我国恶性疟原虫Pfcrt和Pfmdr1基因多态性及与氯喹敏感性关系的研究

目的了解我国恶性疟原虫分离株Pfcrt基因K76T及Pf mdr1基因N86Y和D1246Y的点突变特征及发生率,并分析上述分子标志与恶性疟原虫对氯喹敏感性的关系。方法从我国恶性疟流行区云南和海南省采集恶性疟现症患者血样,根据恶性疟原虫Pfcrt基因和Pf mdr1基因序列设计巢式PCR引物,以血样中的恶性疟原虫DNA为模板,进行巢式PCR-RFLP分析,并对部分PCR产物进行测序验证。采用世界卫生组织制定的体外微量法测定同一批血样中恶性疟原虫对氯喹的敏感性。结果云南、海南省恶性疟原虫Pfcrt基因K76T的突变发生率分别为86.7%和64.3%;云南、海南省恶性疟原虫Pf mdr1 N86Y突变发生率分别为46.5%和3.4%。未发现云南、海南省恶性疟原虫分离株存在Pf mdr1 D1246Y突变。体外微量测定法显示Pfcrt 76T突变发生率氯喹抗性株与敏感株间差异有统计学意义（χ^2=24.70,P〈0.01）。Pf mdr1 86Y突变发生率氯喹抗性株与敏感株间差异无统计学意义（χ^2=0.20,P=0.65）。结论在云南省和海南省现场未发现恶性疟原虫Pf mdr1 D1246Y点突变,抗氯喹恶性疟原虫的Pf mdr1 N86Y突变发生率与敏感株间无显著差异。Pfcrt K76T作为分子标志在我国恶性疟原虫氯喹抗性监测中具有应用价值。

Relationships between chloroquine resistance and polymorphisms in Pfcrt and Pfmdrl in Plasmodium falciparum isolated from China

Objective To investigate the prevalence of the point mutations in Pfcrt K76, Pfmdrl N86Y and D1246Y in Plasmodiurn falciparum isolated from Hainan and Yunnan Provinces, and determine the correlation between the prevalence of mutations and the level of chloroquine resistance of P. falciparum isolates by in vitro microtest. Methods Blood samples were taken from the falciparum malaria cases in Yunnan and Hainan Provinces. Nested PCR were designed based on the sequence of Pfcrt and Pfmdrl to amplify the fragments of Pfcrt gene including the 76^th condon as well as Pfmdrl gene including N86Y and D1246Y condon. Restriction fragment length polymorphism （RFLP） was used to detect the point mutations. DNA sequencing was carried out for some PCR products to verify the PCR-RFLP results. Chloroquine resistance of the same isolates was measured by the in vitro microtest recommended by the World Health Organization （WHO）. Results For P. falciparum isolates from Yunnan and Hainan, the prevalence for Pfcrt 76T was 88.0% and 64.3%. The prevalence of Pfmdrl 86Y was 46.5% and 3.4% in P. falciparum isolates from Yunnan and Hainan, re spectively. No point mutation in Pfmdrl at codon 1246 was found in isolates from the P. falciparum from Yunan and Hainan. There was a significant difference between susceptible and resistant isolates carrying Pfcrt 76T mutant codon （χ^2 =24.70, P〈0.01）. There was no significant difference between susceptible and resistant isolates carrying Pfmdrl 86Y mutant codon （χ^2=0.20, P〈0.65）. Conclusion No point mutation in Pfmdrl at codon 1246 is found in P. falciparum from China; There was no significant difference between susceptible and resistant isolates carrying Pfmdrl N86Y mutant codon; Pfcrt K76T may be used as a molecular marker in the surveillance of the changes of chloroquuine resisitance and the imported cases infected with resistant strains to a area.