Analysis of genes associated with antifolate drug resistance in Plasmodium vivax from different infection sources

Abstract

Abstract:

Objective To analyze the mutation characteristics of dihydrofolate reductase gene (Pvdhfr) and dihydropterroate synthase gene (Pvdhps), which are associated with antifolate drug resistance, in Plasmodium vivax from different infection sources in Yunnan Province. Methods Dry blood spots on filter papers were collected from vivax malaria patients reported through the Reporting System of Chinese Center for Disease Control and Prevention from August 2012 to December 2016 and the corresponding epidemiology data of the patients were collected as well. DNA was extracted from P. vivax, and the Pvdhfr and Pvdhps fragments were amplified with nest-PCR. The PCR products were sequenced, and all sequences were aligned with the reference sequences (GenBank ID: X98123 and PVX_123230). The haplotype number, expected heterozygsity (He), and genetic differentiation (Fixation index) of the two genes in different Plasmodium populations were analyzed with MEGA 5.04 and Arlequin 3.5.1 softwares. The haplotype media evolution tree was constructed with the Network 4.6.0 software. Results A total of 1 203 blood samples were collected, including 1 060 with an infection source in Myanmar, 70 in Africa and 64 from indigenous patients in Yunnan. The PCR results showed that Pvdhfr and Pvdhps were amplified and sequenced in 272 and 708 samples, respectively. There were 53 haplotyes for Pvdhfr in the 272 DNA sequences, with an He of 0.243, and the Yunnan indigenous isolates had the highest He value (0.667). The case of all 12 loci of Pvdhfr gene being wild-type had a frequency of 8.1% (22/272). All the other 52 haplotypes harbored various types of missense mutations at the 12 loci, including single-locus, double-loci, triple-loci, quadruple-loci, quintuple-loci and sixfold-loci mutations. There were 35 haplotyes in 708 DNA sequences of Pvdhps gene, with an He of 0.153, and the Myanmar-source isolates had the highest He (0.142). The case of all the 5 loci of Pvdhps gene being wild-type had a frequency of 36.2% (256/708). All the other 34 haplotypes harbored 29 types of missense mutations at the 5 loci, including single-locus, double-loci, triple-loci, and quadruple-loci mutations. The media network diagram constructed from all the haplotypes of the two P. vivax genes showed that the haplotypes occurred initially as wild-type, and then evolved from single mutation to multiple mutations. In blood samples with wild-type and mutated Pvdhfr, the percentages of Pvdhps mutations were 18.2% (4/22) and 65.6% (147/224), respectively. The blood samples confirmed with Pvdhps mutations and Pvdhfr mutations were from 14 and 9 prefectures, respectively, and predominated by the Myanmar source samples that constituted 97.3% (442/452) and 95.4% (144/151), respectively. Conclusion Both Pvdhfr and Pvdhps have a high mutation frequency and a high degree of mutation in P. vivax from different infection sources in Yunnan Province.

Key words: Plasmodium vivax, Antifolate drug, Dihydrofolate reductase, Dihydropterroate synthase, Resistance-associated gene, Mutation, Analysis

CLC Number:

R382.31

Ying DONG, Yan DENG, Meng-ni CHEN, Yan-chun XU, Xiang-hua MAO, Jian WANG, Ai-ming SUN, Jing-bo XUE. Analysis of genes associated with antifolate drug resistance in Plasmodium vivax from different infection sources[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2018, 36(2): 103-111.

