dioulasso. Cases were SMC-eligible children 6-59 months of age diagnosed with malaria. Two controls were enrolled per case: SMC-eligible children without malaria; and older (5-10 years old), SMC-ineligible children with malaria. We measured SP-AQ drug levels among SMC-eligible children and SP-AQ resistance markers among parasitemic children. Conditional logistic regression was used to compute odds ratios (ORs) comparing drug levels between cases and controls. RESULTS: Compared to SMC-eligible controls, children with malaria were less likely to have any detectable SP or AQ (OR, 0.33 [95% confidence interval, .16-.67]; P = .002) and have lower drug levels (P < .05). Prevalences of mutations mediating high-level SP resistance were rare (0%-1%) and similar between cases and SMC-ineligible controls (P > .05). CONCLUSIONS: Incident malaria among SMC-eligible children was likely due to suboptimal levels of SP-AQ, resulting from missed cycles rather than increased antimalarial resistance to SP-AQ.

Susceptibility of Ugandan Plasmodium falciparum Isolates to the Antimalarial Drug Pipeline.

Malaria, especially Plasmodium falciparum infection, remains an enormous problem, and its treatment and control are seriously challenged by drug resistance. New antimalarial drugs are needed. To characterize the Medicines for Malaria Venture pipeline of antimalarials under development, we assessed the ex vivo drug susceptibilities to 19 compounds targeting or potentially impacted by mutations in P. falciparum ABC transporter I family member 1, acetyl-CoA synthetase, cytochrome b, dihydroorotate dehydrogenase, elongation factor 2, lysyl-tRNA synthetase, phenylalanyl-tRNA synthetase, plasmepsin X, prodrug activation and resistance esterase, and V-type H+ ATPase of 998 fresh P. falciparum clinical isolates collected in eastern Uganda from 2015 to 2022. Drug susceptibilities were assessed by 72-h growth inhibition (half-maximum inhibitory concentration [IC50]) assays using SYBR green. Field isolates were highly susceptible to lead antimalarials, with low- to midnanomolar median IC50s, near values previously reported for laboratory strains, for all tested compounds. However, outliers with decreased susceptibilities were identified. Positive correlations between IC50 results were seen for compounds with shared targets. We sequenced genes encoding presumed targets to characterize sequence diversity, search for polymorphisms previously selected with in vitro drug pressure, and determine genotype-phenotype associations. We identified many polymorphisms in target genes, generally in <10% of isolates, but none were those previously selected in vitro with drug pressure, and none were associated with significantly decreased ex vivo drug susceptibility. Overall, Ugandan P. falciparum isolates were highly susceptible to 19 compounds under development as next-generation antimalarials, consistent with a lack of preexisting or novel resistance-conferring mutations in circulating Ugandan parasites. IMPORTANCE Drug resistance necessitates the development of new antimalarial drugs. It is important to assess the activities of compounds under development against parasites now causing disease in Africa, where most malaria cases occur, and to determine if mutations in these parasites may limit the efficacies of new agents. We found that African isolates were generally highly susceptible to the 19 studied lead antimalarials. Sequencing of the presumed drug targets identified multiple mutations in these genes, but these mutations were generally not associated with decreased antimalarial activity. These results offer confidence that the activities of the tested antimalarial compounds now under development will not be limited by preexisting resistance-mediating mutations in African malaria parasites.

Decreased susceptibility of Plasmodium falciparum to both dihydroartemisinin and lumefantrine in northern Uganda.

