【作者】 李海；

【导师】 杨莉；

【作者基本信息】 广西医科大学 ， 流行病与卫生统计学， 2012， 博士

【摘要】 [目的]评估基于6KD早期分泌靶抗原(early secreting antigen target 6KD, ESAT-6)和10KD培养滤液蛋白(culture filtrate protein 10KD, CFP-10)抗原肽的酶联免疫斑点技术(Enzyme-Linked Immunospot Assay, ELISPOT)在高发病率国家诊断活动性结核病的真实性、可靠性和诊断价值。探讨ELISPOT快速诊断痰液结核分枝杆菌培养和痰涂片抗酸杆菌检查(Acid Fast Bacilli,AFB)阴性结核病、儿童结核病和肺外结核病的可行性。[方法]整群选取在2011年3月至8月期间到广西南宁市第四人民医院医院住院治疗的痰液细菌学培养阳性或组织病理学确诊的活动性结核病人140例作为结核病组,其中细菌学培养阳性90例,阴性36例。整群选取同期到该医院住院治疗的非结核病人98例作为对照组。收集研究对象的外周血单核细胞进行ELISPOT (T SPOT.TB; OxfordImmunotec Ltd., Abingdon, UK)检测,同时进行结核菌素皮肤试验(Tuberculin skin test, TST)。结合最终临床诊断和微生物学的诊断,应用灵敏度、特异度、阳性预测值(Positive predictive value, PPV),阴性预测值(Negative predictive value,NPV)、阳性似然比(Positive Likelihood Ratio)、阴性似然比(Negative Likelihood Ratio)、约登指数、诊断OR值和ELISPOT和TST的检测结果一致性等指标评价ELISPOT和TST在常规临床实践中诊断活动性结核病的真实性、诊断价值和可靠性。应用单因素和多因素非条件Logistic回归模型分析ELISPOT和TST灵敏度和特异度的影响因素。[结果](1)ELISPOT斑点形成细胞(Spot forming cells, SFCs)分布：①结核组ESAT-6形成的SFCs数的中位数(74个/2.5×105PBMCs)显著高于对照组(11个/2.5×105PBMCs)(P<0.001),结核组CFP-10形成的SFCs数的中位数(100个/2.5×105PBMCs)也显著高于对照组(9个/2.5×105PBMCs)(P<0.001)。②涂阳和涂阴结核组ESAT-6形成的SFCs数的中位数(92个和74个/2.5×105PBMCs)均高于对照组(11个/2.5×105PBMCs),差异有统计学意义(P<0.001),涂阳和涂阴结核组CFP-10-ELISPOT形成的SFCs数的中位数(150个和100个/2.5×105PBMCs)均高于对照组(9个/2.5×105PBMCs),差异有统计学意义(P<0.001)。③结核病例ESAT-6和CFP-10抗原肽形成SFCs数与年龄、BMI指数和抗结核治疗时间等影响因素存在关联(P均<0.05),与性别、BCG接种、既往结核病史、家庭接触结核病人和免疫抑制等其他影响因素均无关联(P均>0.05)。(2)TST硬结直径分布：①病例组和对照组TST硬结直径中位数分别为7.00mm(0～27.00mm)和4.50mm(0～15.00mm),差异有统计学意义(P=0.007)。涂阳结核病组TST的硬结直径中位数是8.0mm(0～14.0mm),涂阴结核病组硬结直径中位数是7.0mm(0～27.0mm),对照组TST的硬结直径中位数是4.5mm(0～15.0mm),三组病人TST硬结直径分布比较,差异有统计学意义(χ2=7.912,P=0.023)。涂阳和涂阴结核病组TST硬结直径分布差异无统计学意义(P>0.05),涂阳结核病组和涂阴结核病组硬结直径均大于对照组,分布差异有统计学意义(P值均<0.05)。②在14例儿童结核病例和16例儿童对照者中,病例组TST的硬结直径中位数是10.8mm(0～21.0mm),对照组TST的硬结直径中位数是3.5mm(2.0～15.0mm),病例组的硬结直径显著大于对照组(P=0.008)。(3)ELISPOT与TST总体诊断真实性与诊断价值：①ELISPOT诊断结核病的总体灵敏度为89.3%(95%CI：83.1-93.4),特异度为82.7%(95%CI：74.0-88.9),约登指数为0.72,PPV为88.0%(95%CI：81.7-92.4),NPV为84.4%(95%CI：75.8-90.3)。②TST(≥5mm)诊断活动性结核病的总体灵敏度为68.3%(95%CI：59.6-75.9),特异度为51.0%(95%CI：41.3-60.7),约登指数为0.193,PPV为63.6%(95%CI：55.2-71.4),NPV为56.2%(95%CI：45.8-66.0)。TST(≥10mm)诊断结核病的总体灵敏度为35.8%(95%CI：27.9-44.6),特异度为91.8%(95%CI：84.7-95.8),约登指数为0.276,PPV为84.6%(95%CI：72.5-91.2),NPV为53.3%(95%CI：45.7-60.6)。痰涂片抗酸染色检查的灵敏度为22.1%(95%CI：15.9-29.7)。③ELISPOT的灵敏度、特异度、PPV和NPV均显著高于TST(≥5mm)(P值均<0.001),ELISPOT的灵敏度和NPV显著高于TST(≥10mm)(P<0.001),特异度低于TST(≥10mm), PPV高于TST(≥10mm),但差异均无统计学意义(P>0.05)。④LISPOT、TST(≥5mm)和TST(≥10mm)阳性检测结果的活动性结核病诊断OR值分别为39.71(95%CI：18.78-83.93)、2.24(95%CI：1.30-3.88)和6.27(95%CI：2.78～14.11)。(4)菌阳和菌阴结核病人ELISPOT和TST诊断灵敏度：①菌阳结核病人中,ELISPOT和TST诊断灵敏度分别为97.8%(45/46,95%CI：88.7-99.6)和90.0%(27/30,95%CI：74.4-96.5),差异无统计学意义(P=0.33)。②菌阴结核病人中,ELISPOT和TST诊断灵敏度分别为86.7%(78/90,95%CI：78.1-92.2)和59.6%(53/89,95%CI：49.2-69.2),差异有统计学意义(P<0.001)。(5)肺外结核病人ELISPOT和TST诊断灵敏度：28例肺外结核病例中,ELISPOT阳性灵敏度(85.7%)高于TST(70.8%),差异无统计学意义(P=0.19)。4例结核性脑膜炎患者的ELISPOT结果都是阳性(100%),但只有2例(50%)TST阳性。(6)儿童结核病人ELISPOT和TST诊断真实性与诊断价值：①14个儿童活动性结核病例和16个儿童对照中,ELISPOT灵敏度为85.7%(95%CI：60.1～95.0),特异度为93.8%(95%CI：71.7～98.9)；②TST灵敏度为71.4%(95%CI：45.4～88.3),特异度为56.3%(95%CI：33.2～76.9)。③LISPOT的灵敏度高于TST,但无统计学意义(P>0.05)；ELISPOT的特异度显著高于TST(P<0.05)。