丙型病毒性肝炎临床诊断核酸检测策略评估

发布时间：2018-06-03 02:19

本文选题：丙型肝炎病毒 + 检测策略　；参考：《中国疾病预防控制中心》2014年硕士论文

【摘要】：研究背景丙型肝炎病毒(Hepatitis C virus, HCV)主要通过血液传播,引起的丙型病毒性肝炎(以下简称“丙型肝炎”)呈世界范围流行。由于丙型肝炎发病隐匿、症状不典型,因此其临床诊断主要依靠实验室的检测结果。HCV特异性抗体是丙型肝炎临床诊断中实验室检测的主要指标,针对HCV抗体的检测主要分为筛查试验和补充试验两部分。抗体的筛查试验,筛查试验一般采用酶联免疫吸附法(Enzyme Linked Immunosorbent Assay, ELISA),对于筛查实验结果呈阳性反应的样本采用特异性更好的补充试验对结果进行确证,补充实验通常采用重组免疫印迹试验(Recombinant Immunoblot Assay,RIBA),筛查实验和补充实验配合使用,形成一系列的检测策略,从而为临床诊断提供可靠的结果。我国各级临床实验室虽然已经广泛开展了HCV相关检测,但是仍然没有明确的检测策略以及对应的结果解释。对此,卫生与计划生育委员会委托中国疾控中心编写了《丙型肝炎病毒实验室检测技术规范》(试行)(以下简称《规范》),其中对HCV临床诊断的检测策略做了详细的要求,提出了HCV临床诊断抗体筛查策略、HCV临床诊断核酸检测策略等。HCV临床诊断核酸检测策略是对HCV抗体筛查阳性反应的样本采用核酸扩增检(Nucleric acid Amplification Test, NAT)和RIBA组合使用的检测方法。任何一种疾病检测策略在应用之前,都必须经过严密的评估,确定其可用性、准确性、可靠性后方可推广使用。我国新颁发的《规范》中规定的临床诊断实验室检测策略己完成了抗体检测策略的评估,本研究主要围绕核酸检测策略部分进行评估。研究目的 1.对后续将用于HCV临床i诊断核酸检测策略中的HCV核酸检测试剂的性能进行评估。 2.在国家艾滋病参比实验室进行HCV临床诊断核酸检测策略的实验室小样本范围评估。 3.在多个现场试点进行HCV临床诊断核酸检测策略应用的评估,进行证明性研究,提供一种可推广到全国多点使用的、合理的、可靠的检测策略。 4.为国家级策略评估三阶段中第三阶段的HCV临床诊断检测策略实施后的评估奠定基础。为进一步修订《规范》中HCV检测策略提供依据,使我国丙型肝炎的临床诊断实验室检测流程更规范。研究方法 1.方法学评价部分：建立HCV核酸检测基础血清盘、HCV分亚型血清盘、HCV RNA线性血清盘、HCV RNA灵敏度血清盘、参比室干扰样本血清盘、HCV核酸精密度血清盘。用HCV核酸检测试剂对己建立的血清盘进行盲法检测。对检测结果进行计算求得待评估试剂的阳性符合率、阴性符合率、亚型检出能力、线性、分析灵敏度、分析特异性、精密度等指标以评价检测方法的总体性能。 2.临床诊断核酸检测策略评估部分的研究包括两个阶段：第一阶段,建立一套本底信息明确的血清盘用于HCV临床诊断核酸检测策略在国家艾滋病参比实验室范围内的评估。第二阶段,选择北京、天津两地作为试点,对所选的试点现场进行丙型肝炎临床诊断核酸检测策略的大样本评估,参与评估的样本分别来自2013年HCV监测哨点和HIV监测哨点的吸毒人群以及北京市肾透析丙型肝炎流行病学调查的肾透析人群。两个评估阶段均与抗体检测补充策略即RIBA单独作为筛查阳性样本补充实验的检测策略相比较,计算检测策略的符合率等指标,评估检测策略的应用效果,同时对核酸检测策略在不同人群、不同地点、不同样本量检测的总体性能进行比较。结果 1. HCV RNA检测试剂的阳性符合率为49/50(98%)；阴性符合率为50/50(100%),总符合率为99/100(99%)。对各种可能对临床检测产生干扰的样本的分析特异性为100%。对HCV1到6个型别共9种亚型均具有检出能力。对HCV RNA浓度分别为低、中、高(102、104、106IU/mL)三份样本的检测中,除低浓度样本外对中、高浓度样本的天内精密度均小于6%,天间精密度分别为9.28%、5.03%、1.53%,总精密度分别为11.55%、4.45%、3.08%。磁珠提取HCV核酸检测试剂与Roche COBAS AmpliPrep/COBAS TaqMan HCV Test定量结果间的线性相关系数r值为0.9335。 2.HCV临床诊断核酸检测策略在参比室小样本及现场大量样本进行应用的检测敏感性、特异性、阳性预测值、阴性预测值均能达到100%。HCV临床诊断核酸检测策略的整体检测性能在吸毒人群和肾透析人群中无统计学差异(P0.05)。策略的整体检测性能在应用于参比实验室的小样本与试点现场大样本之间有强一致性,两者之间无统计学差异。结论 1.HCV核酸检测试剂有较高的阳性符合率和阴性符合率,且精密度、灵敏度都较高,对各亚型均由检出能力,适合用于HCV临床诊断核酸检测策略的评估。 2.我国《丙型肝炎病毒实验室检测技术规范》(试行)中的临床诊断核酸检测策略在参比实验室的小范围内及多个试点现场的应用效果均较好,对不同HCV感染率人群的检测效力差异不明显,具有普遍适用性,可有效降低检测的假阳性率,并显著降低检测成本。
[Abstract]:Research background
