潜伏结核感染相关蛋白抗原的研究进展

结核病是由结核分枝杆菌(Mtb)所致的以呼吸系统感染为主的慢性传染病,潜伏结核感染(LTBI)高危者是结核病预防的关键人群,针对LTBI的早期诊断和高危人群的预防性治疗能有效减少活动性结核病(ATB)发病率.但现有的诊断方法诊断LTBI的特异度较低,因此建立快速、敏感、高效的LTBI诊断方法仍是目前结核病控制工作中面临...     

结核分枝杆菌潜伏感染相关抗原的研究进展

结核病是全世界范围内危害人类健康的主要传染病之一。结核分枝杆菌潜伏感染者是结核病的主要来源,早期发现、诊断并有效治疗潜伏感染是有效控制结核病蔓延的重要措施之一。目前,结核分枝杆菌潜伏感染抗原主要包括与结核分枝杆菌缺氧、营养缺乏及与结核分枝杆菌复苏、再激活相关的蛋白。有些潜伏感染相关抗原具有良好的T细胞免疫能力,尤其更易在被潜伏感染人群中识别,有望成为新型的结核分枝杆菌潜伏感染诊断标志物及潜伏感染候选疫苗;有些潜伏感染相关抗原对B淋巴细胞具有较强免疫原性,在结核病体液免疫诊断方面有潜在诊断价值。这些潜伏感染相关蛋白在未来结核分枝杆菌潜伏疫苗及诊断试剂开发中具有良好的应用前景。     

NAP3在结核病诊断中的价值

目的：研究肺结核患者外周血单个核细胞(PBMCs)经结核特异性抗原刺激后中性粒细胞激活蛋白3(NAP3)的表达情况并与结核潜伏感染(latent tuberculosis infection,LTBI)者及非结核感染健康对照者进行比较。方法提取6例活动期肺结核患者和6例 LTBI 个体 PBMCs,用结核分枝杆菌特异性抗原肽刺激后收集细胞,采用基因芯片分析方法比较两组 NAP3表达情况。然后提取60例活动期肺结核患者和60名 PPD 阳性健康个体(根据γ干扰素表达情况将其分成 LTBI 组和非结核感染健康对照组)的 PBMCs,用结核分枝杆菌特异性抗原肽刺激后,采用酶联免疫吸附试验(ELISA)定量检测刺激前、后细胞培养液上清中 NAP3的浓度。结果基因芯片分析发现活动期肺结核患者 NAP3表达明显高于 LTBI 组(U =1.000,P =0.0043)。ELISA 分析结果发现特异性抗原刺激前细胞培养液上清中3组的 NAP3浓度差异无统计学意义(F =0.7341,P =0.4821),而刺激后只有活动期肺结核组和非结核感染健康对照组之间的 NAP3浓度相比差异有统计学意义(P =0.002)。结论高表达的 NAP3可作为诊断结核的指标之一,但是无法区分潜伏感染和活动期感染病例。     

结核分枝杆菌Rv3425-Rv1168c蛋白融合表达与血清学评价

目的 原核表达结核分枝杆菌Rv3425-Rv1168c融合蛋白并进行纯化,通过酶联免疫吸附试验(ELISA)方法评价重组蛋白在结核病血清学诊断中的价值。方法 以结核分枝杆菌H37Rv基因组为模板,通过重叠PCR扩增得到Rv3425-Rv1168c全核酸序列克隆至表达载体pET-24b中,转入大肠杆菌BL21(DE3)进行诱导、表达和纯化,通过ELISA检测100份确诊结核病人、20例非结核呼吸疾病患者、100份健康人血清,评价融合蛋白进行临床血清学诊断的可行性。结果 Rv3425-Rv1168c融合蛋白在原核系统内获得高表达,纯化的融合蛋白经ELISA方法测定,在血清学实验中敏感性为54%,特异性为95%。结论 Rv3425-Rv1168c融合蛋白具有较高的抗原特异性和免疫原性,在结核病血清学诊断方面具有很大的应用价值。     