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References 36

[1]	Watkins WM, Mosobo M.Treatment of Plasmodium falciparum malaria with pyrimethamine-sulfadoxine: selective pressure for resistance is a function of long elimination half-life[J]. Trans R Soc Trop Med Hyg, 1993, 87(1): 75-78.
[2]	潘梅, 陆艳. 磺胺多辛乙胺嘧啶片微生物限度检查方法的建立[J]. 生命科学仪器, 2008, 6(5): 45-47.
[3]	Compaoré R, Yameogo MWE, Millogo T, et al. Evaluation of the implementation fidelity of the seasonal malaria chemoprevention intervention in Kaya health district, Burkina Faso[J]. PLoS One, 2017, 12(11): e0187460.
[4]	Ntirushwa D.A strategic framework for malaria prevention and control during pregnancy in the African region[Z]. Geneva: World Health Organization, 2004.
[5]	Hernandez T, Myatt AV, Coatney GR, et al. Studies in human malaria. XXXIV. acquired resistance to pyrimethamine (daraprim) by the shesson strain of Plasmodium vivax[J]. Am J Trop Med Hyg, 1953, 2(5): 797-804.
[6]	Young MD, Burgess RW.Pyrimethamine resistance in Plasmodium vivax malaria[J]. Bull World Health Organ, 1959, 20(1): 27-36.
[7]	Walker PG, Floyd J, Ter Kuile F, et al. Estimated impact on birth weight of scaling up intermittent preventive treatment of malaria in pregnancy given sulphadoxine-pyrimethamine resistance in Africa: a mathematical model[J]. PLoS Med, 2017, 14(2): e1002243.
[8]	de Oliveira TC, Rodrigues PT, Menezes MJ, et al. Genome-wide diversity and differentiation in New World populations of the human malaria parasite Plasmodium vivax[J]. PLoS Negl Trop Dis, 2017, 11(7): e0005824.
[9]	Saralamba N, Nakeesathit S, Mayxay M, et al. Geographic distribution of amino acid mutations in DHFR and DHPS in Plasmodium vivax isolates from Lao PDR, India and Colombia[J]. Malar J, 2016, 15(1): 484.
[10]	Ndiaye YD, Diédhiou CK, Bei AK, et al. High resolution melting: a useful field-deployable method to measure dhfr and dhps drug resistance in both highly and lowly endemic Plasmodium populations[J]. Malar J, 2017, 16: 153.
[11]	Vinayak S, Alam MT, Mixson-Hayden T, et al. Origin and evolution of sulfadoxine resistant Plasmodium falciparum[J]. PLoS Pathog, 2010, 6(3): e1000830.
[12]	Barnadas C, Tichit M, Bouchier C, et al. Plasmodium vivax dhfr and dhps mutations in isolates from Madagascar and therapeutic response to sulphadoxine-pyrimethamine[J]. Malar J, 2008, 7: 35.
[13]	Asih PB, Marantina SS, Nababan R, et al. Distribution of Plasmodium vivax pvdhfr and pvdhps alleles and their association with sulfadoxine-pyrimethamine treatment outcomes in Indonesia[J]. Malar J, 2015, 14: 365.
[14]	Triglia T, Wang P, Sims PF, et al. Allelic exchange at the endogenous genomic locus in Plasmodium falciparum proves the role of dihydropteroate synthase in sulfadoxine-resistant malaria[J]. EMBO J, 1998, 17(14): 3807-3815.
[15]	Maghsoodloorad S, Haghighi A, Sharifi Sarasiabi K, et al. Genetic diversity of dihydropteroate synthetase gene (dhps) of Plasmodium vivax in Hormozgan Province, Iran[J]. Iran J Parasitol, 2016, 11(1): 98-103.
[16]	Das S, Banik A, Hati AK, et al. Low prevalence of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) quadruple and quintuple mutant alleles associated with SP resistance in Plasmodium vivax isolates of West Bengal, India[J]. Malar J, 2016, 15(1): 395.
[17]	Ganguly S, Saha P, Chatterjee M, et al. Prevalence of polymorphisms in antifolate drug resistance molecular marker genes pvdhfr and pvdhps in clinical isolates of Plasmodium vivax from Kolkata, India[J]. Antimicrob Agents Chemother, 2013, 58(1): 196-200.
[18]	Raza A, Ghanchi NK, Khan MS, et al. Prevalence of drug resistance associated mutations in Plasmodium vivax against sulphadoxine-pyrimethamine in southern Pakistan[J]. Malar J, 2013, 12: 261.
[19]	车立刚, 张有林, 李兴亮, 等. 云南勐腊县恶性疟原虫对复方乙胺嘧啶敏感性观察[J]. 中国寄生虫病防治杂志, 1992, 5(3): 217.
[20]	车立刚, 张有林, 李兴亮, 等. 控制抗药性恶性疟暴发的现场研究[J]. 中国公共卫生学报, 1993, (5): 288.
[21]	杨恒林, 杨品芳, 杨亚明, 等. 云南南部恶性疟原虫对甲氟喹、奎宁、氨酚喹、氯喹、磺胺多辛/乙胺嘧啶及咯萘啶敏感性的体外测定[J]. 中国寄生虫学与寄生虫病杂志, 1994, 12(2): 140-142.
[22]	Koepfli C, Rodrigues PT, Antao T, et al. Plasmodium vivax diversity and population structure across four continents[J]. PLoS Negl Trop Dis, 2015, 9(6): e0003872.
[23]	Hupalo DN, Luo Z, Melnikov A, et al. Population genomics studies identify signatures of global dispersal and drug resistance in Plasmodium vivax[J]. Nat Genet, 2016, 48(8): 953-958.
[24]	Lo E, Hemming-Schroeder E, Yewhalaw D, et al. Transmission dynamics of co-endemic Plasmodium vivax and P. falciparum in Ethiopia and prevalence of antimalarial resistant genotypes[J]. PLoS Negl Trop Dis, 2017, 11(7): e0005806.
[25]	董莹, 毛祥华, 陈梦妮, 等. 2012-2014年云南省疟疾实验室诊断质量分析[J]. 中国寄生虫学与寄生虫病杂志, 2015, 33(3): 191-195.
[26]	董莹, 孙艾明, 陈梦妮, 等. 云南省输入性及本地感染间日疟原虫裂殖子表面蛋白1基因5区序列的多态性分析[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(1): 1-7.
[27]	Ding S, Ye R, Zhang D, et al. Anti-folate combination therapies and their effect on the development of drug resistance in Plasmodium vivax[J]. Sci Rep, 2013, 3: 1008.
[28]	Korsinczky M, Fischer K, Chen N, et al. Sulfadoxine resistance in Plasmodium vivax is associated with a specific amino acid in dihydropteroate synthase at the putative sulfadoxine-binding site[J]. Antimicrob Agents Chemother, 2004, 48(6): 2214-2222.
[29]	Barnadas C, Senn N, Iga J, et al. Plasmodium falciparum and Plasmodium vivax genotypes and efficacy of intermittent preventive treatment in Papua New Guinea[J]. Antimicrob Agents Chemother, 2014, 58(11): 6958-6961.
[30]	Huang F, Zhou S, Zhang S, et al. Monitoring resistance of Plasmdium vivax: point mutations in dihydrofolate reductase gene in isolates from Central China[J]. Parasit Vectors, 2011, 4: 80.
[31]	Mohapatra PK, Prakash A, Taison K, et al. Evaluation of chloroquine (CQ) and sulphadoxine/pyrimethamine (SP) therapy in uncomplicated falciparum malaria in Indo-Myanmar border areas[J]. Trop Med Int Health, 2005, 10(5): 478-483.
[32]	Mishra N, Kaitholia K, Srivastava B, et al. Declining efficacy of artesunate plus sulphadoxine-pyrimethamine in northeastern India[J]. Malar J, 2014, 13: 284.
[33]	White NJ.Determinants of relapse periodicity in Plasmodium vivax malaria[J]. Malar J, 2011, 10: 297.
[34]	董莹, 孙艾明, 邓艳, 等. 云南省恶性疟原虫氯喹及青蒿素抗性相关基因的联合突变分析[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(3): 202-208.
[35]	朱垚吉, 陈梦妮, 徐艳春, 等. 云南省恶性疟原虫Pfcrt基因exon2区72~76编码序列多态性的分析[J]. 中国寄生虫学与寄生虫病杂志, 2016, 34(3): 227-232, 234.
[36]	孙艾明, 董莹, 陈梦妮, 等. 云南省恶性疟原虫青蒿素耐药性相关基因K13 kelch结构域序列多态性的分析[J]. 中国寄生虫学与寄生虫病杂志, 2016, 34(4): 339-345.

