特异性T细胞IFN-γ流式检测对儿童结核感染诊断价值研究
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摘要
目的：
建立流式细胞术胞内细胞因子(cytokine flow cytometry,CFC)检测法,检测结核病组、潜伏结核感染(latent tuberculosis infection,LTBI)组、非结核病组三组分泌r-干扰素(IFN-γ)的结核杆菌特异性CD4+T淋巴细胞比例。计算各组敏感性及特异性并进行比较；同时将CFC检测法结果与结核菌素试验(即PPD皮试)、结核抗体结果进行对比分析,从而探讨流式细胞术胞内细胞因子检测法检测结核杆菌特异性T细胞—IFN-γ对儿童结核病及潜伏结核感染的诊断价值。
方法：
连续收集2008年01月～2010年01月苏州大学附属儿童医院门诊及住院部0-14岁疑似结核病患儿86例；潜伏结核感染患儿75例,健康对照组20例。所有入选对象均已接种BCG,并记录详细的临床资料。综合临床资料及相关检查,最终诊断为结核病者41例,其中肺结核病17例,肺外结核病24例；非结核病者45例；潜伏结核感染者75例,进行以下分组：A组：结核病组,41例；A1组：肺结核病组,17例；A2组：肺外结核病组,24例；B组：潜伏结核感染组(LTBI),75例；C组：非结核病组,45例；D组：健康对照组,20例。
根据结核杆菌特异的效应T淋巴细胞在特异抗原的刺激下分泌γ-干扰素,检测IFN-γ可以确定体内是否存在对结核杆菌反应的效应T淋巴细胞的原理,结合流式细胞仪(flow cytomeytry,FCM)、胞内细胞因子(intracellular cytokine,ICK)染色法建立流式细胞术胞内细胞因子检测法检测结核菌感染的方法。每组研究对象均进行PPD试验、结核抗体检测以及流式细胞术胞内因子检测(以下简称CFC检测法)并进行全面的临床评估,随访3～6个月。CFC检测法简要步骤：肝素钠抗凝真空管采集入选患儿外周血1ml,取三只流式测定管：①阳性对照管：加入0.2ml肝素钠抗凝全血,加入佛波酯(PMA20ng／ml)、离子酶素(IN01μg／ml)共刺激,加入10μl 0.5mg／mlBrefeldin(BFA)：②刺激管：加入0.2ml肝素钠抗凝全血,加入结核杆菌特异性抗原(ESAT-6+CFP-10各50μl)刺激,加入10μl 0.5mg／ml BFA阻断剂；③未刺激管：加入0.2ml肝素钠抗凝全血,加入10μl 0.5mg／ml BFA阻断剂；5% CO2、37℃,阳性刺激管温箱培养6小时,抗原多肽刺激管培养24小时(温箱,松盖)。然后每管均加入固定剂、破膜剂,并加入荧光标记抗体,上流式细胞仪检测结核杆菌特异性T细胞—IFN-r分泌情况。计算流式检测结果的灵敏度及特异度。并与PPD试验、结核抗体检测结果进行对比分析。
结果：
1、结核病组及潜伏结核感染组分泌IFN-γ的结核杆菌特异性CD4+T细胞比例均高于健康对照组及非结核感染组(P<0.05)；而结核病组与潜伏结核感染组相比较,非结核感染组与健康对照组相比较,分泌IFN-γ的结核杆菌特异性CD4+T细胞均无明显差异(P>0.05)；
2、用CFC法检测,41例结核病患儿90.24%阳性(37／41),9.76%阴性(4／41)；潜伏结核感染患儿85.33%阳性(64／75),14.67%阴性(11／75),结核病组与潜伏结核感染组相比,CFC检测法阳性率无明显差异(P>0.05)；而非结核感染组患儿100%阴性(45／45),阳性率为0；故结核病组及潜伏结核感染组CFC检测法阳性率均高于非结核病组(P<0.05)；健康对照组1例阳性,说明该法特异性为95%(19／20)。
3、潜伏结核感染组CFC检测法阳性率为85.33%,与PPD皮试有较好的一致性(85.33%)；而结核抗体检测阳性率为28%,两者相比较,差异有显著统计学意义(P<0.01)。非结核病组PPD假阳性率为40%(18／45),而CFC法假阳性率为0,即对该组来讲,PPD皮试特异性为60%,CFC法特异性为100%,二者比较,差异有统计学意义(P<0.05)。
4、结核病组CFC检测法阳性率为90.24%,结核抗体检测阳性率为56.10%,二者相比较,差异有统计学意义(P<0.05)；PPD皮试阳性率为70.73%,二者相比差异有统计学意义(P<0.05)；肺结核病组CFC检测法阳性率为94.12%,肺外结核组为87.5%,二者比较,无明显差异(P>0.05)。
结论：
1、结核杆菌特异性T细胞—IFN-γ流式检测法敏感性强；且由于ESAT-6、CDP-10两种特异性抗原的应用,特异度高。
2、对于潜伏结核感染患儿,结核杆菌特异性T细胞—IFN-γ流式检测法可区分BCG接种反应,故适合我国儿童普遍接种BCG的国情；且所需时间短,一般24小时即可出结果,可避免主观因素对结果的影响,可以快速、准确诊断儿童潜伏结核感染；
3、对于结核病患儿,结核杆菌特异性T细胞—IFN-γ流式检测法的敏感性、特异性与PPD皮试、结核抗体检测相比,均高于后者,差异有统计学意义,故该法同样可作为儿童结核病诊断的重要辅助手段。
Objects:To measure the proportion of tuberculosis-antigen-specific CD4+T cells excreted cellular interferon-gamma by flow cytometry (CFC) in tuberculosis group, latent tuberculosis group, non-tuberculosis group. Statistic the positivity and specificity of each group, and then compared them. Meanwhile comparing the results of cytokine flow cytometry test, tuberculin skin test and tuberculosis antibody test to discuss the diagnosis value of antigen-specific-T cells IFN-gamma by flow cytometry in children with tuberculosis.
