Okitwi Martin, Orena Stephen, Tumwebaze Patrick K, Katairo Thomas, Taremwa Yoweri, Byaruhanga Oswald, Tukwasibwe Stephen, Nsobya Samuel L, Legac Jennifer, Cooper Roland A, Conrad Melissa D, Rosenthal Philip J
medRxiv. 2025 Jan 1:2024.12.31.24319821. doi: 10.1101/2024.12.31.24319821.
The treatment and control of malaria in Africa is challenged by drug resistance, including transporter, folate pathway, and PfK13 mutations that mediate resistance to aminoquinolines, antifolates, and artemisinins, respectively. Characterization of drug susceptibility informs optimal control strategies.
We characterized ex vivo susceptibilities to nine drugs of isolates collected from individuals presenting with uncomplicated falciparum malaria in eastern (2019-2024) and northern (2021-2024) Uganda using a growth inhibition assay and the dihydroartemisinin (DHA) ring survival assay (RSA). Genetic polymorphisms were characterized by molecular inversion probe and dideoxy sequencing. We assessed drug susceptibilities over time and evaluated associations between susceptibilities and potential resistance markers for samples studied since 2016.
Of 1,297 collected, 724/828 eastern and 390/469 northern Uganda isolates were successfully evaluated for ex vivo drug susceptibilities. Median half-maximal inhibitory concentrations (IC s) were low-nanomolar for chloroquine, monodesethylamodiaquine, piperaquine, pyronaridine, lumefantrine, mefloquine, and DHA, but higher for quinine and pyrimethamine. Over time, susceptibilities improved for chloroquine, decreased for lumefantrine, mefloquine, and DHA, and were unchanged for other drugs. Changes in prevalences of known markers of altered drug susceptibility followed the same patterns. Genotypes associated with drug susceptibility were those previously identified for aminoquinolines and pyrimethamine. For lumefantrine, susceptibility was decreased with wild-type PfCRT K76T or PfMDR1 N86Y, mutant PfK13 C469Y or A675V, the newly identified PfCARL D611N mutation, which increased in prevalence over time, and a number of other polymorphisms. For DHA, RSA results were not associated with PfK13 mutations, but susceptibilities based on IC s were decreased in parasites with the PfK13 C469Y or A675V mutations and the newly identified PfMDR1 Y500N mutation.
Susceptibilities to antimalarial drugs were mostly excellent, but decreased activities of lumefantrine and DHA over time suggest potential loss of efficacies of leading regimens.
National Institutes of Health, Medicines for Malaria Venture, Gates Foundation.
We searched PubMed for combinations of the terms "antimalarial resistance", "malaria", " ", "Africa", "ex vivo", "pfmdr1", "pfcrt", "kelch", or "K13" and identified papers published between Jan 1, 2020, and Dec 30, 2024 on antimalarial drug sensitivity and resistance in Africa. A prior identical search was conducted for papers published from Jan 1, 2000 to Dec 31, 2020 in preparation for an earlier publication. We reviewed and included any relevant articles cited in those references. Our search identified many studies on antimalarial drugs and molecular markers of resistance, but few combining ex vivo drug susceptibility with genotyping results. This study provides a comprehensive assessment of ex vivo susceptibility of Ugandan parasites to nine antimalarial drugs from July, 2019 to June, 2024. It also characterized genotype-phenotype associations based on these ex vivo data and sequencing of 80 genes identified as potential resistance mediators. Our findings add value to the existing literature by providing comprehensive data on antimalarial drug susceptibility in Uganda, including ex vivo drug susceptibilities for >1100 isolates from two regions of the country, description of changes in drug susceptibilities over time, and characterisation of genotype-phenotype associations, considering genetic polymorphisms previously associated with resistance to various antimalarials and potential novel resistance mediators. Malaria parasites circulating in eastern and northern Uganda over the past five years were mostly sensitive to commonly used antimalarial drugs. However, parasite genotypes and phenotypes have changed over time. Most importantly, susceptibilities to dihydroartemisinin and lumefantrine, the components of the first-line antimalarial therapy in Uganda, have decreased over time, although the magnitudes of these decreases are modest, and the clinical implications of the results are uncertain. Continued performance of parasitological and genomic surveillance for evidence of antimalarial drug resistance and institution of policy changes to limit resistance selection and treatment failure should be high priorities.