Artemisinin partial resistance may facilitate selection of Plasmodium falciparum resistant to combination therapy partner drugs. We evaluated 99 P. falciparum isolates collected in 2021 from northern Uganda, where resistance-associated PfK13 C469Y and A675V mutations have emerged, and eastern Uganda, where these mutations are uncommon. With the ex vivo ring survival assay, isolates with the 469Y mutation (median survival 7.3% for mutant, 2.5% mixed, and 1.4% wild type) and/or mutations in Pfcoronin or falcipain-2a, had significantly greater survival; all isolates with survival >5% had mutations in at least one of these proteins. With ex vivo growth inhibition assays, susceptibility to lumefantrine (median IC50 14.6 vs. 6.9 nM, p < 0.0001) and dihydroartemisinin (2.3 vs. 1.5 nM, p = 0.003) was decreased in northern vs. eastern Uganda; 14/49 northern vs. 0/38 eastern isolates had lumefantrine IC50 > 20 nM (p = 0.0002). Targeted sequencing of 819 isolates from 2015-21 identified multiple polymorphisms associated with altered drug susceptibility, notably PfK13 469Y with decreased susceptibility to lumefantrine (p = 6 × 10-8) and PfCRT mutations with chloroquine resistance (p = 1 × 10-20). Our results raise concern regarding activity of artemether-lumefantrine, the first-line antimalarial in Uganda.

Susceptibilities of Ugandan Plasmodium falciparum Isolates to Proteasome Inhibitors.

The proteasome is a promising target for antimalarial chemotherapy. We assessed ex vivo susceptibilities of fresh Plasmodium falciparum isolates from eastern Uganda to seven proteasome inhibitors: two asparagine ethylenediamines, two macrocyclic peptides, and three peptide boronates; five had median IC50 values <100 nM. TDI8304, a macrocylic peptide lead compound with drug-like properties, had a median IC50 of 16 nM. Sequencing genes encoding the ß2 and ß5 catalytic proteasome subunits, the predicted targets of the inhibitors, and five additional proteasome subunits, identified two mutations in ß2 (I204T, S214F), three mutations in ß5 (V2I, A142S, D150E), and three mutations in other subunits. The ß2 S214F mutation was associated with decreased susceptibility to two peptide boronates, with IC50s of 181 nM and 2635 nM against mutant versus 62 nM and 477 nM against wild type parasites for MMV1579506 and MMV1794229, respectively, although significance could not be formally assessed due to the small number of mutant parasites with available data. The other ß2 and ß5 mutations and mutations in other subunits were not associated with susceptibility to tested compounds. Against culture-adapted Ugandan isolates, two asparagine ethylenediamines and the peptide proteasome inhibitors WLW-vinyl sulfone and WLL-vinyl sulfone (which were not studied ex vivo) demonstrated low nM activity, without decreased activity against ß2 S214F mutant parasites. Overall, proteasome inhibitors had potent activity against P. falciparum isolates circulating in Uganda, and genetic variation in proteasome targets was uncommon.

The Multistage Antimalarial Compound Calxinin Perturbates P. falciparum Ca2+ Homeostasis by Targeting a Unique Ion Channel.

Malaria elimination urgently needs novel antimalarial therapies that transcend resistance, toxicity, and high costs. Our multicentric international collaborative team focuses on developing multistage antimalarials that exhibit novel mechanisms of action. Here, we describe the design, synthesis, and evaluation of a novel multistage antimalarial compound, 'Calxinin'. A compound that consists of hydroxyethylamine (HEA) and trifluoromethyl-benzyl-piperazine. Calxinin exhibits potent inhibitory activity in the nanomolar range against the asexual blood stages of drug-sensitive (3D7), multidrug-resistant (Dd2), artemisinin-resistant (IPC4912), and fresh Kenyan field isolated Plasmodium falciparum strains. Calxinin treatment resulted in diminished maturation of parasite sexual precursor cells (gametocytes) accompanied by distorted parasite morphology. Further, in vitro liver-stage testing with a mouse model showed reduced parasite load at an IC50 of 79 nM. A single dose (10 mg/kg) of Calxinin resulted in a 30% reduction in parasitemia in mice infected with a chloroquine-resistant strain of the rodent parasite P. berghei. The ex vivo ookinete inhibitory concentration within mosquito gut IC50 was 150 nM. Cellular in vitro toxicity assays in the primary and immortalized human cell lines did not show cytotoxicity. A computational protein target identification pipeline identified a putative P. falciparum membrane protein (Pf3D7_1313500) involved in parasite calcium (Ca2+) homeostasis as a potential Calxinin target. This highly conserved protein is related to the family of transient receptor potential cation channels (TRP-ML). Target validation experiments showed that exposure of parasitized RBCs (pRBCs) to Calxinin induces a rapid release of intracellular Ca2+ from pRBCs; leaving de-calcinated parasites trapped in RBCs. Overall, we demonstrated that Calxinin is a promising antimalarial lead compound with a novel mechanism of action and with potential therapeutic, prophylactic, and transmission-blocking properties against parasites resistant to current antimalarials.