Methods:Total of 86 consecutive cases of children aged from 0-14 years who were being assessed for suspected tuberculosis,75 cases for latent tuberculosis,20 cases for healthy control were enrolled in this study from patients' room or outpatient in Children's Hospital Affiliated Soochow University during 2008.01 to 2010.01.everyone had BCG vaccination history,and received full clinical assessment.at last,final diagnosis of 41cases were tuberculosis, including 17 pulmonary tuberculosis,24 extrapulmonary tuberculosis.45 cases were not tuberculosis infection.we divide all the children into:group A:tuberculosis,41cases. Including group A1:pulmonary tuberculosis,17cases; group A2: extrapulmonary tuberculosis,24cases; group B:latent tuberculosis infection,75 cases (short for LTBI); group C:non-tuberculosis,45cases; group D:healthy control,20cases. All of them received CFC test, PPD test and tuberculosis-antibody test. According the stimulation of tuberculosis specific antigen ESAT-6, CFP-10, the specific T lymphocytes in whole blood will excrete cellular interferon-gamma, combined the flow cytometry and intracellular cytokine, we create cytokine flow cytometry (CFC).the brief procedures are as following:1ml of blood was collected in Na heparin tubes.samples were divided into three 0.2-ml aliquots. One was incubated with ESAT-6 and CFP-10,one with PMA and ION,the final aliquot served as a non-stimulated control.co-stimulatory antibodies were added to all the aliquots.the blood was incubated at 37℃for several hours in the presence of the Golgi transport inhibitor brefeldin A added during the last 6h. Then fixed, washed, stained the blood for flow cytometry evaluation.at last,statistic the positivity and the specificity of each group,and then compared them.meanwhile comparing the results of cytokine flow cytometry test, tuberculin skin test and tuberculosis antibody test.
Results:1、whether in groupA or groupB, the proportion of tuberculosis-antigen-specific CD4+T cells excreted cellular interferon-gamma are higher than groupD (P<0.05),while there is no obvious statistical difference between groupD and groupC(P>0.05);
2、By CFC,the positivity is 90.24%(37/41), negativity is 9.76%(4/41)in groupA; the positivity is 85.33%(64/75), negativity is 14.67%(11/75) in groupB; the positivity is 13.33%(6/45), negativity is 86.67%(39/45) in groupC. So the positivity in groupA and groupB is higher than groupC (P<0.05). While there is no statistical difference between groupA and groupB (P>0.05).
3、The positivity is 85.33% of CFC in groupB, has a good concordance with PPD test. While the positivity is only 28% of tuberculosis antibody test in this group, compared with the former, has significantly statistical difference (P<0.01).
4、In groupA, the positivity is 90.24% by CFC, compared with the positivity 50.10% of tuberculosis antibody test, there is statistical difference between them (P<0.05).the positivity is 70.73% by PPD test,compared with CFC's positivity, has statistical difference(P<0.05).but the positivity between groupA1 and groupA2 has no statistical difference(P>0.05).
Conclusins:1、the method of antigen-specific-T cells IFN-gamma measured by flow cytometry has higher sensitivity,and because of the tuberculosis-specific-antigen ESAT-6,CFP-10,has better specificity.
2、The identification by flow cytometry of antigen-specific T lymphocytes upon antigen stimulation of whole blood has a better positive predictive value than PPD test, because this test enables the identification of BCG vaccination. And could represent a further tool in the diagnosis of LTBI.
3、As to tuberculosis, this test has a better positive predictive value than PPD test and tuberculosis antibody test. Because it has higher sensitivity and specificity. And also could be a important tool in the diagnosis of tuberculosis in future.
