scholarly journals Phenotypic Changes in Artemisinin-Resistant Plasmodium falciparum LinesIn Vitro: Evidence for Decreased Sensitivity to Dormancy and Growth Inhibition

2011 ◽  
Vol 56 (1) ◽  
pp. 428-431 ◽  
Author(s):  
Franka Teuscher ◽  
Nanhua Chen ◽  
Dennis E. Kyle ◽  
Michelle L. Gatton ◽  
Qin Cheng
Keyword(s):  
Plasmodium Falciparum ◽  
Growth Arrest ◽  
Artemisinin Resistance ◽  
Mature Stage ◽  
Ring Stage ◽  
Content Type ◽  
Resistance Phenotype ◽  
Temporary Growth

ABSTRACTThe appearance ofPlasmodium falciparumparasites with decreasedin vivosensitivity but no measurablein vitroresistance to artemisinin has raised the urgent need to characterize the artemisinin resistance phenotype. Changes in the temporary growth arrest (dormancy) profile of parasites may be one aspect of this phenotype. In this study, we investigated the link between dormancy and resistance, using artelinic acid (AL)-resistant parasites. Our results demonstrate that the AL resistance phenotype has (i) decreased sensitivity of mature-stage parasites, (ii) decreased sensitivity of the ring stage to the induction of dormancy, and (iii) a faster recovery from dormancy.

scholarly journals The Spiroindolone KAE609 Does Not Induce Dormant Ring Stages in Plasmodium falciparum Parasites

2016 ◽  
Vol 60 (9) ◽  
pp. 5167-5174 ◽  
Author(s):  
Marina Chavchich ◽  
Karin Van Breda ◽  
Kerryn Rowcliffe ◽  
Thierry T. Diagana ◽  
Michael D. Edstein
Keyword(s):  
Plasmodium Falciparum ◽  
Growth Arrest ◽  
Artemisinin Resistance ◽  
Multidrug Resistant ◽  
The Novel ◽  
Ring Stage ◽  
Content Type ◽  
Artemisinin Derivatives ◽  
Temporary Growth

ABSTRACTIn vitrodrug treatment with artemisinin derivatives, such as dihydroartemisinin (DHA), results in a temporary growth arrest (i.e., dormancy) at an early ring stage inPlasmodium falciparum. This response has been proposed to play a role in the recrudescence ofP. falciparuminfections following monotherapy with artesunate and may contribute to the development of artemisinin resistance inP. falciparummalaria. We demonstrate here that artemether does induce dormant rings, a finding which further supports the class effect of artemisinin derivatives in inducing the temporary growth arrest ofP. falciparumparasites. In contrast and similarly to lumefantrine, the novel and fast-acting spiroindolone compound KAE609 does not induce growth arrest at the early ring stage ofP. falciparumand prevents the recrudescence of DHA-arrested rings at a low concentration (50 nM). Our findings, together with previous clinical data showing that KAE609 is active against artemisinin-resistant K13 mutant parasites, suggest that KAE609 could be an effective partner drug with a broad range of antimalarials, including artemisinin derivatives, in the treatment of multidrug-resistantP. falciparummalaria.

scholarly journals Modification of an atypical clathrin-independent AP-2 adaptin complex of Plasmodium falciparum reduces susceptibility to artemisinin

2019 ◽  
Author(s):  
Ryan C. Henrici ◽  
Rachel L. Edwards ◽  
Martin Zoltner ◽  
Donelly A. van Schalkwyk ◽  
Melissa N. Hart ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Artemisinin Resistance ◽  
Conditional Knockout ◽  
Parental Line ◽  
Ring Stage ◽  
Intracellular Traffic ◽  
Reduced Ring ◽  
Kelch Domain

