Polymorphism analysis of K13 gene of Plasmodium ovale isolates from Africa

doi:10.12140/j.issn.1000-7423.2019.02.009

Abstract

Abstract:

Objective To analyze the polymorphism of the K13 protein-encoding gene of Plasmodium ovale curtisi and wallikeri isolates from Africa so as to provide molecular basis for monitoring artemisinin resistance of P. ovale in Africa. Methods The blood samples were collected from imported malaria patients with P. ovale identified by microscopic and epidemiological investigation from 2012 to 2016 in Jiangsu Province. DNA was extracted from the blood samples and the P. ovale K13 gene was amplified by nested PCR. The subspecies of P. ovale were further determined by Real-time TaqMan^R PCR. The acquired DNA sequences were aligned with reference sequences from P. ovale curtisi subspecies (PlasmoDB: PocGH01_12019400) and the wallikeri subspecies (GenBank: LT594516.1) using BioEdit software. The mutations of the obtained DNA sequences were analyzed using DNAstar software. The polymorphism of K13 gene was analyzed by DnaSP software. The phylogenetic tree was constructed using MEGA software. Results A total of 168 P. ovale infected blood samples were collected from malaria patients during 2012-2016 in Jiangsu Province. It was confirmed that the sources of infection were from Central African (95 cases), South Africa (37 cases), West Africa (34 cases) and one case from East and North Africa. A 1 500 bp fragment of K13 gene was successfully amplified from all samples and sequenced. The sequence results identified that half of the samples were P. ovale curtisi subspecies (84) or wallikeri subspecies (84). The nucleotide diversity index π of P. ovale K13 gene was 0.000 02. The haplotype diversity index Hd was 0.024. The K13 genes of two subspecies contained two haplotypes and one single nucleotide polymorphism site. There is only one nucleotide polymorphism within each P. ovale subspecies. The P. ovale curtisi had a A/G polymorphism at nucleotide 717 (amino acid 239E) and the P. ovale wallikeri had a T/A polymorphism at the nucleotide 1998 (amino acid 666P), both of them were synonymous mutations without the change of encoded amino acids. The two mutations identified in this study are not related to P. ovale artemisinin resistance. Neutral test was performed on the DNA sequences of both subspecies of P. ovale without statistical difference. The mutation in P. ovale K13 gene in this study is in accordance with the neutral evolution model. The phylogenetic tree shows that the two haplotypes of P. ovale curtisi subspecies are clustered into one branch, and the two haplotypes of P. ovale wallikeri subspecies are clustered into another branch. Conclusion No nonsynonymous mutation is found in K13 gene of P. ovale curtisi and wallikeri subspecies in this study. K13 gene has a low level of genetic polymorphism in Africa isolations and no mutation is related to P. ovale artemisinin resistance.

Key words: Plasmodium ovale, K13 gene, Artemisinin resistance, Genetic polymorphism

CLC Number:

R382.319

Jing CHEN, Yao-bao LIU, Feng TANG, Feng LU, Jian-xia TANG, Jun CAO. Polymorphism analysis of K13 gene of Plasmodium ovale isolates from Africa[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2019, 37(2): 167-172.

