HIV/HCV合并感染者和HCV单纯感染者NS3/4A蛋白酶、NS5A抑制剂的天然耐药变异分析

邓浩辉; 李水凤; 冯倩嫦; 李凌华

doi:10.3969/j.issn.1001-5256.2022.02.015

HIV/HCV合并感染者和HCV单纯感染者NS3/4A蛋白酶、NS5A抑制剂的天然耐药变异分析

DOI: 10.3969/j.issn.1001-5256.2022.02.015

广州医科大学附属市八医院感染病中心，广州 510060

基金项目:

广东省医学科学技术研究基金项目 (B2021302);

国家科技部“十三五”重大专项 (2017ZX10202101-003);

国家自然科学基金项目 (82072265);

广州市基础研究计划民生科技专题项目 (202002020005)

利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。

作者贡献声明：邓浩辉负责课题设计，资料分析，撰写论文；李水凤、冯倩嫦参与收集临床数据；李凌华负责拟定写作思路，指导撰写文章并最后定稿。

详细信息

通信作者:
李凌华，llheliza@126.com

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出版历程
- 收稿日期: 2021-05-04
- 录用日期: 2021-06-05
- 出版日期: 2022-02-20

Naturally occurring resistance-associated variants to NS3/4A protease and NS5A inhibitors in patients with HIV/HCV co-infection or HCV infection alone

Infectious Disease Center, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou 510060, China

Research funding:

Medical Scientific Research Foundation of Guangdong Province, China (B2021302);

Chinese 13th Five-Year National Science and Technology Major Project (2017ZX10202101-003);

National Natural Science Foundation of China (82072265);

Guangzhou Basic Research Program on People's Livelihood Science and Technology (202002020005)

