长沙市122例输入性恶性疟原虫多药抗性基因1拷贝数变异分析

doi:10.12140/j.issn.1000-7423.2022.01.021

摘要/Abstract

摘要：

为了解输入性恶性疟原虫多药抗性基因1（Pfmdr1）拷贝数变异（CNV）情况,对长沙市2016—2019年输入的恶性疟患者滤纸干血斑样品中Pfmdr1与恶性疟原虫微管蛋白基因（Pftub）进行实时荧光定量PCR检测,以Pftub基因为参比基因,计算获得样品的Pfmdr1 CNV值。采用SPSS 23.0统计学软件对检测结果和治疗信息进行统计分析。结果显示,122份血样中有18份发生Pfmdr1 CNV变异,变异率为14.8%（18/122）。2016—2019年血样Pfmdr1 CNV均值分别为1.020 ± 0.076、1.136 ± 0.403、1.387 ± 0.657和1.142 ± 0.349。变异血样输入地来源为赤道几内亚、安哥拉、贝宁、塞拉利昂、刚果民主共和国、尼日利亚、喀麦隆、加纳和刚果共和国。输入地为东非、西非和中非的患者血样Pfmdr1 CNV均值分别为0.999 ± 0.073、1.150 ± 0.368和1.249 ± 0.448。东非和西非、中非相比差异有统计学意义（t = 2.663、3.995,P < 0.05）。变异血样的患者平均用药时长为（5.93 ± 0.94）d,长于非变异血样患者的（3.21 ± 1.23）d（t = 8.930,P < 0.01）。治疗结束后1个月变异血样的患者再燃2例,非变异血样的患者再燃1例（似然比χ² = 3.831,P < 0.05）。治疗结束后1年变异血样的患者再燃2例,非变异血样的患者再燃1例（似然比χ² = 5.372,P < 0.05）。血涂片中存在配子体的变异血样5例,非变异血样3例（似然比χ² = 10.599,P < 0.01）。长沙市输入性恶性疟原虫存在Pfmdr1 CNV变异,变异会造成患者治疗时间延长和原虫不能完全清除。

关键词: 疟疾, 恶性疟原虫, 多药抗性基因1基因, 拷贝数变异, 抗性虫株

Abstract:

To understand the copy number variation (CNV)of Plasmodium falciparum multidrug resistence-1 (Pfmdr1) gene among imported Plasmodium falciparum, real-time fluorescence quantitative PCR was used to detect Pfmdr1 and β-tubulin (Pftub) gene from dried blood samples on filter paper collected from imported falciparum malaria cases in Changsha City from 2016 to 2019, with Pftub as the reference gene, for calculating the Pfmdr1 CNV in the samples examined. SPSS 23.0 statistic software was used to analyze the detection results and individuals’ clinical treatment information. The gene detection indicated that among 122 samples, 18 (14.8%) had Pfmdr1 CNV, with the variation rate 14.8% (18/122). The average of Pfmdr1 CNV in the samples collected from 2016 to 2019 was 1.020 ± 0.076, 1.136 ± 0.403, 1.387 ± 0.657 and 1.142 ± 0.349, respectively. The blood samples with CNV of the falciparum malaria cases were imported from Equatorial Guinea, Angola, Benin, Sierra Leone, Democratic Republic of Congo, Nigeria, Cameroon, Ghana and The Republic of Congo. The average Pfmdr1 CNV of the blood samples of the cases imported from East, West and Central Africa were 0.999 ± 0.073, 1.150 ± 0.368 and 1.249 ± 0.448, respectively, with significant difference between the regions (t = 2.663, 3.995, P < 0.05). The average medication duration for the cases with Pfmdr1 varied CNV was (5.93 ± 0.94) d, which was longer than that from non-varied CNV [(3.21 ± 1.23) d] (t = 8.930, P < 0.01). One month after medication, recrudescence occurred in two cases with varied CNV Pfmdr1, but in one case with non-varied CNV (likelyhood ratio χ ² = 3.831, P < 0.05). One year after treatment ended, recrudescence occurred in two cases with varied CNV, whereas in one case with non-varied CNV （likelyhood ratio χ ² = 5.372,P < 0.05). Gametocytes were detected in the blood smears of 5 cases with varies CNV, while in 3 cases with non-varied CNV （likelyhood ratio χ² = 10.599, P < 0.01). Pfmdr1 gene CNV was found in the imported P. falciparum parasite in Changsha, and the variation may result in longer treatment duration for the patients and unable to completely clear up the parasites.

