酶联免疫斑点试验在脊柱结核辅助诊断中的应用研究
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摘要
研究背景
目前,结核病(tuberculosis, TB)仍然是当今世界严重危害人类健康的传染性疾病之一,是单一致病菌所致的传染性疾病中死亡率最高的疾病,同样也是发展中国家的头号传染性杀手,是全球共同关注的一个严重公共卫生问题。由于结核致病菌其复杂的生物学特性和全球不同地域的社会因素,加之近几十年来各国对结核病防治策略的忽视,流动人口增多、多耐药结核菌增多以及艾滋病的流行,使得结核病在全球范围内死灰复燃,流行逐渐加剧,大有泛滥之势,导致结核病疫情呈全球性的回升,这引起了国际社会广泛的高度重视,世界卫生组织(World Health Organization, WHO)也将结核病作为重点控制的传染病之一。据WHO的最新报告,2010年全球有大约880万新发结核病例,145万例死于该病。我国同样是是全球TB高负担国家之一,受不同地域人群居住状况、社会经济发展水平、人群卫生习惯及文化素质等因素的影响,结核病发病率居高不下。目前我国结核病患者数量居全球第2位,同时也是耐多药TB患者数较多的国家之一。广东省结核病防治任务面临的形势同样非常严峻。目前,结核病人报告数始终位居全省甲乙类传染病报告发病人数前列,根据相关耐药性基线调查和2010年全省结核病流行病学抽样调查,全省每年新发生菌阳肺结核病人估算在4.6万例左右,其中耐多药患者数约0.2万例。肺结核报告发病人数中70%以上为农民,且以男性青壮年为主。
骨关节结核定义为结核分枝杆菌感染骨、关节、滑膜和脊柱所引起的疾患。其发病特点包括：发病隐匿缓慢,病程长,合并症多,常因骨骺和关节遭到结核分枝杆菌侵蚀,而影响其骨骼发育,严重者导致残疾。骨关节结核是发病率最高的肺外结核部位,约占结核总发病率的3-5%,肺外结核发病率的35-50%以上。而脊柱结核占全身骨结核的首位,约占1/3以上,发病年龄以20-30岁的青年为高峰。脊柱结核的典型症状如腰背部僵硬、疼痛、脊柱局部叩击痛,全身中毒症状,如盗汗、食欲不振、潮热、乏力等；一旦出现脊柱后凸成角畸形、神经功能(运动、感觉)障碍或窦道形成已为脊柱结核后期改变。相对于这些晚期脊柱结核患者而言,目前需要关注的问题是随着生活水平的提高及结核菌的变异,脊柱结核患者的症状往往不典型,起病隐匿,发病缓慢,早期临床症状、影像学诊断不明显。因此,如何在早期实现对脊柱结核的诊断,是所有脊柱外科医生需要面对的问题与难题?
众所周知,脊柱结核如能早期诊断、早期治疗,不仅可以制止病变发展、大大缩短疗程、避免或减少畸形发生、而且可以保全功能、甚至可以不用手术而达到治愈的目的,这样也可以为国家和患者节约大量的医疗成本。文献报道,非典型性脊柱结核发生率为2.1%-12.0%,当前脊柱结核延误诊治超过一年的病例约40%,脊柱结核误诊率为32.2%。细菌学检查仍然是结核病确诊的重要手段,是结核病诊断治疗及评价治疗效果最可靠的标准。结核病人的细菌学检查,是发现传染源的主要途径和手段,是确定结核病诊断和化疗方案的重要依据,也是考核疗效,评价治疗效果的可靠标准,在结核病防治工作中起着不可缺少的重要作用。但由于结核菌的遗传特性决定其生长周期长,致使常规细菌学检查方法存在着灵敏度低、操作复杂,需时间较长和影响因素较多,不易标准化等原因,使细菌学度实现对脊柱结核患者的早期诊断并根据具体情况加以治疗,可以大大缩短治疗的疗程,避免或减少畸形发生、而且可以保全功能、甚至可以不用手术而达到治愈的目的,为可能要接受脊柱结核手术治疗的患者节约大量费用,这样也可以为国家和社会节约大量的医疗成本。
近年来,一种应用在结核感染诊断中的新技术——酶联免疫斑点技术(enzyme-linked immunospot assay, ELISPOT)越来越得到许多学者与专家的关注。结核感染的免疫应答反应以细胞免疫为主。作为免疫应答的一部分,T细胞受结核抗原刺激致敏,形成活化的效应T细胞,这些T淋巴细胞在体外受到结核分枝杆菌特异性抗原刺激后可分泌细胞因子γ干扰素(Interferon-y, INF-y)。 ELISPOT技术即酶联免疫斑点技术,该技术起源于酶联免疫吸附试验,该技术利用体外特异性抗原刺激淋巴细胞后,检测分泌特异性抗体细胞数量和功能,随着技术发展用于检测单细胞水平分泌特异性细胞因子的功能以及细胞数量。目前ELISPOT技术在肺结核的诊断应用方面取得了良好的效果,但将其应用于脊柱结核的相关应用研究比较缺乏。
目的
1.探讨CFP-10/ESAT-6融合蛋白-ELISPOT方法在脊柱结核辅助诊断中的应用价值。
2.探讨T-SPOT.TB试剂盒在脊柱结核辅助诊断中的应用价值。
3.探讨非典型性脊柱结核的临床特点及T-SPOT.TB试验对其诊断的应用价值。
4.探讨ELISPOT试验对脊柱结核手术前后疗效的监测作用。方法
1. CFP-10/ESAT-6融合蛋白-ELISPOT在脊柱结核辅助诊断中的应用研究
2011年5月～2012年7月前瞻性纳入南方医科大学南方医院脊柱骨科和广州市胸科医骨科收治的可疑脊柱结核病例。自行设计受试者信息采集表,收集受试者基本临床信息：性别、年龄、合并症、影像学诊断、病理学结果、细菌学结果等,并对受试者出院进一步随访(≥6个月)。采集研究对象的外周血单核细胞进行CFP-10/ESAT-6融合蛋白-ELISPOT检测,同时对所有纳入者行结核菌素皮肤试验(PPD)。结合最终临床诊断和微生物学的诊断,应用灵敏度、特异度、阳性预测值和阴性预测值等指标来评价CFP-10/ESAT-6融合蛋白-ELISPOT在脊柱结核辅助诊断中的应用价值,并且进一步探讨CFP-10/ESAT-6融合蛋白-ELISPOT试验结果与临床因素的相关性。
2.探讨T-SPOT.TB试剂盒在脊柱结核辅助诊断中的应用价值
