收稿日期: 2023-12-26
修回日期: 2024-03-14
网络出版日期: 2024-04-28
基金资助
上海市公共卫生体系建设三年行动计划(GWV-10.1-XK13)
Polymorphism of sulfadoxine-pyrimethemine resistant gene of Plasmodium falciparum in China-Myanmar border area from 2010 to 2018
Received date: 2023-12-26
Revised date: 2024-03-14
Online published: 2024-04-28
Supported by
Three-Year Public Health Action Plan of Shanghai(GWV-10.1-XK13)
目的 分析云南中缅边境地区恶性疟原虫抗叶酸类药物磺胺多辛-乙胺嘧啶抗性基因的多态性和抗性回复突变趋势。方法 收集2010—2018年中缅边境恶性疟病例滤纸血样品，巢式PCR扩增恶性疟原虫二氢叶酸还原酶（pfdhfr）基因和恶性疟原虫二氢蝶酸合酶（pfdhps）基因，采用Geneious Prime软件比对测序结果，Microsoft Excel 2016和GraphPad prism 8.0.2软件分析突变频率和单体型分布差异，卡方检验分析抗性相关基因位点单核苷酸多态性特征和不同年份单体型分布差异，Haploview软件分析pfdhfr和pfdhps基因连锁不平衡情况。结果 共收集中缅边境地区190份恶性疟样品，PCR扩增出pfdhfr 180份、pfdhps 178份。测序结果显示，pfdhfr和pfdhps均出现等位基因的多重感染，其中pfdhfr检出17个多重感染，突变位点分别位于第51、59、108、164位氨基酸密码子，其表型为51I/59R/108N/164L；pfdhps检出4个多重感染，突变位点分别位于第436、437、540、581位氨基酸密码子，其表型为436A/437A/540E\N/581G（“\”表示并列关系）。pfdhfr第N51I、C59R、S108N、I164L位点的突变率分别为57.8%（108/187）、90.1%（172/191）、93.3%（168/180）、59.6%（109/183）；pfdhps第S436A\C、A437G、K540E\N、A581G位点抗性突变率分别为54.7%（99/181）、86.5%（154/178）、84.9%（152/177）、28.7%（51/178）。pfdhfr单体型主要集中在三点、四点突变型（51I59R108N/59R108N164L、51I59R108N164L），分别占44.9%（84/187）和36.9%（69/187）。pfdhfr和pfdhps野生型在2010年均未检出。2011年的pfdhfr三点、四点突变单体型（同上）分布频率分别为35.9%（23/64）、37.5%（24/64），均小于2010年的46.2%（30/65）、49.23%（32/65）（P < 0.05），与2014—2018年的53.4%（32/58）、22.4%（13/58）比较差异有统计学意义（P < 0.05）。2014—2018年的pfdhps三点突变单体型（436A\C437G540E\N，437G540E\N581G）分布频率为62.1%（36/58），小于2010年的82.3%（51/62）和2011年的78.7%（48/61）（均P < 0.05）。连锁不平衡分析显示，pfdhfr C59R与S108N基因关联性强（D’= 1，r2 = 0.8），pfdhps S436A与A581G关联性强（D’= 1，r2 = 0.6），且S108N与 K540E存在较强关联性（D’= 0.8，r2 = 0.2）。结论 云南中缅边境恶性疟原虫pfdhfr和pfdhps基因的抗性分子突变单体型仍是优势基因，且随着停药时间延长突变频率逐渐降低。
燕贺 , 黄芳 , 丰俊 , 尹建海 , 夏志贵 , 曹建平 . 2010—2018年云南中缅边境地区恶性疟原虫抗磺胺多辛-乙胺嘧啶药物基因多态性分析[J]. 中国寄生虫学与寄生虫病杂志, 2024 , 42(2) : 153 -159 . DOI: 10.12140/j.issn.1000-7423.2024.02.004
Objective To analyze the polymorphism of sulfadoxine-pyrimethemine (SP) resistant gene and resistance recovery mutation trend of Plasmodium falciparum in Yunnan China-Myanmar border area. Methods P. falciparum 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. Haploview 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 varying distribution of haplotypes in different years. Results A total of 190 P. falciparum blood filter samples were collected in China-Myanmar border area. Specifically, 180 pfdhfr and 178 pfdhps gene fragments were successfully amplified 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 frequency 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 association 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 haplotype 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.
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