Decreased Susceptibility to Dihydrofolate Reductase Inhibitors Associated With Genetic Polymorphisms in Ugandan Plasmodium falciparum Isolates.

BACKGROUND: The Plasmodium falciparum dihydrofolate reductase (PfDHFR) inhibitors pyrimethamine and cycloguanil (the active metabolite of proguanil) have important roles in malaria chemoprevention, but drug resistance challenges their efficacies. A new compound, P218, was designed to overcome resistance, but drug-susceptibility data for P falciparum field isolates are limited. METHODS: We studied ex vivo PfDHFR inhibitor susceptibilities of 559 isolates from Tororo and Busia districts, Uganda, from 2016 to 2020, sequenced 383 isolates, and assessed associations between genotypes and drug-susceptibility phenotypes. RESULTS: Median half-maximal inhibitory concentrations (IC50s) were 42 100 nM for pyrimethamine, 1200 nM for cycloguanil, 13000 nM for proguanil, and 0.6 nM for P218. Among sequenced isolates, 3 PfDHFR mutations, 51I (100%), 59R (93.7%), and 108N (100%), were very common, as previously seen in Uganda, and another mutation, 164L (12.8%), had moderate prevalence. Increasing numbers of mutations were associated with decreasing susceptibility to pyrimethamine, cycloguanil, and P218, but not proguanil, which does not act directly against PfDHFR. Differences in P218 susceptibilities were modest, with median IC50s of 1.4 nM for parasites with mixed genotype at position 164 and 5.7 nM for pure quadruple mutant (51I/59R/108N/164L) parasites. CONCLUSIONS: Resistance-mediating PfDHFR mutations were common in Ugandan isolates, but P218 retained excellent activity against mutant parasites.

Drug susceptibility of Plasmodium falciparum in eastern Uganda: a longitudinal phenotypic and genotypic study.

