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.).

Varied prevalence of antimalarial drug resistance markers in different populations of newly arrived refugees in Uganda.

Newly arrived refugees offer insights into malaria epidemiology in their countries of origin. We evaluated asymptomatic refugee children within 7 days of arrival in Uganda from South Sudan and the Democratic Republic of Congo (DRC) in 2022 for parasitemia, parasite species, and Plasmodium falciparum drug resistance markers. Asymptomatic P. falciparum infections were common in both populations. Co-infection with P. malariae was more common in DRC refugees. Prevalences of markers of aminoquinoline resistance (PfCRT K76T, PfMDR1 N86Y) were much higher in South Sudan refugees, of antifolate resistance (PfDHFR C59R and I164L, PfDHPS A437G and K540E) much higher in DRC refugees, and of artemisinin partial resistance (ART-R; PfK13 C469Y and A675V) moderate in both populations. Prevalences of most mutations differed from those seen in Ugandans attending health centers near the refugee centers. Refugee evaluations yielded insights into varied malaria epidemiology and identified markers of ART-R in two previously little-studied countries.

Ex vivo drug susceptibility and resistance mediating genetic polymorphisms of Plasmodium falciparum in Bobo-Dioulasso, Burkina Faso.

Malaria remains a leading cause of morbidity and mortality in Burkina Faso, which utilizes artemether-lumefantrine as the principal therapy to treat uncomplicated malaria and seasonal malaria chemoprevention with monthly sulfadoxine-pyrimethamine plus amodiaquine in children during the transmission season. Monitoring the activities of available antimalarial drugs is a high priority. We assessed the ex vivo susceptibility of Plasmodium falciparum to 11 drugs in isolates from patients presenting with uncomplicated malaria in Bobo-Dioulasso in 2021 and 2022. IC50 values were derived using a standard 72 h growth inhibition assay. Parasite DNA was sequenced to characterize known drug resistance-mediating polymorphisms. Isolates were generally susceptible, with IC50 values in the low-nM range, to chloroquine (median IC5010 nM, IQR 7.9-24), monodesethylamodiaquine (22, 14-46) piperaquine (6.1, 3.6-9.2), pyronaridine (3.0, 1.3-5.5), quinine (50, 30-75), mefloquine (7.1, 3.7-10), lumefantrine (7.1, 4.5-12), dihydroartemisinin (3.7, 2.2-5.5), and atovaquone (0.2, 0.1-0.3) and mostly resistant to cycloguanil (850, 543-1,290) and pyrimethamine (33,200, 18,400-54,200), although a small number of outliers were seen. Considering genetic markers of resistance to aminoquinolines, most samples had wild-type PfCRT K76T (87%) and PfMDR1 N86Y (95%) sequences. For markers of resistance to antifolates, established PfDHFR and PfDHPS mutations were highly prevalent, the PfDHPS A613S mutation was seen in 19% of samples, and key markers of high-level resistance (PfDHFR I164L; PfDHPS K540E) were absent or rare (A581G). Mutations in the PfK13 propeller domain known to mediate artemisinin partial resistance were not detected. Overall, our results suggest excellent susceptibilities to drugs now used to treat malaria and moderate, but stable, resistance to antifolates used to prevent malaria.

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.

Asymptomatic School-Aged Children Are Important Drivers of Malaria Transmission in a High Endemicity Setting in Uganda.

Achieving malaria elimination requires a better understanding of the transmissibility of human infections in different transmission settings. This study aimed to characterize the human infectious reservoir in a high endemicity setting in eastern Uganda, using gametocyte quantification and mosquito feeding assays. In asymptomatic infections, gametocyte densities were positively associated with the proportion of infected mosquitoes (ß = 1.60; 95% CI, 1.32-1.92; P < .0001). Combining transmissibility and abundance in the population, symptomatic and asymptomatic infections were estimated to contribute to 5.3% and 94.7% of the infectious reservoir, respectively. School-aged children (5-15 years old) contributed to 50.4% of transmission events and were important drivers of malaria transmission.

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.

Impact of Short-Term Storage on Ex Vivo Antimalarial Susceptibilities of Fresh Ugandan Plasmodium falciparum Isolates.

We measured susceptibilities of Ugandan Plasmodium falciparum isolates assayed on the day of collection or after storage at 4°C. Samples were incubated with serial dilutions of 8 antimalarials, and susceptibilities were determined from 72-h growth inhibition assays. Storage was associated with decreased growth and lower 50% inhibitory concentration values, but differences between assays beginning on day 0 or after 1 or 2 days of storage were modest, indicating that short-term storage before drug susceptibility determination is feasible.

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.

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.