More Information

Corresponding author: LI Linghua, llheliza@126.com

摘要

摘要: 目的通过对抗病毒治疗前HIV/HCV合并感染者和HCV单纯感染者HCV NS3/4A蛋白酶、NS5A抑制剂相关耐药位点进行检测，探讨上述患者天然耐药变异的差异。方法收集2016年1月—2020年1月在广州医科大学附属市八医院住院或门诊就诊的246例HIV/HCV合并感染者和HCV单纯感染者的血清标本，使用Illumina二代测序平台进行测序，比较已在中国获批准的NS3/4A蛋白酶、NS5A抑制剂相关耐药变异在两组患者的差异，纳入分析的药物包括阿舒瑞韦/达拉他韦(ASV/DCV，基因1b型)，艾尔巴韦/格拉瑞韦(EBR/GZR，基因1b型)和格卡瑞韦/哌仑他韦(GLE/PIB，泛基因型)。符合正态分布的计量资料两组间比较采用t检验，不符合正态分布的计量资料两组间比较采用Mann-Whitney U检验。计数资料两组间比较采用χ²检验或Fisher精确检验。结果本研究纳入的246例患者的血清样本中，239例(97.2%)成功进行PCR扩增并测序，包括102例HIV/HCV合并感染者和137例HCV单纯感染者。对ASV/DCV和EBR/GZR相关耐药变异分析结果提示：HCV 1b型的HIV/HCV合并感染者和HCV单纯感染者中存在Y56F、Q80K/L、S122N/R/T等ASV和GZR相关耐药变异，以及L31M、Y93H等DCV和EBR相关耐药变异；2例HIV/HCV合并感染者(均为Y56F+Y93H)和2例HCV单纯感染者(Q80L+L31M和Y56F+Y93H)对EBR/GZR双靶点同时存在耐药变异，两组患者各耐药变异差异均无统计学意义(P值均＞0.05)。对泛基因型的GLE/PIB相关耐药变异分析结果提示，仅在HCV 3a型的患者中，存在3例PIB相关耐药变异，分别为HIV/HCV合并感染者中2例Y93H耐药变异和HCV单纯感染者中1例Y93H耐药变异(P=0.590)，所有纳入分析的患者均不存在GLE/PIB双靶点耐药变异。结论 HIV/HCV合并感染者和HCV单纯感染者的NS3/4A蛋白酶、NS5A抑制剂相关天然耐药变异无明显差异。
- HIV /
- 肝炎病毒属 /
- 直接抗病毒药物 /
- 耐药变异
Abstract: Objective To investigate the difference in naturally occurring resistance-associated variants (RAVs) between the patients with HIV/HCV co-infection and those with HCV infection alone by detecting the drug resistance loci associated with HCV NS3/4A protease and NS5A inhibitors. Methods A total of 246 patients with HIV/HCV co-infection or HCV infection alone who were hospitalized or attended the outpatient service in Guangzhou Eighth People's Hospital, Guangzhou Medical University, from January 2016 to January 2020 were enrolled in this study. Serum samples were collected and next-generation sequencing (Illumina platform, PE250) was used for sequencing. The two groups of patients were compared in terms of RAVs associated with NS3/4A protease and NS5A inhibitors approved in China, and the drugs for analysis included asunaprevir/daclatasvir (ASV/DCV) and elbasvir/grazoprevir (EBR/GZR) for HCV genotype 1b and glecaprevir/pibrentasvir (GLE/PIB) for pan-genotypes. The t-test was used for comparison of normally distributed continuous data between two groups, and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups; the chi-square test or the Fisher's exact test was used for comparison of categorical data between two groups. Results Among the 246 serum samples included in this study, 239 samples (97.2%) were successfully amplified by PCR and sequenced, with 102 samples from the patients with HIV/HCV co-infection and 137 from the patients with HCV infection alone. The analysis of RAVs associated with ASV/DCV and EBR/GZR showed that Y56F, Q80K/L, and S122N/R/T associated with ASV and GZR and L31M and Y93H associated with DCV and EBR were observed in patients with HIV/HCV (genotype 1b) co-infection or HCV (genotype 1b) infection alone; 2 patients with HIV/HCV co-infection had the RAVs of Y56F+Y93H associated with EBR/GZR, and 2 with HCV infection alone had the RAVs of Q80L+L31M and Y56F+Y93H, respectively, associated with EBR/GZR, with no significant difference in RAVs between the two groups (both P > 0.05). The analysis of RAVs associated with GLE/PIB for pan-genotypes showed that 3 patients with PIB-associated Y93H RAV were observed among the patients with HCV genotype 3a infection, among whom 2 had HIV/HCV co-infection and 1 had HCV infection alone (P=0.590), and in addition, no RAVs associated with GLE/PIB were observed. Conclusion There is no significant difference in naturally occurring RAVs associated with HCV NS3/4A protease and NS5A inhibitors between the patients with HIV/HCV co-infection and those with HCV infection alone.
- HIV /
- Hepacivirus /
- Direct Antiviral Drugs /
- Drug Resistance Variation

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表 1 纳入分析的NS3/4A蛋白酶、NS5A抑制剂相关耐药变异

NS3/4A蛋白酶抑制剂相关耐药变异		NS5A抑制剂相关耐药变异
ASV^[3-5]	V36L，Q80K，S122N/R/T，D168A/H	DCV^[3-5]	L31M/F/V，Y93C/H/N
GZR^[3]	V36L/M，Y56F/H，Q80K/L，R155I/K/L/S，A156G/M/T/V，V158A，D168A/C/E/G/K/N/V/Y	EBR^[3] PIB^[6-7]	L31F/M/V，Y93H
GLE^[6-7]		1a型	Q30D，Y93D/H/N
1a型	A156T，Q89R+A156T	1b型	未发现耐药变异
1b型	A156T/V，P89L+A156T/V，A156V+D168V	2a型	F28S+M31I，P29S+K30G
2a型	A156T/V	3型	Y93H
3型	A156G，Y56H+Q168R	6a型	未发现耐药变异
6a型	D168H/V，D168H+M179T

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表 2 HIV/HCV合并感染者和HCV单纯感染者的一般资料

