ESAT-6/CFP-10融合蛋白的抗原性分析及其对诊断结核临床应用价值

目的 分析ESAT-6/CFP-10融合蛋白作为结核抗原的特性,并探讨以其作为抗原的酶联免疫斑点实验(ELISPOT)在结核诊断中的应用价值.方法 以ESAT-6/CFP- 10融合蛋白为刺激抗原的EIISPOT法(ESAT-6/CFP-10-ELISPOT)检测经结核特异性抗原刺激后分泌γ干扰素的效应T淋巴细胞数量方法,测定65例单纯结核病患者,16例HIV/结核双重感染患者,20例HIV感染患者,32例非结核呼吸道疾病患者,30名健康体检者外周血单个核细胞中结核菌抗原特异的T淋巴细胞的频率.结果 ESAT-6/CFP- 10-ELISPOT与结核菌素皮肤试验(TST)在所有81例结核患者中的比较,ESAT-6/CFP- 10-ELISPOT阳性率98.8％,TST阳性率为56.8％;ESAT-6/CFP-10-ELISPOT在涂阳结核组、涂阴结核组、HIV/结核双重感染组阳性率分别为100.0％、97.1％、100.0％,其敏感性远高于痰涂片检查,且各组结果差异无统计学意义;20例HIV感染组有1例阳性,非结核呼吸道疾病患者组与健康对照组均为阴性,提示ESAT-6/CFP- 10-ELISPOT方法的特异度为98.8％.结论 ESAT-6/CFP-10融合蛋白可以很好的刺激效应T淋巴细胞分泌γ干扰素,适合作为结核诊断中的特异性抗原,因而可以用于ELISPOT的检测,ESAT-6/CFP- 10-ELISPOT对结核诊断有应用价值.     

Screening for tuberculosis among 2381 household contacts of sputum-smear-positive cases in The Gambia

Contact investigation is a key component of tuberculosis (TB) control in developed, but not developing, countries. We aimed to measure the prevalence of TB among household contacts of sputum-smear-positive TB cases in The Gambia and to assess the sensitivity of an enzyme-linked immunospot (ELISPOT) assay in this regard. Household contacts of adult smear-positive TB patients were assessed by questionnaire, purified protein derivative (PPD) skin test, ELISPOT assay, physical examination, chest X-ray and sputum/gastric aspirate. Thirty-three TB cases were identified from 2174 of 2381 contacts of 317 adult smear-positive pulmonary TB patients, giving a prevalence of 1518/100000. The cases identified tended to have milder disease than those passively detected. The sensitivity of ESAT-6/CFP-10 ELISPOT test as a screening test for TB disease was estimated as 71%. Fifty-six per cent of contacts with a PPD skin test result >or=10mm induration had detectable responses to ESAT-6/CFP-10 by ELISPOT; 11% with a negative PPD skin test (<10mm) had a positive ESAT-6/CFP-10 response. Active screening for TB among contacts of TB patients may have a role in TB control in The Gambia. These individuals are a high-risk group, and the disease identified is less advanced than that found through passive case detection. An ELISPOT assay was relatively insensitive as a screening test for TB.     

Analysis of complex formation and immune response of CFP-10 and ESAT-6 mutants

ESAT-6 and CFP-10 form a 1:1 heterodimeric complex which contributes to the virulence of Mycobacterium tuberculosis H37Rv. Based on the structure of CFP-10-ESAT-6 complex, we have selected four point mutations each of CFP-10 and ESAT-6 and have analyzed complex formation for the 25 possible combinations between wild-type and mutant CFP-10 and ESAT-6 proteins. We observed that the mutations L25R or F58R of CFP-10 and L29D or L65D of ESAT-6 lead to disruption of complex formation. We have evaluated the immunogenic responses of the wild-type and mutant CFP-10 and ESAT-6 proteins, the wild-type CFP-10-ESAT-6 complex, six complex-forming and two non-complex-forming combinations of wild-type/mutant CFP-10 and ESAT-6 proteins. CFP-10 mutants I21R, L25R, and W43R were found to have better immunogenic potential than wt-CFP-10, while none of the ESAT-6 mutants were better than wt-ESAT-6. Very interestingly, we have discovered that the non-complex-forming mixture of CFP-10-I21R and ESAT-6-L29D gives a strong immunogenic response.     