Hepatitis C virus (Hepatitis C virus, HCV) is mainly transmitted through blood, causing hepatitis C virus hepatitis (hereinafter referred to as "hepatitis C") in the world. Due to HCV occult, symptoms are not typical, so its clinical diagnosis mainly relies on laboratory test results of.HCV specific antibody is the clinical diagnosis of hepatitis C The main index of detection in the laboratory of HCV is divided into two parts: screening test and supplementary test. Screening test of antibody, screening test is usually used in Enzyme Linked Immunosorbent Assay, ELISA, and it is better to supplement the samples with positive reaction in screening experiment. The results were confirmed by the experiment. The supplementary experiment was usually used in combination with the recombinant immunoblotting test (Recombinant Immunoblot Assay, RIBA), the screening experiment and the supplemental experiment, and a series of detection strategies were formed to provide reliable results for clinical diagnosis.
Although the clinical laboratory at all levels in our country has widely carried out HCV related tests, there is still no clear detection strategy and corresponding interpretation. In this regard, the health and Family Planning Commission has commissioned the China CDC to compile a "hepatitis C virus laboratory test specification" (hereinafter referred to as < standard >), of which HCV The detection strategy of clinical diagnosis has been required in detail. The HCV clinical diagnostic antibody screening strategy, the HCV clinical diagnostic nucleic acid detection strategy, and.HCV clinical diagnostic nucleic acid detection strategy are the detection methods used for the combination of nucleic acid amplification test (Nucleric acid Amplification Test, NAT) and RIBA for the positive reaction of HCV antibody screening. Before application, any kind of disease detection strategy must be carefully evaluated to determine its availability, accuracy and reliability. The clinical diagnosis laboratory testing strategy stipulated in the newly issued < standard > has completed the evaluation of antibody detection strategy. This research focuses on the part of nucleic acid detection strategies. Assessment.
research objective
1. evaluate the performance of the HCV nucleic acid reagents that will be used in the clinical I diagnostic nucleic acid detection strategy of HCV.
2. in the National AIDS reference laboratory, a small sample size assessment of HCV clinical diagnostic nucleic acid detection strategy was conducted.