建立流式细胞术胞内细胞因子(cytokine flow cytometry,CFC)检测法,检测结核病组、潜伏结核感染(latent tuberculosis infection,LTBI)组、非结核病组三组分泌r-干扰素(IFN-γ)的结核杆菌特异性CD4+T淋巴细胞比例。计算各组敏感性及特异性并进行比较；同时将CFC检测法结果与结核菌素试验(即PPD皮试)、结核抗体结果进行对比分析,从而探讨流式细胞术胞内细胞因子检测法检测结核杆菌特异性T细胞—IFN-γ对儿童结核病及潜伏结核感染的诊断价值。
方法：
连续收集2008年01月～2010年01月苏州大学附属儿童医院门诊及住院部0-14岁疑似结核病患儿86例；潜伏结核感染患儿75例,健康对照组20例。所有入选对象均已接种BCG,并记录详细的临床资料。综合临床资料及相关检查,最终诊断为结核病者41例,其中肺结核病17例,肺外结核病24例；非结核病者45例；潜伏结核感染者75例,进行以下分组：A组：结核病组,41例；A1组：肺结核病组,17例；A2组：肺外结核病组,24例；B组：潜伏结核感染组(LTBI),75例；C组：非结核病组,45例；D组：健康对照组,20例。
根据结核杆菌特异的效应T淋巴细胞在特异抗原的刺激下分泌γ-干扰素,检测IFN-γ可以确定体内是否存在对结核杆菌反应的效应T淋巴细胞的原理,结合流式细胞仪(flow cytomeytry,FCM)、胞内细胞因子(intracellular cytokine,ICK)染色法建立流式细胞术胞内细胞因子检测法检测结核菌感染的方法。每组研究对象均进行PPD试验、结核抗体检测以及流式细胞术胞内因子检测(以下简称CFC检测法)并进行全面的临床评估,随访3～6个月。CFC检测法简要步骤：肝素钠抗凝真空管采集入选患儿外周血1ml,取三只流式测定管：①阳性对照管：加入0.2ml肝素钠抗凝全血,加入佛波酯(PMA20ng／ml)、离子酶素(IN01μg／ml)共刺激,加入10μl 0.5mg／mlBrefeldin(BFA)：②刺激管：加入0.2ml肝素钠抗凝全血,加入结核杆菌特异性抗原(ESAT-6+CFP-10各50μl)刺激,加入10μl 0.5mg／ml BFA阻断剂；③未刺激管：加入0.2ml肝素钠抗凝全血,加入10μl 0.5mg／ml BFA阻断剂；5% CO2、37℃,阳性刺激管温箱培养6小时,抗原多肽刺激管培养24小时(温箱,松盖)。然后每管均加入固定剂、破膜剂,并加入荧光标记抗体,上流式细胞仪检测结核杆菌特异性T细胞—IFN-r分泌情况。计算流式检测结果的灵敏度及特异度。并与PPD试验、结核抗体检测结果进行对比分析。
结果：
1、结核病组及潜伏结核感染组分泌IFN-γ的结核杆菌特异性CD4+T细胞比例均高于健康对照组及非结核感染组(P<0.05)；而结核病组与潜伏结核感染组相比较,非结核感染组与健康对照组相比较,分泌IFN-γ的结核杆菌特异性CD4+T细胞均无明显差异(P>0.05)；
2、用CFC法检测,41例结核病患儿90.24%阳性(37／41),9.76%阴性(4／41)；潜伏结核感染患儿85.33%阳性(64／75),14.67%阴性(11／75),结核病组与潜伏结核感染组相比,CFC检测法阳性率无明显差异(P>0.05)；而非结核感染组患儿100%阴性(45／45),阳性率为0；故结核病组及潜伏结核感染组CFC检测法阳性率均高于非结核病组(P<0.05)；健康对照组1例阳性,说明该法特异性为95%(19／20)。
3、潜伏结核感染组CFC检测法阳性率为85.33%,与PPD皮试有较好的一致性(85.33%)；而结核抗体检测阳性率为28%,两者相比较,差异有显著统计学意义(P<0.01)。非结核病组PPD假阳性率为40%(18／45),而CFC法假阳性率为0,即对该组来讲,PPD皮试特异性为60%,CFC法特异性为100%,二者比较,差异有统计学意义(P<0.05)。
4、结核病组CFC检测法阳性率为90.24%,结核抗体检测阳性率为56.10%,二者相比较,差异有统计学意义(P<0.05)；PPD皮试阳性率为70.73%,二者相比差异有统计学意义(P<0.05)；肺结核病组CFC检测法阳性率为94.12%,肺外结核组为87.5%,二者比较,无明显差异(P>0.05)。
结论：
1、结核杆菌特异性T细胞—IFN-γ流式检测法敏感性强；且由于ESAT-6、CDP-10两种特异性抗原的应用,特异度高。
2、对于潜伏结核感染患儿,结核杆菌特异性T细胞—IFN-γ流式检测法可区分BCG接种反应,故适合我国儿童普遍接种BCG的国情；且所需时间短,一般24小时即可出结果,可避免主观因素对结果的影响,可以快速、准确诊断儿童潜伏结核感染；
3、对于结核病患儿,结核杆菌特异性T细胞—IFN-γ流式检测法的敏感性、特异性与PPD皮试、结核抗体检测相比,均高于后者,差异有统计学意义,故该法同样可作为儿童结核病诊断的重要辅助手段。
Objects:To measure the proportion of tuberculosis-antigen-specific CD4+T cells excreted cellular interferon-gamma by flow cytometry (CFC) in tuberculosis group, latent tuberculosis group, non-tuberculosis group. Statistic the positivity and specificity of each group, and then compared them. Meanwhile comparing the results of cytokine flow cytometry test, tuberculin skin test and tuberculosis antibody test to discuss the diagnosis value of antigen-specific-T cells IFN-gamma by flow cytometry in children with tuberculosis.
