中缅边境地区恶性疟原虫Pfcrt、Pfmdr和PfK13基因多态性与体外药物敏感性相关性的分析

doi:10.12140/j.issn.1000-7423.2020.05.009

中国寄生虫学与寄生虫病杂志 ›› 2020, Vol. 38 ›› Issue (5): 580-588.doi: 10.12140/j.issn.1000-7423.2020.05.009

中缅边境地区恶性疟原虫Pfcrt、Pfmdr和PfK13基因多态性与体外药物敏感性相关性的分析

张苍林¹(), 聂仁华¹, 徐丹², 吕高伟³, 王剑⁴, 杨亚明¹, 邓艳¹, 刘言¹, 周红宁¹^,^*()

1 云南省虫媒传染病防控研究重点实验室,云南省疟疾研究中心,云南公共卫生与疾病防控协同创新中心,云南省寄生虫病防治所虫媒传染病防控关键技术省创新团队（培育）,普洱市江陆斌专家工作站,云南省寄生虫病防治所,普洱 665099
2 弥渡县疾病预防控制中心,弥渡 675600
3 普洱市疾病预防控制中心,普洱 665099
4 云南省地方病防治所,大理 671000

收稿日期:2020-04-14 出版日期:2020-10-30 发布日期:2020-11-12
通讯作者: 周红宁
作者简介:张苍林（1979-）,男,硕士,副主任技师,从事寄生虫病病原生物学研究。E-mail:43773923@qq.com
基金资助:
云南公共卫生与疾病防控协同创新中心项目(2014YNPHXT03);国家自然科学基金(81160357);国家自然科学基金(30960327);国家自然科学基金(30660160)

Correlation of Pfcrt, Pfmdr and PfK13 gene polymorphisms and in vitro drug susceptibility of Plasmodium falciparum isolates from China-Myanmar border region

ZHANG Cang-lin¹(), NIE Ren-hua¹, XU Dan², LV Gao-wei³, WANG Jian⁴, YANG Ya-ming¹, DENG Yan¹, LIU Yan¹, ZHOU Hong-ning¹^,^*()

1 Yunnan Provincial Key Laboratory of Vector-borne Diseases Control and Research, Yunnan Provincial Center of Malaria Research, Yunnan Provincial Collaborative Innovation Center for Public Health and Disease Prevention and Control, Yunnan Institute of Parasitic Diseases Innovative Team of Key Techniques for Vector Borne Disease Control and Prevention (Developing), Expert Workstation of Professor Jiang Lubing, Yunnan Institute of Parasitic Diseases, Pu’er 665099, China
2 Midu Center for Disease Control and Prevention, Midu 675600, China
3 Pu’er Center for Disease Control and Prevention, Pu’er 665099, China
4 Yunnan Institute of Endemic Disease Control and Prevention, Dali 671000, China

Received:2020-04-14 Online:2020-10-30 Published:2020-11-12
Contact: ZHOU Hong-ning
Supported by:
Yunnan Provincial Collaborative Innovation Center for Public Health and Disease Prevention and Control(2014YNPHXT03);National Natural Science Foundation of China(81160357);National Natural Science Foundation of China(30960327);National Natural Science Foundation of China(30660160)

