2种实时荧光定量聚合酶链式反应法检测HBeAg阳性慢性乙型肝炎患者血清HBV RNA的一致性分析

王扬; 廖昊; 邓中平; 卞丹丹; 任艳; 蒋莹莹; 刘霜; 陈煜; 鲁凤民; 段钟平; 郑素军

doi:10.3969/j.issn.1001-5256.2022.05.012

2种实时荧光定量聚合酶链式反应法检测HBeAg阳性慢性乙型肝炎患者血清HBV RNA的一致性分析

DOI: 10.3969/j.issn.1001-5256.2022.05.012

王扬¹,
廖昊²,
邓中平^{3, 4},
卞丹丹⁵,
任艳¹,
蒋莹莹¹,
刘霜¹,
陈煜¹,
鲁凤民²,
段钟平¹,
郑素军^1, , ,

1.
首都医科大学附属北京佑安医院疑难肝病与人工肝中心，北京 100069
2.
北京大学基础医学院病原生物学系暨感染病中心，北京 100191
3.
北京大学前沿交叉学科研究院，北京 100871
4.
基因诊断技术湖南省重点实验室，长沙 410205
5.
首都医科大学电力教学医院感染性疾病科，北京 100073

基金项目:

国家科技重大专项“艾滋病和病毒性肝炎等重大传染病防治” (2017ZX10302201-004);

国家科技重大专项“艾滋病和病毒性肝炎等重大传染病防治” (2017ZX10202203-006);

国家科技重大专项“艾滋病和病毒性肝炎等重大传染病防治” (2017ZX10201201);

国家科技重大专项“艾滋病和病毒性肝炎等重大传染病防治” (2017ZX10203201-005)

伦理学声明：本研究经由首都医科大学附属北京佑安医院伦理委员会批准，批号：京佑科伦字〔2019〕056号。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：王扬负责整理、统计数据并撰写初稿；廖昊、邓中平给予试验平台及负责试验操作；卞丹丹、任艳及蒋莹莹负责临床数据及血清样本的收集、整理及分析；刘霜、陈煜负责管理项目实施；鲁凤民、段钟平、郑素军负责研究设计，严格审阅及修订稿件并批准最终版本投稿。所有作者审阅稿件并参与稿件的修订。

详细信息

通信作者:
郑素军，zhengsujun@ccmu.edu.cn

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出版历程
- 收稿日期: 2021-09-21
- 录用日期: 2021-10-29
- 出版日期: 2022-05-20

Comparison of two quantitative real-time PCR methods for serum HBV RNA in patients with HBeAg-positive chronic hepatitis B: A propensity score matching study

1.
Difficult & Complicated Liver Diseases and Artificial Liver Center, Beijing YouAn Hospital, Capital Medical University, Beijing 100069, China
2.
Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China
3.
Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China
4.
Hunan Provincial Key Laboratory of Gene Diagnostic Technology, Changsha 410205, China
5.
Department of Infectious Diseases, Electric Power Teaching Hospital, Capital Medical University, Beijing 100073, China

Research funding:

National Science and Technology Key Project on "Major Infectious Diseases such as HIV/AIDS, Viral Hepatitis Prevention and Treatment" (2017ZX10302201-004);

National Science and Technology Key Project on "Major Infectious Diseases such as HIV/AIDS, Viral Hepatitis Prevention and Treatment" (2017ZX10202203-006);

National Science and Technology Key Project on "Major Infectious Diseases such as HIV/AIDS, Viral Hepatitis Prevention and Treatment" (2017ZX10201201);

National Science and Technology Key Project on "Major Infectious Diseases such as HIV/AIDS, Viral Hepatitis Prevention and Treatment" (2017ZX10203201-005)

More Information

Corresponding author: ZHENG Sujun, zhengsujun@ccmu.edu.cn(ORCID: 0000-0002-6367-5764)

