慢性乙型肝炎患者停用核苷(酸)类似物后循环血清中HBV pgRNA、HBcrAg表达水平与复发的相关性分析

张诗琬; 陈星; 刘娇; 赵敏; 梅小平

doi:10.3969/j.issn.1001-5256.2023.01.009

慢性乙型肝炎患者停用核苷(酸)类似物后循环血清中HBV pgRNA、HBcrAg表达水平与复发的相关性分析

DOI: 10.3969/j.issn.1001-5256.2023.01.009

张诗琬¹,
陈星²,
刘娇³,
赵敏¹,
梅小平^1, , ,

1.
川北医学院附属医院感染科, 四川南充 637000
2.
南充市中心医院感染科, 四川南充 637000
3.
遂宁市中心医院感染科, 四川遂宁 629000

基金项目:

南充市市校合作专项资金 (19SXHZ0058)

伦理学声明：本研究方案于2022年3月8日经由川北医学院附属医院伦理委员会审批，批号：2022ER127-1，所纳入患者均签署知情同意书。

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：张诗琬负责资料分析，撰写论文；陈星、刘娇、赵敏参与标本收集，数据整理；梅小平负责指导撰写文章并最后定稿。

详细信息

通信作者:
梅小平，1124377569@qq.com(ORCID: 0000-0002-5077-1706)

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出版历程
- 收稿日期: 2022-10-14
- 录用日期: 2022-11-28
- 出版日期: 2023-01-20

Expression levels of HBV pregenomic RNA and hepatitis B core-related antigen in circulating serum and their association with recurrence in chronic hepatitis B patients after withdrawal from nucleos(t)ide analogues

1.
Department of Infectious Diseases, Affiliated Hospital of North Sichuan Medical College, Nanchong, Sichuan 637000, China
2.
Department of Infectious Diseases, Nanchong Central Hospital, Nanchong, Sichuan 637000, China
3.
Department of Infectious Diseases, Suining Central Hospital, Suining, Sichuan 629000, China

Research funding:

Special Funds for Municipal School Cooperation of Nanchong (19SXHZ0058)

More Information

Corresponding author: MEI Xiaopin, 1124377569@qq.com (ORCID: 0000-0002-5077-1706)