SummaryThe efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin. In the Mekong region this is associated with mutations in the kelch propeller-encoding domain of pfkelch13, but variants of other parasite proteins are also thought to modulate the response to drug. Evidence from human and rodent studies suggests that the μ-subunit of the AP-2 adaptin trafficking complex is one such protein of interest. We generated transgenic Plasmodium falciparum parasites encoding the I592T variant of pfap2μ, orthologous to the I568T mutation associated with in vivo artemisinin resistance in P. chabaudi. When exposed to a four-hour pulse of dihydroartemisin in the ring-stage survival assay, two P. falciparum clones expressing AP-2μ I592T displayed significant and reproducible survival of 8.0% and 10.3%, respectively, compared to <2% for the 3D7 parental line (P = 0.0011 for each clone). In immunoprecipitation and localisation studies of HA-tagged AP-2μ, we identified interacting partners including AP-2α, AP-1/2β, AP-2σ and a kelch-domain protein encoded on chromosome 10 of P. falciparum, K10. Conditional knockout indicates that the AP-2 trafficking complex in P. falciparum is essential for the fidelity of merozoite biogenesis and membrane organisation in the mature schizont. We also show that while other heterotetrameric AP-complexes and secretory factors interact with clathrin, AP-2 complex subunits do not. Thus, the AP-2 complex may be diverted from a clathrin-dependent endocytic role seen in most eukaryotes into a Plasmodium-specific function. These findings represent striking divergences from eukaryotic dogma and support a role for intracellular traffic in determining artemisinin sensitivity in vitro, confirming the existence of multiple functional routes to reduced ring-stage artemisinin susceptibility. Therefore, the utility of pfkelch13 variants as resistance markers is unlikely to be universal, and phenotypic surveillance of parasite susceptibility in vivo may be needed to identify threats to our current combination therapies.

scholarly journals Laboratory Detection of Artemisinin-Resistant Plasmodium falciparum

2014 ◽  
Vol 58 (6) ◽  
pp. 3157-3161 ◽  
Author(s):  
Kesinee Chotivanich ◽  
Rupam Tripura ◽  
Debashish Das ◽  
Poravuth Yi ◽  
Nicholas P. J. Day ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Parasite Clearance ◽  
Ring Stage ◽  
Clearance Time ◽  
Content Type ◽  
Reduced Ring ◽  
Susceptibility Tests ◽  
Low Sensitivity

ABSTRACTConventional 48-hin vitrosusceptibility tests have low sensitivity in identifying artemisinin-resistantPlasmodium falciparum, defined phenotypically by lowin vivoparasite clearance rates. We hypothesized originally that this discrepancy was explained by a loss of ring-stage susceptibility and so developed a simple field-adapted 24-h trophozoite maturation inhibition (TMI) assay focusing on the ring stage and compared it to the standard 48-h schizont maturation inhibition (WHO) test. In Pailin, western Cambodia, where artemisinin-resistantP. falciparumis prevalent, the TMI test mean (95% confidence interval) 50% inhibitory concentration (IC50) for artesunate was 6.8 (5.2 to 8.3) ng/ml compared with 1.5 (1.2 to 1.8) ng/ml for the standard 48-h WHO test (P= 0.001). TMI IC50s correlated significantly with thein vivoresponses to artesunate (parasite clearance time [r= 0.44,P= 0.001] and parasite clearance half-life [r= 0.46,P= 0.001]), whereas the standard 48-h test values did not. On continuous culture of two resistant isolates, the artemisinin-resistant phenotype was lost after 6 weeks (IC50s fell from 10 and 12 ng/ml to 2.7 and 3 ng/ml, respectively). Slow parasite clearance in falciparum malaria in western Cambodia results from reduced ring-stage susceptibility.

scholarly journals Plasmodium berghei K13 Mutations Mediate In Vivo Artemisinin Resistance That is Reversed by Proteasome Inhibition

2020 ◽  
Author(s):  
Nelson V. Simwela ◽  
Barbara H. Stokes ◽  
Dana Aghabi ◽  
Matt Bogyo ◽  
David A. Fidock ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Gene Editing ◽  
Artemisinin Resistance ◽  
Causal Role ◽  
Ring Stage ◽  
Parasite Resistance ◽  
Mutant Lines