摘要/Abstract

摘要：

目的了解中缅边境地区恶性疟原虫对抗疟药物的敏感性及其抗性相关基因多态性之间的关系,为当地制定合理的疟疾药物策略提供依据。方法 2009年在缅甸拉咱市农日班诊所设立调查点,选取镜检为单一感染恶性疟原虫,2周内未用过抗疟药、磺胺和四环素类药物的现症患者为调查对象,给药前采集患者全血或滤纸血滴,用于体外药物敏感性测定和基因检测。运用Rieckmann体外微量测定法对恶性疟原虫进行体外青蒿素、氯喹、哌喹和咯萘啶敏感性检测。提取全血或滤纸血滴DNA,进行恶性疟原虫氯喹抗性转运基因（Pfcrt）、多药抗性基因（Pfmdr）和Kelch基因（PfK13）抗性相关基因检测。应用SPSS 23.0软件Spearman秩相关系数检验分析恶性疟原虫药物抗性与其携带基因之间的关系。结果共收集镜检为单一感染恶性疟原虫患者血样63份,体外药物敏感测定51例,成功测定42例,占82.4%。恶性疟原虫对氯喹的平均抑制浓度、半数抑制量（ID₅₀）和抗性率较高,分别为880 nmol/L、320.5 nmol/L和95.2%;对咯萘啶平均抑制浓度、青蒿素ID₅₀和哌喹抗性率较低,分别为410 nmol/L、84.8 nmol/L和7.1%。在对氯喹有抗性的40份血样中,对咯萘啶、青蒿素和哌喹的交叉抗性率分别为52.5%（21/40）、37.5%（15/40）和7.5%（3/40）;对哌喹有抗性的3份血样中,对氯喹、青蒿素和咯萘啶的交叉抗性率均为3/3。存在交叉抗性的血样从高到低分别为：氯喹与咯萘啶21份,氯喹与青蒿素15份,青蒿素与咯萘啶13份,哌喹与氯喹、青蒿素和咯萘啶均为3份。Spearman秩相关系数检验分析显示,青蒿素与哌喹和咯萘啶的抗性之间均存在相关性（r = 0.354、0.446,P < 0.05）。基因检测结果显示,Pfcrt基因成功测序37份,均为74~76位点C₇₂V₇₃I₇₄E₇₅T₇₆三重突变基因型,突变率为100%（37/37）。Pfmdr基因在第86和1246位点分别成功测序47和49份血样,突变率分别为N86Y（2.1%,1/47）和D1246Y（100%,49/49）。PfK13基因成功测序20份,存在3种基因突变型,其中F446I突变率较高,占50.0%（10/20）,L492L/S和C580Y基因突变率较低,均为5.0%（1/20）。体外药物敏感性检测为抗性的血样中,Pfcrt基因C₇₂V₇₃I₇₄E₇₅T₇₆、Pfcrt基因N86Y1246、PfK13基因I₄₄₆S₄₉₂C₅₈₀和Pfcrt/Pfmdr基因I₇₄E₇₅T₇₆/Y₁₂₄₆基因型频率较高,分别占各自的100%（37/37）、93.9%（46/49）、50.0%（10/20）和69.0%（29/42）。Spearman秩相关系数检验分析显示,Pfcrt、Pfmdr和PfK13基因突变与体外药物敏感性间无相关性。结论缅甸拉咱市恶性疟原虫对氯喹已普遍产生了抗性,对咯萘啶和青蒿素具有一定抗性,但对哌喹仍然敏感。

关键词: 恶性疟原虫, 药物敏感性, 体外微量测定, 抗性基因, 缅甸拉咱市

Abstract:

Objective To investigate correlation between the susceptibility to antimalarials and the polymorphisms of drug resistance genes of Plasmodium falciparum isolates from China-Myanmar border region, to provide basis for formulating rational strategy for using antimalarial drugs in the local areas. Methods In 2009, an investigation site was set up at the Nongriban Clinic in Lazan City, Myanmar, and the participant patients were selected based on the inclusion criteria: single infection of P. falciparum diagnosed by microscopy; and no chemotherapy history with antimalarials, sulfonamides, or tetracyclines within the past 2 weeks. For in vitro drug sensitivity test and genetic analysis, whole blood or filter paper blood samples were collected from the patients prior to drug treatment. The Rieckmann in vitro microassay method was used to assess the susceptibility of P. falciparum parasites to artemisinin, chloroquine, piperquine and pyronidine. Blood DNA was prepared to test examine the polymorphisms of chloroquine resistant transporter gene (Pfcrt), multiple-drug resistance (Pfmdr) and Kelch gene (PfK13). The associations between the susceptibility to antimalarial drugs and gene polymorphisms were analyzed with Spearman rank correlation coefficient test by SPSS 23.0 software. Results A total of 63 patients with single infection of P. falciparum examined by microscopy were included in this study, of them 51 samples underwent in vitro drug susceptibility test, with 42 samples were completed successfully, accounting for 82.4% (42/51). In the in vitro assay, P. falciparum showed higher mean inhibitory concentration, higher 50% inhibitory dose (ID₅₀) and higher resistance rate to chloroquine, which were 880 nmol/L, 320.5 nmol/L and 95.2%, respectively; and showed lower mean inhibitory concentration to pyronaridine (410 nmol/L), lower ID₅₀ to artemisinin (84.8 nmol/L) and lower resistant rate to piperaquine (7.1%). Of the 40 blood samples resistant to chloroquine, the cross resistance rate to pyronaridine was 52.5% (21/40), to artemisinin 37.5% (15/40) and to piperaquine 7.5% (3/40). Of the 3 blood samples resistant to piperaquine, all were cross-resistant to chloroquine (3/3), artemisinin (3/3) and pyronaridine (3/3). Among the samples displaying cross-resistance, 21 cases showed cross-resistance of chloroquine-pyronaridine, 15 of chloroquine-artemisinin, while 13 were artemisinin-pyronaridine cross-resistant, and respective 3 cases of piperaquine-resistance being cross-reactive with chloroquine, artemisinin and pyronaridine. Spearman rank correlation coefficient test revealed a significant correlation between artemisinin resistance and piperaquine or pyronaridine resistance (r = 0.354, 0.446; P < 0.05). The Pfcrt gene of 37 cases was sequenced and examined, indicative of triple-mutant genotype C₇₂V₇₃I₇₄E₇₅T₇₆ (mutation rate 100%, 37/37) at the loci 74-76. The mutation prevalence of Pfmdr gene at position N86Y and D1246Y were 2.1% (1/47) and 100% (49/49), respectively. The PfK13 gene was successfully sequenced from 20 isolates, and found to harbor 3 different mutations, including 10 cases with mutation at F446I, showing higher prevalence(50.0%, 10/20), one case with mutation at L492L/S and one case at C580Y showing low prevalence (5.0%, 1/20; 5.0%, 1/20; respectively). Among the drug resistant cases determined by in vitro drug test, the mutant prevalence of Pfcrt C₇₂V₇₃I₇₄E₇₅T₇₆, Pfmdr N₈₆Y₁₂₄₆, PfK13 I₄₄₆S₄₉₂C₅₈₀ and Pfcrt/Pfmdr I₇₄E₇₅T₇₆/Y₁₂₄₆ was 100% (37/37), 93.9% (46/49), 50% (10/20) and 69.0% (29/42) respectively. Spearman’s rank correlation coefficient analysis showed that there was no correlation of Pfcrt, Pfmdr and PfK13 gene mutation with the drug sensitivity in vitro. Conclusion The P. falciparum isolates from Laza City of China-Myanmar border region have developed resistance to chloroquine widely, and resistance to pyronaridine and artemisinin to certain extent, but remain usceptible to piperaquine.

Key words: Plasmodium falciparum, Drug sensitivity, In vitro microtest, Resistant gene, Laza City of Myanmar

中图分类号:

R382.312

张苍林, 聂仁华, 徐丹, 吕高伟, 王剑, 杨亚明, 邓艳, 刘言, 周红宁. 中缅边境地区恶性疟原虫Pfcrt、Pfmdr和PfK13基因多态性与体外药物敏感性相关性的分析[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(5): 580-588.

