Ex vivo susceptibilities to ganaplacide and diversity in potential resistance mediators in Ugandan Plasmodium falciparum isolates.

Novel antimalarials are urgently needed to combat rising resistance to available drugs. The imidazolopiperazine ganaplacide is a promising drug candidate, but decreased susceptibility of laboratory strains has been linked to polymorphisms in the Plasmodium falciparum cyclic amine resistance locus (PfCARL), acetyl-CoA transporter (PfACT), and UDP-galactose transporter (PfUGT). To characterize parasites causing disease in Africa, we assessed ex vivo drug susceptibilities to ganaplacide in 750 P. falciparum isolates collected in Uganda from 2017 to 2023. Drug susceptibilities were assessed using a 72-hour SYBR Green growth inhibition assay. The median IC50 for ganaplacide was 13.8 nM, but some isolates had up to 31-fold higher IC50s (31/750 with IC50 > 100 nM). To assess genotype-phenotype associations, we sequenced genes potentially mediating altered ganaplacide susceptibility in the isolates using molecular inversion probe and dideoxy sequencing methods. PfCARL was highly polymorphic, with eight mutations present in >5% of isolates. None of these eight mutations had previously been selected in laboratory strains with in vitro drug pressure and none were found to be significantly associated with decreased ganaplacide susceptibility. Mutations in PfACT and PfUGT were found in ≤5% of isolates, except for two frequent (>20%) mutations in PfACT; one mutation in PfACT (I140V) was associated with a modest decrease in susceptibility. Overall, Ugandan P. falciparum isolates were mostly highly susceptible to ganaplacide. Known resistance mediators were polymorphic, but mutations previously selected with in vitro drug pressure were not seen, and mutations identified in the Ugandan isolates were generally not associated with decreased ganaplacide susceptibility.

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. Coinfection 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, K540E, and A581G) 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 2 previously little-studied countries.

The emergence of artemisinin partial resistance in Africa: how do we respond?

Malaria remains one of the most important infectious diseases in the world, with the greatest burden in sub-Saharan Africa, primarily from Plasmodium falciparum infection. The treatment and control of malaria is challenged by resistance to most available drugs, but partial resistance to artemisinins (ART-R), the most important class for the treatment of malaria, was until recently confined to southeast Asia. This situation has changed, with the emergence of ART-R in multiple countries in eastern Africa. ART-R is mediated primarily by single point mutations in the P falciparum kelch13 protein, with several mutations present in African parasites that are now validated resistance mediators based on clinical and laboratory criteria. Major priorities at present are the expansion of genomic surveillance for ART-R mutations across the continent, more frequent testing of the efficacies of artemisinin-based regimens against uncomplicated and severe malaria in trials, more regular assessment of ex-vivo antimalarial drug susceptibilities, consideration of changes in treatment policy to deter the spread of ART-R, and accelerated development of new antimalarial regimens to overcome the impacts of ART-R. The emergence of ART-R in Africa is an urgent concern, and it is essential that we increase efforts to characterise its spread and mitigate its impact.

Emergence, transmission dynamics and mechanisms of artemisinin partial resistance in malaria parasites in Africa.

Malaria, mostly due to Plasmodium falciparum infection in Africa, remains one of the most important infectious diseases in the world. Standard treatment for uncomplicated P. falciparum malaria is artemisinin-based combination therapy (ACT), which includes a rapid-acting artemisinin derivative plus a longer-acting partner drug, and standard therapy for severe P. falciparum malaria is intravenous artesunate. The efficacy of artemisinins and ACT has been threatened by the emergence of artemisinin partial resistance in Southeast Asia, mediated principally by mutations in the P. falciparum Kelch 13 (K13) protein. High ACT treatment failure rates have occurred when resistance to partner drugs is also seen. Recently, artemisinin partial resistance has emerged in Rwanda, Uganda and the Horn of Africa, with independent emergences of different K13 mutants in each region. In this Review, we summarize our current knowledge of artemisinin partial resistance and focus on the emergence of resistance in Africa, including its epidemiology, transmission dynamics and mechanisms. At present, the clinical impact of emerging resistance in Africa is unclear and most available evidence suggests that the efficacies of leading ACTs remain excellent, but there is an urgent need to better appreciate the extent of the problem and its consequences for the treatment and control of malaria.

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.

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.).

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.

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.

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.

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.

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.

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.

House design and risk of malaria, acute respiratory infection and gastrointestinal illness in Uganda: A cohort study.

House construction is rapidly modernizing across Africa but the potential benefits for human health are poorly understood. We hypothesised that improvements to housing would be associated with reductions in malaria, acute respiratory infection (ARI) and gastrointestinal illness in an area of low malaria endemicity in Uganda. Data were analysed from a cohort study of male and female child and adult residents (n = 531) of 80 randomly-selected households in Nagongera sub-county, followed for 24 months (October 4, 2017 to October 31, 2019). Houses were classified as modern (brick walls, metal roof and closed eaves) or traditional (all other homes). Light trap collections of mosquitoes were done every two weeks in all sleeping rooms. Every four weeks, we measured malaria infection (using microscopy and qPCR to detect malaria parasites), incidence of malaria, ARI and gastrointestinal illness. We collected 15,780 adult female Anopheles over 7,631 nights. We collected 13,277 blood samples of which 10.2% (1,347) were positive for malaria parasites. Over 958 person years we diagnosed 38 episodes of uncomplicated malaria (incidence 0.04 episodes per person-year at risk), 2,553 episodes of ARI (incidence 2.7 episodes per person-year) and 387 episodes of gastrointestinal illness (incidence 0.4 episodes per person-year). Modern houses were associated with a 53% lower human biting rate compared to traditional houses (adjusted incidence rate ratio [aIRR] 0.47, 95% confidence interval [CI] 0.32-0.67, p<0.001) and a 24% lower incidence of gastrointestinal illness (aIRR 0.76, 95% CI 0.59-0.98, p = 0.04) but no changes in malaria prevalence, malaria incidence nor ARI incidence. House improvements may reduce mosquito-biting rates and gastrointestinal illness among children and adults. For the health sector to leverage Africa's housing modernization, research is urgently needed to identify the healthiest house designs and to assess their effectiveness across a range of epidemiological settings in sub-Saharan Africa.

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.

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.