ABSTRACTThe recent emergence of Plasmodium falciparum (PF) parasite resistance to the first line antimalarial drug artemisinin is of particular concern. Artemisinin resistance is primarily driven by mutations in the PF K13 protein, which enhance survival of early ring stage parasites treated with the artemisinin active metabolite dihydroartemisinin in vitro and associate with delayed parasite clearance in vivo. However, association of K13 mutations with in vivo artemisinin resistance has been problematic due to the absence of a tractable model. Herein, we have employed CRISPR/Cas9 genome editing to engineer selected orthologous PF K13 mutations into the K13 gene of an artemisinin-sensitive, P. berghei (PB) rodent model of malaria. Introduction of the orthologous PF K13 F446I, M476I, Y493H and R539T mutations into PB K13 produced gene-edited parasites with reduced susceptibility to dihydroartemisinin in the standard 24-hour in vitro assay and increased survival in an adapted in vitro ring-stage survival assay. Mutant PB K13 parasites also displayed delayed clearance in vivo upon treatment with artesunate and achieved faster recrudescence upon treatment with artemisinin. Orthologous C580Y and I543T mutations could not be introduced into PB while the equivalent of the M476I and R539T mutations resulted in significant growth defects. Furthermore, a Plasmodium-selective proteasome inhibitor strongly synergized dihydroartemisinin action in these PB K13 mutant lines, providing further evidence that the proteasome can be targeted to overcome ART resistance. Taken together, our work provides clear experimental evidence for the involvement of K13 polymorphisms in mediating susceptibility to artemisinins in vitro, and most importantly under in vivo conditions.IMPORTANCERecent successes in malaria control have been seriously threatened by the emergence of Plasmodium falciparum parasite resistance to the frontline artemisinin drugs in Southeast Asia. P. falciparum artemisinin resistance is associated with mutations in the parasite K13 protein, which associates with a delay in the time required to clear the parasites upon treatment with the drug. Gene editing technologies have been used to validate the role of several candidate K13 mutations in mediating P. falciparum artemisinin resistance in vitro under laboratory conditions. Nonetheless, the causal role of these mutations under in vivo conditions has been a matter of debate. Here, we have used CRISPR/Cas9 gene editing to introduce K13 mutations associated with artemisinin resistance into the related rodent-infecting parasite, P. berghei. Phenotyping of these P. berghei K13 mutant parasites provides evidence of their role in mediating artemisinin resistance in vivo, which supports in vitro artemisinin resistance observations. However, we were unable to introduce some of the P. falciparum K13 mutations (C580Y, I543T) into the corresponding amino acid residues, while other introduced mutations (M476I, R539T equivalents) carried a pronounced fitness cost. Our study provides evidence of a clear causal role of K13 mutations in modulating susceptibility to artemisinins in vitro and in vivo using the well-characterized P. berghei model. We also show that inhibition of the P. berghei proteasome offsets parasite resistance to artemisinins in these mutant lines.

scholarly journals Modification of pfap2μ and pfubp1 Markedly Reduces Ring-Stage Susceptibility of Plasmodium falciparum to Artemisinin In Vitro

2019 ◽  
Vol 64 (1) ◽  
Author(s):  
Ryan C. Henrici ◽  
Donelly A. van Schalkwyk ◽  
Colin J. Sutherland
Keyword(s):  
Plasmodium Falciparum ◽  
Artemisinin Resistance ◽  
Antimalarial Drugs ◽  
Ring Stage ◽  
Content Type ◽  
Adaptive Mechanisms ◽  
Transgenic Parasites ◽  
Survival Assay

ABSTRACT Management of uncomplicated malaria worldwide is threatened by the emergence in Asia of Plasmodium falciparum carrying variants of the pfk13 locus and exhibiting reduced susceptibility to artemisinin. Mutations in two other genes, ubp1 and ap2μ, are associated with artemisinin resistance in rodent malaria and with clinical failure of combination therapy in African malaria patients. Transgenic P. falciparum clones, each carrying orthologues of mutations in pfap2μ and pfubp1 associated with artemisinin resistance in Plasmodium chabaudi, were derived by Cas9 gene editing. Susceptibility to artemisinin and other antimalarial drugs was determined. Following exposure to 700 nM dihydroartemisinin in the ring-stage survival assay, we found strong evidence that transgenic parasites expressing the I592T variant (11% survival), but not the S160N variant (1% survival), of the AP2μ adaptin subunit were significantly less susceptible than the parental wild-type parasite population. The V3275F variant of UBP1, but not the V3306F variant, also displayed reduced susceptibility to dihydroartemisinin (8.5% survival versus 0.5% survival). AP2μ and UBP1 variants did not elicit reduced susceptibility to 48 h of exposure to artemisinin or to other antimalarial drugs. Therefore, variants of the AP2 adaptor complex μ-subunit and of the ubiquitin hydrolase UBP1 reduce in vitro artemisinin susceptibility at the early ring stage in P. falciparum. These findings confirm the existence of multiple pathways to perturbation of either the mode of action of artemisinin, the parasite’s adaptive mechanisms of resistance, or both. The cellular role of UBP1 and AP2μ in Plasmodium parasites should now be elucidated.

