论文部分内容阅读
目的分析云南省恶性疟病例中恶性疟原虫氯喹抗性转运蛋白(Plasmodium falciparum chloroquine resistant transporter,Pfcrt)基因exon2区72~76编码(简称72~76编码)序列的多态性,为了解云南省抗氯喹恶性疟的流行现状提供依据。方法 2012年8月至2015年9月,从云南省除迪庆、文山、昭通以外的13个州(市)收集恶性疟病例的滤纸血样和相关信息,通过流行病学调查判定感染来源地,根据中国疾病预防控制中心传染病报告信息管理系统疫情登记确认病例发现地。用巢式PCR扩增患者血样中的恶性疟原虫Pfcrt基因exon2区并测序,用MEGA 5.04分析72~76编码序列的多态性,计算变异位点、序列间遗传距离等。用IBM SPSS Statistics 21软件对分类变量72~76编码多态在各人群中的构成比进行分析。结果从2012年8月至2015年9月,共收集恶性疟病例血样232份,病例感染来源地有云南、非洲、缅甸等流行区。其中210份血样巢式PCR扩增阳性,序列分析显示,72~76编码序列存在CVMNK氯喹敏感型和CVIET、SVMNT、CVMNT氯喹抗性型,所占比例分别为15.2%(32/210)、76.2%(160/210)、6.7%(14/210)和1.9%(4/210)。氯喹敏感性型CVMNK在19~55岁、农民、感染地为东南亚(缅甸161例、柬埔寨1例)等病例中检出的比例分别为100%(32/32)、46.9%(15/32)和59.4%(19/32),高于同组其它病例的0、31.3%(10/32)和37.5%(12/32)(χ~2=13.674,8.478,6.292,P<0.05);氯喹抗性型CVIET、SVMNT在感染地为缅甸和柬埔寨的病例中的比例分别为81.3%(130/160)和78.6%(11/14),高于云南感染病例的6.3%(10/160)和21.4%(3/14)(χ~2=6.519,6.620,P<0.05);非洲感染病例中的CVIET比例为12.5%(20/160),未检测到SVMNT型。210条Pfcrt基因exon2区DNA序列的同源位点为145 bp,保守位点占95.2%(138/145),变异位点占4.8%(7/145),序列间遗传距离为0.000~0.036,平均为(0.012±0.005),3种氯喹抗性型CVIET、SVMNT、CVMNT与敏感型CVMNK的遗传距离平均分别为(0.029±0.015)、(0.021±0.013)和(0.014±0.001);检出氯喹抗性型疟原虫的178例病例分布在云南的13个州(市),其中,与缅甸接壤的德宏、保山、临沧3个州(市)以及云南中部的昆明市,检出氯喹抗性型的比例分别为51.7%(92/178)、24.7%(44/178)、5.6%(10/178)和4.5%(8/178),居全省前4位。结论云南省恶性疟病例感染恶性疟原虫的Pfcrt基因exon2区72~76编码序列存在3种氯喹抗性型,氯喹抗性型恶性疟原虫的病例分布在全省81.3%(13/16)的州(市)。
Objective To analyze the polymorphism of sequence 72-76 (code 72-76) in exon2 of Plasmodium falciparum chloroquine resistant transporter (Pfcrt) gene in Plasmodium falciparum chloroquine resistant transporter (Pfcrt) Chloroquine falciparum malaria epidemic status quo provide basis. Methods From August 2012 to September 2015, blood samples and relevant information of malaria cases were collected from 13 states (cities) except Diqing, Wenshan and Zhaotong in Yunnan Province. The epidemiological investigation was used to determine the origin of infection, According to the Chinese Center for Disease Control and infectious disease reporting information management system to confirm the case of epidemics found. The nested PCR was used to amplify and sequence the exon2 region of Pfcrt gene of Plasmodium falciparum in blood samples. The polymorphisms of 72-76 coding sequence were analyzed by MEGA 5.04 to calculate the variation loci and the genetic distance between sequences. Using IBM SPSS Statistics 21 software, the constituent ratios of the coding variants 72-76 of the categorical variables in each population were analyzed. Results A total of 232 blood samples of falciparum malaria cases were collected from August 2012 to September 2015. The source of cases was endemic to Yunnan, Africa and Myanmar. Among them, 210 samples were positive for nested PCR amplification. Sequence analysis showed that there were CVMNK chloroquine sensitive and CVIET, SVMNT and CVMNT chloroquine resistance in 72 ~ 76 coding sequences, accounting for 15.2% (32/210), 76.2 % (160/210), 6.7% (14/210) and 1.9% (4/210). The percentage of chloroquine sensitive CVMNK detected in cases of 19-55 years old, farmer, Southeast Asia (161 cases in Burma, 1 case in Cambodia) were 100% (32/32) and 46.9% (15/32), respectively And 59.4% (19/32), respectively, which were higher than those in other cases in the same group (0, 31.3% and 37.5%, respectively) (χ ~ 2 = 13.674, 8.478 and 6.292, The proportion of resistant CVIET and SVMNT in infected areas of Myanmar and Cambodia was 81.3% (130/160) and 78.6% (11/14) respectively, which was higher than that of 6.3% (10/160) and 21.4% (3/14) (χ ~ 2 = 6.519, 6.620, P <0.05). The proportion of CVIET in African infection was 12.5% (20/160), and no SVMNT was detected. The DNA sequence homology of exon2 of the 210 Pfcrt genes was 145 bp, 95.2% (138/145) conservative sites and 4.8% (7/145) conservative sites. The genetic distance between sequences was 0.000 ~ 0.036, (0.012 ± 0.005). The average genetic distance of three chloroquine resistant CVIETs, SVMNTs, CVMNTs and sensitive CVMNKs were (0.029 ± 0.015), (0.021 ± 0.013) and (0.014 ± 0.001) One hundred and eighty-eight cases of resistant Plasmodium were found in 13 states (cities) of Yunnan. Of the three states (Dehong, Baoshan and Lincang) bordering Burma and Kunming in central Yunnan, chloroquine resistance Sexual proportions were 51.7% (92/178), 24.7% (44/178), 5.6% (10/178) and 4.5% (8/178), respectively, ranking the first 4 provinces. Conclusions There are three kinds of chloroquine resistance in the coding sequence of exon2 of Pfcrt gene of Plasmodium falciparum infected by P. falciparum in Yunnan Province. The cases of chloroquine resistance Plasmodium falciparum are distributed in 81.3% (13/16) of the province (city).