Methods:Total of 86 consecutive cases of children aged from 0-14 years who were being assessed for suspected tuberculosis,75 cases for latent tuberculosis,20 cases for healthy control were enrolled in this study from patients' room or outpatient in Children's Hospital Affiliated Soochow University during 2008.01 to 2010.01.everyone had BCG vaccination history,and received full clinical assessment.at last,final diagnosis of 41cases were tuberculosis, including 17 pulmonary tuberculosis,24 extrapulmonary tuberculosis.45 cases were not tuberculosis infection.we divide all the children into:group A:tuberculosis,41cases. Including group A1:pulmonary tuberculosis,17cases; group A2: extrapulmonary tuberculosis,24cases; group B:latent tuberculosis infection,75 cases (short for LTBI); group C:non-tuberculosis,45cases; group D:healthy control,20cases. All of them received CFC test, PPD test and tuberculosis-antibody test. According the stimulation of tuberculosis specific antigen ESAT-6, CFP-10, the specific T lymphocytes in whole blood will excrete cellular interferon-gamma, combined the flow cytometry and intracellular cytokine, we create cytokine flow cytometry (CFC).the brief procedures are as following:1ml of blood was collected in Na heparin tubes.samples were divided into three 0.2-ml aliquots. One was incubated with ESAT-6 and CFP-10,one with PMA and ION,the final aliquot served as a non-stimulated control.co-stimulatory antibodies were added to all the aliquots.the blood was incubated at 37℃for several hours in the presence of the Golgi transport inhibitor brefeldin A added during the last 6h. Then fixed, washed, stained the blood for flow cytometry evaluation.at last,statistic the positivity and the specificity of each group,and then compared them.meanwhile comparing the results of cytokine flow cytometry test, tuberculin skin test and tuberculosis antibody test.
Results:1、whether in groupA or groupB, the proportion of tuberculosis-antigen-specific CD4+T cells excreted cellular interferon-gamma are higher than groupD (P<0.05),while there is no obvious statistical difference between groupD and groupC(P>0.05);
2、By CFC,the positivity is 90.24%(37/41), negativity is 9.76%(4/41)in groupA; the positivity is 85.33%(64/75), negativity is 14.67%(11/75) in groupB; the positivity is 13.33%(6/45), negativity is 86.67%(39/45) in groupC. So the positivity in groupA and groupB is higher than groupC (P<0.05). While there is no statistical difference between groupA and groupB (P>0.05).
3、The positivity is 85.33% of CFC in groupB, has a good concordance with PPD test. While the positivity is only 28% of tuberculosis antibody test in this group, compared with the former, has significantly statistical difference (P<0.01).
4、In groupA, the positivity is 90.24% by CFC, compared with the positivity 50.10% of tuberculosis antibody test, there is statistical difference between them (P<0.05).the positivity is 70.73% by PPD test,compared with CFC's positivity, has statistical difference(P<0.05).but the positivity between groupA1 and groupA2 has no statistical difference(P>0.05).
Conclusins:1、the method of antigen-specific-T cells IFN-gamma measured by flow cytometry has higher sensitivity,and because of the tuberculosis-specific-antigen ESAT-6,CFP-10,has better specificity.
2、The identification by flow cytometry of antigen-specific T lymphocytes upon antigen stimulation of whole blood has a better positive predictive value than PPD test, because this test enables the identification of BCG vaccination. And could represent a further tool in the diagnosis of LTBI.
3、As to tuberculosis, this test has a better positive predictive value than PPD test and tuberculosis antibody test. Because it has higher sensitivity and specificity. And also could be a important tool in the diagnosis of tuberculosis in future.
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[16]屠德华,万利亚.结核病控制新技术的研究进展.现代结核病控制理论与实践.2009；324
[17]河福金,谢彦,李吉友,等。多参数流式细胞术在非霍奇金淋巴瘤诊断中的应用。中华病理学杂志,2006,35(4)：203-208
[18]魏熙胤,牛瑞芳。流式细胞仪的发展历史及其原理和应用进展。现代仪器,2006,4：8-11
[19]Pietro PL, Tracy HL, Peter JM. Flow cytometrick measurement of in-tracellular cytokines[J]. Imm unolM ethods,2000,243:107-124.
[20]Fat hy MM, Asaad A, Mansour M, et al. Cellular interferon-gamma based assay for diagnosis of pulmonary tuberculosis[J]. Egypt J Immunol,2007,14 (1):33241.
[21]Diel R, Loddenkemper R, Meywald-Walter K, et al. Predictive value of a whole blood IFN-gamma assay for the development of active tuberculosis disease after recent infection with Mycobacterium tuberculosis [J]. Am J Respir Crit Care Med,2008,177 (10):116421170.
[22]Tsiouris SJ, Austin J, Toro P, et al. Result s of a tuberculosis-specific IFN-gamma assay in children at high risk for tuberculosis infection[J]. Int J Tuberc Lung Dis,2006,10(8):939-941.
[23]LalvaniA,PathanAA,McShaneH, etal. Rapid detection of Mycobacte-rium tuberculosis infection by enumeration of antigen-specific T cells [J]. Am J Respir Crit CareM ed,2001,163 (4):824-828.
[24]蔡茹,王健,李朝品,等.结核病患者外周血T细胞亚群、mIL22R与SIL2R的测定.中华微生物和免疫学杂志,2003,23：488.