2011年5月～2012年8月前瞻性纳入南方医科大学南方医院脊柱骨科和广州市胸科医院骨科收治的可疑脊柱结核病例。自行设计调查量表收集受试者信息,采集研究对象的外周血单核细胞同时行T-SPOT.TB试验、CFP-10/ESAT-6融合蛋白-ELISPOT、Rv2041c-ELISPOT、Rv1419-ELISPOT和Rv0057-1352-ELISPOT检测,将上述几种抗原刺激ELISPOT试验结果两两比较。结合最终临床诊断和微生物学的诊断,应用灵敏度、特异度、阳性预测值和阴性预测值等指标来评价T-SPOT.TB试验在脊柱结核辅助诊断中的应用价值,探讨Rv2041c、Rv1419和Rv0057-1352在ELISPOT上的应用价值。
3.探讨非典型性脊柱结核的临床特点及T-SPOT.TB对其诊断的应用价值
选取2011年3月～2012年9月南方医科大学南方医院脊柱骨科、广州市胸科医骨科、广东省中医院骨科收治的非典型脊柱结核患者29例。由经培训的脊柱外科医生采用自行设计的非典型性脊柱结核信息采集表收集相关人口学信息、临床特点(症状与体征)、实验室数据及影像学检查。分析非典型脊柱结核的临床特征及影像学特点。采集研究对象的外周血行T-SPOT.TB试验,根据具体情况同时对所纳入的研究对象行PPD试验、抗酸杆菌涂片检查、病灶病理学检查和病灶结核分枝杆菌培养等,结合最终临床诊断和微生物学的诊断,来评价T-SPOT.TB试验在非典型性脊柱结核诊断中的应用价值。
4.探讨ELISPOT试验对脊柱结核手术前后的疗效监测作用
连续选取2012年3月～2012年6月南方医科大学南方医院脊柱骨科接受外科手术治疗的脊柱结核患者12例。自行设计调查量表收集受试者信息,分别在患者入院时、手术前2天和手术后2天采集受试患者的外周血行T-SPOT.TB试验,观察脊柱结核患者手术前后体内效应T细胞的反应情况。
结果
1.CFP-10/ESAT-6融合蛋白-ELISPOT在脊柱结核辅助诊断中的应用研究
①标本获取及检测情况
在纳入的102例患者中,有11例因未能完成ELISPOT试验或者失访被剔除出此项研究。其中确诊为脊柱结核组的有52例,确诊为非脊柱结核疾病组的有39例。最终纳入的91例患者均接受了CFP-10/ESAT-6融合蛋白-ELISPOT方法、PPD皮肤试验和血清抗结核抗体检测。52例脊柱结核患者中共有47例接受活检穿刺病理检测,42例患者行抗酸杆菌涂片检查,40例患者行结核分枝杆菌培养。
②脊柱结核组和非脊柱结核疾病组ELISPOT形成SFC数量的比较
CFP-10/EAST6融合抗原-ELISPOT形成SFC数量在脊柱结核组和非脊柱结核疾病组间存在极显著差异,CFP-10/EAST6融合抗原-ELISPOT在脊柱结核组形成SFC数量显著高于非脊柱结核疾病组形成的SFC数量。
③ELISPOT方法与临床常用诊断方法阳性率比较
在整个研究过程中,PPD皮肤试验的灵敏度、特异度、阳性预测值和阴性预测值分别为61.5%、46.2%、60.4%和47.4%；血清抗结核抗体试验的灵敏度、特异度、阳性预测值和阴性预测值分别为55.8%、61.5%、65.9%和51.1%；CFP-10/EAST6融合抗原-ELISPOT试验的灵敏度、特异度、阳性预测值和阴性预测值分别为82.7%、87.2%、89.6%和79.1%。脊柱结核组中,ELISPOT试验总体阳性率高于PPD皮肤试验和血清抗结核抗体试验(P<0.05)。
④脊柱结核组ELISPOT试验与PPD皮肤试验、抗结核抗体试验和病理学检查的配对比较
经过χ2检验,CFP-10/EAST6融合抗原-ELISPOT试验检测结果与PPD皮肤试验结果比较有统计学意义(P<0.05),与血清抗结核抗体试验结果比较有统计学意义(P<0.05),与病理学检测结果比较无统计学差异(P>0.05)。病理学检查结果与ELISPOT试验结果有着较好的一致性。
⑤脊柱结核组ELISPOT试验与临床特征的相关性
研究显示年龄、营养状况与SFC数量及ELISPOT最终阳性率有着密切联系。
2.探讨T-SPOT.TB试剂盒在脊柱结核辅助诊断中的应用价值
①研究对象及检测标本情况
在纳入的70例患者中,有5例失访被剔除出此项研究。其中确诊为脊柱结核组的有34例,确诊为非脊柱结核疾病组的有31例。
②T-SPOT.TB试剂盒与常规结核检测方法的比较
在整个研究过程中,PPD皮肤试验的灵敏度、特异度、阳性预测值和阴性预测值分别为61.8%、58.1%、61.8%和58.1%；血清抗结核抗体试验的灵敏度、特异度、阳性预测值和阴性预测值分别为58.8%、70.9%、68.9%和61.1%；T-SPOT.TB试验的灵敏度、特异度、阳性预测值和阴性预测值分别为85.3%、77.4%、80.6%和82.8%。脊柱结核组中,T-SPOT.TB试验总体灵敏度高于PPD皮肤试验和血清抗结核抗体试验。
③脊柱结核组T-SPOT.TB试剂盒与PPD皮肤试验、抗结核抗体试验和病理学检查的配对比较
脊柱结核组中,经过χ2检验,ELISPOT试验检测结果与PPD皮肤试验结果比较有统计学意义(P<0.05),与血清抗结核抗体试验结果比较有统计学意义(P<0.05),与病理学检测结果比较无统计学差异(P>0.05)。病理学检查结果与T-SPOT.TB试验结果有着较好的一致性(一致率：93.8%；Kappa值：0.714,P<0.05)。
④Rv2041c-ELISPOT、Rv1419-ELISPOT和Rv0057-1352-ELISPOT技术检测脊柱结核病人反应情况与试验条件的确定
通过对10例脊柱结核患者的预实验观察,并与CFP-10/ESAT-6-ELISPOT的试验结果相比,Rv1419-ELISPOT试验的反应情况较好,而Rv2041c-ELISPOT和Rv0057-1352-ELSPPOT的反应较弱,在以上研究基础上,选取2.0×105个细胞加入96孔培养板,孵育时间分别为20h,结果显示20μg/ml和40μg/ml蛋白浓度斑点强度相似,最终选取20μg/ml为Rv1419-ELISPOT的终浓度。
⑤脊柱结核组中不同抗原ELISPOT诊断灵敏度的比较