指标	HIV/HCV合并感染者(n=102)	HCV单纯感染者(n=137)	统计值	P值
男/女(例)	83/19	109/28	χ²=0.121	0.728
年龄(岁)	43(39~51)	45(38~51)	Z=0.546	0.585
TBil(μmol/L)	12.2(8.8~17.4)	13.3(10.9~18.5)	Z=-1.374	0.169
ALT(U/L)	44(29~65)	47(30~84)	Z=-1.351	0.177
HCV RNA(log₁₀ IU/mL)	6.4±0.9	6.3±1.0	t=-0.747	0.456
肝硬化代偿期[例(%)]	25(24.5)	35(25.5)	χ²=0.351	0.554
HCV感染的危险因素[例(%)]
静脉吸毒	56(54.9)	23(16.8)	χ²=38.382	＜0.001
未保护性行为	20(19.6)	15(10.9)	χ²=3.507	0.061
输血	2(2.0)	65(47.4)	χ²=59.957	＜0.001
未知	24(23.5)	34(24.8)	χ²=0.053	0.818
HCV基因型[例(%)]
1a	2(2.0)	4(2.9)	χ²=0.003	0.960
1b	24(23.5)	67(48.9)	χ²=15.968	＜0.001
2a	2(2.0)	9(6.6)	χ²=1.918	0.166
3a	11(10.8)	12(8.8)	χ²=0.270	0.600
3b	12(11.8)	9(6.6)	χ²=2.482	0.115
6a	51(50.0)	36(26.3)	χ²=14.213	＜0.001

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表 3 两组HCV 1b型患者ASV和GZR天然耐药变异比较(NS3/4A蛋白酶抑制剂)

耐药变异	HIV/HCV合并感染者(n=24)	HCV单纯感染者(n=67)	χ²值	P值
V36L/M	0	0
Y56F/H	F∶8	F∶9	3.390	0.063
Q80K/L¹⁾	K∶1	K∶2，L∶1	＜0.001	1.000
S122N/R/T	N∶4	N∶2，R∶1，T∶1	1.364	0.243
R155I/K/L/S	0	0
A156G/M/T/V	0	0
V158A	0	0
D168A/C/E/G/K/N/V/Y/H	0	0
注：1)ASV耐药变异，Q80K；GZR耐药变异，Q80K/L。

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表 4 两组HCV 1b型患者DCV和EBR天然耐药变异比较(NS5A抑制剂)

耐药变异	HIV/HCV合并感染者(n=24)	HCV单纯感染者(n=67)	χ²值	P值
L31F/M/V	0	M∶4	0.415	0.520
Y93C/H/N	H∶3	H∶11	0.016	0.899

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表 5 两组患者GLE天然耐药变异比较(NS3/4A蛋白酶抑制剂)

HCV基因型	例数(合并感染/单纯感染)	Y56H	Q89R/L	A156T/V	Q168R/H/V	M179T
1a	2/4	NR	0/0	0/0	NR	NR
1b	24/67	NR	0/0	0/0	0/0	NR
2a	2/9	NR	NR	0/0	NR	NR
3a	11/12	0/0	NR	0/0	0/0	NR
3b	12/9	0/0	NR	0/0	0/0	NR
6a	51/36	NR	NR	NR	0/0	0/0
注：NR，该位点变异与耐药不相关。

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表 6 两组患者PIB天然耐药变异比较(NS5A抑制剂)

HCV基因型	例数(合并感染/单纯感染)	F28S	P29S	Q30D	M31I	Y93D/H/N
1a	2/4	NR	NR	0/0	NR	NR
1b	24/67	NR	NR	NR	NR	NR
2a	2/9	0/0	0/0	0/0	0/0	NR
3a	11/12	NR	NR	NR	NR	H：2/1
3b	12/9	NR	NR	NR	NR	0/0
6a	51/36	NR	NR	NR	NR	NR
注：NR，该位点变异与耐药不相关。

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