结核分枝杆菌模拟抗原的筛选及初步应用

目的 通过对甲期分泌抗原-6(ESAT-6)及培养基滤过蛋白-10(CFP-10)肽库的结核分枝杆菌模拟抗原的筛选,旨在建立一个临床鉴别诊断结核分枝杆菌感染的新方法.方法 基于蛋白ESAT-6和CFP 10的序列,随机设计合成一组1 9肽库,通过γ-干扰素释放试验筛选特异性结核分枝杆菌抗原模拟多肽,并研究确立其工作浓度.结果 经过多轮筛选获得3条特异性模拟多肽,分别是P1MG、P8NV、P11LD,灵敏度分别为93.3％、90.0％、80.0％,特异性均＞90.0％;工作浓度均为2 μg/ml;将3种模拟多肽等浓度混合作为刺激原,初步临床验证试验表明,其灵敏度为95.3％,特异性为96.2％.结论 基于蛋白ESAT-6和CFP-10的序列设计、筛选、制备的混合型模拟多肽抗原,有望建立一种临床结核分枝杆菌鉴别诊断的新方法.     

结核感染T淋巴细胞γ干扰素释放试验在骨关节结核诊断中的应用

目的探讨结核感染T淋巴细胞γ干扰素释放试验(斑点试验,T-SPOT.TB)在骨关节结核中的应用价值。 方法回顾性分析2016年1月至2018年3月在我院骨外科行T-SPOT.TB检测的127疑似骨关节结核感染病例,分析T-SPOT.TB在骨关节结核中的诊断性能;比较在不同部位骨关节结核的阳性率差异;同时比较以早期分泌抗原靶6 (early secreted antigenic target 6,ESAT-6)和培养滤液蛋白10 (culture filtrate protein 10,CFP-10)诊断骨关节结核的阳性率差异。 结果T-SPOT.TB检测的正确率、灵敏度、特异度、阳性预测值、阴性预测值分别为86.61%(110/127)、83.33%(35/42)、88.24%(75/85)、77.78%(35/45)、91.46%(75/82);且在不同类型骨关节结核的阳性率差别无统计学意义(χ²=1.72,P>0.05);单独以ESAT-6和CFP-10抗原的点数诊断骨关节结核阳性率为69.04%、61.90%,差异无统计学意义(χ²=0.27,P>0.05),两者联合诊断阳性率(83.33%)均高于ESAT-6和CFP-10抗原单独诊断的阳性率,差异有统计学意义(χ²=4.16、7.11,P<0.05)。 结论T-SPOT.TB在骨关节结核诊断中具有重要的应用价值,值得临床推广。     

优化T-SPOT. TB在区分脊柱结核与其他脊柱感染中的诊断效能

目的 探讨结核感染T细胞斑点试验(T-SPOT.TB)在脊柱结核(STB)鉴别诊断中的效能,并通过受试者工作特征(ROC)曲线最佳截断值优化诊断效能。方法 收集2010年1月—2019年5月某院脊柱感染患者的临床资料,包括术前T-SPOT.TB检测结果、白细胞计数、C-反应蛋白、血沉、降钙素原和结核抗体等相关数据,根据诊断标准进行临床诊断,分析T-SPOT.TB在术前诊断STB与其他脊柱感染中的灵敏度和特异度,评价优化后的T-SPOT.TB指标的诊断效能。结果 共纳入132例患者,其中78例(59.09%)为STB,54例(40.91%)为非结核脊柱感染。T-SPOT.TB在鉴别诊断STB方面的灵敏度为67.68%,特异度为66.67%。单因素logistic回归分析显示,与非结核脊柱感染比较,T-SPOT.TB检测诊断STB的OR值为4.188(95%CI:1.847～9.974,P<0.001)。优化T-SPOT.TB评价指标,通过绘制ROC曲线,确定ESAT-6、CFP-10、CFP-10+ESAT-6在STB和非结核脊柱感染鉴别诊断中的最佳截断值,分别为12.5、19.5...     