CFP-10/ESAT-6-ELISPOT的灵敏度、特异度、阳性预测值和阴性预测值为：82.4%、83.9%、84.8%和71.9%。T-SPOT.TB试验的灵敏度、特异度、阳性预测值和阴性预测值分别为：85.3%、77.4%、74.4%和80.8%,且两种检测结果有着较好的一致性。
3.非典型性脊柱结核的临床特点及T-SPOT.TB试验对其诊断的应用价值
①非典型性脊柱结核患者的临床症状和体征
从研究纳入的29例非典型性脊柱结核患者临床特点中,可以获知腰背痛是最主要的临床主诉(24例,82.8%)。
②非典型性脊柱结核患者的影像学特征
29例患者主要的影像学特征为：(1)软骨终板终板虫蚀样破坏：本组5例,在MRI上见椎体软骨终板虫蚀样破坏,见于椎体型或以椎体型为主的跳跃型脊柱结核；(2)单一椎体的中心性塌陷或者单一椎体轻度压缩改变：本组5例；(3)多发性脊柱结核包括连续性和跳跃性脊柱结核：本组15例；(4)影像学局部破坏重,而临床全身症状轻,即冷脓肿形成,但无明显骨破坏：本组4例。
③T-SPOT.TB试验与常规结核检测方法的比较
非典型性脊柱结核组中病灶抗酸杆菌涂片和结核分枝杆菌培养的阳性率分别为37.5%和78.3%。在整个研究过程中,PPD皮肤试验的阳性率为58.6%,血清抗结核抗体试验的阳性率为62.1%,T-SPOT.TB试验的阳性率为82.8%。
4. ELISPOT试验对脊柱结核手术前后疗效监测的作用
①患者基本情况分析
本次研究共纳入12例脊柱结核患者,其中有3例脊柱结核患者行单纯结核病灶清除术,5例脊柱结核患者行一期前路病灶清除植骨融合内固定术,4例脊柱结核患者行一期前路病灶清除椎间植骨并后路椎弓根螺钉系统固定术。
②脊柱结核患者入院时,手术前后SFC数目的比较分析
12例脊柱结核患者入院时,手术前后对应ESAT-6的SFC平均数目分别为35.0±22.4、36.7±23.8、30.1±16.2；对应CFP-10的SFC平均数目分别为55.1±68.5、57.4±68.3、44.1±47.9。经过配对样本t检验,患者入院时与手术前、手术前与手术后ESAT-6和CFP-10刺激形成的SFC数量比较均无统计学差异(P>0.05)。
结论
1.以CFP-10/ESAT-6融合蛋白作为抗原的ELISPOT法应用于脊柱结核诊断有着良好的灵敏度和特异度,能为脊柱结核感染患者提供有效的辅助诊断。
2. T-SPOT.TB试验在脊柱结核的诊断中有着较高的灵敏度(尤其是非典型性脊柱结核的诊断),可能成为脊柱结核感染早期诊断的一项重要的筛查工具,并辅助脊柱结核病的诊断。
3.结核分枝杆菌Rv1419融合抗原具有较高的脊柱结核免疫学诊断价值。
Backgroud
Tuberculosis (TB) is one of the most important infectious disease in the world. TB is a disease that can result in very high mortality, especially in developing countries. The incidence of TB has become increasingly serious because of several reasons: the biological characteristics of tuberculosis, the floating of the population and an epidemic of Multi-drug resistant and the AIDS. What's more, delayed diagnosis and treatment can increase the risk for dissemination of M. tuberculosis and decrease survival for some subgroups of TB patients. TB has been an important public health problem, which will become a priority of disease control. According to the World Health Organization (WHO) surveillance study, there were about8.8million new TB cases around the world in2010. According to recent statistics, China is one of the leading countries with a high incidence of TB. Due to of people's living conditions, social and economic development, the differences of the health habits, the morbidity of tuberculosis is high. TB prevention and control work in Guangdong province has made considerable progress and achievements, but also very difficult task. The number of the TB patients always stands in the top list of A or B class infectious diseases in the whole province. According to recent statistics,46thousand people were infected by TB in Guangdong province2010, with about2000reported multidrug resistant. The first group is farmer, occupied70%of TB patients.
