酶联免疫斑点法在丙型肝炎合并潜伏结核感染诊断中的应用性研究
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摘要
丙型肝炎病毒（Hepatitis C Virus，HCV）是丙型肝炎的主要致病原，全世界至少有1亿7千万患者。人在感染HCV后，肝脏炎症逐渐进展，各种症状逐渐显现，肝脏功能改变，从而发展为肝硬化甚至肝癌。事实上，临床上各个年龄段的丙型肝炎病人都存在，而且常常并发其他疾病，其中不乏合并结核感染病人。
干扰素是治疗丙型肝炎的常用抗病毒药物，干扰素的抗病毒增殖作用可抑制蛋白质合成，导致机体白细胞减少；而结核病患者蛋白质消耗较多，本身即可出现白细胞减少的表现。干扰素的流感样反应可加重结核病患者的发热症状，加速结核病病程。因此新发HCV病人，特别是HCV-RNA阳性的病人需要干扰素抗病毒治疗前，把筛查结核病作为常规检查是非常必要的。
结核菌素皮肤试验作为最常用LTBI的检测方法，在临床广泛使用的同时，人们也逐渐认识到它的局限性，该诊断方法的特异性较低。结核菌素皮肤试验中使用结核分枝杆菌（Mycobacterium Tuberculosis，MTB）纯蛋白衍生物（Purified ProteinDerivatives，PPD），是在结核分枝杆菌中粗提的抗原混合物，这一混合物中有200种以上抗原成分与卡介苗（Bacillus Chalmette-Guerin Vaccine，BCG）和非结核分枝杆菌的抗原成分相同，容易发生交叉反应。我国是结核病高流行国家，BCG普遍接种，非结核分枝杆菌感染率也较高，使用结核菌素皮肤试验方法时出现了较高的假阳性率。另外由于机体免疫力低下、应用免疫抑制剂、合并HIV感染等，也常常引起结果的假阴性，因此该方法诊断特异性较低。
酶联免疫斑点（Enzyme Linked-Immunospot Assay，ELISPOT）技术是利用酶联免疫吸附实验（Enzyme Linked-Immunosorbent Assay，ELISA）原理建立的，当T淋巴细胞受到结核分枝杆菌抗原刺激活化成效应T淋巴细胞，分泌IFN-γ（Interferon-γ）。利用ELISPOT方法检测活化的T细胞分泌的IFN-γ，通过对形成斑点的计数可推测体内是否存在对结核分枝杆菌反应的效应T淋巴细胞，对结核分枝杆菌感染进行辅助诊断。
设计思路：
利用ELISPOT方法检测活化的T细胞分泌的IFN-γ，通过对形成斑点的计数可推测体内是否存在对结核分枝杆菌反应的效应T淋巴细胞，对结核分枝杆菌感染进行诊断。
创新性:
应用ELISPOT法对丙型肝炎合并潜伏结核感染诊断的敏感性、特异性进行研究；应用国产ELISPOT-TB诊断试剂盒对丙型肝炎合并潜伏结核感染、活动性结核进行鉴别诊断；应用国产ELISPOT-TB诊断试剂盒对丙型肝炎合并潜伏结核感染、活动性结核临床化疗疗效进行评价。
意义：
ELISPOT法可以早期诊断丙型肝炎合并潜伏结核感染，为丙型肝炎患者的临床干扰素治疗方案的选择提供参考价值。
主要内容与结果：
内容：
1、ESAT-6、CFP-10蛋白酶联免疫斑点法活性检测的效果评价：比较进口T-SPOT-TB试剂盒、TST试验、国产ELISPOT-TB试剂盒诊断结核菌感染的敏感度、特异性；摸索国产ELISPOT-TB试剂盒的最佳检测条件；
2、国产ELISPOT-TB试剂盒对丙型肝炎病人中潜伏结核感染诊断作用的研究：利用国产ELISPOT-TB试剂盒检测丙型肝炎合并潜伏结核感染；比较TST实验、国产ELISPOT-TB试剂盒诊断丙型肝炎合并潜伏结核感染的敏感度、特异性；比较国产ELISPOT-TB试剂盒检测丙型肝炎合并活动性结核病和潜伏结核感染的效果；
3、酶联免疫斑点法在丙型肝炎合并结核感染中的临床应用：利用国产ELISPOT-TB试剂盒对丙型肝炎合并潜伏结核感染和活动性结核病进行鉴别诊断；利用国产ELISPOT-TB试剂盒对丙型肝炎合并潜伏结核感染和活动性结核病化疗疗效进行评估；
4、丙型肝炎合并潜伏结核感染和活动性结核患者全血中相关细胞因子变化的研究：利用Real time-PCR及ELISA法检测丙型肝炎合并潜伏结核感染、丙型肝炎合并活动性结核病人血液中FOXP3、IL-2、IFN-γ细胞因子表达水平的变化。
结果：
1、ELISPOT-TB试剂盒可以有效地诊断丙型肝炎合并潜伏结核感染；
2、ELISPOT-TB试剂盒有效地对丙型肝炎合并潜伏结核感染、活动性结核病进行鉴别诊断；
3、ELISPOT-TB试剂盒可以有效地监测丙型肝炎合并潜伏结核感染的化疗疗效。
4、丙型肝炎病人中潜伏结核感染进展为活动性结核可能与血中FOXP3、IL-2、IFN-γ含量增加有关。
Hepatitis C virus (HCV) is a major public health issue, and approximately200million people are infected with HCV worldwide. Surveys have found that the patientswith chronic hepatitis C have a high incidence of latent tuberculosis infection (LTBI).Interferon is the immune enhancers with broad-spectrum anti-viral effect, which caninhibit viral replication and block the spread of hepatitis viruses. Interferon canstimulate the body's own anti-viral effect of interferon system, but also can improve thebody's natural anti-viral ability to change the body's immune response to hepatitis virusinfection, to reduce or block viral pathological immune hepatitis injury. Interferontreatment of hepatitis C was considered the only effective drug, but not all patients areapplicable to treat with interferon, when a patient sufferred from TB and LTBI, theinterferon could speed up TB's condition. It is necessary to determine whether thepatients with chronic hepatitis C suffered from latent tuberculosis infection at the timeof hepatitis detection or whether the LTBI was directly related to the treatment ofhepatitis C. Interferon is an effective treatment for hepatitis C. However, interferon mayconvert latent tuberculosis into active tuberculosis if hepatitis C patients also sufferfrom LTBI. Interferon therapies may accelerate the progression of tuberculosis.Therefore, it is very important to diagnose latent tuberculosis in hepatitis C patients.