IL-8 mRNA定量检测在活动性结核病鉴别诊断中的价值

目的研究活动性肺结核患者外周血单个核细胞(PBMCs)经结核特异性抗原刺激后白介素-8(interleukin-8,IL-8)的mRNA表达情况并与结核潜伏感染(latent tuberculosis infection,LTBI)及非结核感染健康对照组进行比较。方法提取研究对象的PBMCs,经特异性抗原肽刺激后,收集细胞并提取总RNA然后经实时荧光定量PCR检测技术比较各组IL-8 mRNA表达情况。然后以敏感性(sensitivity)为纵坐标,1-特异性(1-specificity)为横坐标绘制结核组和LTBI组相比较的ROC曲线。结果经结核特异性抗原刺激后,结核组PBMCs中IL-8基因mRNA的相对表达量明显高于LTBI和健康对照组,差异有统计学意义(P0.05)。ROC曲线下面积为0.72。以3.985为临界值,鉴别活动性结核病和LTBI的敏感性和特异性分别为54.17%和90.00%,此时阳性似然比等于5.42,64.7%的病例诊断准确。结论 IL-8有可能作为新的活动性结核病诊断标志物,有助于活动性结核病与LTBI的鉴别诊断。     

结核分枝杆菌Rv2660c蛋白原核重组表达及其免疫活性研究

目的利用原核表达系统表达、纯化结核分枝杆菌潜伏感染相关蛋白Rv2660c,评价其在血清学方面用于结核病诊断的价值。方法合成Rv2660c全基因核酸序列,构建pET28a-Rv2660c融合表达载体,在原核系统内进行重组表达,亲和层析纯化重组Rv2660c蛋白,采用Western-Blot、ELISA实验分析重组蛋白的免疫反应。结果pET28a-Rv2660c在大肠杆菌内成功表达重组Rv2660c蛋白,SDS-PAGE电泳鉴定重组蛋白分子量为10.2 KD,与预期一致,Western-Blot结果表明重组蛋白Rv2660c只能与活动性结核患者血清反应,出现一条杂交条带;ELISA结果发现重组Rv2660c蛋白与结核患者血清能产生较强的反应,结核病菌阳患者组、菌阴患者组、潜伏感染组以及健康对照组A450平均值分别为0.52、0.48、0.28和0.25。结论重组结核分枝杆菌Rv2660c蛋白具有一定的免疫原性,对活动性结核病的诊断具有一定的价值,但对结核分枝杆菌潜伏感染的诊断没有价值。     

高危人群结核分枝杆菌潜伏感染检测及预防性治疗专家共识

主动发现和预防性治疗是全球终结结核病流行策略下,结核病防治行动的重要举措。我国是全球结核病高负担国家之一,要实现全球终止结核病目标,加强结核分枝杆菌潜伏感染(latent tuberculosis infection,LTBI)者的主动发现,对结核病患者密切接触者进行筛查,对新近感染和免疫力低下LTBI人群给予预防性治疗,是降低LTBI者发病的重要措施。然而,当前业内人士对预防性治疗的适用对象、化学药物预防性治疗方案和预防性治疗效果尚存在较多疑虑和争议,同时,新的诊断技术和预防性治疗方法也在不断研究和应用。有鉴于此,中国防痨协会组织专家编写了《高危人群结核分枝杆菌潜伏感染检测及预防性治疗专家共识》,从LTBI检测原理,以及预防性治疗对象、诊断方法、化学预防和免疫预防等方面进行论述,以供我国结核病防治工作者借鉴和参考。     

活动性结核病候选血清学诊断标志物的研究进展

结核病是由结核分枝杆菌感染引起的慢性传染病.我国是结核病高负担国家,目前新发结核病患者人数已位居全球第三.活动性结核病(ATB)患者是结核病的主要传染源,因此快速、准确地诊断ATB对于控制疾病传播和提高治疗效果尤为重要.相对于影像学和细菌学检查,血清学检测具有特异度更高、耗时更短等优点,而寻找可靠的血清学诊断标志物对于...     