Immunogenicity and efficacy analyses of EPC002, ECA006, and EPCP009 protein subunit combinations as tuberculosis vaccine candidates

Tuberculosis (TB) is the leading cause of death from infectious diseases worldwide, and developing a new TB vaccine is a priority for TB control. Combining multiple immunodominant antigens to form a novel multicomponent vaccine with broad-spectrum antigens to induce protective immune responses is a trend in TB vaccine development. In this study, we used T-cell epitope-rich protein subunits to construct three antigenic combinations: EPC002, ECA006, and EPCP009. Fusion expression of purified protein EPC002f (CFP-10-linker-ESAT-6-linker-nPPE18), ECA006f (CFP-10-linker-ESAT-6-linker-Ag85B), and EPCP009f (CFP-10-linker-ESAT-6-linker-nPPE18-linker-nPstS1) and recombinant purified protein mixtures EPC002m (mix of CFP-10, ESAT-6, and nPPE18), ECA006m (mix of CFP-10, ESAT-6, and Ag85B), and EPCP009m (mix of CFP-10, ESAT-6, nPPE18, and nPstS1) were used as antigens, formulated with alum adjuvant, and the immunogenicity and efficacy were analyzed using immunity experiments with BALB/c mice. All protein-immunized groups elicited higher levels of humoral immunity, including IgG and IgG1. The IgG2a/IgG1 ratio of the EPCP009m-immunized group was the highest, followed by that of the EPCP009f-immunized group, which was significantly higher than the ratios of the other four groups. The multiplex microsphere-based cytokine immunoassay revealed that EPCP009f and EPCP009m induced the production of a wider range of cytokines than EPC002f, EPC002m, ECA006f, and ECA006m, which included Th1-type (IL-2, IFN-γ, TNF-α), Th2-type (IL-4, IL-6, IL-10), Th17-type (IL-17), and other proinflammatory cytokines (GM-CSF, IL-12). The enzyme-linked immunospot assays demonstrated that the EPCP009f- and EPCP009m-immunized groups had significantly higher amounts of IFN-γ than the other four groups. The in vitro mycobacterial growth inhibition assay demonstrated that EPCP009m inhibited Mycobacterium tuberculosis (Mtb) growth most strongly, followed by EPCP009f, which was significantly better than that of the other four vaccine candidates. These results indicated that EPCP009m containing four immunodominant antigens exhibited better immunogenicity and Mtb growth inhibition in vitro and may be a promising candidate vaccine for the control of TB.     

7种结核分枝杆菌重组蛋白的纯化及血清学特性

目的分离纯化结核分枝杆菌重组蛋白38-kDa、16-kDa、Mtb81、ESAT-6、CFP10、Rv3425、Rv3391,并比较7种蛋白的免疫学特性,评价其在结核血清学诊断中的应用价值。方法利用间接酶联免疫吸附试验评价确诊结核病人、非结核病人、健康人血清中7种重组蛋白的抗原性。结果成功分离纯化了7种重组蛋白;酶联免疫吸附试验结果表明,7种抗原均能有效区分确诊结核病人、非结核病人和健康人,其中阳性率最高的是ESAT-6为47.3%,特异性最高的是Rv3391为98.3%。联合抗原中阳性率和特异性最高的是CFP10+ESAT-6+Rv3391抗原组合。结论单一抗原中ESAT-6具有较高的免疫原性,联合抗原中CFP10+ESAT-6+Rv3391抗原具有很高的阳性率和特异性,在结核诊断具有很高的诊断潜力。     

难治性肺结核患者PBMCs结核分枝杆菌特异性Th1细胞因子反应低下

目的研究难治性肺结核患者外周血单个核细胞（PBMCs）经结核分枝杆菌特异性抗原肽（ESAT-6,CFP．10）刺激后代表性Thl细胞因子IFN一叫、IL-2和TNF．d的表达特点及意义。方法纳入67例初治肺结核患者、28例难治性肺结核患者和25位结核分枝杆菌潜伏感染者,分离外周血单个核细胞（PBMCs）,用ESAT-6,CFP-10肽库刺激PBMCs,采用FlowCytomix流式技术检测细胞培养液上清中细胞因子IFN-γ、IL-2和TNF-α的浓度。结果PBMCs经结核分枝杆菌特异性抗原肽刺激后,IFN-γ、IL-2和TNF-α均因刺激而升高,初治组的三个因子的水平均显著高于难治组和潜伏感染组（与难治组比较,Mann-WhitneyU值=119．0,282．5、339．5,P均〈0．01;与潜伏感染组比较,Mann-WhitneyU值=387．0,477．0、374．5,P=0．0002,0．0033、0．0001）,难治组IFN-γ和IL-2的水平显著低于潜伏感染组（Mann-WhitneyU值=105．5、162．5,P〈0．01）,TNF-α的水平在难治组和潜伏感染组间的差异无统计学意义（Mann-WhitneyU值=265．0,P=0．2695）。结论难治性肺结核患者结核分枝杆菌特异性Thl细胞因子反应低下,针对结核病的免疫治疗可能对难治性肺结核发挥更大的作用。     