非洲疟疾的治疗和控制面临耐药性挑战，包括转运体、叶酸途径以及PfK13突变，这些突变分别介导对氨基喹啉、抗叶酸药物和青蒿素的耐药性。药物敏感性特征有助于制定最佳控制策略。
我们使用生长抑制试验和双氢青蒿素（DHA）环生存试验（RSA），对2019 - 2024年从乌干达东部和2021 - 2024年从乌干达北部出现非复杂性恶性疟原虫疟疾的个体中收集的分离株对九种药物的体外敏感性进行了特征分析。通过分子倒置探针和双脱氧测序对基因多态性进行了特征分析。我们评估了不同时间的药物敏感性，并评估了自2016年以来所研究样本的敏感性与潜在耐药性标志物之间的关联。
在收集的1297份样本中，828份乌干达东部分离株中的724份和469份乌干达北部分离株中的390份成功进行了体外药物敏感性评估。氯喹、单去乙基阿莫地喹、哌喹、咯萘啶、本芴醇、甲氟喹和DHA的半数最大抑制浓度（IC ）中位数为低纳摩尔，但奎宁和乙胺嘧啶的则较高。随着时间推移，氯喹的敏感性提高，本芴醇、甲氟喹和DHA的敏感性降低，其他药物的敏感性不变。已知药物敏感性改变标志物的流行率变化遵循相同模式。与药物敏感性相关的基因型是先前针对氨基喹啉和乙胺嘧啶鉴定出的那些。对于本芴醇，野生型PfCRT K76T或PfMDR1 N86Y、突变型PfK13 C469Y或A675V、新鉴定出的PfCARL D611N突变（其流行率随时间增加）以及一些其他多态性会降低敏感性。对于DHA，RSA结果与PfK13突变无关，但基于IC 的敏感性在具有PfK13 C469Y或A675V突变以及新鉴定出的PfMDR1 Y500N突变的寄生虫中降低。
对抗疟药物的敏感性大多良好，但随着时间推移本芴醇和DHA活性降低表明主要治疗方案的疗效可能丧失。
美国国立卫生研究院、疟疾药物事业会、盖茨基金会。
我们在PubMed上搜索了“抗疟药物耐药性”、“疟疾”、“ ”、“非洲”、“体外”、“pfmdr1”、“pfcrt”、“kelch”或“K13”等术语的组合，并确定了2020年1月1日至2024年12月30日期间发表的关于非洲抗疟药物敏感性和耐药性的论文。为准备早期出版物，之前对2000年1月1日至2020年12月31日发表的论文进行了相同的搜索。我们审查并纳入了这些参考文献中引用的任何相关文章。我们的搜索发现了许多关于抗疟药物和耐药性分子标志物的研究，但很少有将体外药物敏感性与基因分型结果相结合的研究。本研究全面评估了2019年7月至2024年6月乌干达疟原虫对九种抗疟药物的体外敏感性。它还基于这些体外数据以及对80个被鉴定为潜在耐药介导因子的基因进行测序，对基因型 - 表型关联进行了特征分析。我们的研究结果通过提供乌干达抗疟药物敏感性的全面数据，包括该国两个地区1100多个分离株的体外药物敏感性、药物敏感性随时间的变化描述以及基因型 - 表型关联的特征分析，为现有文献增添了价值，同时考虑了先前与各种抗疟药物耐药性相关的基因多态性以及潜在的新型耐药介导因子。过去五年在乌干达东部和北部传播的疟原虫大多对常用抗疟药物敏感。然而，寄生虫基因型和表型随时间发生了变化。最重要的是，乌干达一线抗疟治疗药物双氢青蒿素和本芴醇的敏感性随时间降低，尽管降低幅度不大，且结果的临床意义尚不确定。持续开展寄生虫学和基因组监测以获取抗疟药物耐药性证据以及制定政策变化以限制耐药性选择和治疗失败应成为高度优先事项。