The tuberculin skin test (TST) is the predominant means of diagnosing LTBI;however, the specificity of this test is limited because the purified protein derivativeused as the antigen broadly cross-reacts with antigens derived from a number ofmycobacterial species, including the vaccine strain Bacillus Chalmette-Guerin (BCG)and other non-tuberculosis mycobacteria (NTM). The TST has poor specificity and themisdiagnosis rates are very high in diagnosing LTBI. Therefore, there is an urgent need for a more sensitive and specific immunological tool for the diagnosis of latenttuberculosis infections.
An IFN-γ ELISPOT assay is based on detecting the interferon-γ (IFN-γ) released byactivated T lymphocytes (T cells) that are stimulated by a number of secretary proteinsencoded by the RD1locus of the Mycobacterium tuberculosis complex but are absentfrom the majority of environmental isolates, including BCG strains. The early secretaryantigenic target6(ESAT-6) and culture filtrate protein10(CFP-10) proteins wereinitially tested and shown to elicit a strong T cell response in subjects with active TB orLTBI. Therefore, in this study, we evaluated the clinical value of this ELISPOT assayfor the diagnosis of LTBI in hepatitis C patients.
Innovation:
We used ELISPOT assay to study the diagnostic sensitivity and specificity ofhepatitis C combined with tuberculosis latent infection; the ELISPOT assay was used todo the differential diagnosis between hepatitis C combined with LTBI and TB; theELISPOT assay was used to evaluate the curative effects of hepatitis C combined withLTBI and TB.
Meaning:
The ELISPOT assay could provide useful support in diagnosing LTBI in hepatitis Cpatients and may provide guidance regarding the treatment of LTBI and hepatitis Cco-infection.
The main contents and results:
Contents:
1. The evaluation of the ELISPOT assay: the abroad T-SPOT-TB kit, TST, the domesticELISPOT-TB kit were used to do the diagnostic sensitivity and specificity of hepatitis Ccombined with TB; the multiple experimental conditions of the domestic ELISPOT-TBkit.
2. Diagnostic value of an ELISPOT assay to detect latent tuberculosis infection inhepatitis C patients: The domestic ELISPOT-TB kit were used to diagnose LTBI inhepatitis C patients; TST and the domestic ELISPOT-TB kit were used to do thediagnostic sensitivity and specificity of LTBI in hepatitis C patients.
3. The clinical applications of ELISPOT assay in LTBI in hepatitis C patients: TheELISPOT assay was used to do the differential diagnosis between hepatitis C combinedwith LTBI and TB; the ELISPOT assay was used to evaluate the curative effects ofLTBI and TB in hepatitis C patients.
4. The study on the changes of CK in hepatitis C combined with LTBI and TB: Realtime-PCR and ELISA were used to detect the changes of FOXP3、IL-2、IFN-γ inhepatitis C combined with LTBI and TB.