According to traditional chinese medicine, osteoarticular TB was called "Gulao".Osteoarticular TB usually presents in a slowly indolent manner with nonspecific clinical presentations, so the diagnosis of tuberculous arthritis is often difficult and delayed, and remains a great challenge for clinicians. Osteoarticular TB accounts for3-5%of all forms of TB and approximately35-50%of cases with extrapulmonary TB. Spinal TB is found in1%to3%of all TB cases and in50%to60%cases of osteoarticular TB. The typical clinical presentations of spinal TB includes:lower back stiffness, night sweats, anorexia, hot flushes and weakness. At physical examination of the patients, kyphosis and neurological deficit was also seen in the late change of the spinal TB. Actually, spinal TB are a great challenge to physicians because of the nonspecific and wide spectrum of clinical presentations that result in delay of diagnosis and the risk of significant potential morbidity and mortality due to several complications. Early diagnosis and treatment is the key to avoiding this long-term disability.
As we know, if the doctor can make an early diagnosis and treatment for spinal TB, they could cure the TB patients without surgery and help the country save a lot of medical costs. According to recent statistics, the morbidity of atypical spinal TB was approximately2.1-12%of cases with spinal TB. The misdiagnosis rate of this disease was32.2%. Definite diagnosis is generally considered to require microbiological confirmation; however, the bacteriological gold standard result, including solid media-based techniques using Lowenstein-Jensen slants and Middlebrook7H10/7H11agar and liquid media-based methods, such as with the BACTEC960MGIT system, usually takes several weeks to be available. These may lead to delay in diagnosis and initiation of treatment. Therefore, we urgently need a faster, more sensitive, and specific test for the diagnosis of spinal TB in clinical practice.
Recently, an enzyme-linked immunospot (ELISPOT) assay using pools of early secretory antigenic target6(ESAT-6) and culture filtrate protein10(CFP-10) peptides was manufactured and commercialized. It is based on the detection of interferon-gamma (IFN-y) released by activated T lymphocytes. The ELISPOT assay has been demonstrated to be useful for the diagnosis of pulmonary TB. However, few studies have investigated the sensitivity and specificity of this assay for diagnosing atypical spinal TB.