(7)ELISPOT和TST诊断结果一致性：①LISPOT和TST总一致性较差(κ=0.24)。在活动型结核病组中,ELISPOT和TST一致性较差(κ=0.071),类似地,在对照组中,ELISPOT和TST一致性也较差(κ=0.15)。不一致的检测结果在结核病组中常见ELISPOT阳性而TST阴性(33例),而在对照组中常见于ELISPOT阴性而TST阳性(36例)。②在14个儿童活动型结核病例和16个儿童对照者中,ELISPOT和TST,总一致性较差(κ=0.35)。在结核病组中,ELISPOT和TST一致性较差(κ=0.18),类似地,在对照组中,ELISPOT和TST一致性也较差(κ=0.16)。不一致的检测结果在病例组中常见ELISPOT阳性而TST阴性(3例),而在对照组中常见于ELISPOT阴性而TST阳性(7例)。(8)ELISPOT和TST灵敏度影响因素：①多因素分析显示,ELISPOT灵敏度未受研究对象性别、年龄、BMI指数、接种卡介苗、既往结核病史、家庭接触结核病人、免疫抑制等因素的影响(P值均>0.05)。②多因素分析显示BMI指数<18.5(0R=0.22,95%CI：0.08～0.59)、接种卡介苗(OR=4.36,95%CI：1.25～15.26)、家庭接触结核病人(OR=6.61,95%CI：1.69～25.93)、有免疫抑制(OR=0.076,95%CI：0.016～0.37)等4个因素(P值均<0.05)是TST灵敏度的独立影响因素。提示营养不良、有免疫抑制更易使TST灵敏度降低,而接种卡介苗和家庭接触结核病人更易使TST灵敏度升高。(9)ELISPOT和TST特异度影响因素：①多因素分析显示,ELISPOT假阳性率未受研究对象性别、年龄、BMI指数、接种卡介苗、免疫抑制等因素的影响(P值均>0.05)。但既往结核病史(多因素分析OR=7.89,95%CI：1.15-54.02)、家庭接触结核病人(多因素分析OR=12.71,95%CI：2.56～63.22)是ELISPOT假阳性率的影响因素(P值均<0.05)。提示家庭接触结核病人和既往结核病史更易使ELISPOT特异度降低。②多因素分析显示BMI指数<18.5(OR=0.18,95%CI：0.04～0.82)、接种卡介苗(OR=12.73,95%CI：3.49～46.50)、既往结核病史(OR=83.85,95%CI：6.77～1038.38)、家庭接触结核病人(OR=21.69,95%CI：2.24～210.29)等4个因素(P值均<0.05)是TST假阳性率的独立影响因素。提示营养不良更易使TST特异度升高,而接种卡介苗、既往结核病史和家庭接触结核病人更易使TST特异度降低。[结论](1)基于ESAT-6和CFP-10抗原肽的ELISPOT技术在高发病率背景下诊断活动性结核病具有较高的诊断灵敏度、特异度、阳性预测值、阴性预测值和诊断OR值,ELISPOT诊断活动性结核病的真实性、可靠性和诊断价值均高于TST。TST诊断灵敏度与特异度均较低,对活动性结核病的诊断的参考意义有限。(2)ELISPOT的灵敏度不受研究参与者年龄、卡介苗接种、结核病接触史、既往结核病史和免疫抑制状况的显著影响,特异度也未受接种卡介苗的影响而降低,检测结果比TST更为可靠。(3)ELISPOT也有明显的缺点,例如不能很好区分活动性结核病患者和潜伏性结核感染者,家庭接触结核病人和既往结核病史更易使ELISPOT特异度降低；由于人类白细胞抗原限制性,无法完全排除假阴性结果；T-SPOT-TB试剂盒价格较昂贵,难以在经济发展水平较低的地区推广应用,用于大规模普查结核病也是不现实的。对于TST硬结直径≥10mm、痰涂片抗酸杆菌检查阳性结核病人可不做ELISPOT检测。(4)ELISPOT适合作为高发病率背景下临床实践中诊断活动性结核病,特别是涂阴结核病、肺外结核病和儿童结核病的可靠的辅助诊断手段,这有助于病例得到更快速的诊断和治疗,减少病人痛苦和不必要的医疗费用。更多还原

【Abstract】 [Objective]To assess the clinical diagnostic validity, reliability, and diagnostic value of enzyme-linked immunospot assay (ELISPOT) based on ESAT-6 and CFP-10 peptide and tuberculin skin test (TST) in the rapid diagnosis of active tuberculosis(TB) in a developing country with high prevalence. To approcach feasibility of ELISPOT in rapid diagnosis of active TB patients with negative sputumn culture of MTB and negative acid fast bacilli(AFB) smear results, children TB patients and extra-pulmonary patients.[Methods]A prospective study was conducted from March to August 2011 at the Nanning Fourth Hospital(NFH), Guangxi province, an area with high prevalence of TB.140 patients with bacteriologically or biopsy specimen confirmed TB(90 cases were culture positive and 36 were culture negative), and 98 patients with non-TB illness attending the NFH during the study period were tested by ELISPOT on peripheral blood mononuclear cells(PBMCs) and TST (Tuberculin skin test) simultaneously. Sensitivity, specificity, positive predictive value(PPV), negative predictive value(NPV), Positive Likelihood Ratio, and Negative Likelihood Ratio were used to assess the diagnostic validity and diagnostic value of ELISPOT and TST based on final clinical and micrological diagnosis. Concordance between test results were used to assess the reliability of ELISPOT and TST. Non conditional univariate and multivariate logistic regression was used to analysis effective factors for sensitivity and specificity of ELISPOT and TST.