摘要

摘要: 目的检测慢性乙型肝炎(CHB)患者停用核苷(酸)类似物(NUC)后循环血清中HBV前基因组RNA(pgRNA)以及乙型肝炎核心相关抗原(HBcrAg)的表达水平，分析停药后CHB患者循环血中不同时段HBV pgRNA、HBcrAg水平与复发之间的相关性。方法选取2019年12月—2022年7月川北医学院附属医院门诊就诊者中抗HBV治疗至少5年到达完全应答并满足2017年版欧洲肝病学会指南停药标准的CHB患者108例，根据停药时间分为停药后4、12、24周组；根据复发情况分为复发组与未复发组。运用实时荧光定量PCR法检测CHB患者循环血清中HBV pgRNA水平，运用ELISA检测研究对象静脉血中HBcrAg的表达水平，采用实时荧光定量PCR法高精度检测HBV DNA载量。计量资料两组间比较采用t检验；多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。采用Pearson相关检验分析循环血中各指标间的相关性。结果 CHB患者停药后在随访4~12周时复发率17.1%，24周后累积复发率达到29.3%，其中单独HBV DNA复阳者各占64.3%、60.0%，HBeAg单独复阳者各占28.6%、20.0%，HBV DNA、HBeAg同时复阳者各占7.1%、20.0%。停药24周时，CHB患者循环血清中HBV pgRNA、HBcrAg、HBV DNA表达水平较停药时及停药后4周时明显高表达，不同时段比较差异具有统计学意义(P值均＜0.05)。复发组循环血清中HBV pgRNA、HBcrAg、HBV DNA水平较未复发组显著高表达(t值分别2.549、8.654、27.429，P值均＜0.05)；对复发组进一步分析发现，在12~24周时复发组患者循环血清中HBV pgRNA、HBcrAg与HBV DNA水平较4~12周时高表达(P值均＜0.05)。复发组停药时循环血清中HBV pgRNA、HBcrAg的表达水平较未复发组停药时显著高表达(t值分别为18.561、6.152，P值均＜0.001)。相关性分析显示，CHB患者停药后复发组循环血清中HBV pgRNA、HBcrAg与HBV DNA呈正相关(r值分别为0.82、0.66，P值均＜0.001)；未复发组循环血清中HBV pgRNA、HBcrAg与HBV DNA均无相关性(r值分别为0.14、0.04，P值均＞0.05)。结论停药时复发组HBV pgRNA、HBcrAg水平较未复发组高表达，提示未复发组在停药时的HBV pgRNA、HBcrAg水平可能作为CHB患者可安全停药的参考临界值指标，HBV pgRNA、HBcrAg水平检测可能是未来抗HBV治疗终点选择的潜在参考指标之一。
- 慢性乙型肝炎 /
- HBV前基因组RNA /
- 乙型肝炎核心相关抗原 /
- 复发
Abstract: Objective To investigate the expression levels of HBV pregenomic RNA (pgRNA) and hepatitis B core-related antigen (HBcrAg) in circulating serum of chronic hepatitis B (CHB) patients after withdrawal from nucleos(t)ide analogues (NUC), as well as the correlation of HBV pgRNA and HBcrAg levels in circulating blood in different periods of time with recurrence in CHB patients after drug withdrawal. Methods Among the patients who attended the outpatient service of Affiliated Hospital of North Sichuan Medical College from December 2019 to July 2022, a total of 108 CHB patients who received anti-HBV therapy for at least 5 years and met the criteria for drug withdrawal in 2017 EASL Guidelines were enrolled. According to the time of drug withdrawal, the patients were divided into 4-, 12-, and 24-week groups after drug withdrawal, and according to the presence or absence of recurrence, they were divided into recurrence group and non-recurrence group. Quantitative real-time PCR was used to measure the level of HBV pgRNA in circulating serum of CHB patients; ELISA was used to measure the expression level of HBcrAg in peripheral venous blood; quantitative real-time PCR was used to measure HBV DNA load with high accuracy. The t-test was used for comparison of continuous data between two groups. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. The Pearson correlation test was used to investigate the correlation between the indices in circulating blood. Results For the CHB patients after drug withdrawal, the recurrence rate was 17.1% at 4-12 weeks, cumulative recurrence rate reached 29.3% after 24 weeks of follow-up, the patients with positive HBV DNA alone accounted for 64.3% and 60.0%, respectively, those with positive HBeAg alone accounted for 28.5% and 20.0%, respectively, and those with positive HBV DNA and HBeAg accounted for 7.1% and 20.0%, respectively. The expression levels of HBV pgRNA, HBcrAg, and HBV DNA in circulating serum of CHB patients at 24 weeks after drug withdrawal were significantly higher than those at the time of drug withdrawal and at 4 weeks after drug withdrawal, and there was a significant difference between groups at different time points (all P < 0.05). Compared with the non-recurrence group, the recurrence group had significantly higher expression levels of HBV pgRNA, HBcrAg, and HBV DNA in circulating serum (t=2.549, 8.654, and 27.429, all P < 0.05), and further analysis of the recurrence group showed that the levels of HBV pgRNA, HBcrAg, and HBV DNA in circulating serum at 12-24 weeks were significantly higher than those at 4-12 weeks (all P < 0.05). At the time of drug withdrawal, the recurrence group had significantly higher expression levels of HBV pgRNA and HBcrAg in circulating serum than the non-recurrence group (t=18.561 and 6.152, both P < 0.001). The Pearson correlation analysis showed that in the recurrence group after drug withdrawal, HBV pgRNA and HBcrAg were positively correlated with HBV DNA in circulating serum (r=0.82 and 0.66, both P < 0.001), while no such correlation was observed in the non-recurrence group (r=0.14 and 0.04, both P > 0.05). Conclusion The recurrence group had significantly higher expression levels of HBV pgRNA and HBcrAg than the non-recurrence group at the time of drug withdrawal, suggesting that the levels of HBV pgRNA and HBcrAg in the CHB patients of the non-recurrence group at the time of drug withdrawal may be used as the reference thresholds for safe drug withdrawal in CHB patients, and measurement of HBV pgRNA and HBcrAg may be one of the potential reference indicators for the selection of anti-HBV treatment endpoints in the future.
- Hepatitis B, Chronic /
- HBV Pregenomic RNA /
- Hepatitis B Core Related Antigen /
- Recurrence

