RESUMO
Objective: To understand the endemic situation of chloroquine-resistant falciparum malaria in Yunnan Province by analyzing the polymorphism of the 72-76 amino-acid coding sequence within exon 2 region of Plasmodium falciparum chloroquine resistant transporter (Pfcrt) gene ï¼referred to as the 72-76 coding regionï¼ in malaria patients. Methods: The filter paper blood samples and relative information of falciparum malaria cases were collected in 13 prefectures of Yunnan Province ï¼excluding Diqing, Wenshan, Zhaotong prefecturesï¼ from August 2012 to September 2015. The source of infection was determined by epidemiological investigation and the place of case discovery was confirmed according to the endemic registration in the Infectious Diseases Reporting Manage System, Chinese Center for Disease Control and Prevention. The exon2 region of Pfcrt gene was amplified by nested PCR and sequenced. The polymorphism of the 72-76 coding region was analyzed with MEGA 5.04. The variable sites and genetic distance between sequences were calculated. The constituent ratio of the polymorphism in sub-populations was analyzed with IBM SPSS Statistics 21 software. Results: Two hundred and thirty-two blood samples were collected in the period and source of infection included Yunnan of China, Africa and Myanma. Nested-PCR resulted in positive products in 210 samples. Sequence analysis showed the presence of chloroquine-sensitive genotypeï¼CVMNKï¼ï¼15.2%, 32/210ï¼ and mutated chloroquine-resistant genotypeï¼CVIET, SVMNT and CVMNTï¼ï¼76.2%, 160/210; 6.7%, 14/210; 1.9%, 4/210ï¼ 72-76 coding regions. The proportion of the CVMNK type was 100%ï¼32/32ï¼ in cases with the range of 19-55 years, 46.9% ï¼15/32ï¼ in farmers, and 59.4% ï¼19/32ï¼ in patients with infection source in Southeast Asia, all significantly higher than those of other cases in the same groupsï¼0; 31.3%, 10/32; and 37.5%, 12/32 respectively, χ2=13.674, 8.478, 6.292, P<0.05ï¼. The proportion of the CVIET and SVMNT genotypes in patients with infection source in Myanma and Cambodia was 81.3%ï¼130/160ï¼ and 78.6%ï¼11/14ï¼ respectively, significantly higher than those in patients with infection source in Yunnan Provinceï¼6.3%, 10/160; 21.4%, 3/14ï¼ï¼χ2=6.519 and 6.620, P<0.05ï¼. In samples with Africa infection source, the proportion of CVIET was 12.5%ï¼20/160ï¼, with no detection of SVMNT. There was a 145 bp homologous locus among the 210 exon2 regions, of which the conservative sites accounted for 95.2%ï¼138/145ï¼ and variable sites for 4.8%ï¼7/145ï¼. The genetic distance between the 210 sequences ranged 0.000-0.036ï¼0.012±0.005ï¼. The genetic distances from genotypes CVIET, SVMNT and CVMNT to the chloroquine-sensitive genotype CVMNK wereï¼0.029±0.015ï¼, ï¼0.021±0.013ï¼ and ï¼0.014±0.001ï¼ respectively. 178 cases with chloroquine-resistant P. falciparum distributed in all the 13 prefectures. Among them, the regions with top detection rate of chloroquine-resistant genotypes were Dehongï¼51.7%, 92/178ï¼, Baoshanï¼24.7%, 44/178ï¼ and Lincangï¼5.6%, 10/178ï¼ bordering on Myanmar and Kunming (4.5%, 8/178). Conclusion: There are three chloroquine-resistant genotypes of the 72-76 coding region in falciparum malaria cases in Yunnan Province, which distribute in 81.3%ï¼13/16ï¼ of prefectures in the Province.