scholarly journals Artemisinin-Resistant Plasmodium falciparum Parasites Exhibit Altered Patterns of Development in Infected Erythrocytes

2015 ◽  
Vol 59 (6) ◽  
pp. 3156-3167 ◽  
Author(s):  
Amanda Hott ◽  
Debora Casandra ◽  
Kansas N. Sparks ◽  
Lindsay C. Morton ◽  
Geocel-Grace Castanares ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Artemisinin Resistance ◽  
Drug Pressure ◽  
Content Type ◽  
Phenotypic Resistance ◽  
Artemisinin Derivatives ◽  
Stage Of Development ◽  
Clinical Resistance

ABSTRACTArtemisinin derivatives are used in combination with other antimalarial drugs for treatment of multidrug-resistant malaria worldwide. Clinical resistance to artemisinin recently emerged in southeast Asia, yetin vitrophenotypes for discerning mechanism(s) of resistance remain elusive. Here, we describe novel phenotypic resistance traits expressed by artemisinin-resistantPlasmodium falciparum. The resistant parasites exhibit altered patterns of development that result in reduced exposure to drug at the most susceptible stage of development in erythrocytes (trophozoites) and increased exposure in the most resistant stage (rings). In addition, a novelin vitrodelayed clearance assay (DCA) that assesses drug effects on asexual stages was found to correlate with parasite clearance half-lifein vivoas well as with mutations in the Kelch domain gene associated with resistance (Pf3D7_1343700). Importantly, all of the resistance phenotypes were stable in cloned parasites for more than 2 years without drug pressure. The results demonstrate artemisinin-resistantP. falciparumhas evolved a novel mechanism of phenotypic resistance to artemisinin drugs linked to abnormal cell cycle regulation. These results offer insights into a novel mechanism of drug resistance inP. falciparumand new tools for monitoring the spread of artemisinin resistance.

scholarly journals K13 Propeller Mutations in Plasmodium falciparum Populations in Regions of Malaria Endemicity in Vietnam from 2009 to 2016

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
Nguyen Thuy-Nhien ◽  
Nguyen Kim Tuyen ◽  
Nguyen Thanh Tong ◽  
Nguyen Tuong Vy ◽  
Ngo Viet Thanh ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Artemisinin Resistance ◽  
Parasite Clearance ◽  
Single Strain ◽  
Content Type ◽  
Reduced Ring ◽  
Resistance Markers ◽  
Multiple Strains

ABSTRACT The spread of artemisinin-resistant Plasmodium falciparum compromises the therapeutic efficacy of artemisinin combination therapies (ACTs) and is considered the greatest threat to current global initiatives to control and eliminate malaria. This is particularly relevant in Vietnam, where dihydroartemisinin-piperaquine (DP) is the recommended ACT for P. falciparum infection. The propeller domain gene of K13, a molecular marker of artemisinin resistance, was successfully sequenced in 1,060 P. falciparum isolates collected at 3 malaria hot spots in Vietnam between 2009 and 2016. Eight K13 propeller mutations (Thr474Ile, Tyr493His, Arg539Thr, Ile543Thr, Pro553Leu, Val568Gly, Pro574Leu, and Cys580Tyr), including several that have been validated to be artemisinin resistance markers, were found. The prevalences of K13 mutations were 29% (222/767), 6% (11/188), and 43% (45/105) in the Binh Phuoc, Ninh Thuan, and Gia Lai Provinces of Vietnam, respectively. Cys580Tyr became the dominant genotype in recent years, with 79.1% (34/43) of isolates in Binh Phuoc Province and 63% (17/27) of isolates in Gia Lai Province carrying this mutation. K13 mutations were associated with reduced ring-stage susceptibility to dihydroartemisinin (DHA) in vitro and prolonged parasite clearance in vivo. An analysis of haplotypes flanking K13 suggested the presence of multiple strains with the Cys580Tyr mutation rather than a single strain expanding across the three sites.

