目的 了解中缅边境地区恶性疟原虫对抗疟药物的敏感性及其抗性相关基因多态性之间的关系,为当地制定合理的疟疾药物策略提供依据。方法 2009年在缅甸拉咱市农日班诊所设立调查点,选取镜检为单一感染恶性疟原虫,2周内未用过抗疟药、磺胺和四环素类药物的现症患者为调查对象,给药前采集患者全血或滤纸血滴,用于体外药物敏感性测定和基因检测。运用Rieckmann体外微量测定法对恶性疟原虫进行体外青蒿素、氯喹、哌喹和咯萘啶敏感性检测。提取全血或滤纸血滴DNA,进行恶性疟原虫氯喹抗性转运基因（Pfcrt）、多药抗性基因（Pfmdr）和Kelch基因（PfK13）抗性相关基因检测。应用SPSS 23.0软件Spearman秩相关系数检验分析恶性疟原虫药物抗性与其携带基因之间的关系。结果 共收集镜检为单一感染恶性疟原虫患者血样63份,体外药物敏感测定51例,成功测定42例,占82.4%。恶性疟原虫对氯喹的平均抑制浓度、半数抑制量（ID₅₀）和抗性率较高,分别为880 nmol/L、320.5 nmol/L和95.2%;对咯萘啶平均抑制浓度、青蒿素ID₅₀和哌喹抗性率较低,分别为410 nmol/L、84.8 nmol/L和7.1%。在对氯喹有抗性的40份血样中,对咯萘啶、青蒿素和哌喹的交叉抗性率分别为52.5%（21/40）、37.5%（15/40）和7.5%（3/40）;对哌喹有抗性的3份血样中,对氯喹、青蒿素和咯萘啶的交叉抗性率均为3/3。存在交叉抗性的血样从高到低分别为：氯喹与咯萘啶21份,氯喹与青蒿素15份,青蒿素与咯萘啶13份,哌喹与氯喹、青蒿素和咯萘啶均为3份。Spearman秩相关系数检验分析显示,青蒿素与哌喹和咯萘啶的抗性之间均存在相关性（r = 0.354、0.446,P < 0.05）。基因检测结果显示,Pfcrt基因成功测序37份,均为74~76位点C₇₂V₇₃I₇₄E₇₅T₇₆三重突变基因型,突变率为100%（37/37）。Pfmdr基因在第86和1246位点分别成功测序47和49份血样,突变率分别为N86Y（2.1%,1/47）和D1246Y（100%,49/49）。PfK13基因成功测序20份,存在3种基因突变型,其中F446I突变率较高,占50.0%（10/20）,L492L/S和C580Y基因突变率较低,均为5.0%（1/20）。体外药物敏感性检测为抗性的血样中,Pfcrt基因C₇₂V₇₃I₇₄E₇₅T₇₆、Pfcrt基因N86Y1246、PfK13基因I₄₄₆S₄₉₂C₅₈₀和Pfcrt/Pfmdr基因I₇₄E₇₅T₇₆/Y₁₂₄₆基因型频率较高,分别占各自的100%（37/37）、93.9%（46/49）、50.0%（10/20）和69.0%（29/42）。Spearman秩相关系数检验分析显示,Pfcrt、Pfmdr和PfK13基因突变与体外药物敏感性间无相关性。结论 缅甸拉咱市恶性疟原虫对氯喹已普遍产生了抗性,对咯萘啶和青蒿素具有一定抗性,但对哌喹仍然敏感。

Objective To investigate correlation between the susceptibility to antimalarials and the polymorphisms of drug resistance genes of Plasmodium falciparum isolates from China-Myanmar border region, to provide basis for formulating rational strategy for using antimalarial drugs in the local areas. Methods In 2009, an investigation site was set up at the Nongriban Clinic in Lazan City, Myanmar, and the participant patients were selected based on the inclusion criteria: single infection of P. falciparum diagnosed by microscopy; and no chemotherapy history with antimalarials, sulfonamides, or tetracyclines within the past 2 weeks. For in vitro drug sensitivity test and genetic analysis, whole blood or filter paper blood samples were collected from the patients prior to drug treatment. The Rieckmann in vitro microassay method was used to assess the susceptibility of P. falciparum parasites to artemisinin, chloroquine, piperquine and pyronidine. Blood DNA was prepared to test examine the polymorphisms of chloroquine resistant transporter gene (Pfcrt), multiple-drug resistance (Pfmdr) and Kelch gene (PfK13). The associations between the susceptibility to antimalarial drugs and gene polymorphisms were analyzed with Spearman rank correlation coefficient test by SPSS 23.0 software. Results A total of 63 patients with single infection of P. falciparum examined by microscopy were included in this study, of them 51 samples underwent in vitro drug susceptibility test, with 42 samples were completed successfully, accounting for 82.4% (42/51). In the in vitro assay, P. falciparum showed higher mean inhibitory concentration, higher 50% inhibitory dose (ID₅₀) and higher resistance rate to chloroquine, which were 880 nmol/L, 320.5 nmol/L and 95.2%, respectively; and showed lower mean inhibitory concentration to pyronaridine (410 nmol/L), lower ID₅₀ to artemisinin (84.8 nmol/L) and lower resistant rate to piperaquine (7.1%). Of the 40 blood samples resistant to chloroquine, the cross resistance rate to pyronaridine was 52.5% (21/40), to artemisinin 37.5% (15/40) and to piperaquine 7.5% (3/40). Of the 3 blood samples resistant to piperaquine, all were cross-resistant to chloroquine (3/3), artemisinin (3/3) and pyronaridine (3/3). Among the samples displaying cross-resistance, 21 cases showed cross-resistance of chloroquine-pyronaridine, 15 of chloroquine-artemisinin, while 13 were artemisinin-pyronaridine cross-resistant, and respective 3 cases of piperaquine-resistance being cross-reactive with chloroquine, artemisinin and pyronaridine. Spearman rank correlation coefficient test revealed a significant correlation between artemisinin resistance and piperaquine or pyronaridine resistance (r = 0.354, 0.446; P < 0.05). The Pfcrt gene of 37 cases was sequenced and examined, indicative of triple-mutant genotype C₇₂V₇₃I₇₄E₇₅T₇₆ (mutation rate 100%, 37/37) at the loci 74-76. The mutation prevalence of Pfmdr gene at position N86Y and D1246Y were 2.1% (1/47) and 100% (49/49), respectively. The PfK13 gene was successfully sequenced from 20 isolates, and found to harbor 3 different mutations, including 10 cases with mutation at F446I, showing higher prevalence(50.0%, 10/20), one case with mutation at L492L/S and one case at C580Y showing low prevalence (5.0%, 1/20; 5.0%, 1/20; respectively). Among the drug resistant cases determined by in vitro drug test, the mutant prevalence of Pfcrt C₇₂V₇₃I₇₄E₇₅T₇₆, Pfmdr N₈₆Y₁₂₄₆, PfK13 I₄₄₆S₄₉₂C₅₈₀ and Pfcrt/Pfmdr I₇₄E₇₅T₇₆/Y₁₂₄₆ was 100% (37/37), 93.9% (46/49), 50% (10/20) and 69.0% (29/42) respectively. Spearman’s rank correlation coefficient analysis showed that there was no correlation of Pfcrt, Pfmdr and PfK13 gene mutation with the drug sensitivity in vitro. Conclusion The P. falciparum isolates from Laza City of China-Myanmar border region have developed resistance to chloroquine widely, and resistance to pyronaridine and artemisinin to certain extent, but remain usceptible to piperaquine.