结核分枝杆菌H37Rv株重组蛋白CFP-10的表达及纯化研究

目的表达、纯化结核分枝杆菌H37Rv株重组蛋白CFP-10(rCFP-10),为结核病血清学诊断价值的研究及亚单位疫苗的研制提供物质基础。方法重组质粒pET23b-CFP-10转化E.coli表达菌株BL21(DE3)pLysE,IPTG诱导rCFP-10表达。经SDS-PAGE电泳和Western印迹法鉴定后,优化表达条件用镍离子螯合亲和层析柱HisTrapTMHP纯化重组蛋白,最后用斑点金免疫渗滤法初步评价纯化蛋白的免疫反应性。结果成功构建了重组质粒pET23b-CFP-10,并且rCFP-10以可溶性形式高效表达,表达量约占菌体总蛋白的10%。经亲和层析后得到高纯度有免疫反应性的重组蛋白(纯度约为98.5%)。结论高纯度有免疫反应性的rCFP-10为新型亚单位疫苗的研发及其结核病血清学诊断价值的研究奠定了基础。     

Immunogenicity of a recombinant Mycobacterium bovis bacille Calmette-Guèrin expressing malarial and tuberculosis epitopes

Recombinant Mycobacterium bovis bacille Calmette-Guèrin (rBCG) expressing the malarial epitopes F2R(II)EBA and (NANP)3 as well as two T cell epitopes of the M. tuberculosis ESAT-6 antigen, generated in favour of mycobacterium codon usage elicited specific immune response against these epitopes. Immunised Balb/c mice demonstrated an increase in almost all of the IgG subclasses against both malarial epitopes and enhanced splenocyte proliferative response against the malarial epitopes as well as selected peptides of ESAT-6. Furthermore, flow cytometric analyses showed elevated numbers of CD4+ lymphocytes expressing IFN-gamma and IL-2 against the ESAT-6 peptides, suggesting a specific Th1-mediated response. This study demonstrated that expressing malarial and TB epitopes in a single rBCG construct induced appropriate humoral and cellular immune response against immunogenic epitopes from both organisms.     

活动性肺结核病患者外周血ESAT6、Ag85B水平与细胞因子相关性分析

目的 检测结核病患者外周血血清中结核分枝杆菌特异性分泌蛋白ESAT-6、Ag85B及细胞因子IL-10、TGF-β1、IL-35的水平,探讨分析ESAT-6、Ag85B水平与Treg细胞的增殖之间的相关性。 方法 ELISA方法检测活动性结核病患者和健康志愿者外周血中结核分枝杆菌特异性分泌蛋白ESAT-6、Ag85B及细胞因子IL-10、TGF-β1、IL-35的表达水平,并用统计学方法分析血清中特异性分泌蛋白ESAT-6、Ag85B的浓度与Treg细胞刺激分泌的细胞因子IL-10、TGF-β1、IL-35的水平的相关性。 结果 活动性结核病患者血清中特异性分泌蛋白ESAT-6、Ag85B和细胞因子IL-10、TGF-β1的浓度与正常对照组相比均显著升高(P<0.001),但IL-35的浓度与正常对照组相比却显著降低,差异有统计学意义(P<0.001)。活动性结核病患者血清中结核分枝杆菌特异性分泌蛋白ESAT-6和Ag85B的水平与Treg细胞刺激活化分泌的细胞因子IL-10、TGF-β1、IL-35之间的相关性分析结果显示差异无统计学意义(P>0.05)。 结论 活动性结核病患者血清中结核分枝杆菌特异性分泌蛋白ESAT-6和Ag85B的水平均明显高于健康对照组,提示活动性结核病患者Treg细胞的高水平表达可能与结核分枝杆菌毒力系统有关。     