BACKGROUND: Treatment and control of malaria depends on artemisinin-based combination therapies (ACTs) and is challenged by drug resistance, but thus far resistance to artemisinins and partner drugs has primarily occurred in southeast Asia. The aim of this study was to characterise antimalarial drug susceptibility of Plasmodium falciparum isolates from Tororo and Busia districts in Uganda. METHODS: In this prospective longitudinal study, P falciparum isolates were collected from patients aged 6 months or older presenting at the Tororo District Hospital (Tororo district, a site with relatively low malaria incidence) or Masafu General Hospital (Busia district, a high-incidence site) in eastern Uganda with clinical symptoms of malaria, a positive Giemsa-stained blood film for P falciparum, and no signs of severe disease. Ex-vivo susceptibilities to ten antimalarial drugs were measured using a 72-h microplate growth inhibition assay with SYBR Green detection. Relevant P falciparum genetic polymorphisms were characterised by molecular methods. We compared results with those from earlier studies in this region and searched for associations between drug susceptibility and parasite genotypes. FINDINGS: From June 10, 2016, to July 29, 2019, 361 P falciparum isolates were collected in the Busia district and 79 in the Tororo district from 440 participants. Of 440 total isolates, 392 (89%) successfully grew in culture and showed excellent drug susceptibility for chloroquine (median half-maximal inhibitory concentration [IC50] 20·0 nM [IQR 12·0-26·0]), monodesethylamodiaquine (7·1 nM [4·3-8·9]), pyronaridine (1·1 nM [0·7-2·3]), piperaquine (5·6 nM [3·3-8·6]), ferroquine (1·8 nM [1·5-3·3]), AQ-13 (24·0 nM [17·0-32·0]), lumefantrine (5·1 nM [3·2-7·7]), mefloquine (9·5 nM [6·6-13·0]), dihydroartemisinin (1·5 nM [1·0-2·0]), and atovaquone (0·3 nM [0·2-0·4]). Compared with results from our study in 2010-13, significant improvements in susceptibility were seen for chloroquine (median IC50 288·0 nM [IQR 122·0-607·0]; p<0·0001), monodesethylamodiaquine (76·0 nM [44·0-137]; p<0·0001), and piperaquine (21·0 nM [7·6-43·0]; p<0·0001), a small but significant decrease in susceptibility was seen for lumefantrine (3·0 nM [1·1-7·6]; p<0·0001), and no change in susceptibility was seen with dihydroartemisinin (1·3 nM [0·8-2·5]; p=0·64). Chloroquine resistance (IC50>100 nM) was more common in isolates from the Tororo district (11 [15%] of 71), compared with those from the Busia district (12 [4%] of 320; p=0·0017). We showed significant increases between 2010-12 and 2016-19 in the prevalences of wild-type P falciparum multidrug resistance protein 1 (PfMDR1) Asn86Tyr from 60% (391 of 653) to 99% (418 of 422; p<0·0001), PfMDR1 Asp1246Tyr from 60% (390 of 650) to 90% (371 of 419; p<0·0001), and P falciparum chloroquine resistance transporter (PfCRT) Lys76Thr from 7% (44 of 675) to 87% (364 of 417; p<0·0001). INTERPRETATION: Our results show marked changes in P falciparum drug susceptibility phenotypes and genotypes in Uganda during the past decade. These results suggest that additional changes will be seen over time and continued surveillance of susceptibility to key ACT components is warranted. FUNDING: National Institutes of Health and Medicines for Malaria Venture.

Associations between Varied Susceptibilities to PfATP4 Inhibitors and Genotypes in Ugandan Plasmodium falciparum Isolates.

Among novel compounds under recent investigation as potential new antimalarial drugs are three independently developed inhibitors of the Plasmodium falciparum P-type ATPase (PfATP4): KAE609 (cipargamin), PA92, and SJ733. We assessed ex vivo susceptibilities to these compounds of 374 fresh P. falciparum isolates collected in Tororo and Busia districts, Uganda, from 2016 to 2019. Median IC50s were 65 nM for SJ733, 9.1 nM for PA92, and 0.5 nM for KAE609. Sequencing of pfatp4 for 218 of these isolates demonstrated many nonsynonymous single nucleotide polymorphisms; the most frequent mutations were G1128R (69% of isolates mixed or mutant), Q1081K/R (68%), G223S (25%), N1045K (16%), and D1116G/N/Y (16%). The G223S mutation was associated with decreased susceptibility to SJ733, PA92, and KAE609. The D1116G/N/Y mutations were associated with decreased susceptibility to SJ733, and the presence of mutations at both codons 223 and 1116 was associated with decreased susceptibility to PA92 and SJ733. In all of these cases, absolute differences in susceptibilities of wild-type (WT) and mutant parasites were modest. Analysis of clones separated from mixed field isolates consistently identified mutant clones as less susceptible than WT. Analysis of isolates from other sites demonstrated the presence of the G223S and D1116G/N/Y mutations across Uganda. Our results indicate that malaria parasites circulating in Uganda have a number of polymorphisms in PfATP4 and that modestly decreased susceptibility to PfATP4 inhibitors is associated with some mutations now present in Ugandan parasites.

Balanced impacts of fitness and drug pressure on the evolution of PfMDR1 polymorphisms in Plasmodium falciparum.

