云南省疟原虫多重药物抗性基因分布特征及不同类群疟原虫的基因条形码鉴定研究

The malaria epidemic was still the most serious area in 2015 in Yunnan Province, and the number of patients had an increase 25% over the previous year. Due to the lack of the drug resistance monitoring within the province and the etiology judgment of malaria infection sources, the anti-malaria measures were accurately implemented during the stage of malaria elimination in Yunnan Province. We have found that the detection rates of chloroquine-resistant and artemisinin-resistant gene were 85% and 26% from the case of Yunnan malaria epidemic, and these resistant strains were found in the region of 75% and 81% in Yunnan Province, This is the direct evidence of drug-resistance Plasmodium in Yunnan Province, which was not acquired by observation the malaria cases of Myanmar territory bordering with Yunnan Province in the past. We also found that the polymorphism structure was not the same between the different parasite in Pfhrp2, Pvmsp gene, which appearing the early identification as a potential source of infection. With the advantages of obtaining malaria acute cases blood samples covering the whole Yunnan Province and a good foundation work,inIn this study, we will use the gene polymorphism detection and the gene barcode data management technology. At last, through analyses the biological information, we can could get the polymorphism of parasite genetic structure and the accumulation accumulate a large number ofof DNA sequences. All these can help us revealing the multiple resistant malaria and building the DNA barcoding database of Plasmodium. More importantly, we can provide the judgement basis for antimalarial drug policy and malaria source timely, and contributeprovidean identification tool for the evaluation of therapeutic effect in Yunnan provinceProvince and its surrounding areas.

云南省2015年的疟疾疫情仍属全国最严重地区，且发病人数较上一年增加25%，由于缺乏对全省范围内的药物抗性监测和对疟疾传染来源的病原学判断，云南疟疾消除阶段的抗疟措施精准落实步履艰难。我们已发现，云南疟疾病例中的氯喹、青蒿素抗性基因检出率达85%和26%，81%和75%的地区存在抗性虫株，这是以往靠观察缅甸境内疟疾病例无法获取的云南省抗疟药抗性直接数据；也发现疟原虫Pfhrp2、Pvmsp等基因多态在不同类群疟原虫间存在差异，作为感染来源鉴别标识的潜力初现端倪。本课题借助可以获取全省疟疾病例血样的优势，在原有基础上，拟采用基因检测、生物信息分析和条形码数据管理技术，通过对疫情病例的疟原虫基因进行多态分析和DNA序列积累，以揭示云南省多重抗性疟疾的流行和构建具有针对性的疟原虫DNA条形码鉴定数据库，为云南省抗疟药政策及时调整提供依据，为云南及周边的疟疾传染来源判断、治疗效果评价提供鉴定工具。

“云南省疟原虫多重药物抗性基因分布特征及不同类群疟原虫的基因条形码鉴定研究”自然科学基金资助下，以服务云南省的疟疾消除目标为导向，着重开展了云南省疟疾消除阶段的疟原虫基因组生物信息数据库建立及其运，由项目组提供的疟疾订正诊断结果、病例感染地溯源等内容是云南省开展精准治疗疟疾病例和开展消除疟疾认证的重要依据。研究中，项目组首次采用核酸检测技术复检了云南省2016－2020年的全部疟疾病例，是云南省订正确诊每例疟疾病例的唯一权威依据。另外，项目研究首次建立起了“疟原虫基因进化分析数据库”，收集的逾6000条溯源基因序列为云南省追溯疟疾病例感染地、辨别病例性质（输入性、本土性）提供了比对资源。其中，子孢子表面蛋白基因（csp）的分子溯源效果稳定；裂殖子表面蛋白1/3基因（msp1/3）在不同种群间的同源性较好，适宜用作免疫诊断的靶蛋白；首次发现云南省存在富组氨酸蛋白2基因（pfhrp2）完全缺失的缺陷性恶性疟原虫，为改良疟疾免疫诊断试剂盒提供了依据。再者，首次建立起“疟原虫耐药性分析模板数据库”，收集了近5000条耐药性标记基因序列是云南省实时分析抗疟药筛选压力的基础数据。分析显示：氯喹耐药性标记基因（crt）突变程度深、积累严重；青蒿素耐药性标记基因（k13）单点突变骤然增多、呈种群扩张趋势；磺胺多辛耐药性标记基因（dhfr）和乙胺嘧啶耐药性标记基因（dhps）常见多位点联合突变。第四，首次建立起“云南省疟疾消除阶段的疟原虫形态图片数据库”和创建了“云南疟疾病例诊断信息管理数据库”网站，为云南省医学寄生虫生物学大数据分析提供了共享平台。最后，项目组依托研究成果，建立起了系统性、连续性俱佳的“云南省消除疟疾阶段的疟疾病例虫株种质资源库”，发表论13篇（SCI收录4篇），“云南省疟原虫基因组生物信息数据库建立及其运用”获云南省科技进步二等奖（公示期已过，尚未发证书）和云南省卫生科技成果二等奖（证书：2019-GW-2020-09-2-6-01），培养硕士研究生4名，课题组内3人晋升高级职称。