Objectives
1.To assess performance of a laboratory-developed recombinant CFP-10/ESAT-6fusion protein (rCFP-10/ESAT-6)-based ELISPOT assay in diagnosis of spinal tuberculosis.
2.To assess clinical value of an enzyme-linked immunospot assay (T-SPOT. TB) for the diagnosis of spinal tuberculosis.
3.To analyze the clinical characteristics of the atypical spinal TB and assess the diagnostic value of an enzyme-linked immunospot (T-SPOT.TB) assay in clinically suspected cases of atypical spinal TB.
4. To assess clinical value of an enzyme-linked immunospot assay (T-SPOT. TB) for the monitoring efficacy of surgical treatment of spinal TB.
Methods
1.To assess performance of a laboratory-developed recombinant CFP-10/ESAT-6fusion protein (rCFP-10/ESAT-6)-based ELISPOT assay in diagnosis of spinal tuberculosis.
Suspected spinal TB patients were prospectively recruited recruited in two hospitals (Nanfang Hospital, Southern Medical University; Guangzhou thoracic hospital) from May2011to July2012. Data on clinical characteristics of the patients and conventional laboratory results were collected. The specific data were collected on age, sex, underlying diseases, radiographic image examination, lymphocyte count, pathology, microbiological results, and follow-up observations.4-5ml of venous blood samples from the volunteers were taken in heparinized glass tubes, and peripheral blood mononuclear cells (PBMC) were isolated and quantified the second day after the patients went into hospital. This study aimed to assess performance of a laboratory-developed rCFP-10/ESAT-6-ELISPOT assay in diagnosis of spinal TB.
2. To assess clinical value of an enzyme-linked immunospot assay (T-SPOT.TB) for the diagnosis of spinal TB.
Suspected spinal TB patients were prospectively recruited recruited in two hospitals (Nanfang Hospital, Southern Medical University; Guangzhou thoracic hospital) from May2011to August2012. Data on clinical characteristics of the patients and conventional laboratory results were collected. The specific data were collected on age, sex, underlying diseases, radiographic image examination, lymphocyte count, pathology, microbiological results, and follow-up observations. In addition to conventional tests for TB, we used ELISPOT assays to measure the IFN-y response to ESAT-6, CFP-10, rCFP-10/ESAT-6, Rv2041c, Rv1419and Rv0057-1352in T-cells in samples of peripheral blood mononuclear cells (PBMC).
3. To analyze the clinical characteristics of the atypical spinal TB and assess the diagnostic value of an enzyme-linked immunospot (T-SPOT.TB) assay in clinically suspected cases of atypical spinal TB.
From March2011to September2012, a total of29patients with atypical spinal TB were enrolled. Data on clinical characteristics of the patients and conventional laboratory results were collected, and blood samples were obtained for T-SPOT.TB assay. The aim of this study was to assess the diagnostic value of T-SPOT.TB assay in clinically atypical spinal TB.
4. To assess clinical value of an enzyme-linked immunospot assay (T-SPOT. TB) for the monitoring efficacy of surgical treatment of spinal TB.
A sequential subgroup of spinal TB patients was enrolled for evaluation of responses of T-SPOT.TB assay on admission, two days before and after surgery. All these spinal TB patients underwent focal cleaning or one-stage anterior debridement, bone graft plus anterior or posterior instrumentation.
Results
1. The performance of a laboratory-developed rCFP-10/ESAT-6-based ELISPOT assay in diagnosis of spinal TB.
①Patient characteristics
A total of102patients with suspected spinal TB were prospectively recruited during the study period.11patients were excluded from the study, among which5did not complete the rCFP-10/ESAT-6-ELISPOT assay and6had no final diagnosis. The remaining91patients were ultimately included for ELISPOT analyses. The spinal TB group contained52cases. The non-TB disease group contained39subjects.47patients with spinal TB had available biopsy or surgical specimens for histopathological examination.42patients with spinal TB had AFB staining and40patients had cultures for M. tuberculosis, respectively.
②Spread of SFCs above the negative control/2.5×105peripheral blood mononuclear cells (PBMCs) in response to recombinant CFP-10/ESAT-6fusion protein in spinal TB patients and non-spinal TB patients.
The mean number of SFC in spinal TB patients was significantly higher than that of non-spinal TB patients.
③Clinical diagnostic value of the ELISPOT assay for spinal TB
Among the spinal TB patients and non-TB disease patients, the overall sensitivity, specificity, positive predictive value, and negative predictive value for spinal TB diagnosis by the PPD skin test were61.5%,46.2%,60.4%and47.4%, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value for spinal TB diagnosis by the Antibody detection were55.8%,61.5%,65.9%and51.1%. By comparison, the sensitivity, specificity, positive predictive value and negative predictive value for spinal TB diagnosis by the ELISPOT assay were82.7%,87.2%,89.6%and79.1%, respectively. The overall sensitivity of ELISPOT is higher than that of PPD skin test and antibody detection test (P<0.05).