Key words: Malaria, Plasmodium falciparum, Multidrug resistance-1 gene, Copy number variations, Resistant strain

中图分类号:

R383.3

田斌, 廖瑜, 文岚, 肖芳, 张兵, 申晓君. 长沙市122例输入性恶性疟原虫多药抗性基因1拷贝数变异分析[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(1): 127-131.

TIAN Bin, LIAO Yu, WEN Lan, XIAO Fang, ZHANG Bin, SHEN Xiao-jun. Analysis on the copy number variation of multidrug resistance-1 gene in 122 imported cases of falciparum malaria in Changsha[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2022, 40(1): 127-131.

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基因	序列	产物大小/bp
Pfmdr1	F: 5′-TGCATCTATAAAACGATCAGACAAA-3′	87
	R: 5′-TCGTGTGTTCCATGTGACTGT-3′
	FAM: 5′-TTTAATAACCCTGATCGAAATGG-AACCTTTG-3′ TAMRA
Pftub	F: 5′-TGATGTGCGCAAGTGATCC-3′	79
	R: 5′-TCCTTTGTGGACATTCTTCCTC-3′
	JOE: 5′-TAGCACATGCCGTTAAATATCTTC-CATGTCT-3′ TAMRA

区域	国别	患者数		$\bar{x}±s$	t 值	P值
区域	国别	CNV ≤ 1.5	CNV > 1.5	$\bar{x}±s$	t 值	P值
东非	坦桑尼亚	1	0	1.218 ± 0.023	1.674	> 0.05
	埃塞俄比亚	4	0	1.040 ± 0.125	3.094	< 0.05
	马维拉	1	0	1.002 ± 0.012	7.017	< 0.01
	肯尼亚	1	0	0.812 ± 0.028	12.706	< 0.01
	莫桑比克	2	0	0.950 ± 0.074	5.839	< 0.01
	乌干达	4	0	0.941 ± 0.258	2.972	< 0.05
	马达加斯加	2	0	1.067 ± 0.126	1.876	> 0.05
	布隆迪	1	0	1.002 ± 0.024	6.295	< 0.01
	小计	16	0	0.999 ± 0.073	3.919	< 0.01
西非	尼日利亚	13	2	1.083 ± 0.323	1.682	> 0.05
	加纳	8	1	1.043 ± 0.357	1.476	> 0.05
	贝宁	1	1	1.976 ± 0.348	4.376	< 0.01
	塞拉利昂	6	2	1.415 ± 0.595	1.957	> 0.05
	利比里亚	2	0	0.922 ± 0.046	8.373	< 0.01
	科特迪瓦	5	0	1.128 ± 0.116	1.185	> 0.05
	布基纳法索	1	0	0.867 ± 0.023	1.184	> 0.05
	马里	5	0	0.962 ± 0.072	7.067	< 0.01
	小计	41	6	1.150 ± 0.368	0.314	> 0.05
中非	刚果共和国	13	1	1.066 ± 0.357	1.497	> 0.05
	几内亚	2	0	0.960 ± 0.025	8.354	< 0.01
	刚果民主共和国	14	4	1.228 ± 0.384	0.858	> 0.05
	喀麦隆	7	1	1.111 ± 0.547	1.498	> 0.05
	赤道几内亚	2	3	1.747 ± 0.507	4.849	< 0.01
	安哥拉	3	3	1.600 ± 0.703	2.552	< 0.05
	加蓬	3	0	1.260 ± 0.176	1.378	> 0.05
	小计	44	12	1.249 ± 0.448	1.239	> 0.05
亚洲	巴基斯坦	2	0	0.986 ± 0.030	7.089	< 0.01
大洋洲	巴布亚新几内亚	1	0	1.114 ± 0.023	2.195	< 0.05

分组	用药方案	用药平均时长/d	治疗结束后1月病原学检查结果/份		治疗结束后1年病原学检查结果/份
分组	用药方案	用药平均时长/d	阳性	阴性	阳性	阴性
CNV > 1.5 （n = 18）	ACT口服药或注射剂（n = 7）	4.86 ± 0.98	1	7	2	4
	ACT口服药 + 注射剂（n = 10）	6.97 ± 1.76	0	9	0	4
	其他（n = 1）	3.00 ± 0.00	1	0	0	0
小计		5.93 ± 0.94	2	16	2	8
CNV ≤ 1.5 （n = 104）	ACT口服药或注射剂（n = 93）	3.11 ± 1.07	0	84	0	72
	ACT口服药 + 注射剂（n = 7）	4.65 ± 2.29	0	2	0	2
	其他（n = 4）	3.10 ± 0.64	1	2	1	1
小计		3.21 ± 1.23	1	88	1	75