云南及其境外周边科泰复治疗恶性疟原虫无性体转阴时间延长分子基础研究

Artemisinin is the most efficacious drug for malaria treatment in the world. However, significantly delayed asexual parasite clearance times have been confirmed along China-Myanmar border. In the Great Mekong Sub-region (GMS), five countries had confirmed artemisinin resistance except China. In order to contain development and spread of artemisinin resistance, the project proposes to conduct clinically therapeutic efficacy of dihydroartemisinin-piperaquine (DHA-PPQ), establishing laboratory strains of Plasmodium falciparum, in-vitro survival assays of P. falciparum after respectively exposing to single DHA and PPQ. These researches will make clear the changing sensitivity of P. falciparum to DHA-PPQ in time, the cause of delayed asexual parasite clearance times in treatment of Plasmodium falciparum malaria with DHA-PPQ, i.e. reduced sensitivity of DHA or PPQ or both DHA and PPQ in Yunnan and neighboring areas of other countries. Furthermore, polymerase chain reaction (PCR) test and sequencing of DNA will be conducted to type and look for molecular markers for the delayed asexual parasite clearance times in treatment of Plasmodium falciparum malaria with DHA-PQ. The results of these researches will contribute to eliminating malaria and maintaining achievement of malaria elimination by providing scientific evidences and technical support in China and other areas of GMS.

青蒿素是目前全世界治疗疟疾最有效的药物，但中缅边境地区科泰复治疗恶性疟原虫无性体转阴时间已显著延长，大湄公河次区域（GMS）除中国外，其余5国恶性疟原虫对青蒿素抗性已被证实。为有效遏制青蒿素抗性发展和扩散，本研究拟通过双氢青蒿素哌喹片临床疗效试验、建立恶性疟原虫实验室虫株，然后开展双氢青蒿素和哌喹单药分别处理体外培养恶性疟原虫环状体存活实验，实时掌握云南及境外周边恶性疟原虫对科泰复敏感性变化，研究清楚云南及境外周边无性体清除时间延长原因，即是双氢青蒿素还是哌喹单药敏感性下降，还是两者均下降；并进一步通过PCR实验和DNA测序，找到治疗恶性疟原虫无性体转阴时间延长分子标志，为中国和GMS疟疾消除及消除成果巩固提供科学依据和技术支撑。

青蒿素抗性被认为是阻碍全球疟疾控制和消除重大威胁之一，为掌握云南及境外周边恶性疟对所使用双氢青蒿素哌喹片（科泰复）疗效和敏感性变化，本项目开展了科泰复临床疗效试验、用PCR实验检测可能与抗性相关的基因突变，以寻找恶性疟原虫无性体转阴时间延长分子基础，并尝试建立恶性疟原虫实验室虫株以开展相关研究。项目共收治恶性疟120例，完成42天随访116例，治愈率为94.17%（95%可信区间CI：88.35-97.62%），临床疗效试验结果证明科泰复仍敏感，可继续用于治疗恶性疟，但49.80 ±19.22小时的疟原虫无性体转阴时间，较2008-2013年间有所延长，科泰复对配子体无杀灭作用，用药15天后配子体才完全自然消失。PCR成功检测了140例病人恶性疟原虫基因，67（47.48%，95%CI: 38.95-56.12%)份样本有K13基因突变，共有突变点10个，其中48（40.29%，95%CI:26.68-43.06)份样本有F446I突变。把疟原虫无性体转阴时间≥72小时定义为疟原虫清除时间延长，并把疟原虫无性体转阴时间作结果变量，多因素Logistic回归分析显示疟原虫清除时间延长与K13基因总突变和各单一位点突变均无显著相关（比值比OR的95%CI包含1，且P>0.05），也没有发现其它与疟原虫无性体清除时间延长的基因突变。研究结果提示，在目前不断报告与青蒿素抗性相关分子标志，但青蒿素临床治疗恶性疟仍有效的背景下，要澄清此问题的话，需结合临床疗效对其分子基础进一步开展深入研究。