BACKGROUND: Anti-malarial drug resistance may be limited by decreased fitness in resistant parasites. Important contributors to resistance are mutations in the Plasmodium falciparum putative drug transporter PfMDR1. METHODS: Impacts on in vitro fitness of two common PfMDR1 polymorphisms, N86Y, which is associated with sensitivity to multiple drugs, and Y184F, which has no clear impact on drug sensitivity, were evaluated to study associations between resistance mediators and parasite fitness, measured as relative growth in competitive culture experiments. NF10 P. falciparum lines engineered to represent all PfMDR1 N86Y and Y184F haplotypes were co-cultured for 40 days, and the genetic make-up of the cultures was characterized every 4 days by pyrosequencing. The impacts of culture with anti-malarials on the growth of different haplotypes were also assessed. Lastly, the engineering of P. falciparum containing another common polymorphism, PfMDR1 D1246Y, was attempted. RESULTS: Co-culture results were as follows. With wild type (WT) Y184 fixed (N86/Y184 vs. 86Y/Y184), parasites WT and mutant at 86 were at equilibrium. With mutant 184 F fixed (N86/184F vs. 86Y/184F), mutants at 86 overgrew WT. With WT N86 fixed (N86/Y184 vs. N86/184F), WT at 184 overgrew mutants. With mutant 86Y fixed (86Y/Y184 vs. 86Y/184F), WT and mutant at 86 were at equilibrium. Parasites with the double WT were in equilibrium with the double mutant, but 86Y/Y184 overgrew N86/184F. Overall, WT N86/mutant 184F parasites were less fit than parasites with all other haplotypes. Parasites engineered for another mutation, PfMDR1 1246Y, were unstable in culture, with reversion to WT over time. Thus, the N86 WT is stable when accompanied by the Y184 WT, but incurs a fitness cost when accompanied by mutant 184F. Culturing in the presence of chloroquine favored 86Y mutant parasites and in the presence of lumefantrine favored N86 WT parasites; piperaquine had minimal impact. CONCLUSIONS: These results are consistent with those for Ugandan field isolates, suggest reasons for varied haplotypes, and highlight the interplay between drug pressure and fitness that is guiding the evolution of resistance-mediating haplotypes in P. falciparum.

Changing Prevalence of Potential Mediators of Aminoquinoline, Antifolate, and Artemisinin Resistance Across Uganda.

BACKGROUND: In Uganda, artemether-lumefantrine is recommended for malaria treatment and sulfadoxine-pyrimethamine for chemoprevention during pregnancy, but drug resistance may limit efficacies. METHODS: Genetic polymorphisms associated with sensitivities to key drugs were characterized in samples collected from 16 sites across Uganda in 2018 and 2019 by ligase detection reaction fluorescent microsphere, molecular inversion probe, dideoxy sequencing, and quantitative polymerase chain reaction assays. RESULTS: Considering transporter polymorphisms associated with resistance to aminoquinolines, the prevalence of Plasmodium falciparum chloroquine resistance transporter (PfCRT) 76T decreased, but varied markedly between sites (0-46% in 2018; 0-23% in 2019); additional PfCRT polymorphisms and plasmepsin-2/3 amplifications associated elsewhere with resistance to piperaquine were not seen. For P. falciparum multidrug resistance protein 1, in 2019 the 86Y mutation was absent at all sites, the 1246Y mutation had prevalence ≤20% at 14 of 16 sites, and gene amplification was not seen. Considering mutations associated with high-level sulfadoxine-pyrimethamine resistance, prevalences of P. falciparum dihydrofolate reductase 164L (up to 80%) and dihydropteroate synthase 581G (up to 67%) were high at multiple sites. Considering P. falciparum kelch protein propeller domain mutations associated with artemisinin delayed clearance, prevalence of the 469Y and 675V mutations has increased at multiple sites in northern Uganda (up to 23% and 41%, respectively). CONCLUSIONS: We demonstrate concerning spread of mutations that may limit efficacies of key antimalarial drugs.