结核分枝杆菌分泌蛋白MPT64的克隆表达和纯化

目的构建结核分枝杆菌分泌蛋白MPT64原核表达载体并进行表达和纯化。方法以结核分枝杆菌标准株H37Rv基因组DNA为模板扩增出MPT64基因,产物经纯化回收后与载体pMD-T连接转化,酶切鉴定,克隆到pET21a原核表达载体,测序鉴定插入序列完全正确者转化大肠杆菌BL21,诱导表达MPT64融合蛋白,利用亲和层析纯化表达产物,SDS-PAGE电泳进行鉴定。结果成功构建MPT64表达载体,SDS-PAGE电泳鉴定成功表达MPT64蛋白。并以此蛋白进行结核抗体的检测,其特异性和敏感性分别为96%和43%。结论成功表达结核分枝杆菌MPT64蛋白,并进行特异性和敏感性的检测,证明重组的MPT64蛋白有希望成为结核病血清学诊断的组合抗原的候选者之一。     

结核分枝杆菌潜伏感染的治疗:利大于弊还是弊大于利

结核病仍然是全球十大死因之一。结核分枝杆菌潜伏感染(LTBI)是指结核分枝杆菌感染人体后,未发生活动性结核病,但具有发展为活动性结核病的风险。文章针对LTBI治疗充分阐述利弊,并结合中国结核病现实状况,提出在中国需要进行逐步的完善相关人群的预防治疗,逐渐由点到面推广LTBI的治疗,把结核病消灭在LTBI状态,降低结核病发病率,达到世界卫生组织提出的2035年消灭结核病的目标。     

Recommendations for the diagnosis and treatment of latent and active tuberculosis in inflammatory joint diseases candidates for therapy with tumor necrosis factor alpha inhibitors: March 2008 update

The Portuguese Society of Rheumatology and the Portuguese Society of Pulmonology have updated the guidelines for the diagnosis and treatment of latent tuberculosis infection (LTBI) and active tuberculosis (ATB) in patients with inflammatory joint diseases (IJD) that are candidates to therapy with tumour necrosis factor alpha (TNFalpha) antagonists. In order to reduce the risk of tuberculosis (TB) reactivation and the incidence of new infections, TB screening is recommended to be done as soon as possible, ideally at the moment of IJD diagnosis, and patient assessment repeated before starting anti-TNFalpha therapy. Treatment for ATB and LTBI must be done under the care of a TB specialist. When TB treatment is indicated, it should be completed prior to starting anti-TNFalpha therapy. If the IJD activity justifies the need for immediate treatment, anti-TNFalpha therapy can be started two months after antituberculous therapy has been initiated, in the case of ATB, and one month after in the case of LTBI. Chest X-ray is mandatory for all patients. If Gohn s complex is present, the patient should be treated for LTBI; healed lesions require the exclusion of ATB. In cases of suspected active lesions ATB should be excluded/confirmed and adequate therapy initiated. Tuberculin skin test, with two units of RT23, should be performed in all patients. If the induration is <5 mm, the test should be repeated within 1 to 2 weeks, on the opposite forearm, and will be considered negative only if the result is again <5 mm. Positive TST implicates LTBI treatment, unless previous proper treatment was provided. If TST is performed in immunossuppressed IJD patients, LTBI treatment should be offered to the patient before starting anti-TNFalpha therapy, even in the presence of a negative test, after risk/benefit assessment.     

Aberrant expression of long non-coding RNAs in peripheral blood mononuclear cells response to tuberculosis in children

We aimed to identify long non-coding RNAs (lncRNAs) aberrantly expressed in peripheral blood mononuclear cells (PBMCs) triggered by active tuberculosis (ATB), latent tuberculosis infection (LTBI), and healthy controls (HC). We examined lncRNAs expression in PBMCs isolated from children with ATB and LTBI, and from HC using RNA sequencing. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were used to explore the biological processes and signaling pathways of aberrantly expressed mRNAs. A total of 348 and 205 lncRNAs were differentially expressed in the ATB and LTBI groups, respectively, compared to the HC group. Compared to the LTBI group, 125 lncRNAs were differentially expressed in the ATB group. Compared to the HC group, 2317 mRNAs were differentially expressed in the ATB group, and 1093 mRNAs were differentially expressed in the LTBI group. Compared to the LTBI group, 2328 mRNAs were differentially expressed in the ATB group. The upregulated mRNAs were mainly enriched in neutrophil activation, neutrophil-mediated biological processes, and positive regulation of immune response in tuberculosis (TB), whereas the downregulated mRNAs were enriched in signaling pathways and structural processes, such as the Wnt signaling pathway and rDNA heterochromatin assembly. This is the first study on the differential expression of lncRNAs in PBMCs of children with TB. We identified significant differences in the expression profiles of lncRNAs and mRNAs in the PBMCs of children with ATB, LTBI, and HC, which has important implications for exploring lncRNAs as novel biomarkers for the diagnosis of TB. In addition, further experimental identification and validation of lncRNA roles could help elucidate the underlying mechanisms of Mycobacterium tuberculosis infection in children.     