Age-Related Changes in Malaria Clinical Phenotypes During Infancy Are Modified by Sickle Cell Trait.

BACKGROUND: Infants are protected against Plasmodium falciparum malaria. Mechanisms that drive this protection remain unclear due to a poor understanding of malaria clinical phenotypes during infancy. METHODS: We enrolled a birth cohort of 678 infants in Busia, Uganda, an area of high malaria transmission. We followed infants through 12 months of age and quantified protection against parasitemia and clinical disease. RESULTS: Symptomatic malaria incidence increased from 1.2 to 2.6 episodes per person-year between 0 and <6 months and between 6 and 12 months of age, while the monthly probability of asymptomatic parasitemia given infection decreased from 32% to 21%. Sickle cell trait (HbAS) was protective against symptomatic malaria (incidence rate ratio  = 0.57 comparing HbAS vs hemoglobin AA (HbAA); 95% confidence interval, 0.44-0.74; P < .001), but age modified this relationship (Pint = <0.001), with nonlinear protection that waned between 0 and 9 months of age before increasing. Increasing age was associated with higher parasite densities at the time of infection and, in infants with HbAS, a reduced ability to tolerate high parasite densities without fever. CONCLUSIONS: Age-dependent changes in HbAS protective efficacy in infancy were accompanied by differential loss of antiparasite and antidisease protection among HbAS and HbAA infants. This provides a framework for investigating the mechanisms that underlie infant protection against malaria. CLINICAL TRIALS REGISTRATION: NCT02793622.

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.

Deletions of pfhrp2 and pfhrp3 genes were uncommon in rapid diagnostic test-negative Plasmodium falciparum isolates from Uganda.

BACKGROUND: Rapid diagnostic tests (RDTs) play a key role in malaria case management. The most widely used RDT identifies Plasmodium falciparum based on immunochromatographic recognition of P. falciparum histidine-rich protein 2 (PfHRP2). Deletion of the paralogous pfhrp2 and pfhrp3 genes leads to false-negative PfHRP2-based RDTs, and has been reported in P. falciparum infections from South America and Africa. However, identification of pfhrp2/pfhrp3 deletions has usually been based only on failure to amplify these genes using PCR, without confirmation based on PfHRP2 protein expression, and understanding of the true prevalence of deletions is incomplete. METHODS: Deletions of pfhrp2/pfhrp3 in blood samples were investigated from cross-sectional surveys in 2012-13 in three regions of varied malaria transmission intensity in Uganda. Samples with positive Giemsa-stained thick blood smears, but negative PfHRP2-based RDTs were evaluated by PCR amplification of conserved subunit ribosomal DNA for Plasmodium species, PCR amplification of pfhrp2 and pfhrp3 genes to identify deletions, and bead-based immunoassays for expression of PfHRP2. RESULTS: Of 3516 samples collected in cross-sectional surveys, 1493 (42.5%) had positive blood smears, of which 96 (6.4%) were RDT-negative. Of these 96 RDT-negative samples, P. falciparum DNA was identified by PCR in 56 (58%) and only non-falciparum plasmodial DNA in 40 (42%). In all 56 P. falciparum-positive samples there was a failure to amplify pfhrp2 or pfhrp3: in 25 (45%) pfhrp2 was not amplified, in 39 (70%) pfhrp3 was not amplified, and in 19 (34%) neither gene was amplified. For the 39 P. falciparum-positive, RDT-negative samples available for analysis of protein expression, PfHRP2 was not identified by immunoassay in only four samples (10.3%); these four samples all had failure to amplify both pfhrp2 and pfhrp3 by PCR. Thus, only four of 96 (4.2%) smear-positive, RDT-negative samples had P. falciparum infections with deletion of pfhrp2 and pfhrp3 confirmed by failure to amplify the genes by PCR and lack of expression of PfHRP2 demonstrated by immunoassay. CONCLUSION: False negative RDTs were uncommon. Deletions in pfhrp2 and pfhrp3 explained some of these false negatives, but most false negatives were not due to deletion of the pfhrp2 and pfhrp3 genes.

Associations between Malaria-Preventive Regimens and Plasmodium falciparum Drug Resistance-Mediating Polymorphisms in Ugandan Pregnant Women.