[25]Grotzke JE,Lewinsohn DM. Role of CD8+ T lymphocytes in control of mycobacterium tuberculosis infection.Microbes Infect,2005,7:776-788.
[26]Lazarevic V,Nolt D,Flynn JL.Long-term control of Mycobacterium tuberculosis infection is mediated by dynamic immune responses. J Immunol,2005,175:1107-1117.
[27]Barrera SDL,Aleman M,Musella R,et al.Il-10 down-regulates costimulatory molecules on Mycobacterium tuberculosis-pulsed macrophages and impairs the lytic activity of CD+4 CD+8CTL in tuberculosis patients. Clin Exp Immunol,2004,138:128-138.
[28]Tully QKortsik C,Hohn H,et al.Highly focused Tcell responses in latent human pulmonary Mycobacterium tuberculosis infection. J Immunol,2005,174:2174-2184.
[29]Ewer K,Millington KA,Deeks JJ,et al.Dynamic antigen-specific T-cell responses after point-source exposure to Mycobacterium tuberculosis.Am J Respir Crit Care Med,2006,174:831-839.
[30]Veenstra H,Baumann R,Carroll NM,et al.Changes in leucocyte and lymphocyte subsets during tuberculosis treatment;prominence of CD3dim CD56+ natural killer Tcells in fast treatment responders. Clin Exp Immunol,2006,145:252-260.
[31]Serbina NV,Lazarevic V,Flynn JL,et al. CD+4 T cells are required for the development of cytotoxic CD+8 T cells during Mycobacterium tuberculosis infection. J Immunol,2001,167:6991-7000.
[32]Rodrigues DSS,Medeiros EAS,Weckx LY,et al.Immunophenotypic characterization of peripheral T lymphocytes in M. tuberculosis infection and disease.Clin Exp Immunol,2002,128:149-154.
[33]Barcelos W,Martins-Filho OA,Guimars TMPD,et al.Peripheral blood mononuclear cells immunophenotyping in pulmonary tuberculosis patients before and after treatment.Microbiol Immunol,2006,50:597-605.
[34]Ashtekar MD, Samue AM,Kadiv GV,etal. T lymphocytes in pulmonary tuberculosis.Clin Exp Immunol,2003,97:14-17
[35]Serbina NV, Liu CC, Scanga CA, et al. CD+8 cytotoxic T lymphocytes from lungs of M. tuberculosis infected mice express perforin in vivo and lyse infected macrohages. J Immunol,2000,165:353-363.
[36]Carranza C,Juarez E,Torres M,et al.Mycobacterium tuberculosis growth control by iung macrophages and CD8 cells from patient contacts. Am J Respir Crit Care Med,2006,173:238-245.
[1]Mason CM,Ali J.Immunity against mycobacteria.Semin Respir Crit Care Med,2004,25(1):53～59
[2]Starke JR. Tuberculosis in children.Respir Crit Care Med,2004,25(3):353～354.
[3][EB/OL].http://www.moh.gv.cn/publicfiles/business/htmlfiles/wsb/xwfb/200804/28004.htm.2007-03-20.我国结核病防治工作稳步推进
[4]Lobne P.Catanzaro diagnosing tuberculosis:facts and fauacies[J].JRespir Dis,2002, 21(6):377.
[5]范永琛.儿童结核病的诊断与治疗[J].中国实用儿科杂志,2003,18(7)：389.
[6]Nahid P, Pai M, Hopewell PC. Advances in the diagnosis andtreatment of tuberculosis [J].Proc Am Thorac Soc,2006,3:103-110.
[7]WHO. Guidance for National Tuberculosis Programmes on the Management of Tuberculosis in Children. Geneva:WHO,2006.
[8]Gie R. Diagnostic Atlas of Intrathoracic Tuberculosis in Children:A Guide for Low Income Countries [J]. Paris:International Union against Tuberculosis and Lung Disease,2003. www.iualtd.org (last accessed July 2007)
[9]Marais BJ, Gie RP, Schaaf HS, et al. The natural history of disease of childhood intra-thoracic tuberculosis:a critical review of the prechemotherapy literature [J]. Int J Tuberc Lung Dis,2004,8:392-402.
[10]Swingler GH, du Toit G, van der Merwe L, et al. Diagnostic accuracy of chest radiography in detecting mediastinal lymphadenopathyin suspected pulmonary tuberculosis [J].Arch Dis Child,2005,90:1153-1156.
[11]Andronikou S, Joseph E, Lucas S, et al. CT scanning for the detection of tuberculous mediastinal and hilar lymphadenopathy in children [J]. Pediatr Radiol,2004,34: 232-236.
[12]SinghM,Moosa NV, Kumar L, SharmaMlRole of gastric lavage and bronchoalveolar lavage in the bacteriological diagnosis of childhood ulmonary tuberculosis[J]1 Indian Pediatr,2000,37 (9):947～511
[13]A12Marri MR, Kirkpatrick MB1Erythrocyte sedimentation rate in childhood tuberculosis:is it still worthwhile [J] 1 Int J Tuberc Lung Dis,2000,4 (2):237～91
[14]赵雁林,尚美.我国结核病实验室诊断的现状[J].中华检验医学杂志,2007,30(7)：725-728.