结核休眠菌的治疗及控制策略

目前全球结核病疫情严峻,全世界约有1/3的人口存在潜伏结核菌感染。目前主要采用长疗程化疗来彻底清除结核病患者体内的结核菌,但机体仍可能有少量休眠菌潜伏下来,成为以后结核病复发的根源。所以控制潜伏感染的休眠菌至关重要。根据休眠菌在体内的生长周期,将控制休眠菌的方法分为药物直接杀灭潜伏期的结核菌、疫苗阻止休眠菌复苏和制剂或药物主动复苏休眠菌后再予以杀灭,其中主动复苏休眠菌后再予以杀灭时间短,易控制,将是未来控制潜伏感染的主要方法。     

结核分枝杆菌Rv3621c原核表达及免疫功能研究

目的 以结核分枝杆菌(Mycobacterium tuberculosis,M.tb)H37Rv基因组为模板,构建、纯化及鉴定原核表达质粒pPROEX-Rv3621c,通过人群、小鼠试验进行免疫原性评价。方法 构建重组质粒pPROEX-Rv3621c,并以全血干扰素释放分析技术(Whole-blood IFN-γ release assay,WBIA)检测其能否被安徽省淮南市M.tb感染者T细胞特异性识别。rRv3621c混合佐剂MTM[母牛分枝杆菌(M.vaccae),人工合成海藻糖-6'6,二分枝菌酸(TDB),单磷酰脂质A(MPLA)]免疫小鼠后,检测血清中特异性抗体分泌水平、脾细胞中抗原特异性Th1型细胞因子分泌水平及肺脏细胞因子mRNA表达水平。结果 成功构建重组质粒pPROEX-Rv3621c,并使之诱导表达、纯化和鉴定。在rRv3621c蛋白诱导下,活动性结核(Active tuberculosis, ATB)患者外周血淋巴细胞释放的IFN-γ水平明显较高(t=4.813, P<0.01),且ATB患者产生的IFN-γ水平高于潜伏性结核(Latent tuberculosis infection, LTBI)人群(t=4.442, P<0.01)。BCG+Rv3621c/MTM组小鼠产生的特异性抗体滴度水平明显高于Rv3621c/MTM组(P<0.01)和BCG组(P<0.01),Rv3621c/MTM组和BCG+Rv3621c/MTM组小鼠的IgG2a/IgG1比值大于1,明显高于MTM组和BCG组。BCG+Rv3621c/MTM组小鼠均分泌高水平IFN-γ、TNF-α和IL-2。Rv3621c/MTM组小鼠肺脏组织中IFN-γ、TNF-α及iNOS表达水平较高。结论 M.tb感染者外周血T细胞可特异性识别rRv3621c蛋白,rRv3621c混合佐剂MTM可以诱导较强烈的抗原特异性Th1型免疫应答。     

