Polymorphism of sulfadoxine-pyrimethemine resistant gene of Plasmodium falciparum in China-Myanmar border area from 2010 to 2018
Objective To analyze the polymorphism of sulfadoxine-pyrimethemine(SP)resistant gene and resis-tance recovery mutation trend of Plasmodium falciparum in Yunnan China-Myanmar border area.Methods P.falci-parum blood filter paper samples were collected from China-Myanmar border area in 2010 to 2018.The target gene fragments P.falciparum dihydrofolate reductase(pfdhfr)and dihydropteroate synthase(pfdhps)were amplified by nest-PCR,of which the DNA sequences were aligned by Geneious Prime software.Microsoft Excel 2016 and GraphPad Pism 8.0.2 software were used to analyze the differences in mutation frequency and distribution of haplotype.Haplo-view software was used to analyze the linkage disequilibrium of gene 180 pfdhfr and 178 pfdhps,chi-square test was used to analyze the characteristics of single nucleotide polymorphisms at resistance-associated gene loci and the vary-ing distribution of haplotypes in different years.Results A total of 190 P.falciparum blood filter samples were col-lected in China-Myanmar border area.Specifically,180 pfdhfr and 178 pfdhps gene fragments were successfully ampli-fied and sequenced.The mutated amino acid codon locus in pfdhfr 51,59,108 and 164 was changed as N51I,C59R,S108N,I164L;which in pfdhps 436,437,540 and 581 was changed as S436 A and C,A437G,K540E and N,A581G.The resistant site frequency in Pfdhfr N51I,C59R,S108N and I164L was respectively 57.8％(108/187),90.1％(172/191),93.3％(168/180),59.6％(109/183),while in pfdhps S436A\C,A437G,K540E\N,A581G the resistant site fre-quency was respectively 54.7％(99/181),86.5％(154/178),84.9％(152/177)and 28.7％(51/178).The majority of pfdhfr genotypes were focused on triple and quadruple site mutations(51I59R108N/59R108N164L,51I59R108N164L),with 44.9％(84/187)and 36.9％(69/187),respectively.The wild types of two genes were not detected in 2010.The triple and quadruple mutated haplotypes frequency(51I59R108N/59R108N164L,51I59R108N164L)with 35.9％(23/64)and 37.5％(24/64)in 2011 was obviously less than in 2010 with 46.2％(30/65),49.23％(32/65)and 53.4％(32/58),22.4％(13/58)from the year 2014 to 2018(P＜0.05).The pfdhps triple mutated haplotypes distribution frequency 62.1％(36/58)(436A\C437G540E\N,437G540E\N/581G)after the year 2014 was significantly less than 82.3％(51/62)in 2010 and 78.7％(48/61)in 2011(P＜0.05).The Linkage disequilibrium analysis between the two loci C59R and S108N in pfdhfr showed a strong association with D'=1 and r2=0.8,S436A and A581G in pfdhps gene showed a strong asso-ciation with D'=1 and r2=0.6.Meanwhile,S108N in pfdhfr and K540E in pfdhps were associated with D'=0.8,r2=0.2.Conclusion It demonstrated that P.falciparum resistant gene pfdhfr and pfdhps presented with mutated haplo-type as the dominant gene in Yunnan,China-Myanmar border area,furthermore,its mutation frequency may reduce as the medication discontinuation time for the host extended.
Plasmodium falciparumAntifolate drug resistancepfdhfr,pfdhpsChina-Myanmar border
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