Antimalarial Trioxolanes with Superior Drug-Like Properties and In Vivo Efficacy.

The emergence of artemisinin resistance, combined with certain suboptimal properties of ozonide agents arterolane and artefenomel, has necessitated the search for new drug candidates in the endoperoxide class. Our group has focused on trioxolane analogues with substitution patterns not previously explored. Here, we describe the enantioselective synthesis of analogues bearing a trans-3″ carbamate side chain and find these to be superior, both in vitro and in vivo, to the previously reported amides. We identified multiple analogues that surpass the oral efficacy of arterolane in the Plasmodium berghei model while exhibiting drug-like properties (logD, solubility, metabolic stability) similar or superior to next-generation clinical candidates like E209 and OZ609. While the preclinical assessment of new analogues is still underway, current data suggest the potential of this chemotype as a likely source of future drug candidates from the endoperoxide class.

Falcipain Inhibitors Based on the Natural Product Gallinamide A Are Potent in Vitro and in Vivo Antimalarials.

A library of analogues of the cyanobacterium-derived depsipeptide natural product gallinamide A were designed and prepared using a highly efficient and convergent synthetic route. Analogues were shown to exhibit potent inhibitory activity against the Plasmodium falciparum cysteine proteases falcipain 2 and falcipain 3 and against cultured chloroquine-sensitive (3D7) and chloroquine-resistant (W2) strains of P. falciparum. Three lead compounds were selected for evaluation of in vivo efficacy against Plasmodium berghei infection in mice on the basis of their improved blood, plasma, and microsomal stability profiles compared with the parent natural product. One of the lead analogues cured P. berghei-infected mice in the Peters 4 day-suppressive test when administered 25 mg kg-1 intraperitoneally daily for 4 days. The compound was also capable of clearing parasites in established infections at 50 mg kg-1 intraperitoneally daily for 4 days and exhibited moderate activity when administered as four oral doses of 100 mg kg-1.

Modeling Prevention of Malaria and Selection of Drug Resistance with Different Dosing Schedules of Dihydroartemisinin-Piperaquine Preventive Therapy during Pregnancy in Uganda.

Dihydroartemisinin-piperaquine (DHA-PQ) is under study for intermittent preventive treatment during pregnancy (IPTp), but it may accelerate selection for drug resistance. Understanding the relationships between piperaquine concentration, prevention of parasitemia, and selection for decreased drug sensitivity can inform control policies and optimization of DHA-PQ dosing. Piperaquine concentrations, measures of parasitemia, and Plasmodium falciparum genotypes associated with decreased aminoquinoline sensitivity in Africa (pfmdr1 86Y, pfcrt 76T) were obtained from pregnant Ugandan women randomized to IPTp with sulfadoxine-pyrimethamine (SP) or DHA-PQ. Joint pharmacokinetic/pharmacodynamic models described relationships between piperaquine concentration and the probability of genotypes of interest using nonlinear mixed effects modeling. An increase in the piperaquine plasma concentration was associated with a log-linear decrease in risk of parasitemia. Our models predicted that higher median piperaquine concentrations would be required to provide 99% protection against mutant infections than against wild-type infections (pfmdr1: N86, 9.6 ng/ml; 86Y, 19.6 ng/ml; pfcrt: K76, 6.5 ng/ml; 76T, 19.6 ng/ml). Comparing monthly, weekly, and daily dosing, daily low-dose DHA-PQ was predicted to result in the fewest infections and the fewest mutant infections per 1,000 pregnancies (predicted mutant infections for pfmdr1 86Y: SP monthly, 607; DHA-PQ monthly, 198; DHA-PQ daily, 1; for pfcrt 76T: SP monthly, 1,564; DHA-PQ monthly, 283; DHA-PQ daily, 1). Our models predict that higher piperaquine concentrations are needed to prevent infections with the pfmdr1/pfcrt mutant compared to those with wild-type parasites and that, despite selection for mutants by DHA-PQ, the overall burden of mutant infections is lower for IPTp with DHA-PQ than for IPTp with SP. (This study has been registered at ClinicalTrials.gov under identifier NCT02282293.).