[Results](1)SFCs distribution of ELISPOT:①The numbers of ESAT-6 SFCs in PBMC were significantly different between patients with active TB and controls (median number of SFCs was 74 and 11 out of 250,000 PBMCs, respectively, P＜0.001). The numbers of CFP-10 SFCs in PBMC were significantly different between patients with active TB and controls (median number of SFCs was 100 and 9 out of 250,000 PBMCs, respectively, P＜0.001).②The numbers of ESAT-6 SFCs in PBMC were significantly different between patients with positive and negative AFB active TB and controls (median number of SFCs was 92,74 and 11 out of 250,000 PBMCs, respectively, P<0.001). The numbers of CFP-10 SFCs in PBMC were significantly different between patients with positive and negative AFB active TB and controls (median number of SFCs was 150,100 and 9 out of 250,000 PBMCs, respectively, P＜0.001).③FCs produced by ESAT-6 and CFP-10 were associated with age, Body mass Index and anti-TB treatment significantly(P＜0.05), but not with sex, BCG vaccination, previous history of TB, household contact with TB patients, and immunosuppress(P＞0.05).(2)Distribution of TST induration:①Size of TST induration were significantly different between patients with active TB and controls (median number of induration was 7.00mm and 4.50mm respectively, P=0.007). The size of TST induration for active TB patients with AFB positive, active TB patients with AFB negative and control was 8.0mm(0～14.0mm),7.0mm(0～27.0mm) and 4.5mm(000～15.0mm), respectively. Distribution of TST induration between active TB patients with AFB smear positive, active TB patients with AFB smear negative and control were significantly different(x2=7.912, P=0.023). However, distribution of TST induration between active TB patients with AFB positive and active TB patients with AFB smear negative were not significantly different(P>0.05). Distribution of TST induration between active TB patients with AFB smear positive and control, and between active TB patients with AFB smear negative and control were were significantly different(P＜0.05).②In 14 TB patients and 16 controls who were children, size of TST induration were significantly different between patients with active TB and controls (median number of induration was 10.8mm and 3.5mm, respectively, P=0.008).(3)Dignostic validity and value of ELISPOT and TST:①The sensitivites, specificity, Youden’s index, PPV and NPV of ELISPOT were 89.3%(95%CI: 83.1-93.4),82.7%(95%CI:74.0-88.9),0.72,88.0%(95%CI:81.7-92.4) and 84.4%(95%CI:75.8-90.3), respectively.②The sensitivites, specificity, Youden’s index, PPV and NPV of TST(≥5mm) were 68.3%(95%CI:59.6-75.9), 51.0%(95%CI:41.3-60.7),0.193,63.6%(95%CI:55.2-71.4), and 56.2%(95%CI: 45.8-66.0, respectively; The sensitivites, specificity, Youden’s index, PPV and NPV of TST(≥10mm) were 35.8%(95%CI:27.9-44.6),91.8%(95%CI: 84.7-95.8),0.276,84.6%(95%CI:72.5-91.2) and 53.3%(95%CI:45.7-60.6), respectively.