ZHANG Cang-lin, NIE Ren-hua, XU Dan, LV Gao-wei, WANG Jian, YANG Ya-ming, DENG Yan, LIU Yan, ZHOU Hong-ning. Correlation of Pfcrt, Pfmdr and PfK13 gene polymorphisms and in vitro drug susceptibility of Plasmodium falciparum isolates from China-Myanmar border region[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2020, 38(5): 580-588.

图/表 6

表1

表2

表3

图1

图2

表4

2009年中缅边境恶性疟原虫Pfcrt、Pfmdr和PfK13不同基因型在体外药物敏感性试验中的频率分布情况

基因类型^a Genotype^a	血样数量 No. isolates	百分比/% Percentage/%	体外检测对各药物产生抗性的血样数 Cases of in vitro resistance to drugs
基因类型^a Genotype^a	血样数量 No. isolates	百分比/% Percentage/%	青蒿素 Artemisinin		氯喹 Chloroquine		哌喹 Piperaquine	咯萘啶 Pyronaridine
Pfcrt基因（体外,n = 35） Pfcrt gene (in vitro, n = 35)
三重基因突变C₇₂V₇₃I₇₄E₇₅T₇₆ Triple-mutant genotype C₇₂V₇₃I₇₄E₇₅T₇₆	37	100	12	28		3			16
Pfmdr基因（体外,86位点n = 45,1246位点n = 47） Pfmdr gene (in vitro, n = 45 at codon 86, n = 47 at codon 1246）
单基因突变Y₁₂₄₆ Single-mutant genotype Y₁₂₄₆	2	4.1	0		2		0	1
单基因突变N₈₆Y₁₂₄₆ Single-mutant genotype N₈₆Y₁₂₄₆	46	93.9	16		34		3	21
双重突变Y₈₆Y₁₂₄₆ Double-mutant genotype Y₈₆Y₁₂₄₆	1	2.0	0		1		0	0
PfK13基因（体外, n = 20） PfK13 gene (in vitro, n = 20)
野生型F₄₄₆S₄₉₂C₅₈₀ Wild-type genotype F₄₄₆S₄₉₂C₅₈₀	8	40.0	3		5		2	3
单基因突变I₄₄₆S₄₉₂C₅₈₀ Single-mutant genotype I₄₄₆S₄₉₂C₅₈₀	10	50.0	4		9		1	8
单基因突变F₄₄₆L/S₄₉₂C₅₈₀ Single-mutant genotype F₄₄₆L/S₄₉₂C₅₈₀	1	5.0	1		1		0	1
单基因突变F₄₄₆S₄₉₂Y₅₈₀ Single-mutant genotype F₄₄₆S₄₉₂Y₅₈₀	1	5.0	1		1		0	1
Pfcrt/Pfmdr 多基因突变I₇₄E₇₅T₇₆/Y₁₂₄₆ Pfcrt/Pfmdr multi-mutant genotypes I₇₄E₇₅T₇₆/Y₁₂₄₆	29	69.0	8		21		2	10
Pfcrt/Pfmdr多基因突变I₇₄E₇₅T₇₆/Y₈₆Y₁₂₄₆ Pfcrt/Pfmdr multi-mutant genotypes I₇₄E₇₅T₇₆/Y₈₆Y₁₂₄₆	1	2.4	0		1		0	0
Pfcrt/Pfmdr/PfK13多基因突变I₇₄E₇₅T₇₆/Y₁₂₄₆/I₄₄₆ Pfcrt/Pfmdr/PfK13 multi-mutant genotypes I₇₄E₇₅T₇₆/Y₁₂₄₆/I₄₄₆	6	14.3	3		5		1	5
Pfcrt/Pfmdr/PfK13多基因突变I₇₄E₇₅T₇₆/Y₁₂₄₆/L/S₄₉₂ Pfcrt/Pfmdr/PfK13 multi-mutant genotypes I₇₄E₇₅T₇₆/Y₁₂₄₆/L/S₄₉₂	1	2.4	1		1		0	1
Pfmdr/PfK13多基因突变Y₁₂₄₆/I₄₄₆ Pfmdr/PfK13 multi-mutant genotypes Y₁₂₄₆/I₄₄₆	4	9.5	1		4		0	3
Pfmdr/PfK13多基因突变Y₁₂₄₆/Y₅₈₀ Pfmdr/ PfK13 multi-mutant genotypes Y₁₂₄₆/Y₅₈₀	1	2.4	1		1		0	1