多态位点 Polymorphic loci																不同感染地 Different infection sources
单倍型Haplotye	等位形式Alleles	14	15	18	57	58	61	99	117	131	156	173	185	计数No.	频率/%Frequency/%	云南Yunnan	缅甸Myanmar	非洲Africa
Hap-1	M	C	A	K	I	R	M	H	T	R	D	I	R	29	10.7	1	28	0
Hap-2	W	C	A	K	F	S	T	H	S	R	D	I	R	22	8.1	0	21	1
Hap-3	M	C	A	K	I	R	M	H	T	R	N	I	R	43	15.8	1	41	1
Hap-4	M	C	A	K	F	R	T	H	N	R	D	I	R	19	7.0	1	18	0
Hap-5	M	C	A	K	L	R	M	H	T	R	D	I	R	18	6.6	0	18	0
Hap-6	M	C	A	K	F	R	T	H	N	R	N	I	R	16	5.9	0	14	2
Hap-7	M	C	A	K	F	S	T	S	S	R	N	I	R	20	7.4	0	20	0
Hap-8	M	C	A	K	F	S	T	H	S	R	N	I	R	19	7.0	0	19	0
Hap-9	M	C	A	K	L	R	M	H	T	R	N	I	R	16	5.9	0	16	0
Hap-10	M	C	A	K	F	S	T	H	N	R	N	I	R	2	0.7	0	2	0
Hap-11	M	Y	A	K	L	R	M	H	T	R	D	I	R	2	0.7	0	2	0
Hap-12	M	C	A	I	L	R	M	H	T	R	D	I	R	4	1.5	0	4	0
Hap-13	M	C	A	K	F	S	T	S	S	R	D	I	R	3	1.1	0	3	0
Hap-14	M	C	A	I	F	S	T	H	S	R	N	I	R	3	1.1	0	3	0
Hap-15	M	C	A	K	I	R	M	H	T	R	N	I	R	2	0.7	0	2	0
Hap-16	M	C	A	I	F	S	T	S	S	R	N	I	R	8	2.9	0	8	0
Hap-17	M	Y	A	K	F	S	T	H	S	R	D	I	R	2	0.7	0	2	0
Hap-22	M	C	A	K	L	R	M	H	T	R	N	I	R	3	1.1	0	3	0
Hap-23	M	C	A	K	L	R	T	H	S	R	D	I	R	2	0.7	0	2	0
Hap-24	M	C	A	I	L	R	M	H	T	R	N	I	R	2	0.7	0	2	0
Hap-29	M	C	A	K	L	R	M	H	T	R	N	I	R	2	0.7	0	2	0
Hap-31	M	C	A	I	I	R	M	H	T	R	N	I	R	3	1.1	0	3	0
Hap-44	M	C	A	I	F	S	T	H	S	R	D	I	R	2	0.7	0	2	0
其它Others^a	M	-	-	-	-	-	-	-	-	-	-	-	-	30	11.0	0	28	2
合计Total														272	100	3	263	6
He																0.667	0.070	0.619
Fst																0.070^b	0.223^c	0.106^d
Va/%																6.9	12.3	10.6
Vb/%																93.1	87.7	89.4