scholarly journals Plasmodium berghei K13 Mutations Mediate In Vivo Artemisinin Resistance That Is Reversed by Proteasome Inhibition

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Nelson V. Simwela ◽  
Barbara H. Stokes ◽  
Dana Aghabi ◽  
Matt Bogyo ◽  
David A. Fidock ◽  
...  
Keyword(s):  
Plasmodium Berghei ◽  
Gene Editing ◽  
Artemisinin Resistance ◽  
Ring Stage ◽  
Parasite Resistance ◽  
Content Type ◽  
Mutant Lines

ABSTRACT The recent emergence of Plasmodium falciparum parasite resistance to the first line antimalarial drug artemisinin is of particular concern. Artemisinin resistance is primarily driven by mutations in the P. falciparum K13 protein, which enhance survival of early ring-stage parasites treated with the artemisinin active metabolite dihydroartemisinin in vitro and associate with delayed parasite clearance in vivo. However, association of K13 mutations with in vivo artemisinin resistance has been problematic due to the absence of a tractable model. Herein, we have employed CRISPR/Cas9 genome editing to engineer selected orthologous P. falciparum K13 mutations into the K13 gene of an artemisinin-sensitive Plasmodium berghei rodent model of malaria. Introduction of the orthologous P. falciparum K13 F446I, M476I, Y493H, and R539T mutations into P. berghei K13 yielded gene-edited parasites with reduced susceptibility to dihydroartemisinin in the standard 24-h in vitro assay and increased survival in an adapted in vitro ring-stage survival assay. Mutant P. berghei K13 parasites also displayed delayed clearance in vivo upon treatment with artesunate and achieved faster recrudescence upon treatment with artemisinin. Orthologous C580Y and I543T mutations could not be introduced into P. berghei, while the equivalents of the M476I and R539T mutations resulted in significant growth defects. Furthermore, a Plasmodium-selective proteasome inhibitor strongly synergized dihydroartemisinin action in these P. berghei K13 mutant lines, providing further evidence that the proteasome can be targeted to overcome artemisinin resistance. Taken together, our findings provide clear experimental evidence for the involvement of K13 polymorphisms in mediating susceptibility to artemisinins in vitro and, most importantly, under in vivo conditions. IMPORTANCE Recent successes in malaria control have been seriously threatened by the emergence of Plasmodium falciparum parasite resistance to the frontline artemisinin drugs in Southeast Asia. P. falciparum artemisinin resistance is associated with mutations in the parasite K13 protein, which associates with a delay in the time required to clear the parasites upon drug treatment. Gene editing technologies have been used to validate the role of several candidate K13 mutations in mediating P. falciparum artemisinin resistance in vitro under laboratory conditions. Nonetheless, the causal role of these mutations under in vivo conditions has been a matter of debate. Here, we have used CRISPR/Cas9 gene editing to introduce K13 mutations associated with artemisinin resistance into the related rodent-infecting parasite, Plasmodium berghei. Phenotyping of these P. berghei K13 mutant parasites provides evidence of their role in mediating artemisinin resistance in vivo, which supports in vitro artemisinin resistance observations. However, we were unable to introduce some of the P. falciparum K13 mutations (C580Y and I543T) into the corresponding amino acid residues, while other introduced mutations (M476I and R539T equivalents) carried pronounced fitness costs. Our study provides evidence of a clear causal role of K13 mutations in modulating susceptibility to artemisinins in vitro and in vivo using the well-characterized P. berghei model. We also show that inhibition of the P. berghei proteasome offsets parasite resistance to artemisinins in these mutant lines.

scholarly journals Role of Plasmodium falciparum Kelch 13 Protein Mutations in P. falciparum Populations from Northeastern Myanmar in Mediating Artemisinin Resistance

