Department of Immunology and Medicine, US Army Medical Corps, Armed Forces Research Institute of Medical Sciences (USAMC-AFRIMS), Bangkok, Thailand.
Malar J. 2012 Sep 13;11:325. doi: 10.1186/1475-2875-11-325.
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.
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.
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.
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.
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。
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。
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。
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。
