Polymorphism of sulfadoxine-pyrimethemine resistant gene of Plasmodium falciparum in China-Myanmar border area from 2010 to 2018

doi:10.12140/j.issn.1000-7423.2024.02.004

Abstract

Abstract:

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.

Key words: Plasmodium falciparum, Antifolate drug resistance, pfdhfr, pfdhps, China-Myanmar border

CLC Number:

R382.312

YAN He, HUANG Fang, FENG Jun, YIN Jianhai, XIA Zhigui, CAO Jianping. Polymorphism of sulfadoxine-pyrimethemine resistant gene of Plasmodium falciparum in China-Myanmar border area from 2010 to 2018[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2024, 42(2): 153-159.

Figures/Tables 6

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引物名称 Primer	序列 Sequence	扩增长度/bp
pfdhfrNF	TTTATGATGGAACAAGTCTGC	648
pfdhfrNR	AGTATATACATCGCTAACAGA
pfdhfrF	ACAAGTCTGCGACGTTTTCGATATTTATG	594
pfdhfrR	ATTCATATGTACTATTTATTCTAGT
pfdhpsNF	AGCAATTGCTAATCCACCTG	838
pfdhpsNR	GATTCTTTTTCAGATGGAGG
pfdhpsF	TAATCCACCTGAAAAGAAATAC	706
pfdhpsR	ATTTTTGTTGAACCTAAACGTGCTGTTCA
DHFR^a	ATTCATATGTACTATTTATTCTAGT	-
DHPS1^a	TTTTTAAACACACCACATATATT	-
DHPS2^a	ATTTCTGGATTATTTGTACAAGCAC	-

基因Gene	单倍体型 Haplotype	样品数/份 No. Isolate	频率/% Frequency/%
pfdhfr	NCSI	11	5.9
	ICSI	1	0.5
	NCNI	1	0.5
	IRSI	1	0.5
	NRNI	18	9.6
	ICNI	2	1.1
	IRNI	42	22.5
	NRNL	42	22.5
	IRNL	69	36.9
pfdhps	SAKA	15	8.3
	CAKA	1	0.6
	SAEA	3	1.7
	SGKA	2	1.1
	AAKA	1	0.6
	AAEA	4	2.2
	AGKA	4	2.2
	SGKG	3	1.7
	SGEA	12	6.7
	SAEG	1	0.6
	AGEA	78	43.3
	AGNA	8	4.4
	SGEG	40	22.2
	SGNG	7	3.9
	AGEG	1	0.6