3. the evaluation of the application of the HCV clinical diagnostic nucleic acid detection strategy was conducted in several field trials, and a demonstration study was carried out to provide a reasonable and reliable detection strategy that could be extended to the national multipoint use.
4. lay the foundation for the assessment of the implementation of the third stage HCV clinical diagnosis strategy in the three stage of the national strategy assessment, and to provide a basis for further revising the HCV detection strategy in the < standard > to make the laboratory testing flow of hepatitis C in China more standardized.
research method
The 1. methodology evaluation part: establish HCV nucleic acid test basal blood disks, HCV subtype blood disks, HCV RNA linear sera disc, HCV RNA sensitivity sera disc, reference room interfering sample sera disc, HCV nucleic acid precision blood disks. The serum disk entry blind method was detected by HCV nucleic acid detection reagent. The test results were calculated and evaluated. The positive coincidence rate, negative coincidence rate, subtype detection ability, linearity, analysis sensitivity, specificity and precision were used to evaluate the overall performance of the method.
2. the study of the 2. clinical diagnostic nucleic acid detection strategy includes two stages: the first stage, the establishment of a set of background information clear serum plates for the HCV clinical diagnostic nucleic acid detection strategy in the National AIDS reference laboratory assessment. The second stage, the choice of Beijing, Tianjin as a pilot, the selected pilot site into the site. A large sample assessment of nucleic acid detection strategies for the clinical diagnosis of hepatitis C was conducted, and the samples participated in the assessment were from the drug users in the HCV sentinel and HIV sentinel points in 2013 and the renal dialysis population in the epidemiological investigation of HCV in Beijing. The two assessment stages were all screened by the anti physical examination supplementation strategy, RIBA alone. The test strategy of positive samples was compared, the coincidence rate of the detection strategy was calculated, and the application effect of the detection strategy was evaluated. At the same time, the overall performance of the detection strategy of nucleic acid detection strategy in different population, different locations and different sample quantities was compared.
Result
The positive coincidence rate of the 1. HCV RNA detection reagents was 49/50 (98%); the negative coincidence rate was 50/50 (100%) and the total coincidence rate was 99/100 (99%). The specificity of the analytical specificity for various possible samples that could interfere with clinical detection was detected by 100%. on 9 subtypes from HCV1 to 6 types. The RNA concentration of HCV was low, medium and high (102104106IU/mL), respectively. In the detection of three samples, in addition to the low concentration samples, the precision of the high concentration samples was less than 6%, the day precision was 9.28%, 5.03%, 1.53%, respectively, the total precision was 11.55%, 4.45%, 3.08%. magnetic beads extracted HCV nucleic acid detection reagent and the linear correlation coefficient r value between Roche COBAS AmpliPrep/ COBAS TaqMan HCV Test quantitative results. For 0.9335.
The detection sensitivity, specificity, positive predictive value and negative predictive value of the 2.HCV clinical diagnostic nucleic acid detection strategy used in the small sample of the reference room and the large number of samples in the field can reach the overall detection performance of the 100%.HCV clinical diagnostic nucleic acid detection strategy. There is no statistical difference between the drug addicts and the renal dialysis population (P0.05). There was no statistical difference in the detection performance between the small samples used in the reference laboratory and the large sample sites at the pilot site.
conclusion
The 1.HCV nucleic acid detection reagent has high positive coincidence rate and negative coincidence rate, and the precision is high, and the sensitivity is high. All the subtypes are detected. It is suitable for the evaluation of nucleic acid detection strategy in the clinical diagnosis of HCV.
2. the clinical diagnostic nucleic acid detection strategy in the laboratory test of hepatitis C virus (HCV) in China has better application effect in the small range of the reference laboratory and in several pilot sites. The difference of detection effectiveness for people with different HCV infection rates is not obvious, and it is universally applicable, which can effectively reduce the false positive rate of detection. Significantly reduce the cost of detection.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.63

【参考文献】