Pyrimethamine was first introduced for the treatment of malaria in Asia and Africa during the early 1980s, replacing chloroquine, and has become the first line of drugs in many countries. In recent years, development of pyrimethamine resistance in Plasmodium vivax has become a barrier to effective malaria control strategies. Here, we describe the use of meta-barcoded deep amplicon sequencing technology to assess the evolutionary origin of pyrimethamine resistance by analysing the flanking region of dihydrofolate reductase (dhfr) locus. The genetic modelling suggests that 58R and 173L single mutants and 58R/117N double mutants are present on a single lineage; suggesting a single origin of these mutations. The triple mutants (57L/58R/117N, 58R/61M/117N and 58R/117N/173L) share the lineage of 58R/117N, suggesting a common origin. In contrast, the 117N mutant is present on two separate lineages suggesting that there are multiple origins of this mutation. We characterised the allele frequency of the P. vivax dhfr locus. Our results support the view that the single mutation of 117N and double mutations of 58R/117N arise commonly, whereas the single mutation of 173L and triple mutations of 57L/58R/117N, 58R/61M/117N and 58R/117N/173L are less common. Our work will help to inform mitigation strategies for pyrimethamine resistance in P. vivax.

乙胺嘧啶在1980年代初期首次引入用于治疗亚洲和非洲的疟疾，取代了氯喹，并已成为许多国家的第一线药物。近年来，间日疟原虫对乙胺嘧啶的耐药性发展已成为有效控制疟疾策略的障碍。在这里，我们描述了通过分析二氢叶酸还原酶（dhfr）基因座的侧翼区域，使用亚条形码深扩增子测序技术评估乙胺嘧啶抗性的进化起源。遗传模型表明58R和173L单突变体和58R / 117N双突变体存在于单个谱系中。提示这些突变的单一来源。三重突变体（57L / 58R / 117N，58R / 61M / 117N和58R / 117N / 173L）共有58R / 117N谱系，表明其共有来源。相反，117N突变体存在于两个不同的谱系中，表明该突变体有多个起源。我们表征了间日疟原虫dhfr基因座的等位基因频率。我们的结果支持这样的观点，即117N单突变和58R / 117N双突变普遍出现，而173L单突变和57L / 58R / 117N，58R / 61M / 117N和58R / 117N / 173L三突变较少共同。我们的工作将有助于为间日疟原虫的乙胺嘧啶抗性提供缓解策略。58R / 61M / 117N和58R / 117N / 173L不太常见。我们的工作将有助于为间日疟原虫的乙胺嘧啶抗性提供缓解策略。58R / 61M / 117N和58R / 117N / 173L不太常见。我们的工作将有助于为间日疟原虫的乙胺嘧啶抗性提供缓解策略。