结核分枝杆菌感染T细胞斑点试验在疑诊肺结核患者中的临床应用

摘要

目的 探讨结核分枝杆菌感染T细胞斑点试验(T-SPOT.TB)在肺结核诊断和鉴别诊断中的应用价值.方法 纳入2014年4月—2016年12月新疆维吾尔自治区胸科医院疑似肺结核患者700例,于入院次日清晨采集肘正中静脉血用于T-SPOT.TB检测;完善胸部CT检查;取痰液或经支气管镜刷检取样后行涂片抗酸染色,对痰液和灌洗液行结核杆菌及普通细菌培养;必要时取活检行组织病理学检查和诊断性抗结核、抗感染治疗.T-SPOT.TB检测按试剂盒说明书操作,取血5ml分离单个核细胞(PBMC),在预包被抗人γ-干扰素抗体的孔中加入2.5× 105个PBMCs,分别与两种结核分枝杆菌特异性抗原,即早期分泌靶抗6(ESAT-6)和培养过滤蛋白10(CFP-10)共同孵育,计数斑点形成细胞(SFCs).本研究金标准:①结核分枝杆菌涂片或培养阳性;②临床诊断.满足任何一条即为阳性.观察T-SPOT.TB对活动性肺结核的诊断效能,确定T-SPOT.TB诊断活动性肺结核的最佳临界值.将患者分为活动性肺结核组与非肺结核病组,再将活动性肺结核患者分为初治肺结核与复治肺结核亚组;结核分枝杆菌涂片或培养阳性(简称菌阳)与结核分枝杆菌涂片或培养阴性(简称菌阴)亚组.比较各组患者T-SPOT.TB检测A、B抗原所得SFCs的差异.结果 700例疑诊肺结核患者中624例获得确诊,其中528例(84.6％)确诊为活动性肺结核纳入活动性肺结核组,96例(15.4％)排除肺结核纳入非肺结核组.活动性肺结核组中414例T-SPOT.TB检测结果为阳性,非肺结核组47例T-SPOT.TB检测结果为阴性,T-SPOT.TB检测灵敏度78.4％,特异度49.0％,阳性预测值89.4％,阴性预测值29.2％,阳性似然比为1.537,阴性似然比为0.441.绘制受试者工作特征曲线(ROC),可见当A抗原取值16.0 SFCs/2.5×105 PBMC、B抗原取值7.0 SFCs/2.5×105 PBMC进行并联检测时,T-SPOT.TB的特异度提高至62.5％,灵敏度为72.7％.活动性肺结核组A、B抗原的SFCs显著高于非肺结核组(P＜0.01),菌阳肺结核组B抗原的SFCs高于菌阴肺结核组(P＜0.05),其余各组差异无统计学意义.结论 T-SPOT.TB在结核高流行、高感染地区对活动性肺结核诊断的灵敏度较高、特异度低,需结合临床表现进行综合判定.较高的斑点数对判断活动性肺结核有一定的提示意义.%Objective To explore the application value of T-spot test of Mycobacterium tuberculosis infection (T-SPOT.TB) on diagnosis and differential diagnosis of pulmonary tuberculosis.Methods From Apr.2014 to Dec.2016,700 patients with suspected pulmonary tuberculosis were collected,venous blood (5ml) was drawn off and sputum was collected from each patient separately for T-SPOT.TB and pathogens identification (including TB).Chest CT,bronchoscopy brush or biopsy histopathological examination were followed up,cultivation of My.tuberculosis and of common bacteria with sputum or lavage fluid when needed.T-SPOT.TB test was performed according to the kit instruction operation.2.5 × 105 peripheral blood mononuclear cells (PBMCs) were added into the pre-coated anti-human γ-interferon antibody,and co-incubated separately with two specific My.tuberculosis antigens,namely early secretory targeting 6 (ESAT-6) and culture filtration protein 10 (CFP-10),and then the spot forming cells (SFCs) were counted.The gold standard for present study were set as follows:1) My.tuberculosis smear positive or culture positive;2) Clinical diagnosis (meet any one is positive).The efficacy of T-SPOT.TB on diagnosing active TB was observed,and then the optimal critical value for diagnosing active TB was determined.Patients diagnosed as active TB were divided into 4 subgroups:initial treatment group,retreatment group,smear or culture positive group,and smear or culture negative group.T-SPOT.TB was carried out to detect A and B antigen,and the difference of formed SFCs was then compared.The present study was approved by the Ethics Committee of Xinjiang Uygur Autonomous Region Chest Hospital.Results Of 700 cases suspected of pulmonary tuberculosis enrolled in present study,528 out of 624 definite cases (84.6％) were finally diagnosed as active tuberculosis (active TB group) and 96 cases (15.4％) were as without TB infection (non-TB group).Positive results of T-SPOT.TB test were found in 414 cases in active TB group,and 47 cases in non-TB group were reported with T-SPOT.TB negative.The sensitivity and specificity of T-SPOT.TB test for diagnosing active TB were 78.4％ and 49％,respectively.The positive predictive value,negative predictive value,positive likelihood ratio and negative likelihood ratio were 89.4％,29.2％,1.537 and 0.441,respectively.ROC curve showed that the specificity increased significantly (from 49％ to 62.5％) while the sensitivity decreased (from 78.4％ to 72.7％) when antigen A (cut-off:16.0 SFCs/2.5 × 105 PBMC) was combined with antigen B (cut-off:7.0 SFCs/2.5 × 105 PBMC) for analysis.In addition,the number of A and B antigen spots in active TB group was significantly higher than that in non-TB group (P＜0.01).The number of B antigen spots in positive TB group was significantly higher than that in negative TB group (P＜0.05).There was no significant difference among the other groups.Conclusions Since the high sensitivity and low specificity of T-SPOT.TB in the diagnosis of active TB,the final diagnosis should be combined with clinical manifestations.When the A antigen is 16.0 SFCs/2.5 × 105 PBMC and the B antigen is 7.0 SFCs/2.5 × 105 PBMC,the specificity of T-SPOT.TB will be improved.Higher number of spots has a certain reference diagnostic value for active TB.