[15]张诩,卢建平.四种结核分枝杆菌检测方法的临床应用评价[J].中国防痨杂志,2006,28(1)：16-17.
[16]梁晓海,黄平东.实时荧光定量聚合酶链反应法检测肺结核患者痰标本价值的对照研究[J].现代中西医结合杂志,2006,15(3)：287.
[17]胡海利.荧光双标记定量检测对诊断结核病的临床意义[J].上海医药,2002,23(9)：411-412.
[18]张灵霞,庄玉辉,杨华卫,等.两种DNA探针杂交检测结核分枝杆菌的研究[J].中华检验医学杂志,2001,2(24)：106
[19]张津,赵顺英,迟巍,等.结核分支杆菌多抗原蛋白芯片对儿童结核病的诊断价值[J].中国循证儿科杂志,2008：3(1)：9-14.
[20]Lalbani A. Spotting latent infection:the path to better tuberculosis control.Thorax,2003,58:916～918
[21]Hernandez-Garduno E. Predictive value of the tuberculin skin test and theQuantiFERON-TB GOLD In-Tube assay for the development of active tuberculosis disease.Am J Respir Crit Care Med.2008 Dec15;178(12):1282
[22]Manuel O, Kumar D. QuantiFERON-TB Gold assay for the diagnosis of latent tuberculosis infection.Expert Rev Mol Diagn.2008 May; 8(3):247～56
[23]Kanunfre KA,Leite OH,Lopes MI,et al. Enhancement of diagnosis of latent tuberculosis infection release assay for pulmonary tuberculosis.Clin Vaccine Immunol. 2008 Jun; 15(6):1028-30
[24]Song KH,Jeon JH,Park WB,et al. Usefulness of the whole-blood interferon-gamma release assay for diagnosis of extrapulmonary tuberculosis.Dian Microbiol Infect Dis.2008 Dec 11
[25]Liebeschuetz S, Bamber S, Ewer K, et al. Diagnosis of tuberculosis in South African children with a T-cell-based assay:a prospective cohort study [J].Lancet,2004, 364 (9452):2196-2203.
[26]Zar HJ, Hanslo D, Apolles P, et al. Induced sputum versus gastric lavage for microbiological confirmation of pulmonary tuberculosis in infants and young children: aprospective study. Lancet,2005,365:130～134.
[27]Nicol MP, Pienaar D, Wood K, et al.Enzyme-linked immunospot assay response to early secretory antigenic target 6,culture filtrate protein 10 and purified protein derivative among children with tuberculosis:implications for diagnosis and monitoring of therapy [J]. Clin Infect Dis,2005,40 (9):1301-1308.
[28]张卫平,韩文,董菊,等.酶联免疫斑点技术对小儿结核病的诊断价值[J].实用儿科临床杂志,2009,24(4)：277-279.
[29]孙琳,焦伟伟,赵顺英,等.ELISPOT检测技术在儿童结核病诊断中的应用[J].标记免疫分析与临床,2008,15(6)：349-351.
[2]Garg SK,Tiwari RP,Tiwari D,et al. Diagnosisi of tuberculosis:vailable technologies, limitations,and possibilities[J].J Clin Labnal,2003,17:155-163.
[3]Starke JR. Tuberculosis in children.Respir Crit Care Med,2004,25(3):353～354.
[4]乐军,梁莉,李苏辉,等.酶联免疫斑点试验快速诊断结核分枝杆菌感染的临床应用价值.中华检验医学杂志[J],2006,29(11)：1005-1008.
[5]全国结核流行病学抽样调查技术指导组.2000年全国结核流行病学抽样调查资料汇编[M].北京：人民卫生出版社,2003：153-162.
[6]江载芳,易著文.实用小儿结核病学[M].北京：人民卫生出版社,2006.
[7]Leung WL, Law KL, Leung VS, et al. Comparison of Intracellular Cytokine Flow Cytometry and an Enzyme Immunoassay for Evaluation of Cellular Immune Response to Active Tuberculosis. Clin Vac Immunol.2009,16(3):344-351
[8]Hernandez-Garduno E. Predictive value of the tuberculin skin test and theQuantiFERON-TB GOLD In-Tube assay for the development of active tuberculosis disease.Am J Respir Crit Care Med.2008 Dec15;178(12):12
[9]Leung WL, Law KL, Leung VS, et al. Comparison of Intracellular Cytokine Flow Cytometry and an Enzyme Immunoassay for Evaluation of Cellular Immune Response to Active Tuberculosis. Clin Vac Immunol.2009,16(3):344-351
[10]中华医学会儿科分会呼吸学组,《中华儿科杂志》编委会,儿童肺结核的临床诊断标准和治疗方案(试行)[J].中华儿科杂志2006,44(4)：249-251.
[11]胡亚美,江载芳.诸福棠实用儿科学上册[M].第7版.北京：人民卫生出版社,2002,991-999.
[12]Dye C,Espinal MA,watt CJ,etal.Worldwide incidence of multidrugresistant Tuberculosis[J] J infect Dis.2002,185(8):197-202
[13]Banerjee.S,Nandyala A,podii.R,etal. Mycobacterium tuberculosis(Mtb) isovitrate dehydrogenases show strong B cell response and distinguish vaccinate controls from TB patients [J].ProeNatlAeadSeiUSA,2004,101(34):12652-12657
[14]Arend SM, Geluk A,VanMeijgaarden KE, et al. Detection of active tuberculosis infection by T cell responses to early-secreted antigenic target 6-kd protein and culture filtrate protein 10. J Infect Dis,2000,181(10),1850-1854.
