A Plasmodium falciparum screening assay for anti-gametocyte drugs based on parasite lactate dehydrogenase detection
A Plasmodium falciparum screening assay for anti-gametocyte drugs based on parasite lactate dehydrogenase detection
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DOI：
10.1093/jac/dkt165
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发表时间：
2013-09-01
影响因子：
5.2
通讯作者：
Taramelli, Donatella
中科院分区：
文献类型：
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作者：
D'Alessandro, Sarah;Silvestrini, Francesco;Taramelli, Donatella
疟原虫配子母细胞负责疟疾寄生虫从人类传播到蚊子，是新的抗疟药物实现疟疾消除/根除的关键目标。我们开发了一种新的抗配子体化合物的比色筛选方法的基础上，寄生虫乳酸脱氢酶（pLDH）测定，已经标准化的无性stages.Gametocytogenesis恶性疟原虫株的3D 7和NF 54在体外诱导和无性寄生虫耗尽与N-乙酰葡萄糖胺，以测量配子体的活力和药物敏感性。在96孔板中用双氢青蒿素、环氧霉素、亚甲蓝、伯氨喹、嘌呤霉素或氯喹处理配子细胞，并使用修改的Makler方案评价pLDH活性。用标准膜饲养试验（SMFA）测定感染力，Giemsa染色法测定配子体血与pLDH活性呈线性相关。药物处理72小时后观察到pLDH活性的浓度依赖性降低，而需要另外72小时的无药物孵育以获得配子体活力的完全抑制。SMFA处理和对照配子体证实，减少pLDH活性转化为减少卵囊发育的蚊子vector.The配子体pLDH测定是快速，易于执行，廉价和可重复的，适合于筛选新的传输阻断化合物，这不需要寄生虫转基因株系。
Plasmodium gametocytes, responsible for malaria parasite transmission from humans to mosquitoes, represent a crucial target for new antimalarial drugs to achieve malaria elimination/eradication. We developed a novel colorimetric screening method for anti-gametocyte compounds based on the parasite lactate dehydrogenase (pLDH) assay, already standardized for asexual stages, to measure gametocyte viability and drug susceptibility.Gametocytogenesis of 3D7 and NF54 Plasmodium falciparum strains was induced in vitro and asexual parasites were depleted with N-acetylglucosamine. Gametocytes were treated with dihydroartemisinin, epoxomicin, methylene blue, primaquine, puromycin or chloroquine in 96-well plates and the pLDH activity was evaluated using a modified Makler protocol. Mosquito infectivity was measured by the standard membrane feeding assay (SMFA).A linear correlation was found between gametocytaemia determined by Giemsa staining and pLDH activity. A concentration-dependent reduction in pLDH activity was observed after 72 h of drug treatment, whereas an additional 72 h of incubation without drugs was required to obtain complete inhibition of gametocyte viability. SMFA on treated and control gametocytes confirmed that a reduction in pLDH activity translates into reduced oocyst development in the mosquito vector.The gametocyte pLDH assay is fast, easy to perform, cheap and reproducible and is suitable for screening novel transmission-blocking compounds, which does not require parasite transgenic lines.