表4

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基因Gene	引物Primer	引物序列（5′→3′） Primer sequence (5′→3′)	PCR轮次 PCR round	产物片段/bp Amplicon size/bp
Pfcrt	76A	GCGCGCGCATGGCTCACGTTTAGGTGGAG	第1轮	206
	76B	GGGCCCGGCGGATGTTACAAAACTATAGTTACC
	76D1	TGTGCTCATGTGTTTAAACTT	第2轮	145
	76D2	CAAAACTATAGTTACCAATTTTG
Pfmdr	86A	GCGCGCGTTGAACAAAAAGAGTACCGCTG	第1轮	450
	86B	GGGCCCTCGTACCAATTCCTGAACTCAC
	86D1	TTTACCGTTTAAATGTTTACCTGC	第2轮	291
	86D2	CCATCTTGATAAAAAACACTTCTT
	1246A	GGGGGATGACAAATTTTCAAGATTA	第1轮	295
	1246B	GGGGGACTAACACGTTTAACATCTT
	1246D1	AATGTAAATGAATTTTCAAACC	第2轮	203
	1246D2	CATCTTCTCTTCCAAATTTGATA
PfK13	PCR_F	CGGAGTGACCAAATCTGGGA	第1轮	2 181
	PCR_R	GGGAATCTGGTGGTAACAGC
	N1_F	GCCAAGCTGCCATTCATTTG	第2轮	849
	N1_R	GCCTTGTTGAAAGAAGCAGA

药物 Drug	测定血样 No. sample tested	抗性率/% Resistance rate/%	平均抑制浓度^a/nmol·L^-1 Mean inhibition concentration^a/nmol·L^-1	ID₅₀ （95%可信限） ID₅₀ (95%CI)	对其他抗疟药的抗性血样（交叉抗性率/%） No. resistance to other antimalarials (Proportion of cross resistance/%)
药物 Drug	测定血样 No. sample tested	抗性率/% Resistance rate/%		ID₅₀ （95%可信限） ID₅₀ (95%CI)	青蒿素 Artemisinin	氯喹 Chloroquine	哌喹 Piperaquine	咯萘啶 Pyronaridine
青蒿素 Artemisinin	42	40.5	640	84.8 (44.2~ 162.7)	-	15（88.2）	3（17.6）	13（76.5）
氯喹^[28] Chloroquine	42	95.2	880	320.5 (57.9~1775)	15（37.5）	-	3（7.5）	21（52.5）
哌喹^[28] Piperaquine	42	7.1	440	128.2 (125.1~ 131.4)	3（100）	3（100）	-	3（100）
咯萘啶^[28] Pyronaridine	42	54.8	410	96.0 (85.4~ 107.9)	13（56.5）	21（91.3）	3（13.0）	-

中缅边境地区恶性疟原虫Pfcrt、Pfmdr和PfK13基因多态性与体外药物敏感性相关性的分析

Correlation of Pfcrt, Pfmdr and PfK13 gene polymorphisms and in vitro drug susceptibility of Plasmodium falciparum isolates from China-Myanmar border region

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药物Drug	浓度/（pmol·井^-1） Concentration/(pmol·well^-1)
药物Drug	A	B	C	D	E	F	G	H
青蒿素Artemisinin	0	0.15	0.5	1.5	5	15	50	150
氯喹Chloroquine	0	1	2	4	8	16	32	64
哌喹Piperaquine	0	2	4	8	16	32	64	128
咯萘啶Pyronaridine	0	2	4	8	16	32	64	128

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