The diagnosis of latent tuberculosis infection (LTBI): currently available tests,future developments,and perspectives to eliminate tuberculosis (TB)

Introduction:Despite great efforts, tuberculosis (TB) is still a major public health threat worldwide. For decades, TB control programs have focused almost exclusively on infectious TB active cases. However, it is evident that this strategy alone cannot achieve TB elimination. To achieve this objective a comprehensive strategy directed toward integrated latent tuberculosis infection (LTBI) management is needed. Recently it has been recognized that LTBI is not a stable condition but rather a spectrum of infections (e.g., intermittent, transient or progressive) which may lead to incipient, then subclinical, and finally active TB disease.Aim:Provide an overview of current available LTBI diagnostic test including updates, future developments and perspectives.Results:There is currently no test for the direct identification of live MT infection in humans. The diagnosis of LTBI is indirect and relies on the detection of an immune response against MT antigens, assuming that the immune response has developed after a contact with the biological agent. Tuberculin skin test (TST) and interferon gamma release assays (IGRAs) are the main diagnostic tools for LTBI, however, both present strengths and limitations. The most ancient diagnostic test (TST) can be associated with several technical errors, has limited positive predictive value, is being influenced by BCG vaccination and several conditions can reduce the skin reactivity. Notwithstanding these limitations, prompt identification of TST conversion, should orientate indications for preventive therapy of LTBI. IGRAs have superior specificity, are not affected by M. bovis, BCG vaccination and other environmental mycobacteria. However, they present some logistical and organisational constraints and are more expensive. Currently, the WHO guidelines recommend that either a TST or an IGRA can be used to detect LTBI in high-income and upper middle-income countries with estimated TB incidences less than 100 per 100,000 population. Two skin tests (C-TB and Diaskintest), using only two specific M. tuberculosis antigens (ESAT-6 and CFP-10) instead of the tuberculin solution, have recently been developed but, to date, none of these tests is available on the European market.Conclusion:Early identification and treatment of individuals with LTBI is an important priority for TB control in specific groups at risk within the population: this is of crucial meaning in recently infected cases both at the community level and in some occupational settings. Currently there is no gold standard test for LTBI: an improved understanding of the available tests is needed to develop better tools for diagnosing LTBI and predicting progression to clinical active disease.Key words: Tuberculosis, TB, latent tuberculosis infection, LTBI, diagnostic tests, elimination, occupational health, public health     

时间分辨荧光免疫法检测脑脊液结核分枝杆菌特异性抗原的诊断价值

目的 建立时间分辨荧光免疫分析法检测脑脊液中结核分枝杆菌(MTU)特异性抗原培养滤液蛋白10(CFP10)和6000-早期分泌性抗原靶(ESAT-6),并对该方法的性能进行评价,提高该抗原在结核性脑膜炎中的诊断价值.方法 收集70例患者的脑脊液,其中23例为结核性脑膜炎,47例为非结核性脑膜炎,建立时间分辨荧光免疫分析法检测脑脊液中ESAT-6-CFP10抗原,并与酶联免疫吸附法等检测MTU或菌体物质进行比较分析.结果 时间分辨荧光免疫分析法检测ESAT-6-CFP10抗原的检测限为0.7 ng/ml；批内和批间CV分别平均为3.2％和4.7％,平均回收率为97.4％,临界值取12.2 ng/ml时,敏感度为91.3％,特异度为95.7％；该方法的敏感度和(或)特异度要高于酶联免疫吸附法、抗酸染色、MTU培养等.结论 应用时间分辨荧光免疫分析法,检测脑脊液中ESAT-6-CFP10抗原对诊断结核性脑膜炎有很高的敏感度和特异度,提高了MTU特异性抗原用于临床结核病诊断的敏感性.     