Results:
1. The ELISPOT assay could diagnose the LTBI in hepatitis C patients.
2. The ELISPOT assay could do the differential diagnosis between hepatitis C combinedwith LTBI and TB.
3. The ELISPOT assay could evaluate the curative effects of LTBI and TB in hepatitis Cpatients.
4. The reason why LTBI can progress to TB could be related with the changes ofFOXP3、IL-2、IFN-γ in hepatitis C.
干扰素是治疗丙型肝炎的常用抗病毒药物，干扰素的抗病毒增殖作用可抑制蛋白质合成，导致机体白细胞减少；而结核病患者蛋白质消耗较多，本身即可出现白细胞减少的表现。干扰素的流感样反应可加重结核病患者的发热症状，加速结核病病程。因此新发HCV病人，特别是HCV-RNA阳性的病人需要干扰素抗病毒治疗前，把筛查结核病作为常规检查是非常必要的。
结核菌素皮肤试验作为最常用LTBI的检测方法，在临床广泛使用的同时，人们也逐渐认识到它的局限性，该诊断方法的特异性较低。结核菌素皮肤试验中使用结核分枝杆菌（Mycobacterium Tuberculosis，MTB）纯蛋白衍生物（Purified ProteinDerivatives，PPD），是在结核分枝杆菌中粗提的抗原混合物，这一混合物中有200种以上抗原成分与卡介苗（Bacillus Chalmette-Guerin Vaccine，BCG）和非结核分枝杆菌的抗原成分相同，容易发生交叉反应。我国是结核病高流行国家，BCG普遍接种，非结核分枝杆菌感染率也较高，使用结核菌素皮肤试验方法时出现了较高的假阳性率。另外由于机体免疫力低下、应用免疫抑制剂、合并HIV感染等，也常常引起结果的假阴性，因此该方法诊断特异性较低。
酶联免疫斑点（Enzyme Linked-Immunospot Assay，ELISPOT）技术是利用酶联免疫吸附实验（Enzyme Linked-Immunosorbent Assay，ELISA）原理建立的，当T淋巴细胞受到结核分枝杆菌抗原刺激活化成效应T淋巴细胞，分泌IFN-γ（Interferon-γ）。利用ELISPOT方法检测活化的T细胞分泌的IFN-γ，通过对形成斑点的计数可推测体内是否存在对结核分枝杆菌反应的效应T淋巴细胞，对结核分枝杆菌感染进行辅助诊断。
设计思路：
利用ELISPOT方法检测活化的T细胞分泌的IFN-γ，通过对形成斑点的计数可推测体内是否存在对结核分枝杆菌反应的效应T淋巴细胞，对结核分枝杆菌感染进行诊断。
创新性:
应用ELISPOT法对丙型肝炎合并潜伏结核感染诊断的敏感性、特异性进行研究；应用国产ELISPOT-TB诊断试剂盒对丙型肝炎合并潜伏结核感染、活动性结核进行鉴别诊断；应用国产ELISPOT-TB诊断试剂盒对丙型肝炎合并潜伏结核感染、活动性结核临床化疗疗效进行评价。
意义：
ELISPOT法可以早期诊断丙型肝炎合并潜伏结核感染，为丙型肝炎患者的临床干扰素治疗方案的选择提供参考价值。
主要内容与结果：
内容：
1、ESAT-6、CFP-10蛋白酶联免疫斑点法活性检测的效果评价：比较进口T-SPOT-TB试剂盒、TST试验、国产ELISPOT-TB试剂盒诊断结核菌感染的敏感度、特异性；摸索国产ELISPOT-TB试剂盒的最佳检测条件；
2、国产ELISPOT-TB试剂盒对丙型肝炎病人中潜伏结核感染诊断作用的研究：利用国产ELISPOT-TB试剂盒检测丙型肝炎合并潜伏结核感染；比较TST实验、国产ELISPOT-TB试剂盒诊断丙型肝炎合并潜伏结核感染的敏感度、特异性；比较国产ELISPOT-TB试剂盒检测丙型肝炎合并活动性结核病和潜伏结核感染的效果；
3、酶联免疫斑点法在丙型肝炎合并结核感染中的临床应用：利用国产ELISPOT-TB试剂盒对丙型肝炎合并潜伏结核感染和活动性结核病进行鉴别诊断；利用国产ELISPOT-TB试剂盒对丙型肝炎合并潜伏结核感染和活动性结核病化疗疗效进行评估；
4、丙型肝炎合并潜伏结核感染和活动性结核患者全血中相关细胞因子变化的研究：利用Real time-PCR及ELISA法检测丙型肝炎合并潜伏结核感染、丙型肝炎合并活动性结核病人血液中FOXP3、IL-2、IFN-γ细胞因子表达水平的变化。
结果：
1、ELISPOT-TB试剂盒可以有效地诊断丙型肝炎合并潜伏结核感染；
2、ELISPOT-TB试剂盒有效地对丙型肝炎合并潜伏结核感染、活动性结核病进行鉴别诊断；
3、ELISPOT-TB试剂盒可以有效地监测丙型肝炎合并潜伏结核感染的化疗疗效。
4、丙型肝炎病人中潜伏结核感染进展为活动性结核可能与血中FOXP3、IL-2、IFN-γ含量增加有关。
Hepatitis C virus (HCV) is a major public health issue, and approximately200million people are infected with HCV worldwide. Surveys have found that the patientswith chronic hepatitis C have a high incidence of latent tuberculosis infection (LTBI).Interferon is the immune enhancers with broad-spectrum anti-viral effect, which caninhibit viral replication and block the spread of hepatitis viruses. Interferon canstimulate the body's own anti-viral effect of interferon system, but also can improve thebody's natural anti-viral ability to change the body's immune response to hepatitis virusinfection, to reduce or block viral pathological immune hepatitis injury. Interferontreatment of hepatitis C was considered the only effective drug, but not all patients areapplicable to treat with interferon, when a patient sufferred from TB and LTBI, theinterferon could speed up TB's condition. It is necessary to determine whether thepatients with chronic hepatitis C suffered from latent tuberculosis infection at the timeof hepatitis detection or whether the LTBI was directly related to the treatment ofhepatitis C. Interferon is an effective treatment for hepatitis C. However, interferon mayconvert latent tuberculosis into active tuberculosis if hepatitis C patients also sufferfrom LTBI. Interferon therapies may accelerate the progression of tuberculosis.Therefore, it is very important to diagnose latent tuberculosis in hepatitis C patients.