[15]Lalvani A, Pathan AA,McShane H, et al. Rapid detection of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells.Am J Resp ir Crit CareMed,2001,163 (21),824-828.
[16]屠德华,万利亚.结核病控制新技术的研究进展.现代结核病控制理论与实践.2009；324
[17]河福金,谢彦,李吉友,等。多参数流式细胞术在非霍奇金淋巴瘤诊断中的应用。中华病理学杂志,2006,35(4)：203-208
[18]魏熙胤,牛瑞芳。流式细胞仪的发展历史及其原理和应用进展。现代仪器,2006,4：8-11
[19]Pietro PL, Tracy HL, Peter JM. Flow cytometrick measurement of in-tracellular cytokines[J]. Imm unolM ethods,2000,243:107-124.
[20]Fat hy MM, Asaad A, Mansour M, et al. Cellular interferon-gamma based assay for diagnosis of pulmonary tuberculosis[J]. Egypt J Immunol,2007,14 (1):33241.
[21]Diel R, Loddenkemper R, Meywald-Walter K, et al. Predictive value of a whole blood IFN-gamma assay for the development of active tuberculosis disease after recent infection with Mycobacterium tuberculosis [J]. Am J Respir Crit Care Med,2008,177 (10):116421170.
[22]Tsiouris SJ, Austin J, Toro P, et al. Result s of a tuberculosis-specific IFN-gamma assay in children at high risk for tuberculosis infection[J]. Int J Tuberc Lung Dis,2006,10(8):939-941.
[23]LalvaniA,PathanAA,McShaneH, etal. Rapid detection of Mycobacte-rium tuberculosis infection by enumeration of antigen-specific T cells [J]. Am J Respir Crit CareM ed,2001,163 (4):824-828.
[24]蔡茹,王健,李朝品,等.结核病患者外周血T细胞亚群、mIL22R与SIL2R的测定.中华微生物和免疫学杂志,2003,23：488.
[25]Grotzke JE,Lewinsohn DM. Role of CD8+ T lymphocytes in control of mycobacterium tuberculosis infection.Microbes Infect,2005,7:776-788.
[26]Lazarevic V,Nolt D,Flynn JL.Long-term control of Mycobacterium tuberculosis infection is mediated by dynamic immune responses. J Immunol,2005,175:1107-1117.
[27]Barrera SDL,Aleman M,Musella R,et al.Il-10 down-regulates costimulatory molecules on Mycobacterium tuberculosis-pulsed macrophages and impairs the lytic activity of CD+4 CD+8CTL in tuberculosis patients. Clin Exp Immunol,2004,138:128-138.
[28]Tully QKortsik C,Hohn H,et al.Highly focused Tcell responses in latent human pulmonary Mycobacterium tuberculosis infection. J Immunol,2005,174:2174-2184.
[29]Ewer K,Millington KA,Deeks JJ,et al.Dynamic antigen-specific T-cell responses after point-source exposure to Mycobacterium tuberculosis.Am J Respir Crit Care Med,2006,174:831-839.
[30]Veenstra H,Baumann R,Carroll NM,et al.Changes in leucocyte and lymphocyte subsets during tuberculosis treatment;prominence of CD3dim CD56+ natural killer Tcells in fast treatment responders. Clin Exp Immunol,2006,145:252-260.
[31]Serbina NV,Lazarevic V,Flynn JL,et al. CD+4 T cells are required for the development of cytotoxic CD+8 T cells during Mycobacterium tuberculosis infection. J Immunol,2001,167:6991-7000.
[32]Rodrigues DSS,Medeiros EAS,Weckx LY,et al.Immunophenotypic characterization of peripheral T lymphocytes in M. tuberculosis infection and disease.Clin Exp Immunol,2002,128:149-154.
[33]Barcelos W,Martins-Filho OA,Guimars TMPD,et al.Peripheral blood mononuclear cells immunophenotyping in pulmonary tuberculosis patients before and after treatment.Microbiol Immunol,2006,50:597-605.
[34]Ashtekar MD, Samue AM,Kadiv GV,etal. T lymphocytes in pulmonary tuberculosis.Clin Exp Immunol,2003,97:14-17
[35]Serbina NV, Liu CC, Scanga CA, et al. CD+8 cytotoxic T lymphocytes from lungs of M. tuberculosis infected mice express perforin in vivo and lyse infected macrohages. J Immunol,2000,165:353-363.
[36]Carranza C,Juarez E,Torres M,et al.Mycobacterium tuberculosis growth control by iung macrophages and CD8 cells from patient contacts. Am J Respir Crit Care Med,2006,173:238-245.
[1]Mason CM,Ali J.Immunity against mycobacteria.Semin Respir Crit Care Med,2004,25(1):53～59
[2]Starke JR. Tuberculosis in children.Respir Crit Care Med,2004,25(3):353～354.
