关键词: 云南省, 恶性原疟虫, 氯喹抗性, 青蒿素抗性, 氯喹转运蛋白基因, 青蒿素抗性相关基因, 联合突变, 分析

Abstract: Objective To analyze the co-mutation property of chloroquine resistant transporter gene(Pfcrt) and artemisinin-resistant gene K13 in Plasmodium falciparum based on reported malaria cases in Yunnan Province, in order to help unveil the mechanisms of multidrug resistance of P. falciparum. Methods The dry blood samples on filter paper were collected from falciparum malaria patients reported through the Reporting System of Chinese Center for Disease Control and Prevention from August 2012 to December 2016. The corresponding epidemiological data were collected as well. DNA was extracted from P. falciparum, and the exon2 region in Pfcrt gene and K13 kelch domain were amplified with nest-PCR. PCR products were sequenced, and the sequences were aligned with the reference sequences 2655199 and PF3D7_1343700. The haplotype number, expected heterozygosity(He), and genetic differentiation(fixation index, Fst) of the exon 2 region of Pfcrt and K13 kelch domain were analyzed by MEGA 5.04 and Arlequin 3.01 softwares. The haplotype media evolution tree was drawn with the Network 4.6.0 software. Data were analyzed with SPSS 21.0 software. Results A total of 234 blood samples were collected. The PCR results showed that the targets were amplified and 218 products of exon2 region of Pfcrt and 192 products of kelch domain of K13 were sequenced. Among them, 192 blood samples were simultaneously analyzed for the genotypes of Pfcrt and K13, including 12 samples of Yunnan local origin, 30 of Africa origin, and 150 of Myanmar origin. There were seven haplotypes for the exon2 region of Pfcrt in 218 samples, with an He of 0.238 5 and 83.0%(181/218) missense mutations. The proportions of single-locus mutation(CVMNT genotype), double-loci mutation (SVMNT genotype) and three-loci mutation(CVIET genotype) in the 72-76 nucleotide locus were 1.8%(4/218), 6.4%(14/218) and 74.8%(163/218), respectively. The media network diagram from the seven haplotypes showed that the haplotype first occurred as 72-76 locus wild-type CVMNK, then evolved a pattern from single mutation to double mutation and to triple mutation. There were 21 haplotypes for the kelch domain of K13 in 192 samples, with an He of 0.044 9 and 35.9%(69/192) missense mutation. Single mutation was found at 9 loci including 16, 446 and 676, with a highest rate for F446I (25.0%, 48/192). The media network diagram from the 21 haplotypes showed a“Satellite” layout. The Fst of Pfcrt gene was smallest between Yunnan local population and Myanmar imported population of P. falciparum(0.020 3). Among blood samples of chloroquine-sensitive CVMNK wild-type, 20.6%(7/34) were found with singe locus mutation in kelch domain, and chloroquine-resistant mutation-types CVMNT and CVIET had 1/4 and 31.7%(51/161), respectively. Conclusion The co-mutation rate of chloroquine-resistant gene and artemisinin-resistant gene of P. falciparum isolates from reported malaria patients in Yunnan Province is 27.1%. The artemisinin-resistant mutation genotypes show a similar pattern as population expansion.

Key words: Yunnan Province, Plasmodium falciparum, Chloroquine resistance, Atemisinin resistance, Co-mutation, Analysis