摘要

摘要: 目的评价S(Shengxiang)公司、X(Xinbo)公司两种实时荧光定量聚合酶链式反应(real-time PCR)方法定量检测HBV RNA结果的一致性。方法从2007年7月—2008年8月组建的108例慢性乙型肝炎(CHB)前瞻性随访患者队列中，选取HBeAg血清学转换患者20例进行1∶ 1倾向性评分匹配未转换患者20例，采用S公司和X公司两种real-time PCR定量检测方法，回顾性检测基线、12周、24周及48周血清样本HBV RNA。符合正态分布的计量资料组间比较采用配对t检验；不符合正态分布的计量资料组间比较采用Mann-Whitney U检验。计数资料采用χ²检验。通过Pearson相关系数、组内相关系数(ICC)和Bland-Altman法评价检测结果的一致性。结果 S试剂检测132份血清样本，X试剂检测154份血清样本，两种试剂对送检样本中HBV RNA检出率均为100%。两种试剂共同检测血清样本131份，在基线、随访12周、24周及48周分别检测34、29、35及33份血清，X试剂检测HBV RNA定量数据分别为(5.75±1.64、5.43±1.73、5.13±1.54、4.76±1.55)log₁₀拷贝/mL，均高于S试剂(4.80±1.48、4.52±1.53、4.10±1.50、3.92±1.43)log₁₀拷贝/mL，差异均有统计学意义(t=8.348、t=5.341、Z=-5.086、Z=-4.762, P值均＜0.001)。两种方法相关性分析中，r及ICC值分别为0.915(95%CI：0.836~0.957)与0.771(95%CI：-0.021~0.931)、0.849(95%CI：0.701~0.927)与0.733(95%CI：0.138~0.902)、0.951(95%CI：0.905~0.975)与0.776(95%CI：-0.058~0.942)、0.933(95%CI：0.867~0.967)与0.804(95%CI：-0.014~0.944) (P值均＜0.05)。Bland-Altman分析表明，两种方法检测结果中96.18%(126/131)的样本差异位于平均差±1.96标准差之间。结论 X试剂的HBV RNA定量结果高于S试剂定量结果，但两种real-time PCR定量检测HBeAg血清学转换和未转换CHB患者HBV RNA结果的一致性良好。
- 乙型肝炎, 慢性 /
- RNA /
- 聚合酶链反应
Abstract: Objective To investigate the consistency between Shengxiang (S) and Xinbo (X) real-time PCR methods in the quantification of HBV RNA. Methods In the prospective follow-up cohort of 108 chronic hepatitis B (CHB) patients established from July 2007 to August 2008, 20 patients with HBeAg seroconversion were selected, and 20 patients without seroconversion were selected by propensity score matching at a ratio of 1∶ 1. The two quantification methods from S and X companies were used, and a retrospective analysis was performed for HBV RNA in serum samples at baseline and weeks 12, 24, and 48. The paired t-test was used for comparison of normally distributed continuous data between groups, and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between groups; the chi-square test was used for comparison of categorical data. The Pearson correlation coefficient, intraclass correlation coefficient (ICC), and the Bland-Altman method were used to evaluate the consistency of the two quantification methods. Results A total of 132 serum samples were tested by S reagent, and 154 were tested by X reagent; the detection rate of HBV RNA was 100% by both reagents. A total of 131 serum samples were tested by both reagents, with 34 samples at baseline and 29, 35, and 33 samples, respectively, at weeks 12, 24, and 48 of follow-up; at these four time points, the HBV RNA quantification data detected by X reagent were significantly higher than those detected by S reagent (5.75±1.64/5.43±1.73/5.13±1.54/4.76±1.55 log₁₀ copies/mL vs 4.80±1.48/4.52±1.53/4.10±1.50/3.92± 1.43 log₁₀ copies/mL, t=8.348, t=5.341, Z=-5.086, Z=-4.762, all P < 0.001). The correlation analysis of the two methods showed a Pearson correlation coefficient of 0.915 (95% confidence interval [CI]: 0.836-0.957) and an ICC of 0.771(95%CI: -0.021 to 0.931) at baseline, a Pearson correlation coefficient of 0.849(95%CI: 0.701-0.927) and an ICC of 0.733(95%CI: 0.138-0.902) at week 12, a Pearson correlation coefficient of 0.951(95%CI: 0.905-0.975) and an ICC of 0.776(95%CI: -0.058 to 0.942) at week 24, and a Pearson correlation coefficient of 0.933(95%CI: 0.867-0.967) and an ICC of 0.804(95%CI: -0.014 to 0.944) at week 48 (all P < 0.05). The Bland-Altman analysis showed that the difference of 96.18%(126/131) samples tested by the two methods was within the mean difference±1.96 standard deviation. Conclusion HBV RNA quantification by X reagent is higher than that by S reagent, while the two real-time PCR quantification methods show a good consistency in CHB patients with HBeAg seroconversion and those without seroconversion.
- Hepatitis B, Chronic /
- RNA /
- Polymerase Chain Reaction

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图 1 两种检测方法在HBeAg转换和未转换患者中测得的HBV RNA定量动态变化

Figure 1. Dynamic changes of HBV RNA quantification measured by two assays in HBeAg seroconversion and non-seroconversion patients

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图 2 Bland-Altman分析

注：用X和S试剂在131份CHB患者临床血清中检测的HBV RNA水平，纵坐标为检测结果的两种试剂检测结果的差值，虚线之间的区域对应于平均差±1.96标准差。

Figure 2. Bland-Altman analysis

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表 1 HBeAg转换与未转换患者基线时一般临床特征

Table 1. Baseline clinical characteristics in HBeAg seroconversion and non-seroconversion patients

