检测外周血特异性结核分枝杆菌抗体对活动性结核病的诊断价值研究

Study on diagnostic value for active tuberculosis by detecting Mycobacterium tuberculosis-specific antibodies in the peripheral blood

目的研究外周血浆细胞分泌特异性结核分枝杆菌抗体对活动性结核病的诊断价值。方法纳入2013年4月至7月期间深圳市第三人民医院肺科门诊活动性结核病患者；健康对照组人员来自体检科门诊志愿者，分为3个组：活动性结核病组（104例）、结核分枝杆菌（Mtb）潜伏感染组（26例）和健康对照组（33名）。分离其外周血单个核细胞（PBMCs）。体外培养4d后收集培养上清液，用ELISA法和蛋白免疫印迹法，ELISA和蛋白免疫印迹法检测外周血浆细胞分泌的特异性结核分枝杆菌抗体；检测各组收集的PBMCs培养上清液中的特异性结核分枝杆菌抗体，比较各组间的差异。所有数据均使用GraphPadPrism5．0进行统计学分析，用受试者工作特征（ROC）曲线评价ELISA法检测的诊断价值，得出敏感度、特异度；多组间的比较采用Kruskal—Wallis检验；两组间比较采用Dunnmultiplecomparison检验；蛋白免疫印迹法用四格表的卡方检验；以P〈0．05为差异有统计学意义。结果ELISA法检测活动性结核病组特异性结核分枝杆菌抗体的A450nm值（0．593±0．206）高于Mtb潜伏感染组（0．342±0．152）和健康对照组（0．246±0．121），差异有统计学意义（H=77．27，P〈0．001）。特异性结核分枝杆菌抗体区分活动性结核病患者和Mtb潜伏感染者、健康对照者曲线下面积（AUC）分别为0．857、0．944，当诊断界限值为0．42时，其区分活动性结核病和Mtb潜伏感染的敏感度为77．9％（81／104），特异度为80．8％（21／26）；区分活动性结核病和健康人的敏感度为77．9％（81／104），特异度为93．9％（31／33）。蛋白免疫印迹法检测活动性结核病和健康人的敏感度、特异度和准确性分别为79．2％（61／77）、100．0％（11／11）、81．8％（72／88）（x2=24．8，P〈0．001）。结论ELISA法和蛋白免疫印迹法检测外周血浆细胞分泌特异性结核分枝杆菌抗体诊断活动性结核病特异度高，可以作为临床结核病实验诊断的辅助方法。

Objective To evaluate the diagnostic value for active tuberculosis （TB） by detecting antibodies secreted from Mycobacterium tuberculosis-specific plasma ceils in peripheral blood. Methods The study period was from April 2013 to July 2013, and the study sample included 3 groups of subjects： 104 patients with active TB; 26 cases with latent TB infection （LTBI） ; and 33 healthy volunteers as a control group. The active TB patients were enrolled from the Outpatient Department of Shenzhen 3rd People＇s Hospital while the LTBI and healthy volunteers were enrolled from Health Examination Clinic of Shenzhen 3rd People＇s Hospital. Peripheral blood mononuclear cells （PBMCs） from all study subjects were isolated and cultured in vitro for 4 days, and then the supernatants of each group were collected. Mycobacterium tuberculosis specific antibodies in lymphocyte supernatant were tested by ELISA and Western blot and the differences among the three groups were analyzed. GraphPad Prism 5.0 was used for data analysis. The diagnostic value of EI.ISA was evaluated by receiver operating characteristic （ROC） curve. The Kruskal-Wallis test was used for comparison among multi-groups and the Dunn muhiple comparison test was used for comparison between two groups. The difference was considered to be statistically significance if the P-value was 〈0.05. Results The A450nm value of Myeobacterium tuberculosis-specific antibodies measured by ELISA was higherin the group of patients with active TB （0. 593±0. 206） than those in the group of cases with I.TBI （0. 342±0. 152） and in the group of healthy persons （0. 246±0. 121） respectively, the differences had statistically significance （H=77.27, P〈0. 001）. Areas under the curve （AUC） of Mycobacterium tuberculosis-specific antibodies which used for distinguish active rib and LTBI, active TB and healthy person were 0. 857 and 0. 944 respectively; when the value of 0.42 was used as a diagnostic threshold, the sensitivity and specificity for districting active TB and LTBI were 77.9% （81/104） and 80. 80% （21/26） respectively, and sensitivity and specificity for distinguishing active TB and health people were 77.9%（81/104） and 93.9% （31/33） respectively. The sensitivity, specificity, and accuracy of Western blot test in detecting active TB patients and healthy person were respectively 79.2 % （61/77）, 100.0% （11/11） and 81.8% （72/88） （x2 =24.8, P〈0. 001）. Conclusion The specificity of both EI.ISA and Western blot tests are high in detecting Mycobacterium tuberculosis specific antibodies secreted from peripheral blood plasma cells, so they can be used for diagnosis of active TB as new methods in clinical laboratory.