单倍型Haplotype	等位形式Alleles	多态位点Polymorphic loci							不同感染地 Different infection sources
单倍型Haplotype	等位形式Alleles	382	383	512	553	571	计数No.	频率Frequency	云南Yunnan	缅甸Myanmar	非洲Africa
Hap-1	W	S	A	K	A	E	256	36.2	5	242	9
Hap-2	M	S	A	K	G	E	91	12.9	2	86	3
Hap-3	M	S	G	K	G	E	171	24.2	1	170	0
Hap-4	M	S	A	E	G	E	8	1.1	0	8	0
Hap-5	M	S	A	K	A	Q	7	1.0	0	7	0
Hap-7	M	A	G	K	G	E	37	5.2	3	34	0
Hap-8	M	A	G	K	A	E	23	3.2	1	22	0
Hap-9	M	S	G	E	G	E	4	0.6	0	4	0
Hap-10	M	S	G	K	A	E	86	12.1	1	85	0
Hap-14	M	S	G	K	A	Q	12	1.7	1	11	0
Hap-23	M	S	G	M	G	E	2	0.3	0	2	0
Hap-24	M	C	G	E	G	E	3	0.4	0	3	0
Hap-27	M	S	A	M	G	E	2	0.3	0	2	0
其它Others^a	M	-	-	-	-	-	6	0.8	0	6	0
合计Total	-						708	100	14	682	12
He	-								0.352	0.119	0.119
Fst	-								0.011^b	0.142^c	0.094^de
Va/%	-								1.1	14.2	9.4
Vb/%	-								98.9	85.8	90.6