③The sensitivites, specificity, PPV and NPV of ELISPOT were higher than that of TST(≥5mm) with significant differences(P＜0.001). Sensitivity and NPV of ELISPOT were higher than that of TST(≥10mm) (P＜0.001), and no significant difference were observed in specificity and PPV between ELISPOT and TST(≥10mm) (P＞0.05).③In patients with a positive ELISPOT, TST(≥5mm) and TST (≥10mm) results, the diagnostic OR for active TB were 39.71(95%CI:18.78-83.93),2.24(95%CI:1.30-3.88) and 6.27(95%CI:2.78-14.11), repectively。 (4)Sensitivity of ELISPOT and TST in TB patients with positive and negative culture results:①The sensitivity in TB patients with positive results of acid fast stain or sputum bacteriological culture for ELISPOT and TST were 97.8%(95%CI,88.7-99.6) and 90.0%(95% CI,74.4-96.5), respectively. Sensitivity for ELISPOT was higher than that of TST with significant differences(P=0.33).②ensitivity for ELISPOT and TST in TB patients with negative results of acid fast stain or sputum bacteriological culture were 86.7% (95% CI,78.1 to 92.2) and 59.6%(95% CI,49.2-69.2), respectively. The sensitivity of ELISPOT was higher than that of TST with significant difference (P＜0.001)in cases who were negative in results of acid fast stain or sputum bacteriological culture.(5)Sensitivity of ELISPOT and TST in extra-pulmonary TB patients: 24/28(85.7%) and 17/24(70.8%) cases of extra-pulmonary TB were positive in ELISPOT and TST respectively. Similarly,4/4 (100%) and 2/4 (50.0%) cases of cerbral tuberculosis were positive in ELISPOT and TST respectively.(6)Sensitivity of ELISPOT and TST in children TB patients:①Among 14 children cases and 16 children controls, the sensitivity for ELISPOT and TST was 85.7%(95%CI:60.1-95.0) and 71.4%(95%C7:45.4-88.3), respectively.②Specificity for ELISPOT and TST was 93.8%(95%CI:71.7-98.9)and 56.3%(95%CI:33.2-76.9), respectively.③Sensitivity of ELISPOT was higher than that of TST, although no significant difference was observed(P＞0.05). Specificity of ELISPOT was higher than that of TST with with significantly different(P＜0.05).(7)Concordance between ELISPOT and TST:①Very poor agreement was observed between ELISPOT and TST in all participants(K=0.24). In patients with active TB, agreement between ELISPOT and TST was poor (κ=0.071), similarly, agreement between ELISPOT and TST was poor (κ=0.15). Disconcordance test results were most often ELISPOT positive yet TST negative in patients with active TB (33 cases), and ELISPOT negative yet TST positive in patients with non TB illness (36 controls).②or agreement was observed between ELISPOT and TST in children participants(K=0.35). In children patients with active TB, agreement between ELISPOT and TST was poor (κ=0.18), similarly, agreement between ELISPOT and TST was poor (κ=0.16) in control children. Disconcordance test results were most often ELISPOT positive yet TST negative in patients with active TB (3 cases), and ELISPOT negative yet TST positive in patients with non TB illness (7 controls).(8)Influence factors of sensitivity of ELISPOT and TST:①Non conditional univariate and multivariate logistic regression analysis showed that sensitivity of ELISPOT was not affected by sex, age, Body Mass Index, BCG vaccination, previous TB history, household contact with patients known to have TB, and immunosupress(P＞0.05).