Abstract：
BACKGROUND: Mutations in the Plasmodium falciparum dhfr gene confer resistance to pyrimethamine, which is widely used for malaria chemoprevention in Africa. We aimed to evaluate the frequency and evolution of dhfr mutations in Plasmodium ovale spp in Africa and their functional consequences, which are incompletely characterised.
METHODS: We analysed dhfr mutations and their frequencies in P ovale spp isolates collected between Feb 1, 2004, and Aug 31, 2023, from the French National Malaria Reference Centre collection and from field studies in Benin, Gabon, and Kenya. Genetic patterns of positive selection were investigated. Full-length recombinant wild-type and mutant DHFR enzymes from both P ovale curtisi and P ovale wallikeri were expressed in bacteria to test whether the most common mutations reduced pyrimethamine susceptibility.
RESULTS: We included 518 P ovale spp samples (314 P ovale curtisi and 204 P ovale wallikeri). In P ovale curtisi, Ala15Ser-Ser58Arg was the most common dhfr mutation (39%; 124 of 314 samples). In P ovale wallikeri, dhfr mutations were less frequent, with Phe57Leu-Ser58Arg reaching 17% (34 of 204 samples). These two mutants were the most prevalent in central and east Africa and were fixed in Kenyan isolates. We detected six and four other non-synonymous mutations, representing 8% (24 isolates) and 2% (five isolates) of the P ovale curtisi and P ovale wallikeri isolates, respectively. Whole-genome sequencing and microsatellite analyses revealed reduced genetic diversity around the mutant pocdhfr and powdhfr genes. The mutant DHFR proteins showed structural changes at the pyrimethamine binding site in-silico, confirmed by a 4-times increase in pyrimethamine half-maximal inhibitory concentration in an Escherichia coli growth assay for the Phe57Leu-Ser58Arg mutant and 50-times increase for the Ala15Ser-Ser58Arg mutant, compared with the wild-type counterparts.
CONCLUSIONS: The widespread use of sulfadoxine-pyrimethamine for malaria chemoprevention might have exerted fortuitous selection pressure for dhfr mutations in P ovale spp. This calls for closer monitoring of dhfr and dhps mutations in P ovale spp.
BACKGROUND: French Ministry of Health, Agence Nationale de la Recherche, and Global Emerging Infections Surveillance branch of the Armed Forces Health Surveillance Division.
METHODS: We analysed dhfr mutations and their frequencies in P ovale spp isolates collected between Feb 1, 2004, and Aug 31, 2023, from the French National Malaria Reference Centre collection and from field studies in Benin, Gabon, and Kenya. Genetic patterns of positive selection were investigated. Full-length recombinant wild-type and mutant DHFR enzymes from both P ovale curtisi and P ovale wallikeri were expressed in bacteria to test whether the most common mutations reduced pyrimethamine susceptibility.
RESULTS: We included 518 P ovale spp samples (314 P ovale curtisi and 204 P ovale wallikeri). In P ovale curtisi, Ala15Ser-Ser58Arg was the most common dhfr mutation (39%; 124 of 314 samples). In P ovale wallikeri, dhfr mutations were less frequent, with Phe57Leu-Ser58Arg reaching 17% (34 of 204 samples). These two mutants were the most prevalent in central and east Africa and were fixed in Kenyan isolates. We detected six and four other non-synonymous mutations, representing 8% (24 isolates) and 2% (five isolates) of the P ovale curtisi and P ovale wallikeri isolates, respectively. Whole-genome sequencing and microsatellite analyses revealed reduced genetic diversity around the mutant pocdhfr and powdhfr genes. The mutant DHFR proteins showed structural changes at the pyrimethamine binding site in-silico, confirmed by a 4-times increase in pyrimethamine half-maximal inhibitory concentration in an Escherichia coli growth assay for the Phe57Leu-Ser58Arg mutant and 50-times increase for the Ala15Ser-Ser58Arg mutant, compared with the wild-type counterparts.
CONCLUSIONS: The widespread use of sulfadoxine-pyrimethamine for malaria chemoprevention might have exerted fortuitous selection pressure for dhfr mutations in P ovale spp. This calls for closer monitoring of dhfr and dhps mutations in P ovale spp.