Snapshot of Quantiferon TB gold testing in Northern Mexico

Most people infected with Mycobacterium tuberculosis have an asymptomatic condition named latent tuberculosis. These people do not have bacilli in the corporal secretions and are hard to diagnose by conventional laboratory tests. Diagnosis of latent tuberculosis infection (LTBI) in México is based on the tuberculin skin test (TST). This test has disadvantages, principally because the vaccine containing the Bacille Calmette-Guérin (BCG) is applied to 99% of this population and causes false positive TST outcomes. Recently, interferon-gamma release assays (IGRA) have been demonstrated to be a good test to detect latent tuberculosis with equal or better sensitivity to TST and without interference from BCG. However, in México the IGRA are an uncommon test due to the higher cost compared to TST. The main objective of this work was demonstrate the potential utility of the Quantiferon TB(?) gold in tube (QTB(?)-GIT) test to detect latent TB in a population from northern México. Samples from 106 subjects with close contact, or without contact, with actively infected TB patients were tested to detect LTBI. Our results show a significant difference between individuals in close contact with active TB patients (39.7%) compared to those without contact (3.2%), p < 0.01. The concordance between TST and QTB(?)-GIT was poor (κ = 0.31). Our preliminary results show that the QTB(?)-GIT has better capacity than TST to detect latent tuberculosis infection.     

Spectrum of manifestations of Mycobacterium tuberculosis infection in primates infected with SIV

To characterize the manifestations of coinfection with M. tuberculosis and SIV infection, we studied 12 SIV-infected rhesus monkeys, six of which were infected intrabronchially with a low dose of Mycobacterium tuberculosis H37Rv. In the six coinfected animals, M. tuberculosis antigen-stimulated lung and blood cells produced high concentrations of IFN-gamma but not IL-4 8-16 weeks after infection. Of the three coinfected animals with high levels of plasma viremia, two developed disseminated tuberculosis and the other died of bacterial peritonitis. Of three coinfected animals with moderate levels of plasma viremia, two had no clinical or radiographic evidence of tuberculosis or progressive SIV infection for 6 months after infection. At neuropsy, pulmonary granulomata were observed and acid-fast organisms or M. tuberculosis were present. These clinical, immunologic and pathologic findings are consistent with those in humans with latent tuberculosis infection (LTBI), and suggest that a model of LTBI in SIV-infected primates can be developed. Such a model will permit delineation of the immunologic and microbial factors that characterize LTBI in HIV-infected persons.     

结核潜伏感染者全血miR-144-3p、miR-146a-5p的表达

目的探讨结核潜伏感染者全血miR-144-3p、miR-146a-5p的表达及其与活动性肺结核患者的差异。方法收集结核潜伏感染者30例,活动性肺结核患者30例。收集全血,提取总RNA,利用反转录-荧光定量PCR方法检测miR-144-3p、miR-146a-5p的表达。两组间比较采用t检验。结果结核潜伏感染者全血中miRNA144-3p的表达(2.014±1.48)比活动性肺结核患者(1.056±0.746)明显提高,差异有统计学意义(P<0.05),而结核潜伏感染者全血中miR-146a-5p的表达(1.937±1.109)与活动性肺结核患者(1.469±0.693)相似,差异无统计学意义(P>0.05)。结论 miR-144-3p可能成为诊断结核潜伏感染者的标志物。     

结核潜伏感染诊断新型候选标志物的筛选及临床验证

目的 筛选及临床验证结核潜伏感染(LTBI)新型候选标志物,为研究LTBI诊断新方法提供科学基础。方法 选择2013年1月至2015年12月就诊于遵义医学院附属医院的活动性肺结核患者(ATB组)、LTBI者(LTBI组)及健康人群(H组)血清。小样本分别独立地与包含4262个结核分枝杆菌(MTB)重组蛋白的Mtbprot^TM芯片反应进行初筛,选择差异较为明显的蛋白质,定制成蛋白小芯片,进行较大样本验证,对有效数据进行归一化处理,根据单个蛋白的P值进行评价,进一步通过SPSS统计软件,优化出最优组合。结果 用Mtbprot^TM芯片,经32份血清(H组7份、LTBI组9份及ATB组16份)试验验证,初筛出前100个可能与LTBI相关蛋白质,用这些蛋白质制成小芯片,分别与204份血清(H组44份,LTBI组60份,ATB组100份)进行进一步验证试验,优选出8个蛋白标志物,分别为Rv1860、Rv2031c、Rv1441c、Rv0494、Rv3301c、Rv0351、Rv1821和Rv0280,其组合诊断LTBI的特异性及敏感性分别为90.9%和83.1%。结论 成功筛选鉴定出8个LTBI新型候选蛋白标志物,其组合应用在LTBI诊断和鉴别诊断上具有较大的潜在应用前景。