[3][EB/OL].http://www.moh.gv.cn/publicfiles/business/htmlfiles/wsb/xwfb/200804/28004.htm.2007-03-20.我国结核病防治工作稳步推进
[4]Lobne P.Catanzaro diagnosing tuberculosis:facts and fauacies[J].JRespir Dis,2002, 21(6):377.
[5]范永琛.儿童结核病的诊断与治疗[J].中国实用儿科杂志,2003,18(7)：389.
[6]Nahid P, Pai M, Hopewell PC. Advances in the diagnosis andtreatment of tuberculosis [J].Proc Am Thorac Soc,2006,3:103-110.
[7]WHO. Guidance for National Tuberculosis Programmes on the Management of Tuberculosis in Children. Geneva:WHO,2006.
[8]Gie R. Diagnostic Atlas of Intrathoracic Tuberculosis in Children:A Guide for Low Income Countries [J]. Paris:International Union against Tuberculosis and Lung Disease,2003. www.iualtd.org (last accessed July 2007)
[9]Marais BJ, Gie RP, Schaaf HS, et al. The natural history of disease of childhood intra-thoracic tuberculosis:a critical review of the prechemotherapy literature [J]. Int J Tuberc Lung Dis,2004,8:392-402.
[10]Swingler GH, du Toit G, van der Merwe L, et al. Diagnostic accuracy of chest radiography in detecting mediastinal lymphadenopathyin suspected pulmonary tuberculosis [J].Arch Dis Child,2005,90:1153-1156.
[11]Andronikou S, Joseph E, Lucas S, et al. CT scanning for the detection of tuberculous mediastinal and hilar lymphadenopathy in children [J]. Pediatr Radiol,2004,34: 232-236.
[12]SinghM,Moosa NV, Kumar L, SharmaMlRole of gastric lavage and bronchoalveolar lavage in the bacteriological diagnosis of childhood ulmonary tuberculosis[J]1 Indian Pediatr,2000,37 (9):947～511
[13]A12Marri MR, Kirkpatrick MB1Erythrocyte sedimentation rate in childhood tuberculosis:is it still worthwhile [J] 1 Int J Tuberc Lung Dis,2000,4 (2):237～91
[14]赵雁林,尚美.我国结核病实验室诊断的现状[J].中华检验医学杂志,2007,30(7)：725-728.
[15]张诩,卢建平.四种结核分枝杆菌检测方法的临床应用评价[J].中国防痨杂志,2006,28(1)：16-17.
[16]梁晓海,黄平东.实时荧光定量聚合酶链反应法检测肺结核患者痰标本价值的对照研究[J].现代中西医结合杂志,2006,15(3)：287.
[17]胡海利.荧光双标记定量检测对诊断结核病的临床意义[J].上海医药,2002,23(9)：411-412.
[18]张灵霞,庄玉辉,杨华卫,等.两种DNA探针杂交检测结核分枝杆菌的研究[J].中华检验医学杂志,2001,2(24)：106
[19]张津,赵顺英,迟巍,等.结核分支杆菌多抗原蛋白芯片对儿童结核病的诊断价值[J].中国循证儿科杂志,2008：3(1)：9-14.
[20]Lalbani A. Spotting latent infection:the path to better tuberculosis control.Thorax,2003,58:916～918
[21]Hernandez-Garduno E. Predictive value of the tuberculin skin test and theQuantiFERON-TB GOLD In-Tube assay for the development of active tuberculosis disease.Am J Respir Crit Care Med.2008 Dec15;178(12):1282
[22]Manuel O, Kumar D. QuantiFERON-TB Gold assay for the diagnosis of latent tuberculosis infection.Expert Rev Mol Diagn.2008 May; 8(3):247～56
[23]Kanunfre KA,Leite OH,Lopes MI,et al. Enhancement of diagnosis of latent tuberculosis infection release assay for pulmonary tuberculosis.Clin Vaccine Immunol. 2008 Jun; 15(6):1028-30
[24]Song KH,Jeon JH,Park WB,et al. Usefulness of the whole-blood interferon-gamma release assay for diagnosis of extrapulmonary tuberculosis.Dian Microbiol Infect Dis.2008 Dec 11
[25]Liebeschuetz S, Bamber S, Ewer K, et al. Diagnosis of tuberculosis in South African children with a T-cell-based assay:a prospective cohort study [J].Lancet,2004, 364 (9452):2196-2203.
[26]Zar HJ, Hanslo D, Apolles P, et al. Induced sputum versus gastric lavage for microbiological confirmation of pulmonary tuberculosis in infants and young children: aprospective study. Lancet,2005,365:130～134.
[27]Nicol MP, Pienaar D, Wood K, et al.Enzyme-linked immunospot assay response to early secretory antigenic target 6,culture filtrate protein 10 and purified protein derivative among children with tuberculosis:implications for diagnosis and monitoring of therapy [J]. Clin Infect Dis,2005,40 (9):1301-1308.
[28]张卫平,韩文,董菊,等.酶联免疫斑点技术对小儿结核病的诊断价值[J].实用儿科临床杂志,2009,24(4)：277-279.
[29]孙琳,焦伟伟,赵顺英,等.ELISPOT检测技术在儿童结核病诊断中的应用[J].标记免疫分析与临床,2008,15(6)：349-351.