An ESAT-6:CFP10 DNA vaccine administered in conjunction with Mycobacterium bovis BCG confers protection to cattle challenged with virulent M. bovis

The potency of genetic immunization observed in the mouse has demonstrated the utility of DNA vaccines to induce cell-mediated and humoral immune responses. However, it has been relatively difficult to generate comparable responses in non-rodent species. The use of molecular adjuvants may increase the magnitude of these suboptimal responses. In this study, we demonstrate that the co-administration of plasmid-encoded GM-CSF and CD80/CD86 with a novel ESAT-6:CFP10 DNA vaccine against bovine tuberculosis enhances antigen-specific cell-mediated immune responses. ESAT-6:CFP10+GM-CSF+CD80/CD86 DNA vaccinated animals exhibited significant (p<0.01) antigen-specific proliferative responses compared to other DNA vaccinates. Increased expression (p< or =0.05) of CD25 on PBMC from ESAT-6:CFP10+GM-CSF+CD80/CD86 DNA vaccinates was associated with increased proliferation, as compared to control DNA vaccinates. Significant (p<0.05) numbers of ESAT-6:CFP10-specific IFN-gamma producing cells were evident from all ESAT-6:CFP10 DNA vaccinated animals compared to control DNA vaccinates. However, the greatest increase in IFN-gamma producing cells was from animals vaccinated with ESAT-6:CFP10+GM-CSF+CD80/CD86 DNA. In a low-dose aerosol challenge trial, calves vaccinated as neonates with Mycobacterium bovis BCG and ESAT-6:CFP10+GM-CSF+CD80/CD86 DNA exhibited decreased lesion severity in the lung and lung-associated lymph nodes following viruluent M. bovis challenge compared to other vaccinated animals or non-vaccinated controls. These data suggest that a combined vaccine regimen of M. bovis BCG and a candidate ESAT-6:CFP10 DNA vaccine may offer greater protection against tuberculosis in cattle than vaccination with BCG alone.     

Mycobacterium tuberculosis PPE protein Rv0256c induces strong B cell response in tuberculosis patients

Tuberculosis (TB) is one of the most important diseases of humans and major public health problem worldwide. Early and accurate diagnosis of TB is necessary for the treatment, prevention, and control of TB. Therefore, it is important to identify suitable antigens that can differentiate active tuberculosis patients from BCG-vaccinated individuals. In the present study, we have used Rv0256c (PPE2) protein of Mycobacterium tuberculosis to screen the sera of infected patients belonging to different clinical TB presentations, and BCG-vaccinated clinically healthy individuals by enzyme immunoassay. Our results demonstrated that Rv0256c displayed stronger and specific immunoreactivity against the sera obtained from clinically active tuberculosis patients compared to PPD and ESAT-6 and could differentiate the TB-patients from the BCG-vaccinated controls. Importantly, Rv0256c was also found to detect even the extrapulmonary and smear-negative pulmonary cases which often are tedious and difficult to detect using conventional diagnostic methods. This study suggests that Rv0256c can be used as a potential marker for the serodiagnosis of tuberculosis patients.     

T细胞酶联免疫斑点法对艾滋病合并结核病患者的诊断价值研究

目的探讨T细胞酶联免疫斑点法(T.SPOT-TB)在艾滋病合并结核病患者中的诊断价值。方法应用T.SPOT-TB对57例诊断明确的艾滋病合并结核病患者的血液样本进行检测并分析不同因素对T.SPOT-TB诊断价值的影响,同时对所有患者做结核菌素皮试(TST)试验进行比较。结果在艾滋病合并结核病的所有患者、肺结核组、肺外结核组、肺结核初治组、肺结核复治组的各组患者中,T.SPOT-TB的阳性检出率分别为80.70%、77.41%、88.46%、72.73%、80.00%;而TST试验的阳性检出率分别为22.81%、25.81%、19.23%、54.54%、10.00%。在患者总体、肺结核组、肺外结核组、复治组中,T.SPOT-TB检测的阳性检出率均高于TST试验(P0.05)。结论对于MTB/HIV双重感染患者,T.SPOT-TB检测的敏感性高于目前常用的TST试验,且不受结核发病部位和初复治类型的影响,适用于我国艾滋病合并结核病的快速诊断。     

