利用参考克隆优化 HRP-2 体外疟疾药物敏感性检测，以提高对恶性疟原虫现场分离株的比较。
Optimizing the HRP-2 in vitro malaria drug susceptibility assay using a reference clone to improve comparisons of Plasmodium falciparum field isolates.
机构信息
Department of Immunology and Medicine, US Army Medical Corps, Armed Forces Research Institute of Medical Sciences (USAMC-AFRIMS), Bangkok, Thailand.
出版信息
Malar J. 2012 Sep 13;11:325. doi: 10.1186/1475-2875-11-325.
BACKGROUND
Apparent emerging artemisinin-resistant Plasmodium falciparum malaria in Southeast Asia requires development of practical tools to monitor for resistant parasites. Although in vitro anti-malarial susceptibility tests are widely used, uncertainties remain regarding interpretation of P. falciparum field isolate values.
METHODS
Performance parameters of the W2 P. falciparum clone (considered artemisinin "sensitive") were evaluated as a reference for the HRP-2 immediate ex vivo assay. Variability in W2 IC50s was assessed, including intra- and inter-assay variability among and between technicians in multiple experiments, over five freeze-thaw cycles, over five months of continuous culture, and before and after transport of drug-coated plates to remote field sites. Nominal drug plate concentrations of artesunate (AS) and dihydroartemisinin (DHA) were verified by LC-MS analysis. Plasmodium falciparum field isolate IC50s for DHA from subjects in an artemisinin-resistant area in Cambodia were compared with W2 susceptibility.
RESULTS
Plate drug concentrations and day-to-day technical assay performance among technicians were important sources of variability for W2 IC50s within and between assays. Freeze-thaw cycles, long-term continuous culture, and transport to and from remote sites had less influence. Despite variability in W2 susceptibility, the median IC50s for DHA for Cambodian field isolates were higher (p <0.0001) than the W2 clone (3.9 nM), both for subjects with expected (less than 72 hours; 6.3 nM) and prolonged (greater or equal to 72 hours; 9.6 nM) parasite clearance times during treatment with artesunate monotherapy.
CONCLUSION
The W2 reference clone improved the interpretability of field isolate susceptibility from the immediate ex vivo HRP-2 assay from areas of artemisinin resistance. Methods to increase the reproducibility of plate coating may improve overall assay interpretability and utility.
背景
东南亚地区明显出现青蒿素耐药恶性疟原虫，需要开发实用工具来监测耐药寄生虫。虽然体外抗疟药敏感性试验被广泛应用，但在解释恶性疟原虫野外分离株数值时仍存在不确定性。
方法
评估 W2 恶性疟原虫克隆（被认为是青蒿素“敏感”）的性能参数，作为 HRP-2 即时离体检测的参考。评估了 W2IC50 的变异性，包括多个实验中不同技术员之间的实验内和实验间变异性，在五个冻融循环、五个月的连续培养、药物包被平板运输到偏远野外地点前后。通过 LC-MS 分析验证青蒿琥酯（AS）和双氢青蒿素（DHA）的名义药物平板浓度。比较柬埔寨青蒿素耐药地区患者的 DHA 野外分离株 IC50 与 W2 敏感性。
结果
平板药物浓度和技术员之间日常技术检测性能是 W2IC50 实验内和实验间变异性的重要来源。冻融循环、长期连续培养以及往返偏远地点的影响较小。尽管 W2 敏感性存在变异性，但柬埔寨野外分离株的 DHA 中位 IC50 值更高（p<0.0001），均高于 W2 克隆（3.9 nM），青蒿琥酯单药治疗时，预计（小于 72 小时；6.3 nM）和延长（大于或等于 72 小时；9.6 nM）寄生虫清除时间的患者。
结论
W2 参考克隆提高了即时离体 HRP-2 检测中抗青蒿素耐药地区野外分离株敏感性的可解释性。增加平板包被重现性的方法可能会提高整体检测可解释性和实用性。
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