④Comparsion of spinal tuberculosis patients detected by PPD skin test, TB antiboby test, Histopathology diagnosis and ELISPOT
When we assessed the concordance between the histopathology detection and the ELISPOT assay using the κ coefficient, we found moderate agreement between the two tests.
⑤Clinical characteristics associated with the ELISPOT results in spinal TB patients
In this study, we found that age and emaciation were associated with the number of SFC or positive rate of ELISPOT in spinal TB patients.
2. Clinical value of an enzyme-linked immunospot assay (T-SPOT.TB) for the diagnosis of spinal tuberculosis.
①Patient characteristics
A total of70patients with suspected spinal TB were prospectively recruited during the study period.5patients were excluded from the study.
The remaining65patients were ultimately included for T-SPOT.TB analyses. The spinal TB group contained34cases. The non-TB disease group contained31subjects.
②Clinical diagnostic value of the T-SPOT.TB assay for spinal TB
Among the spinal TB patients and non-TB disease patients, the overall sensitivity, specificity, positive predictive value, and negative predictive value for spinal TB diagnosis by the PPD skin test were61.8%,58.1%,61.8%and58.1%, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value for spinal TB diagnosis by the Antibody detection were58.8%,70.9%,68.9%,61.1%. By comparison, the sensitivity, specificity, positive predictive value and negative predictive value for spinal TB diagnosis by the T-SPOT.TB assay were85.3%,77.4%,80.6%and82.8%, respectively. The overall sensitivity of T-SPOT.TB is higher than that of PPD skin test and antibody detection test (P<0.05).
③Comparsion of spinal tuberculosis patients detected by PPD skin test, TB antiboby test, Histopathology diagnosis and T-SPOT.TB
When we assessed the concordance between the histopathology detection and the T-SPOT.TB assay using the κ coefficient, we found moderate agreement between the two tests.
④The determination of test condition about Rv2041c-ELISPOT, Rv1419-ELI SPOT and Rv0057-1352-ELISPOT
To change the condition of experiment, and find that the best numbers of cell is2×105, the best incubated time is20hour and the best density of proteinum is20μg/ml.
④Clinical diagnostic value of different antige response to the ELISPOT assay for spinal TB
The sensitivity, specificity, positive predictive value and negative predictive value for spinal TB diagnosis by the CFP-10/ESAT-6-ELISPOT assay were82.4%,83.9%,84.8%and71.9%, respectively. The sensitivity, specificity, positive predictive value and negative predictive value for spinal TB diagnosis by the T-SPOT.TB were85.3%,77.4%,74.4%and80.8%.When we assessed the concordance between these two tests using the κ coefficient, we found moderate agreement between the two tests.
3. To analyze the clinical characteristics of the atypical spinal TB and assess the diagnostic value of an enzyme-linked immunospot (T-SPOT.TB) assay in clinically suspected cases of atypical spinal TB.
①Clinical presentation
Back pain was the most common clinical complaint (24patients,82.8%).
②Imaging study findings
Through the imaging study, worm-eaten destruction of vertebral endplate was seen in5patients. Destruction of centricity of the single vertebral body or concentric collapse of single vertebral body were seen in5patients.15patients presented contiguous or skipped vertebral body destruction. Tuberculous abscess with no identifiable osseous lesion were visible by CT and MRI in4patients
③Clinical diagnostic value of the T-SPOT.TB assay for atypical spinal TB
We found that37.5%,78.3%of patients were positive for AFB smear and cultures for M. tuberculosis, respectively. The overall sensitivity, specificity, positive predictive value, and negative predictive value for atypical spinal TB diagnosis by the PPD skin test were58.6%,59.4%,56.7%and61.3%. By comparison, the sensitivity, specificity, positive predictive value and negative predictive value for atypical spinal TB diagnosis by the T-SPOT.TB assay were82.8%,81.3%,80.0%and83.9%, respectively. The overall sensitivity of the T-SPOT.TB assay was higher than that of PPD skin test (P<0.05).
4. To assess clinical value of an enzyme-linked immunospot assay (T-SPOT. TB) for the monitoring efficacy of surgical treatment of spinal TB.
①Patient characteristics
Additional responses were evaluated in a consecutive subset of12spinal TB patients on two days before and after surgery.3spinal TB patients only underwent focal cleaning. The remaining9spinal TB patients underwent one-stage anterior debridement, bone graft plus anterior or posterior instrumentation.
②Responses to ESAT-6, CFP-10before and after Surgery
The median ESAT-6(30spots per250,000PBMCs) and CFP-10(44spots per250,000PBMCs) response were slightly lower after surgery than those before surgery (ESAT-6:36spots per250,000PBMCs; CFP-10:57spots per250,000PBMCs), but those failed to reach statistical significance (P>0.05).
Conclusions
1. A laboratory-developed rCFP-10/ESAT-6-based ELISPOT assay is a useful adjunct to current tests for diagnosis of spinal TB.