The tuberculin skin test (TST) is the predominant means of diagnosing LTBI;however, the specificity of this test is limited because the purified protein derivativeused as the antigen broadly cross-reacts with antigens derived from a number ofmycobacterial species, including the vaccine strain Bacillus Chalmette-Guerin (BCG)and other non-tuberculosis mycobacteria (NTM). The TST has poor specificity and themisdiagnosis rates are very high in diagnosing LTBI. Therefore, there is an urgent need for a more sensitive and specific immunological tool for the diagnosis of latenttuberculosis infections.
An IFN-γ ELISPOT assay is based on detecting the interferon-γ (IFN-γ) released byactivated T lymphocytes (T cells) that are stimulated by a number of secretary proteinsencoded by the RD1locus of the Mycobacterium tuberculosis complex but are absentfrom the majority of environmental isolates, including BCG strains. The early secretaryantigenic target6(ESAT-6) and culture filtrate protein10(CFP-10) proteins wereinitially tested and shown to elicit a strong T cell response in subjects with active TB orLTBI. Therefore, in this study, we evaluated the clinical value of this ELISPOT assayfor the diagnosis of LTBI in hepatitis C patients.
Innovation:
We used ELISPOT assay to study the diagnostic sensitivity and specificity ofhepatitis C combined with tuberculosis latent infection; the ELISPOT assay was used todo the differential diagnosis between hepatitis C combined with LTBI and TB; theELISPOT assay was used to evaluate the curative effects of hepatitis C combined withLTBI and TB.
Meaning:
The ELISPOT assay could provide useful support in diagnosing LTBI in hepatitis Cpatients and may provide guidance regarding the treatment of LTBI and hepatitis Cco-infection.
The main contents and results:
Contents:
1. The evaluation of the ELISPOT assay: the abroad T-SPOT-TB kit, TST, the domesticELISPOT-TB kit were used to do the diagnostic sensitivity and specificity of hepatitis Ccombined with TB; the multiple experimental conditions of the domestic ELISPOT-TBkit.
2. Diagnostic value of an ELISPOT assay to detect latent tuberculosis infection inhepatitis C patients: The domestic ELISPOT-TB kit were used to diagnose LTBI inhepatitis C patients; TST and the domestic ELISPOT-TB kit were used to do thediagnostic sensitivity and specificity of LTBI in hepatitis C patients.
3. The clinical applications of ELISPOT assay in LTBI in hepatitis C patients: TheELISPOT assay was used to do the differential diagnosis between hepatitis C combinedwith LTBI and TB; the ELISPOT assay was used to evaluate the curative effects ofLTBI and TB in hepatitis C patients.
4. The study on the changes of CK in hepatitis C combined with LTBI and TB: Realtime-PCR and ELISA were used to detect the changes of FOXP3、IL-2、IFN-γ inhepatitis C combined with LTBI and TB.
Results:
1. The ELISPOT assay could diagnose the LTBI in hepatitis C patients.
2. The ELISPOT assay could do the differential diagnosis between hepatitis C combinedwith LTBI and TB.
3. The ELISPOT assay could evaluate the curative effects of LTBI and TB in hepatitis Cpatients.
4. The reason why LTBI can progress to TB could be related with the changes ofFOXP3、IL-2、IFN-γ in hepatitis C.
引文
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