恶性疟原虫体外药敏实验检测方法的比较研究

Comparative study of assay methods for in vitro antimalarial drug efficacy testing in Plasmodium falciparum

目的比较WHO微量测试法（WHO microtest）、疟原虫乳酸脱氢酶（Plasmodial lactate dehydrogenase,p LDH）ELISA测定法、富组蛋白Ⅱ（Histidine-rich proteinⅡ,HRPⅡ）ELISA测定法和SYBR GreenⅠ荧光测定法等4种恶性疟原虫体外药敏实验检测方法,确定一种较为稳定、简便快速且经济的体外药敏实验检测方法,用于后续疟原虫敏感性的监测及新型抗疟药的筛查。方法以恶性疟原虫虫株3D7、FCC、K1、Dd2为对象,分别采用WHO microtest法、p LDH法、HRPⅡ法和SYBR GreenⅠ法测定其对氯喹、哌喹和阿莫地喹的敏感性,并采用Friedman检验、偏相关分析、Pearson相关分析和Bland-Altman分析对4种药敏检测方法的IC50值进行分析。结果在初始密度为0.5%~1.0%时,4种方法的IC50值之间的差异无统计学意义（P〉0.05）,任意2种方法间均呈直线相关关系（P〈0.001）。WHO microtest法技术要求高、耗时长且结果判定受主观影响;HRPⅡ法的敏感性高于p LDH法和SYBR GreenⅠ法,但成本高;SYBR GreenⅠ法技术要求低、耗时短且成本低。结论 SYBR GreenⅠ法是一种较为便捷、经济的检测方法,适用于大规模监测疟原虫药物敏感性及研究新型抗疟药。

Objective To compare four assay methods of in vitro antimalarial drug efficacy testing,including WHO microtest,Plasmodial lactate dehydrogenase（p LDH）,Histidine-rich protein Ⅱ（HRP Ⅱ）and SYBR Green Ⅰ,so as to determine a stable,simple,rapid,and economic method for monitoring the drug sensitivity of malaria parasites and screening new antimalarial drugs. Methods WHO microtest,p LDH,HRP Ⅱ and SYBR Green Ⅰ were applied to test the drug efficacy of chloroquine,piperaquine and amodiaquine against four Plasmodium falciparum reference strains（3D7,FCC,K1 and Dd2）,respectively. The consistency of the 50% inhibitory concentration（IC50）values from the four assay methods were analyzed by Friedman tests,Partial correlation analysis,Pearson＇s correlation analysis and Bland-Altman plots. Results With the initial parasitemia ranged from 0.5% to 1%,there were no statistically significant differences（P 〉0.05）among the IC50 values obtained by the four assay methods,which were correlated well（both P 〈0.001）. WHO microtest was highly labor-intensive,timeconsuming and subjective;although HRP Ⅱ was more sensitive than p LDH and SYBR Green Ⅰ,which was more expensive;SYBR Green Ⅰwas a simple,rapid and economic assay method. Conclusion SYBR Green Ⅰ,as a simple and cost-effective assay method,is suitable for high-throughput malaria drug sensitivity monitoring and research of new antimalarial drug screening.