Changing Molecular Markers of Antimalarial Drug Sensitivity across Uganda.

The potential spread of antimalarial drug resistance to Africa, in particular for artemisinins and key partner drugs, is a major concern. We surveyed Plasmodium falciparum genetic markers associated with drug sensitivity on 3 occasions at ∼6-month intervals in 2016 and 2017 at 10 sites representing a range of epidemiological settings in Uganda. For putative drug transporters, we found continued evolution toward wild-type sequences associated with increased sensitivity to chloroquine. For pfcrt K76T, by 2017 the prevalence of the wild type was >60% at all sites and >90% at 6 sites. For the pfmdr1 N86Y and D1246Y alleles, wild type prevalence ranged from 80 to 100%. We found low prevalence of K13 propeller domain mutations, which are associated with artemisinin resistance in Asia, but one mutation previously identified in northern Uganda, 675V, was seen in 2.0% of samples, including 5.5% of those from the 3 northernmost sites. Amplification of the pfmdr1 and plasmepsin2 genes, associated elsewhere with decreased sensitivity to lumefantrine and piperaquine, respectively, was seen in <1% of samples. For the antifolate targets pfdhfr and pfdhps, 5 mutations previously associated with resistance were very common, and the pfdhfr 164L and pfdhps 581G mutations associated with higher-level resistance were seen at multiple sites, although prevalence did not clearly increase over time. Overall, changes were consistent with the selective pressure of the national treatment regimen, artemether-lumefantrine, with increased sensitivity to chloroquine, and with poor efficacy of antifolates. Strong evidence for resistance to artemisinins was not seen. Continued surveillance of markers that predict antimalarial drug sensitivity is warranted.

Impact of Intermittent Preventive Treatment During Pregnancy on Plasmodium falciparum Drug Resistance-Mediating Polymorphisms in Uganda.

Background: In a recent trial of intermittent preventive treatment in pregnancy (IPTp) in Uganda, dihydroartemisinin-piperaquine (DP) was superior to sulfadoxine-pyrimethamine (SP) in preventing maternal and placental malaria. Methods: We compared genotypes using sequencing, fluorescent microsphere, and quantitative polymerase chain reaction assays at loci associated with drug resistance in Plasmodium falciparum isolated from subjects receiving DP or SP. Results: Considering aminoquinoline resistance, DP was associated with increased prevalences of mutations at pfmdr1 N86Y, pfmdr1 Y184F, and pfcrt K76T compared to SP (64.6% vs 27.4%, P < .001; 93.9% vs 59.2%, P < .001; and 87.7% vs 75.4%, P = .03, respectively). Increasing plasma piperaquine concentration at the time of parasitemia was associated with increasing pfmdr1 86Y prevalence; no infections with the N86 genotype occurred with piperaquine >2.75 ng/mL. pfkelch13 propeller domain polymorphisms previously associated with artemisinin resistance were not identified. Recently identified markers of piperaquine resistance were uncommon and not associated with DP. Considering antifolate resistance, SP was associated with increased prevalence of a 5-mutation haplotype (pfdhfr 51I, 59R, and 108N; pfdhps 437G and 581G) compared to DP (90.8% vs 60.0%, P = .001). Conclusions: IPTp selected for genotypes associated with decreased sensitivity to treatment regimens, but genotypes associated with clinically relevant DP resistance in Asia have not emerged in Uganda.

Altered Plasmodium falciparum Sensitivity to the Antiretroviral Protease Inhibitor Lopinavir Associated with Polymorphisms in pfmdr1.

The HIV protease inhibitor lopinavir inhibits Plasmodium falciparum aspartic proteases (plasmepsins) and parasite development, and children receiving lopinavir-ritonavir experienced fewer episodes of malaria than those receiving other antiretroviral regimens. Resistance to lopinavir was selected in vitro over ∼9 months, with ∼4-fold decreased sensitivity. Whole-genome sequencing of resistant parasites showed a mutation and increased copy number in pfmdr1 and a mutation in a protein of unknown function, but no polymorphisms in plasmepsin genes.