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图 1 复发组HBV pgRNA与HBV DNA的关系

Figure 1. Relationship between HBV pgRNA and HBV DNA in relapse group

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图 2 复发组HBcrAg与HBV DNA的关系

Figure 2. Relationship between HBcrAg and HBV DNA in relapse group

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图 3 未复发组HBV pgRNA与HBV DNA的关系

Figure 3. Relationship between HBV pgRNA and HBV DNA in non relapse group

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图 4 未复发组HBcrAg与HBV DNA的关系

Figure 4. Relationship between HBcrAg and HBV DNA in non relapse group

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表 1 CHB患者停药后随访24周内病毒学复发人数及累积复发率

Table 1. The number of viral recurrences and cumulative recurrence rate of CHB patients within 24 weeks of follow-up after drug withdrawal

组别	复发率(%)
停药4周	0
停药12周	17.1(14/82)
HBV DNA复阳	64.3(9/14)
HBeAg复阳	28.6(4/14)
HBV DNA、HBeAg同时复阳	7.1(1/14)
停药24周	14.7(10/68)
HBV DNA复阳	60.0(6/10)
HBeAg复阳	20.0(2/10)
HBV DNA、HBeAg同时复阳	20.0(2/10)

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表 2 CHB患者停药后各时段循环血清中HBV pgRNA、HBcrAg、HBV DNA表达水平比较

Table 2. Comparison of HBV pgRNA, HBcrAg and HBV DNA expression levels in circulating serum of CHB patients at different periods after drug withdrawal

组别	例数	HBV pgRNA (log₁₀拷贝/mL)	HBcrAg (log₁₀ U/L)	HBV DNA (log₁₀ IU/mL)
停药时	82	1.36±0.12	1.31±0.16	1.30±0.18
停药4周	82	1.37±0.10	1.31±0.17	1.61±0.20
停药12周	82	1.39±0.12	1.40±0.13	2.38±0.26
停药24周	68	1.42±0.14¹⁾²⁾	1.51±0.16¹⁾²⁾	2.50±0.35¹⁾²⁾
F值		10.276	14.359	21.473
P值		0.022	0.013	< 0.001
注：与停药时相比，1)P＜0.05；与停药4周相比，2)P＜0.05。

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表 3 CHB患者停药后复发组与未复发组循环血清中HBV pgRNA、HBcrAg、HBV DNA、HBsAg水平比较

Table 3. Comparison of HBV pgRNA, HBcrAg, HBV DNA and HBsAg levels in circulating serum between relapse group and non relapse group of CHB patients after drug withdrawal

组别	例数	HBV pgRNA (log₁₀拷贝/mL)	HBcrAg (log₁₀ U/L)	HBV DNA (log₁₀ IU/mL)
复发组	24	1.49±0.15	2.02±0.11	3.74±0.39
未复发组	58	1.39±0.13	1.35±0.17	2.24±0.15
t值		2.549	8.654	25.680
P值		0.015	< 0.001	< 0.001

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表 4 停药时复发组与未复发组CHB患者循环血清中HBV pgRNA、HBcrAg表达水平比较

Table 4. Comparison of HBV pgRNA, HBcrAg expression levels in circulating serum of CHB patients in relapse and non relapse groups at the time of drug withdrawal

组别	例数	HBV pgRNA (log₁₀拷贝/mL)	HBcrAg (log₁₀ U/L)
复发组	24	1.42±0.11	1.55±0.20
未复发组	58	1.34±0.07	1.21±0.11
t值		18.561	6.152
P值		< 0.001	< 0.001

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