2. The T-SPOT.TB assay is a promising tool for the clinical evaluation of spinal TB and merits additional assessment in a diagnostic trial.
3. The recombinant Rv1419protein of mycobacterium tuberculosis has relatively high value in the immunological diagnosis of tuberculosis.
目前,结核病(tuberculosis, TB)仍然是当今世界严重危害人类健康的传染性疾病之一,是单一致病菌所致的传染性疾病中死亡率最高的疾病,同样也是发展中国家的头号传染性杀手,是全球共同关注的一个严重公共卫生问题。由于结核致病菌其复杂的生物学特性和全球不同地域的社会因素,加之近几十年来各国对结核病防治策略的忽视,流动人口增多、多耐药结核菌增多以及艾滋病的流行,使得结核病在全球范围内死灰复燃,流行逐渐加剧,大有泛滥之势,导致结核病疫情呈全球性的回升,这引起了国际社会广泛的高度重视,世界卫生组织(World Health Organization, WHO)也将结核病作为重点控制的传染病之一。据WHO的最新报告,2010年全球有大约880万新发结核病例,145万例死于该病。我国同样是是全球TB高负担国家之一,受不同地域人群居住状况、社会经济发展水平、人群卫生习惯及文化素质等因素的影响,结核病发病率居高不下。目前我国结核病患者数量居全球第2位,同时也是耐多药TB患者数较多的国家之一。广东省结核病防治任务面临的形势同样非常严峻。目前,结核病人报告数始终位居全省甲乙类传染病报告发病人数前列,根据相关耐药性基线调查和2010年全省结核病流行病学抽样调查,全省每年新发生菌阳肺结核病人估算在4.6万例左右,其中耐多药患者数约0.2万例。肺结核报告发病人数中70%以上为农民,且以男性青壮年为主。
骨关节结核定义为结核分枝杆菌感染骨、关节、滑膜和脊柱所引起的疾患。其发病特点包括：发病隐匿缓慢,病程长,合并症多,常因骨骺和关节遭到结核分枝杆菌侵蚀,而影响其骨骼发育,严重者导致残疾。骨关节结核是发病率最高的肺外结核部位,约占结核总发病率的3-5%,肺外结核发病率的35-50%以上。而脊柱结核占全身骨结核的首位,约占1/3以上,发病年龄以20-30岁的青年为高峰。脊柱结核的典型症状如腰背部僵硬、疼痛、脊柱局部叩击痛,全身中毒症状,如盗汗、食欲不振、潮热、乏力等；一旦出现脊柱后凸成角畸形、神经功能(运动、感觉)障碍或窦道形成已为脊柱结核后期改变。相对于这些晚期脊柱结核患者而言,目前需要关注的问题是随着生活水平的提高及结核菌的变异,脊柱结核患者的症状往往不典型,起病隐匿,发病缓慢,早期临床症状、影像学诊断不明显。因此,如何在早期实现对脊柱结核的诊断,是所有脊柱外科医生需要面对的问题与难题?
众所周知,脊柱结核如能早期诊断、早期治疗,不仅可以制止病变发展、大大缩短疗程、避免或减少畸形发生、而且可以保全功能、甚至可以不用手术而达到治愈的目的,这样也可以为国家和患者节约大量的医疗成本。文献报道,非典型性脊柱结核发生率为2.1%-12.0%,当前脊柱结核延误诊治超过一年的病例约40%,脊柱结核误诊率为32.2%。细菌学检查仍然是结核病确诊的重要手段,是结核病诊断治疗及评价治疗效果最可靠的标准。结核病人的细菌学检查,是发现传染源的主要途径和手段,是确定结核病诊断和化疗方案的重要依据,也是考核疗效,评价治疗效果的可靠标准,在结核病防治工作中起着不可缺少的重要作用。但由于结核菌的遗传特性决定其生长周期长,致使常规细菌学检查方法存在着灵敏度低、操作复杂,需时间较长和影响因素较多,不易标准化等原因,使细菌学度实现对脊柱结核患者的早期诊断并根据具体情况加以治疗,可以大大缩短治疗的疗程,避免或减少畸形发生、而且可以保全功能、甚至可以不用手术而达到治愈的目的,为可能要接受脊柱结核手术治疗的患者节约大量费用,这样也可以为国家和社会节约大量的医疗成本。