②Multivariate logistic regression analysis showed that Body Mass Index less than 18.5(OR=0.22,95% CI:0.08～0.59), BCG vaccination(OR=4.36,95% CI:1.25～15.26), household contact with patients known to have TB(OR=6.61,95% CI:1.69～25.93), and immunosupress(OR=0.076,95% CI:0.016～0.37) were independent affected factors for sensitivity of TST(P＜0.05). It was suggested that malnutrition and immunosupress tend to decrease sensitivity of TST, however, BCG vaccination and household contact with patients known to have TB tend to increase sensitivity of TST.(9)Influence factors of specificity of ELISPOT and TST:①evious TB history(OR=7.89,95% CI:1.15～54.02) and household contact with patients known to have TB(OR=12.71,95% CI:2.56～63.22) were independent affected factors for false positive rate of ELISPOT according to multivariate logistic regression analysis(P＜0.05). It was suggested that previous TB history and household contact with patients known to have TB tend to decrease specificity of ELISPOT.②Body Mass Index less than 18.5(OR).18,95% CI:0.04～0.82), BCG vaccination(OR=12.73,95% C7:3.49～46.50), previous histroy of TB (OR=83.85,95% CI:6.77～1038.38)and household contact with patients known to have TB(OR=21.69,95%CI:2.24～210.29) were independent affected factors for false positive rate of ELISPOT according to multivariate logistic regression analysis(P＜0.05). It was suggested that malnutrition tend to increase specificity of TST, however, BCG vaccination, previous histroy of TB and household contact with patients known to have TB tend to decrease specificity of TST.[Conclusion](1)High sensitivity, specificity, PPV, NPV and diagnostic OR of ELISPOT based on ESAT-6 and CFP-10 peptide are observed in high prevalance background. Diagnositic validity and value of ELISPOT is higher than that of tuberculin skin test. The diagnositic value of TST is limitted.(2)The sensitivity of ELISPOT is less affected by those factors which included age, malnutrition, history of BCG vaccination, previous history of TB, household contact of TB, or immunolsuppress status, and the specificity is not decreased by BCG vaccination.The detection results of ELISPOT appears to be more reliable than TST.(3)ELISPOT have obviously drawback:it can’t distinguish active TB patients and patients with LTBI obviously, its’specificity is decline by the household contact of TB and previous history of TB, false negative results can not be excluded due to limitation of human leucocyte antigen, and T-SPOT-TB kit is too costly to diagnosis active TB and screen TB in a large scale in area with low income. It is not neccessarly to perform ELISPOT detection for TB patients with TST induration larger than 10 mm and positive results of AFB smear. (4)ELISPOT is a reliable supplementary means for rapid diagnosis of active TB with negative AFB smear results, extra-pulmonary and children cases in country with high prevalence of TB, which would lead to rapid diagnosis and treatment and reduction of medical cost.更多还原