Figures/Tables 5

Fig. 1

Table 1

Fig. 2

Table 2

Fig. 3

References 26

[1]	Stephens JWW.A new malaria parasite of man[J]. Ann Trop Med Parasitol, 1922, 16(4): 383-388.
[2]	Sutherland CJ, Tanomsing N, Nolder D, et al. Two nonrecombining sympatric forms of the human malaria parasite Plasmodium ovale occur globally[J]. J Infect Dis, 2010, 201(10): 1544-1550.
[3]	Nakeesathit S, Saralamba N, Pukrittayakamee S, et al. Limited polymorphism of the Kelch propeller domain in Plasmodium malariae and P. ovale isolates from Thailand[J]. Antimicrob Agents Chemother, 2016, 60(7): 4055-4062.
[4]	Mayxay M, Pukrittayakamee S, Newton PN, et al. Mixed-species malaria infections in humans[J]. Trends Parasitol, 2004, 20(5): 233-240.
[5]	Imwong M, Nakeesathit S, Day NP, et al. A review of mixed malaria species infections in anopheline mosquitoes[J]. Malar J, 2011, 10: 253.
[6]	Noedl H, Se Y, Schaecher K, et al. Evidence of artemisinin-resistant malaria in western Cambodia[J]. N Engl J Med, 2008, 359(24): 2619-2620.
[7]	Phyo AP, Nkomo S, Stepniewska K, et al. Emergence of artemisinin-resistant malaria on the western border of Thailand: a longitudinal study[J]. Lancet, 2012, 379(9830): 1960-1966.
[8]	Ashley EA, Dhorda M, Fairhurst RM, et al. Spread of artemisinin resistance in Plasmodium falciparum malaria[J]. N Engl J Med, 2014, 371(5): 411-423.
[9]	Kyaw MP, Nyunt MH, Chit K, et al. Reduced susceptibility of Plasmodium falciparum to artesunate in southern Myanmar[J]. PLoS One, 2013, 8(3): e57689.
[10]	Ariey F, Witkowski B, Amaratunga C, et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria[J]. Nature, 2014, 505(7481): 50-55.
[11]	Ghorbal M, Gorman M, Macpherson CR, et al. Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system[J]. Nat Biotechnol, 2014, 32(8): 819-821.
[12]	Takala-Harrison S, Jacob CG, Arze C, et al. Independent emergence of artemisinin resistance mutations among Plasmodium falciparum in Southeast Asia[J]. J Infect Dis, 2015, 211(5): 670-679.
[13]	White NJ.Malaria: a molecular marker of artemisinin resistance[J]. Lancet, 2014, 383(9927): 1439-1440.
[14]	Mbengue A, Bhattacharjee S, Pandharkar T, et al. A molecular mechanism of artemisinin resistance in Plasmodium falciparum malaria[J]. Nature, 2015, 520(7549): 683-687.
[15]	Lu F, Culleton R, Zhang M, et al. Emergence of indigenous artemisinin-resistant Plasmodium falciparum in Africa[J]. N Engl J Med, 2017, 376(10): 991-993.
[16]	Talundzic E, Ndiaye YD, Deme AB, et al. Molecular epidemiology of Plasmodium falciparum kelch13 mutations in senegal determined by using targeted amplicon deep sequencing[J]. Antimicrob Agents Chemother, 2017, 61(3): e02116-16.
[17]	Xu C, Wei Q, Yin K, et al. Surveillance of antimalarial resistance Pfcrt, Pfmdr1, and Pfkelch13 polymorphisms in African Plasmodium falciparum imported to Shandong Province, China[J]. Sci Rep, 2018, 8(1): 12951.
[18]	Li X, Zhang D, Hannink M, et al. Crystal structure of the Kelch domain of human Keap1[J]. J Biol Chem, 2004, 279(52): 54750-54758.
[19]	Bauffe F, Desplans J, Fraisier C, Parzy D.Real-time PCR assay for discrimination of Plasmodium ovale curtisi and Plasmodium ovale wallikeri in the Ivory Coast and in the Comoros Islands[J]. Malar J, 2012, 11: 307.
[20]	Ariey F, Witkowski B, Amaratunga C, et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria[J]. Nature, 2014, 505(7481): 50-55.
[21]	罗飞, 周爽, 袁熠, 等. 2011-2015年重庆市境外输入性疟疾流行特征分析及防控策略探讨[J].中国血吸虫病防治杂志, 2017, 29(3): 310-314.
[22]	毛祥华, 邓艳, 董莹,等. 云南省输入性疟疾病例时间分布特征分析[J]. 中国血吸虫病防治杂志, 2017, 29(4): 445-448.
[23]	张丽, 丰俊, 张少森, 等. 2017年全国消除疟疾进展及疫情特征分析[J].中国寄生虫学与寄生虫病杂志, 2018, 36(3): 201-209.
[24]	Maltha J, Gillet P, Jacobs J.Malaria rapid diagnostic tests in travel medicine[J]. Clin Microbiol Infect, 2013, 19(5): 408-415.
[25]	World Health Organization.Emergency response to artemisinin resistance in the Greater Mekong subregion: Regional FrameWork for Action 2013-2015[R]. Geneva: World Health Organization, 2013.
[26]	World Health Organization.Artemisinin and artemisinin-based combination therapy resistance[R]. Geneva: World Health Organization, 2017.

感染来源 Origin of infection	柯氏亚种数量 No. of P. ovale curtisi	沃氏亚种数量 No. of P. ovale wallikeri	合计 Total
南非South Africa
安哥拉Angola	10	22	32
莫桑比克Mozambique	1	2	3
南非共和国 The Republic of South Africa	1	0	1
赞比亚Zambia	1	0	1
小计Subtotal	13	24	37
西非West Africa
几内亚Guinea	1	3	4
加纳Ghana	1	1	2
科特迪瓦Cote d'Ivoire	0	1	1
利比里亚Liberia	2	1	3
尼日尔Niger	1	0	1
尼日利亚Nigeria	13	8	21
萨拉里昂Sierra Leone	1	1	2
小计Subtotal	19	15	34
中非 Central Africa
赤道几内亚Equatorial Guinea	31	26	57
刚果（布）Republic of Congo	7	7	14
刚果（金）Democratic Republic of the Congo	7	2	9
加蓬Gabon	1	4	5
喀麦隆Cameroon	6	4	10
小计Subtotal	52	43	95
东非 East Africa
乌干达Uganda	0	1	1
小计Subtotal	0	1	1
北非 North Africa
南苏丹South Sudan	0	1	1
小计Subtotal	0	1	1
合计Total	84	84	168

亚种Subspecies	样本数 No. of samples	单体型数 No. of haplotypes	核苷酸多样性指数π	单体型多样性指数Hd	单核苷酸多态性SNP	非同义突变数 No. of non-synonymous mutation	同义突变数No. of synonymous mutation	Tajima’s D检验 Tajima’s D test	Fu-Li 检验 Fu-Li’s test	Fay-Wu 检验 Fay-Wu test
柯氏亚种P. ovale curtisi	84	2	0.000 02	0.024	1	0	1	-1.047 26	-1.985 58	-1.984 24
沃氏亚种P. ovale wallikeri	84	2	0.000 02	0.024	1	0	1	-1.047 26	-1.985 58	-1.984 24