Intermittent preventive treatment in pregnancy (IPTp) with monthly sulfadoxine-pyrimethamine (SP) is recommended for malaria-endemic parts of Africa, but efficacy is compromised by resistance, and, in recent trials, dihydroartemisinin-piperaquine (DP) has shown better antimalarial protective efficacy. We utilized blood samples from a recent trial to evaluate selection by IPTp with DP or SP of Plasmodium falciparum genetic polymorphisms that alter susceptibility to these drugs. The prevalence of known genetic polymorphisms associated with altered drug susceptibility was determined in parasitemic samples, including 375 collected before IPTp drugs were administered, 125 randomly selected from those receiving SP, and 80 from those receiving DP. For women receiving DP, the prevalence of mixed/mutant sequences was greater in samples collected during IPTp than that in samples collected prior to the intervention for PfMDR1 N86Y (20.3% versus 3.9%; P < 0.001), PfMDR1 Y184F (73.0% versus 53.0%; P < 0.001), and PfCRT K76T (46.4% versus 24.0%; P < 0.001). Considering SP, prior to IPTp, the prevalence of all 5 common antifolate mutations was over 92%, and this prevalence increased following exposure to SP, although none of these changes were statistically significant. For two additional mutations associated with high-level SP resistance, the prevalence of PfDHFR 164L (13.7% versus 4.0%; P = 0.004), but not PfDHPS 581G (1.9% versus 3.0%; P = 0.74), was greater in samples collected during IPTp compared to those collected before the intervention. Use of IPTp in Uganda selected for parasites with mutations associated with decreased susceptibility to IPTp regimens. Thus, a potential drawback of IPTp is selection of parasites with decreased drug susceptibility.

Identification and characterization of immature Anopheles and culicines (Diptera: Culicidae) at three sites of varying malaria transmission intensities in Uganda.

BACKGROUND: Over the last two decades, there has been remarkable progress in malaria control in sub-Saharan Africa, due mainly to the massive deployment of long-lasting insecticidal nets and indoor residual spraying. Despite these gains, it is clear that in many situations, additional interventions are needed to further reduce malaria transmission. The World Health Organization (WHO) has promoted the Integrated Vector Management (IVM) approach through its Global Vector Control Response 2017-2030. However, prior roll-out of larval source management (LSM) as part of IVM, knowledge on ecology of larval aquatic habitats is required. METHODS: Aquatic habitats colonized by immature Anopheles and culicines vectors were characterized at three sites of low, medium and high malaria transmission in Uganda from October 2011 to June 2015. Larval surveys were conducted along transects in each site and aquatic habitats described according to type and size. Immature Anopheles, culicines and pupae from the described habitats were sampled using standard dipping methods to determine larval and pupae densities. Larvae were identified as anopheline or culicine, and counted. Pupae were not identified further. Binary logistic regression analysis was used to identify factors associated with the presence of immature Anopheles and culicines in each site. RESULTS: A total of 1205 larval aquatic habitats were surveyed and yielded a total of 17,028 anopheline larvae, 26,958 culicine larvae and 1189 pupae. Peaks in larval abundance occurred in all sites in March-May and August-October coinciding with the rainy seasons. Anopheles larvae were found in 52.4% (n = 251) of aquatic habitats in Tororo, a site of high transmission, 41.9% (n = 536) of habitats in Kanungu, a site with moderate malaria transmission, and 15.8% (n = 418) in Jinja, a site with low malaria transmission. The odds of finding larvae was highest in rice fields compared to pools in both Tororo (odds ratio, OR = 4.21, 95% CI 1.22-14.56, p = 0.02) and Kanungu (OR = 2.14, 95% CI 1.12-4.07, p = 0.02), while in Jinja the odd were highest in containers (OR = 4.55, 95% CI = 1.09-19.14, p = 0.03). In Kanungu, larvae were less likely to be found in containers compared to pools (OR = 0.26, 95% CI 0.09-0.66, p = 0.008) and river fringe (OR = 0.19, 95% CI 0.07-0.52, p = 0.001). Medium sized habitats were associated with high odds of finding larvae compared to small habitats (OR = 3.59, 95% CI 1.18-14.19, p = 0.039). CONCLUSIONS: These findings show that immature Anopheles and culicines were common in areas of high and moderate transmission but were rare in areas of low transmission. Although immature Anopheles and culicines were found in all types of water bodies, they were most common in rice fields and less common in open drains and in river fringes. Methods are needed to reduce the aquatic stages of anopheline mosquitoes in human-made habitats, particularly rice fields.

Comparative Efficacy of Artemether-Lumefantrine and Dihydroartemisinin-Piperaquine for the Treatment of Uncomplicated Malaria in Ugandan Children.

