[Polymorphism of 72-76 Coding Sequence within Exon 2 Region of Pfcrt in Yunnan Province].

Abstract

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 （referred to as the 72-76 coding region） in malaria patients.

Methods: The filter paper blood samples and relative information of falciparum malaria cases were collected in 13 prefectures of Yunnan Province （excluding Diqing, Wenshan, Zhaotong prefectures） 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 genotype（CVMNK）（15.2%, 32/210） and mutated chloroquine-resistant genotype（CVIET, SVMNT and CVMNT）（76.2%, 160/210; 6.7%, 14/210; 1.9%, 4/210） 72-76 coding regions. The proportion of the CVMNK type was 100%（32/32） in cases with the range of 19-55 years, 46.9% （15/32） in farmers, and 59.4% （19/32） in patients with infection source in Southeast Asia, all significantly higher than those of other cases in the same groups（0; 31.3%, 10/32; and 37.5%, 12/32 respectively, χ2=13.674, 8.478, 6.292, P<0.05）. The proportion of the CVIET and SVMNT genotypes in patients with infection source in Myanma and Cambodia was 81.3%（130/160） and 78.6%（11/14） respectively, significantly higher than those in patients with infection source in Yunnan Province（6.3%, 10/160; 21.4%, 3/14）（χ2=6.519 and 6.620, P<0.05）. In samples with Africa infection source, the proportion of CVIET was 12.5%（20/160）, 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%（138/145） and variable sites for 4.8%（7/145）. The genetic distance between the 210 sequences ranged 0.000-0.036（0.012±0.005）. The genetic distances from genotypes CVIET, SVMNT and CVMNT to the chloroquine-sensitive genotype CVMNK were（0.029±0.015）, （0.021±0.013） and （0.014±0.001） 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 Dehong（51.7%, 92/178）, Baoshan（24.7%, 44/178） and Lincang（5.6%, 10/178） 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%（13/16） of prefectures in the Province.