BACKGROUND: French Ministry of Health, Agence Nationale de la Recherche, and Global Emerging Infections Surveillance branch of the Armed Forces Health Surveillance Division.
摘要：
背景：恶性疟原虫dhfr基因的突变赋予对乙胺嘧啶的抗性，在非洲广泛用于疟疾化学预防。我们旨在评估非洲卵形疟原虫dhfr突变的频率和进化及其功能后果，不完全表征。
方法：我们分析了2004年2月1日至2023年8月31日从法国国家疟疾参考中心收集和贝宁实地研究中收集的卵卵卵圆菌分离株中的dhfr突变及其频率，加蓬,肯尼亚。研究了阳性选择的遗传模式。在细菌中表达了来自卵圆刀和卵圆刀的全长重组野生型和突变型DHFR酶，以测试最常见的突变是否降低了乙胺嘧啶的敏感性。
结果：我们包括了518个卵圆类样本（314个卵圆类和204个卵圆类）。在POvaleCurtisi,Ala15Ser-Ser58Arg是最常见的dhfr突变(39%;314个样品中的124个)。在POvaleWallikeri,dhfr突变频率较低，Phe57Leu-Ser58Arg达到17%(204个样品中的34个)。这两个突变体在中非和东非最普遍，并固定在肯尼亚的分离株中。我们检测到六个和四个其他非同义突变，代表8％（24个分离株）和2％（5个分离株）的卵卵圆虫和卵圆虫的分离株，分别。全基因组测序和微卫星分析显示，突变pocdhfr和powdhfr基因周围的遗传多样性降低。突变型DHFR蛋白在计算机上的乙胺嘧啶结合位点显示出结构变化，在Phe57Leu-Ser58Arg突变体的大肠杆菌生长测定中，乙胺嘧啶半最大抑制浓度增加了4倍，而Ala15Ser-Ser58Arg突变体增加了50倍，与野生型同行相比。
结论：磺胺多辛-乙胺嘧啶在疟疾化学预防中的广泛使用可能对卵圆虫中的dhfr突变产生了偶然的选择压力。这要求更密切地监测卵卵圆中的dhfr和dhps突变。
背景：法国卫生部，国家机构，和武装部队健康监测司全球新兴感染监测科。
方法：我们分析了2004年2月1日至2023年8月31日从法国国家疟疾参考中心收集和贝宁实地研究中收集的卵卵卵圆菌分离株中的dhfr突变及其频率，加蓬,肯尼亚。研究了阳性选择的遗传模式。在细菌中表达了来自卵圆刀和卵圆刀的全长重组野生型和突变型DHFR酶，以测试最常见的突变是否降低了乙胺嘧啶的敏感性。
结果：我们包括了518个卵圆类样本（314个卵圆类和204个卵圆类）。在POvaleCurtisi,Ala15Ser-Ser58Arg是最常见的dhfr突变(39%;314个样品中的124个)。在POvaleWallikeri,dhfr突变频率较低，Phe57Leu-Ser58Arg达到17%(204个样品中的34个)。这两个突变体在中非和东非最普遍，并固定在肯尼亚的分离株中。我们检测到六个和四个其他非同义突变，代表8％（24个分离株）和2％（5个分离株）的卵卵圆虫和卵圆虫的分离株，分别。全基因组测序和微卫星分析显示，突变pocdhfr和powdhfr基因周围的遗传多样性降低。突变型DHFR蛋白在计算机上的乙胺嘧啶结合位点显示出结构变化，在Phe57Leu-Ser58Arg突变体的大肠杆菌生长测定中，乙胺嘧啶半最大抑制浓度增加了4倍，而Ala15Ser-Ser58Arg突变体增加了50倍，与野生型同行相比。
结论：磺胺多辛-乙胺嘧啶在疟疾化学预防中的广泛使用可能对卵圆虫中的dhfr突变产生了偶然的选择压力。这要求更密切地监测卵卵圆中的dhfr和dhps突变。
背景：法国卫生部，国家机构，和武装部队健康监测司全球新兴感染监测科。