BACKGROUND: In Uganda, artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DHA-PQ) showed excellent treatment efficacy for uncomplicated malaria in prior trials. Because the frequency of resistance to artemisinins and piperaquine is increasing in Southeast Asia and the prevalence of Plasmodium falciparum polymorphisms associated with resistance has changed, we reassessed treatment efficacies at 3 sites in Uganda. METHODS: For this randomized, single-blinded clinical trial, children aged 6-59 months with uncomplicated falciparum malaria were assigned treatment with AL or DHA-PQ and followed for 42 days. Primary end points were risks of recurrent parasitemia, either unadjusted or adjusted to distinguish recrudescence from new infection. We assessed selection by study regimens of relevant P. falciparum genetic polymorphisms associated with drug resistance. RESULTS: Of 599 patients enrolled, 578 completed follow-up. There were no early treatment failures. The risk of recurrent parasitemia was lower with DHA-PQ as compared to AL at all 3 sites at 42 days (26.0% vs 47.0%; P < .001). Recrudescent infections were uncommon in both the DHA-PQ and AL arms (1.1% and 2.2%, respectively; P = .25). Neither regimen selected for pfcrt or pfmdr1 polymorphisms associated with drug resistance. CONCLUSIONS: AL and DHA-PQ remain effective for the treatment of malaria in Uganda. Neither regimen selected for genetic polymorphisms associated with drug resistance. CLINICAL TRIALS REGISTRATION: ISRCTN15793046.

The Diversity of the Plasmodium falciparum K13 Propeller Domain Did Not Increase after Implementation of Artemisinin-Based Combination Therapy in Uganda.

Artemisinin-based combination therapies (ACTs) are the standard of care to treat uncomplicated falciparum malaria. However, resistance to artemisinins, defined as delayed parasite clearance after therapy, has emerged in Southeast Asia, and the spread of resistance to sub-Saharan Africa could have devastating consequences. Artemisinin resistance has been associated in Southeast Asia with multiple nonsynonymous single nucleotide polymorphisms (NS-SNPs) in the propeller domain of the gene encoding the Plasmodium falciparum K13 protein (K13PD). Some K13PD NS-SNPs have been seen in Africa, but the relevance of these mutations is unclear. To assess whether ACT use has selected for specific K13PD mutations, we compared the K13PD genetic diversity in clinical isolates collected before and after the implementation of ACT use from seven sites across Uganda. We detected K13PD NS-SNPs in 16 of 683 (2.3%) clinical isolates collected between 1999 and 2004 and in 26 of 716 (3.6%) isolates collected between 2012 and 2016 (P = 0.16), representing a total of 29 different polymorphisms at 27 codons. Individual NS-SNPs were usually detected only once, and none were found in more than 0.7% of the isolates. Three SNPs (C469F, P574L, and A675V) associated with delayed clearance in Southeast Asia were seen in samples collected between 2012 and 2016, each in a single isolate. No differences in diversity following implementation of ACT use were found at any of the seven sites, nor was there evidence of selective pressures acting on the locus. Our results suggest that selection by ACTs is not impacting on K13PD diversity in Uganda.

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.

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.).

Impact of vector control interventions on malaria transmission intensity, outdoor vector biting rates and Anopheles mosquito species composition in Tororo, Uganda.

BACKGROUND: Long-lasting insecticidal nets (LLINs) and indoor residual spraying of insecticide (IRS) are widely recommended for the prevention of malaria in endemic regions. Data from human landing catches provide information on the impact of vector control on vector populations. Here, malaria transmission indoors and outdoors, before and after mass deployment of LLINs and IRS in Uganda was compared. METHODS: The study took place in Tororo district, a historically high transmission area where universal LLIN distribution was conducted in November 2013 and May 2017 and 6 rounds of IRS implemented from December 2014 to July 2018. Human landing catches were performed in 8 houses monthly from October 2011 to September 2012 (pre-intervention period) and every 4 weeks from November 2017 to October 2018 (post-intervention period). Mosquitoes were collected outdoors from 18:00 to 22:00 h and indoors from 18:00 to 06:00 h. Female Anopheles were tested for the presence of Plasmodium falciparum sporozoites and species identification performed using gross dissection and polymerase chain reaction (PCR). RESULTS: The interventions were associated with a decline in human biting rate from 19.6 to 2.3 female Anopheles mosquitoes per house per night (p < 0.001) and annual entomological inoculation rate from 129 to 0 infective bites per person per year (p < 0.001). The proportion of mosquitoes collected outdoors increased from 11.6 to 49.4% (p < 0.001). Prior to the interventions the predominant species was Anopheles gambiae sensu stricto (s.s.), which comprised an estimated 76.7% of mosquitoes. Following the interventions, the predominant species was Anopheles arabiensis, which comprised 99.5% of mosquitoes, with almost complete elimination of An. gambiae s.s. (0.5%). CONCLUSIONS: Mass distribution of LLINs and 6 rounds of IRS dramatically decreased vector density and sporozoite rate resulting in a marked reduction in malaria transmission intensity in a historically high transmission site in Uganda. These changes were accompanied by a shift in vector species from An. gambiae s.s. to An. arabiensis and a relative increase in outdoor biting.