Analysis of mutations of Plasmodium falciparum multidrug resistance gene 1 and K13 gene in imported Plasmodium falciparum in Henan Province

Abstract

Abstract:

Objective To identify mutations of Plasmodium falciparum multidrug resistance gene 1 （Pfmdr1） and K13 gene in imported P. falciparum in Henan Province during 2013-2015. Methods Blood samples and epidemiological information were collected from 386 workers returning from Africa, who were diagnosed as imported falciparum malaria in Henan Province during 2013-2015. Plasmodium DNA was extracted, followed by nested PCR to amplify the Pfmdr1 and K13 gene. The PCR products were sequenced and sequence alignment was carried out to analyze gene mutations. Results The 386 patients returned from 26 African countries and were predominantly males. The top 3 disease-exporting counties were Angola (22.5%, 87/386), Equatorial Guinea (13.7%, 53/386), and Nigeria (13.2%, 51/386). Pfmdr1 and K13 genes were amplified from all the 386 blood samples. Sequencing results indicated mutation rate of 23.6% (91/386) and 3.1% (12/386) at loci 86 and 1246 of Pfmdr1, respectively, and 5.4% (21/386) for K13 gene. The 21 blood samples with K13 gene mutations were from 10 countries, in which no C580Y mutation was found, but the R539T and P574L mutations (0.3%, 1/386 for each), which are associated with artemisinin resistance, were found in samples from Angola and Equatorial Guinea. The mutation rate for Pfmdr1 locus 86 was 28.8% (36/125), 23.4% (32/137) and 18.6% (23/124) in each year during 2013-2015, respectively (χ² = 6.438, P < 0.05), and that for locus 1246 was 4.0% (5/125), 3.7% (5/137), 1.6% (2/124), respectively (χ² = 1.384, P > 0.05). The mutation rate of K13 gene was 3.2% (4/125), 8.8% (12/137) and 4.0% (5/124) in each year during 2013-2015, respectively (χ² = 4.631, P > 0.05). Conclusion The mutation rate at loci 86 and 1246 of Pfmdr1 is 23.6% (91/386) and 3.1% (12/386), respectively, and that of K13 gene was 5.4% (21/386). Besides, two mutations R539T and P574L associated with artemisinin resistance are detected.

Key words: Imported malaria, Plasmodium falciparum, Plasmodium falciparum multidrug resistance gene 1, K13 gene, Mutation

CLC Number:

R382.312

Cheng-yun YANG, Su-hua LI, Ya-lan ZHANG, Rui-min ZHOU, Ying LIU, Dan QIAN, Yu-ling ZHAO, Bian-li XU, Hong-wei ZHANG, Yan DENG. Analysis of mutations of Plasmodium falciparum multidrug resistance gene 1 and K13 gene in imported Plasmodium falciparum in Henan Province[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2018, 36(2): 97-102.

Figures/Tables 4

Table 1

Fig. 1

Table 2

Mutation distribution of Plasmodium falciparum multidrug resistance gene 1 and K13 gene in imported P. falciparum in Henan Province

基因 Gene	突变位点 Mutation position	突变病例数 No. cases with mutation	来源国（病例数） Source country （No. cases）
K13	R471R	2	安哥拉Angola（2）
	R539T	1	安哥拉Angola（1）
	M579I	1	安哥拉Angola（1）
	Q613E	2	安哥拉Angola（1）, 刚果金Congo, DRC （1）
	N664D	1	津巴布韦Zimbabwe （1）
	A578S	2	乌干达Uganda （1）, 几内亚Guinea （1）
	F662C/N664D	1	贝宁Benin （1）
	N629S	1	利比里亚Liberia （1）
	E556K/D648Y	1	塞拉利昂Sierra Leone （1）
	G496G	1	尼日利亚Nigeria （1）
	K610R	1	尼日利亚Nigeria （1）
	P574L	1	赤道几内亚Equatorial Guinea （1）
	M476I/V589I	1	赤道几内亚Equatorial Guinea （1）
	L663V	1	赤道几内亚Equatorial Guinea （1）
	K658Q	1	赤道几内亚Equatorial Guinea （1）
	A578T/M579I	1	赤道几内亚Equatorial Guinea （1）
	D464N	1	赤道几内亚Equatorial Guinea （1）
	N664N	1	赤道几内亚Equatorial Guinea （1）
Pfmdr1	86位点 Locus 86	91	尼日利亚Nigeria （11）, 苏丹Sudan （2）, 利比亚Libya （2）, 赞比亚Zambia （2）, 安哥拉Angola（11）, 肯尼亚Kenya （1）, 坦桑尼亚Tanzania（1）, 乍得Chad（1）, 马里Mali（1）, 多哥Togo （2）, 贝宁Benin （1）, 喀麦隆Cameroon （7）, 加蓬Gabon （2）, 赤道几内亚Equatorial Guinea （20）, 刚果金Congo, DRC （2）, 刚果布Congo （1）, 利比里亚Liberia （10）, 加纳Ghana （1）, 塞拉利昂Sierra Leone （4）, 几内亚Guinea （8）
	1246位点 Locus 1246	12	利比亚Libya （2）, 肯尼亚Kenya （1）, 利比里亚Liberia （3）, 刚果金Congo, DRC （1）, 喀麦隆Cameroon （2）, 塞拉利昂Sierra Leone （1）, 安哥拉Angola（1）
	86/1246位点 Locus 86/1246	11	利比亚Libya （2）, 肯尼亚Kenya （1）, 利比里亚Liberia （3）, 刚果金Congo, DRC （1）, 喀麦隆Cameroon （2）, 塞拉利昂Sierra Leone （1）
K13/Pfmdr1	K13/86位点 K13/Locus 86	5	利比里亚Liberia （1）, 几内亚Guinea （1）,尼日利亚Nigeria （1）, 赤道几内亚Equatorial Guinea （2）
	K13/86/1246位点 K13/Locus 86/1246	1	利比里亚Liberia （1）

Table 2

Table 3

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基因 Gene	位点 Locus	引物 Primer	引物序列(5′→3′) Sequence (5′→3′)	片段长度/bp Fragment length/bp
Pfmdr1	86	P1	TTAAATGTTTACCTGCACAACATAGAA AATT	520
		P2	CTCCACAATAACTTGCAACAGTTCTTA
		N1	TGTATGTGCTGTATTATCAGGA
		N2	CTCTTCTATAATGGACATGGTA
	1246	P3	GGGGGATGACAAATTTTCAAGATTA	203
		P4	GGGGGACTAACAAGTTTAACATCTT
		N3	AATGTAAATGAATTTTCAAACC
		N4	CATCTTCTCTTCCAAATTTGATA
K13		P5	GCCTTGTTGAAAGAAGCAGAA	751
		P6	CGCCATTTTCTCCTCCTGTA
		N6	GTGGCAGCTCCAAAATTCAT