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Faiza Amber Siddiqui ◽  
Rachasak Boonhok ◽  
Mynthia Cabrera ◽  
Huguette Gaelle Ngassa Mbenda ◽  
Meilian Wang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasmodium Falciparum ◽  
Survival Rates ◽  
Artemisinin Resistance ◽  
Ring Stage ◽  
Content Type ◽  
Stage Development ◽  
Survival Assay ◽  
Kelch 13

ABSTRACT Mutations in the Plasmodium falciparum Kelch 13 (PfK13) protein are associated with artemisinin resistance. PfK13 is essential for asexual erythrocytic development, but its function is not known. We tagged the PfK13 protein with green fluorescent protein in P. falciparum to study its expression and localization in asexual and sexual stages. We used a new antibody against PfK13 to show that the PfK13 protein is expressed ubiquitously in both asexual erythrocytic stages and gametocytes and is localized in punctate structures, partially overlapping an endoplasmic reticulum marker. We introduced into the 3D7 strain four PfK13 mutations (F446I, N458Y, C469Y, and F495L) identified in parasites from the China-Myanmar border area and characterized the in vitro artemisinin response phenotypes of the mutants. We found that all the parasites with the introduced PfK13 mutations showed higher survival rates in the ring-stage survival assay (RSA) than the wild-type (WT) control, but only parasites with N458Y displayed a significantly higher RSA value (26.3%) than the WT control. After these PfK13 mutations were reverted back to the WT in field parasite isolates, all revertant parasites except those with the C469Y mutation showed significantly lower RSA values than their respective parental isolates. Although the 3D7 parasites with introduced F446I, the predominant PfK13 mutation in northern Myanmar, did not show significantly higher RSA values than the WT, they had prolonged ring-stage development and showed very little fitness cost in in vitro culture competition assays. In comparison, parasites with the N458Y mutations also had a prolonged ring stage and showed upregulated resistance pathways in response to artemisinin, but this mutation produced a significant fitness cost, potentially leading to their lower prevalence in the Greater Mekong subregion. IMPORTANCE Artemisinin resistance has emerged in Southeast Asia, endangering the substantial progress in malaria elimination worldwide. It is associated with mutations in the PfK13 protein, but how PfK13 mediates artemisinin resistance is not completely understood. Here we used a new antibody against PfK13 to show that the PfK13 protein is expressed in all stages of the asexual intraerythrocytic cycle as well as in gametocytes and is partially localized in the endoplasmic reticulum. By introducing four PfK13 mutations into the 3D7 strain and reverting these mutations in field parasite isolates, we determined the impacts of these mutations identified in the parasite populations from northern Myanmar on the ring stage using the in vitro ring survival assay. The introduction of the N458Y mutation into the 3D7 background significantly increased the survival rates of the ring-stage parasites but at the cost of the reduced fitness of the parasites. Introduction of the F446I mutation, the most prevalent PfK13 mutation in northern Myanmar, did not result in a significant increase in ring-stage survival after exposure to dihydroartemisinin (DHA), but these parasites showed extended ring-stage development. Further, parasites with the F446I mutation showed only a marginal loss of fitness, partially explaining its high frequency in northern Myanmar. Conversely, reverting all these mutations, except for the C469Y mutation, back to their respective wild types reduced the ring-stage survival of these isolates in response to in vitro DHA treatment.

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

2015 ◽  
Vol 59 (8) ◽  
pp. 5061-5064 ◽  
Author(s):  
Roland A. Cooper ◽  
Melissa D. Conrad ◽  
Quentin D. Watson ◽  
Stephanie J. Huezo ◽  
Harriet Ninsiima ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Gene Polymorphisms ◽  
Artemisinin Resistance ◽  
Ring Stage ◽  
Content Type ◽  
Molecular Assays ◽  
Highly Sensitive ◽  
Survival Assay

ABSTRACTWe evaluated markers of artemisinin resistance inPlasmodium falciparumisolated in Kampala in 2014. By standardin vitroassays, all isolates were highly sensitive to dihydroartemisinin (DHA). By the ring-stage survival assay, after a 6-h DHA pulse, parasitemia was undetectable in 40 of 43 cultures at 72 h. Two of 53 isolates had nonsynonymous K13-propeller gene polymorphisms but did not have the mutations associated with resistance in Asia. Thus, we did not see evidence for artemisinin resistance in Uganda.

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