应用酶联免疫斑点法快速诊断儿童结核分枝杆菌感染的研究

目的:探讨基于ESAT-6和CFP-10抗原肽的酶联免疫斑点技术(Enzyme-link immunospot essay,ELISPOT)在临床实践中诊断儿童结核分枝杆菌感染的临床价值。方法:应用ELISPOT技术检测14例确诊的儿童肺结核病例和16例健康儿童对照的外周血单个核细胞的INF-γ,同时对所有研究对象实施结核菌素皮肤实验(Tuberculin Skin Test,TST),探讨ELIS-POT技术在快速诊断儿童结核分枝杆菌感染的临床应用价值。结果: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)。TST灵敏度为71.4%(95%CI:45.4～88.3),特异度为56.3%(95%CI:33.2～76.9)。ELISPOT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)。ELISPOT和TST检测结果总一致性较差(κ=0.35)。在活动型结核病组中,ELISPOT和TST一致性较差(κ=0.18),类似地,在对照组中,ELIS-POT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)。结论:ELISPOT诊断儿童活动性结核病准确性高于TST。ELISPOT具有较高的诊断灵敏度和特异度,其特异度不受儿童的卡介苗接种的影响,适合作为临床诊断儿童结核分枝杆菌感染的辅助诊断技术,有助于感染的儿童得到更快速的诊断和治疗。     

Immunogenicity of late stage specific peptide antigens of Mycobacterium tuberculosis

Global burden of latent TB infection comprises one-third of the world population. Identifying potential Mycobacterium tuberculosis (Mtb) latency associated antigens that can generate protective immunity against the pathogen is crucial for designing an effective TB vaccine. Usually the immune system responds to a small number of amino acids as MHC Class I or Class II peptides. The precision to trigger epitope specific protective T-cell immune response could therefore be achieved with synthetic peptide-based subunit vaccine.In the present study we have considered an immunoinformatic approach using available softwares (ProPred, IEDB, NETMHC, BIMAS, Vaxijen2.0) and docking and visualizing softwares (CABSDOCK, HEX, Pymol, Discovery Studio) to select 10 peptides as latency antigens from 4 proteins (Rv2626, Rv2627, Rv2628, and Rv2032) of DosR regulon of Mtb. As Intracellular IFN-γ secreted by T cells is the most essential cytokine in Th1 mediated protective immunity, these peptides were verified as potential immunogenic epitopes in Peripheral Blood Mononuclear Cells (PBMCs) of 10 healthy contacts of TB patients (HTB) and 10 Category I Pulmonary TB patients (PTB).The antigen-specific CD4 and CD8 T cells expressing intracellular IFN-γ were analyzed using monoclonal antibodies in all subjects by multi-parameter flow cytometry.Both, PTB and HTB individuals responded to DosR peptides by showing increased frequency of IFN-γ⁺CD4 and IFN-γ⁺CD8 T cells. The T-cell responses were significantly higher in PTB patients in comparision to the HTB individuals. Additionally, our synthetic peptides and pools showed higher frequencies of IFN-γ⁺CD4 and IFN-γ⁺CD8 T cells than the peptides of Ag85B.This pilot study can be taken up further in larger sample size which may support the untapped opportunity of designing Mtb DosR inclusive peptide based post-exposure subunit vaccine.     

结核分枝杆菌CFP10 ESAT6重组融合蛋白抗原血清学诊断价值

目的研究重组CFP10 ESAT6蛋白的抗原性,评价其在结核病血清学诊断中的价值,探寻更有效的结核病诊断试剂。方法应用基因工程技术表达、纯化rCFP10 ESAT6蛋白,通过酶联免疫吸附试验检测192例健康者和210例结核病患者血清与纯化rCFP10 ESAT6的抗体反应。结果 rCFP10 ESAT6蛋白在大肠埃希菌细胞内以包涵体形式高效表达,表达量约占菌体总蛋白的25％,分子量约为28 kD,具有良好的抗原特异性。在210例结核病患者血清中,103例菌阳患者和107例菌阴患者抗rCFP10 ESAT6抗体的阳性率分别为30.10％(31/103)和28.97％(31/107),总的灵敏度为29.52%(62/210);在192例健康者血清中,95例PPD阴性者和97例PPD阳性者抗rCFP10 ESAT6抗体的阳性率分别为2.11%(2/95)和6.19%(6/97),总的特异性为95.83%(184/192)。结论rCFP10 ESAT6蛋白可能成为结核病血清学诊断的组合抗原之一。