PBMC结核抗体分泌试验和酶联免疫斑点试验对活动性结核病临床诊断价值的研究

发布时间：2019-06-20 11:09

【摘要】：目的：研究外周血单个核细胞（PBMC）分泌结核抗体试验和酶联免疫斑点试验（ELISPOT）对活动性结核病（TB）的临床诊断价值。方法：受试者共163例，包括活动性结核病患者104例（菌阳肺结核27例，菌阴肺结核61例，肺外结核16例）、结核分枝杆菌（MTB）潜伏感染者（LTBI）26例和健康对照（HC）33名。采用Ficoll密度梯度离心法分离受试者PBMCs，体外培养4d后收集培养上清液，用间接酶联免疫吸附试验（ELISA）和蛋白免疫印迹法（WB）检测PBMCs培养上清液中抗原特异性结核抗体；用ELISPOT方法检测受试者早期分泌抗原靶6KDa蛋白（ESAT-6）、ESAT-6/培养滤出液蛋白-10（CFP-10）融合抗原刺激后产生γ-干扰素的T细胞水平；比较分析三种方法的敏感度、特异度、阳性预测值及阴性预测值。结果： 1. ELISA法检测TB组特异性结核抗体的OD值高于LTBI组和HC组（p0.001）；特异性结核抗体区分TB和LTBI、HC的曲线下面积（AUC）分别为0.857、0.944，当cut-off值为0.42时，区分TB和LTBI的敏感度为77.9%，，特异度为80.8%，阳性预测值为94.2%，阴性预测值为47.7%；区分TB和HC的敏感度为77.9%，特异度为93.9%，阳性预测值为97.6%，阴性预测值为57.4%。 2. WB法检测活动性结核的敏感度为79.2%，特异度为100.0%，阳性预测值为100.0%，阴性预测值为40.7%（p0.001）。 3. ELISPOT法检测活动性结核的敏感度为82.7%，特异度为87.9%，阳性预测值为95.6%，阴性预测值为61.7%（p0.001）。 4. ELISA、WB和ELISPOT方法诊断活动性结核的敏感度、特异度、阳性预测值进行平行比较，差异均无统计学意义（p0.05）；阴性预测值进行平行比较，WB法的阴性预测值低于ELISA法和ELISPOT法（p0.05）；ELISA和ELISPOT法在菌阳组、菌阴组活动性肺结核的阳性检出率比较，差异均无统计学意义（p0.05）。 5. ELISA、WB、ELISOPT法检测16例肺外结核的阳性率分别为81.3%、56.3%、68.8%。结论： 1. PBMC结核抗体分泌试验的ELISA法，对活动性结核的诊断效能高，并可以区分活动性结核、结核分枝杆菌潜伏感染及健康人。 2. PBMC结核抗体分泌试验的免疫印迹法操作简便，诊断活动性结核的特异度高。 3.PBMC结核抗体分泌试验与ELISPOT法对活动性结核均具有较高的诊断价值，PBMC结核抗体分泌试验可以作为结核病实验室诊断的新方法。
[Abstract]:Objective: to study the clinical diagnostic value of tuberculosis antibody test and enzyme-linked immunosorbent assay (ELISPOT) secreted by peripheral blood mononuclear cells (PBMC) in active tuberculosis (TB). Methods: a total of 163 subjects, including 104 cases of active tuberculosis (27 cases of positive pulmonary tuberculosis, 61 cases of negative pulmonary tuberculosis, 16 cases of extrapulmonary tuberculosis), 26 cases of latent infection of Mycobacterium tuberculosis (MTB) and 33 cases of healthy control (HC). The culture medium of PBMCs, was isolated by Ficoll density gradient centrifugation for 4 days, and the antigen-specific tuberculosis antibody in the culture medium of PBMCs was detected by indirect enzyme-linked immunosorbent assay (ELISA) and Western immunoblotting (WB). ELISPOT method was used to detect the T cell level of interferon gamma induced by early secretion of antigen target 6KDa protein (ESAT-6) and ESAT-6/ culture filtrate protein 10 (CFP-10) fusion antigen, and the sensitivity, specificity, positive predictive value and negative predictive value of the three methods were compared and analyzed. Results: 1. The OD value of specific tuberculosis antibody in TB group was higher than that in LTBI group and HC group (p0.001), and the area (AUC) under the curve of specific tuberculosis antibody distinguishing TB and LTBI,HC was 0.857 and 0.944, respectively. when cut-off value was 0.42, the sensitivity, specificity, positive predictive value and negative predictive value of distinguishing TB and LTBI were 77.9%, 80.8%, 94.2% and 47.7%, respectively. when cut-off value was 0.42%, the sensitivity, specificity, positive predictive value and negative predictive value of TB and LTBI were 77.9%, 80.8%, 94.2% and 47.7%, respectively. The sensitivity, specificity, positive predictive value and negative predictive value of TB and HC were 77.9%, 93.9%, 97.6% and 57.4%, respectively. 2. The sensitivity, specificity, positive predictive value and negative predictive value of WB for the detection of active tuberculosis were 79.2%, 100.0%, 100.0% and 40.7% (p0.001) respectively. 3. The sensitivity, specificity, positive predictive value and negative predictive value of ELISPOT in detecting active tuberculosis were 82.7%, 87.9%, 95.6% and 61.7% (p0.001) respectively. 4. There was no significant difference in the sensitivity, specificity and positive predictive value of ELISA,WB and ELISPOT in the diagnosis of active tuberculosis (p0.05), but the negative predictive value of WB was lower than that of Elisa and ELISPOT (p0.05); ELISA and ELISPOT). There was no significant difference in the positive rate of active pulmonary tuberculosis between the negative group and the negative group (p0.05). 5. The positive rates of 16 cases of extrapulmonary tuberculosis detected by ELISA,WB,ELISOPT were 81.3%, 56.3% and 68.8%, respectively. Conclusion: 1. ELISA method of PBMC tuberculosis antibody secretion test is effective in the diagnosis of active tuberculosis, and can distinguish active tuberculosis, latent infection of Mycobacterium tuberculosis and healthy people. 2. The immunoblotting method of PBMC tuberculosis antibody secretion test is simple and has high specificity in the diagnosis of active tuberculosis. 3.PBMC tuberculosis antibody secretion test and ELISPOT method have high diagnostic value for active tuberculosis. PBMC tuberculosis antibody secretion test can be used as a new method for laboratory diagnosis of tuberculosis.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R52

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