临床参数	HBeAg转换(n=20)	HBeAg未转换(n=20)	统计值	P值
年龄(岁)	38.00±11.60	39.00±8.89	t=-0.306	0.761
男性[例(%)]	15(75.00)	14(70.00)	χ²=0.125	0.723
ETV[例(%)]	10(50.00)	10(50.00)
BMI(kg/m²)	23.74±3.12	24.48±4.67	t=-0.593	0.557
HBV基因型(B/C/其他)¹⁾	0/7/2	2/5/1		0.620
ALT(U/L)	68.85(14.90~264.40)	73.40(12.70~317.30)	Z=-0.379	0.705
AST(U/L)	36.25(13.30~145.00)	53.35(12.90~108.80)	Z=-0.271	0.787
AST/ALT	0.82±0.35	0.72±0.26	t=0.974	0.336
TBil(μmol/L)	15.90(6.70~36.40)	14.95(8.80~27.20)	Z=-0.528	0.598
ALP(U/L)	86.90(46.90~242.70)	86.35(45.00~171.50)	Z=-0.393	0.694
HBsAg(log₁₀ IU/mL)	3.36(2.15~3.79)	3.80(-0.07~4.95)	Z=-1.878	0.060
HBV DNA(log₁₀ IU/mL)	6.02(2.03~9.28)	5.79(2.05~8.49)	Z=-0.611	0.545
PLT(×10⁹/L)	156.89±60.57	165.25±66.02	t=-0.411	0.683
注：1)17例取得基因型患者纳入分析，P值结果为C基因型与非C基因型比较结果。

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表 2 HBV RNA定量检测结果比较

Table 2. Comparison of HBV RNA quantification

随访时间	样本数	S试剂	X试剂	统计值	P值
基线(log₁₀拷贝/mL)	34	4.80±1.48	5.75±1.64	t=8.348	＜0.001
HBeAg转换	16	4.47±1.40	5.47±1.76	t=4.709	＜0.001
HBeAg未转换	18	5.10±1.52	6.00±1.55	t=8.376	＜0.001
12周(log₁₀拷贝/mL)	29	4.52±1.53	5.43±1.73	t=5.341	＜0.001
HBeAg转换	14	4.40±1.71	4.95±1.86	t=3.173	0.007
HBeAg未转换	15	4.63±1.40	5.87±1.54	t=4.695	＜0.001
24周(log₁₀拷贝/mL)	35	4.15(2.30~6.83)	4.72(2.30~7.97)	Z=-5.086	＜0.001
HBeAg转换	16	3.00(2.30~6.60)	4.36(3.08~6.94)	Z=-3.516	＜0.001
HBeAg未转换	19	4.46±1.59	5.51±1.75	t=8.974	＜0.001
48周(log₁₀拷贝/mL)	33	3.81(2.30~7.07)	4.62(2.30~7.82)	Z=-4.762	＜0.001
HBeAg转换	15	3.44±1.12	4.36±1.15	t=5.469	＜0.001
HBeAg未转换	18	4.31±1.57	5.09±1.78	t=6.865	＜0.001
总数(log₁₀拷贝/mL)	131	4.15(2.30~7.83)	5.07(2.30~9.12)	Z=-9.406	＜0.001
HBeAg转换	61	3.81(2.30~7.83)	4.59(2.30~9.12)	Z=-6.213	＜0.001
HBeAg未转换	70	4.67(2.30~7.80)	5.75(2.30~8.41)	Z=-7.084	＜0.001

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表 3 两种方法检测HBV RNA定量结果之间的相关性

Table 3. Correlation between HBV RNA quantification measured by two assays

随访时间	r值	95%CI	P值	ICC	95%CI	P值
基线	0.915	0.836~0.957	＜0.001	0.771	-0.021~0.931	＜0.001
HBeAg转换	0.879	0.678~0.957	＜0.001	0.720	0.001~0.918	＜0.001
HBeAg未转换	0.956	0.882~0.984	＜0.001	0.817	-0.052~0.958	＜0.001
12周	0.849	0.701~0.927	＜0.001	0.733	0.138~0.902	＜0.001
HBeAg转换	0.935	0.803~0.980	＜0.001	0.892	0.499~0.970	＜0.001
HBeAg未转换	0.762	0.410~0.917	0.001	0.567	-0.080~0.857	＜0.001
24周	0.951	0.905~0.975	＜0.001	0.776	-0.058~0.942	＜0.001
HBeAg转换	0.941	0.834~0.980	＜0.001	0.705	-0.067~0.928	＜0.001
HBeAg未转换	0.957	0.890~0.984	＜0.001	0.798	-0.057~0.953	＜0.001
48周	0.933	0.867~0.967	＜0.001	0.804	-0.014~0.944	＜0.001
HBeAg转换	0.837	0.568~0.944	＜0.001	0.636	-0.085~0.894	＜0.001
HBeAg未转换	0.967	0.913~0.988	＜0.001	0.869	0.009~0.969	＜0.001
总数	0.916	0.883~0.940	＜0.001	0.777	-0.003~0.926	＜0.001
HBeAg转换	0.899	0.835~0.938	＜0.001	0.763	0.020~0.920	＜0.001
HBeAg未转换	0.922	0.877~0.951	＜0.001	0.774	-0.031~0.930	＜0.001

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