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

Dihydroartemisinin-piperaquine (DP) offers prolonged protection against malaria, but its impact on Plasmodium falciparum drug sensitivity is uncertain. In a trial of intermittent preventive treatment in schoolchildren in Tororo, Uganda, in 2011 to 2012, monthly DP for 1 year decreased the incidence of malaria by 96% compared to placebo; DP once per school term offered protection primarily during the first month after therapy. To assess the impact of DP on selection of drug resistance, we compared the prevalence of key polymorphisms in isolates that emerged at different intervals after treatment with DP. Blood obtained monthly and at each episode of fever was assessed for P. falciparum parasitemia by microscopy. Samples from 160 symptomatic and 650 asymptomatic episodes of parasitemia were assessed at 4 loci (N86Y, Y184F, and D1246Y in pfmdr1 and K76T in pfcrt) that modulate sensitivity to aminoquinoline antimalarials, utilizing a ligase detection reaction-fluorescent microsphere assay. For pfmdr1 N86Y and pfcrt K76T, but not the other studied polymorphisms, the prevalences of mutant genotypes were significantly greater in children who had received DP within the past 30 days than in those not treated within 60 days (86Y, 18.0% versus 8.3% [P = 0.03]; 76T, 96.0% versus 86.1% [P = 0.05]), suggesting selective pressure of DP. Full sequencing of pfcrt in a subset of samples did not identify additional polymorphisms selected by DP. In summary, parasites that emerged soon after treatment with DP were more likely than parasites not under drug pressure to harbor pfmdr1 and pfcrt polymorphisms associated with decreased sensitivity to aminoquinoline antimalarials. (This study has been registered at ClinicalTrials.gov under no. NCT01231880.).

Antimalarial Benzoxaboroles Target Plasmodium falciparum Leucyl-tRNA Synthetase.

There is a need for new antimalarials, ideally with novel mechanisms of action. Benzoxaboroles have been shown to be active against bacteria, fungi, and trypanosomes. Therefore, we investigated the antimalarial activity and mechanism of action of 3-aminomethyl benzoxaboroles against Plasmodium falciparum Two 3-aminomethyl compounds, AN6426 and AN8432, demonstrated good potency against cultured multidrug-resistant (W2 strain) P. falciparum (50% inhibitory concentration [IC50] of 310 nM and 490 nM, respectively) and efficacy against murine Plasmodium berghei infection when administered orally once daily for 4 days (90% effective dose [ED90], 7.4 and 16.2 mg/kg of body weight, respectively). To characterize mechanisms of action, we selected parasites with decreased drug sensitivity by culturing with stepwise increases in concentration of AN6426. Resistant clones were characterized by whole-genome sequencing. Three generations of resistant parasites had polymorphisms in the predicted editing domain of the gene encoding a P. falciparum leucyl-tRNA synthetase (LeuRS; PF3D7_0622800) and in another gene (PF3D7_1218100), which encodes a protein of unknown function. Solution of the structure of the P. falciparum LeuRS editing domain suggested key roles for mutated residues in LeuRS editing. Short incubations with AN6426 and AN8432, unlike artemisinin, caused dose-dependent inhibition of [(14)C]leucine incorporation by cultured wild-type, but not resistant, parasites. The growth of resistant, but not wild-type, parasites was impaired in the presence of the unnatural amino acid norvaline, consistent with a loss of LeuRS editing activity in resistant parasites. In summary, the benzoxaboroles AN6426 and AN8432 offer effective antimalarial activity and act, at least in part, against a novel target, the editing domain of P. falciparum LeuRS.

Impact of antimalarial treatment and chemoprevention on the drug sensitivity of malaria parasites isolated from ugandan children.

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.