近年来,一种应用在结核感染诊断中的新技术——酶联免疫斑点技术(enzyme-linked immunospot assay, ELISPOT)越来越得到许多学者与专家的关注。结核感染的免疫应答反应以细胞免疫为主。作为免疫应答的一部分,T细胞受结核抗原刺激致敏,形成活化的效应T细胞,这些T淋巴细胞在体外受到结核分枝杆菌特异性抗原刺激后可分泌细胞因子γ干扰素(Interferon-y, INF-y)。 ELISPOT技术即酶联免疫斑点技术,该技术起源于酶联免疫吸附试验,该技术利用体外特异性抗原刺激淋巴细胞后,检测分泌特异性抗体细胞数量和功能,随着技术发展用于检测单细胞水平分泌特异性细胞因子的功能以及细胞数量。目前ELISPOT技术在肺结核的诊断应用方面取得了良好的效果,但将其应用于脊柱结核的相关应用研究比较缺乏。
目的
1.探讨CFP-10/ESAT-6融合蛋白-ELISPOT方法在脊柱结核辅助诊断中的应用价值。
2.探讨T-SPOT.TB试剂盒在脊柱结核辅助诊断中的应用价值。
3.探讨非典型性脊柱结核的临床特点及T-SPOT.TB试验对其诊断的应用价值。
4.探讨ELISPOT试验对脊柱结核手术前后疗效的监测作用。方法
1. CFP-10/ESAT-6融合蛋白-ELISPOT在脊柱结核辅助诊断中的应用研究
2011年5月～2012年7月前瞻性纳入南方医科大学南方医院脊柱骨科和广州市胸科医骨科收治的可疑脊柱结核病例。自行设计受试者信息采集表,收集受试者基本临床信息：性别、年龄、合并症、影像学诊断、病理学结果、细菌学结果等,并对受试者出院进一步随访(≥6个月)。采集研究对象的外周血单核细胞进行CFP-10/ESAT-6融合蛋白-ELISPOT检测,同时对所有纳入者行结核菌素皮肤试验(PPD)。结合最终临床诊断和微生物学的诊断,应用灵敏度、特异度、阳性预测值和阴性预测值等指标来评价CFP-10/ESAT-6融合蛋白-ELISPOT在脊柱结核辅助诊断中的应用价值,并且进一步探讨CFP-10/ESAT-6融合蛋白-ELISPOT试验结果与临床因素的相关性。
2.探讨T-SPOT.TB试剂盒在脊柱结核辅助诊断中的应用价值
2011年5月～2012年8月前瞻性纳入南方医科大学南方医院脊柱骨科和广州市胸科医院骨科收治的可疑脊柱结核病例。自行设计调查量表收集受试者信息,采集研究对象的外周血单核细胞同时行T-SPOT.TB试验、CFP-10/ESAT-6融合蛋白-ELISPOT、Rv2041c-ELISPOT、Rv1419-ELISPOT和Rv0057-1352-ELISPOT检测,将上述几种抗原刺激ELISPOT试验结果两两比较。结合最终临床诊断和微生物学的诊断,应用灵敏度、特异度、阳性预测值和阴性预测值等指标来评价T-SPOT.TB试验在脊柱结核辅助诊断中的应用价值,探讨Rv2041c、Rv1419和Rv0057-1352在ELISPOT上的应用价值。
3.探讨非典型性脊柱结核的临床特点及T-SPOT.TB对其诊断的应用价值
选取2011年3月～2012年9月南方医科大学南方医院脊柱骨科、广州市胸科医骨科、广东省中医院骨科收治的非典型脊柱结核患者29例。由经培训的脊柱外科医生采用自行设计的非典型性脊柱结核信息采集表收集相关人口学信息、临床特点(症状与体征)、实验室数据及影像学检查。分析非典型脊柱结核的临床特征及影像学特点。采集研究对象的外周血行T-SPOT.TB试验,根据具体情况同时对所纳入的研究对象行PPD试验、抗酸杆菌涂片检查、病灶病理学检查和病灶结核分枝杆菌培养等,结合最终临床诊断和微生物学的诊断,来评价T-SPOT.TB试验在非典型性脊柱结核诊断中的应用价值。
4.探讨ELISPOT试验对脊柱结核手术前后的疗效监测作用
连续选取2012年3月～2012年6月南方医科大学南方医院脊柱骨科接受外科手术治疗的脊柱结核患者12例。自行设计调查量表收集受试者信息,分别在患者入院时、手术前2天和手术后2天采集受试患者的外周血行T-SPOT.TB试验,观察脊柱结核患者手术前后体内效应T细胞的反应情况。
结果
1.CFP-10/ESAT-6融合蛋白-ELISPOT在脊柱结核辅助诊断中的应用研究
①标本获取及检测情况
在纳入的102例患者中,有11例因未能完成ELISPOT试验或者失访被剔除出此项研究。其中确诊为脊柱结核组的有52例,确诊为非脊柱结核疾病组的有39例。最终纳入的91例患者均接受了CFP-10/ESAT-6融合蛋白-ELISPOT方法、PPD皮肤试验和血清抗结核抗体检测。52例脊柱结核患者中共有47例接受活检穿刺病理检测,42例患者行抗酸杆菌涂片检查,40例患者行结核分枝杆菌培养。
②脊柱结核组和非脊柱结核疾病组ELISPOT形成SFC数量的比较
CFP-10/EAST6融合抗原-ELISPOT形成SFC数量在脊柱结核组和非脊柱结核疾病组间存在极显著差异,CFP-10/EAST6融合抗原-ELISPOT在脊柱结核组形成SFC数量显著高于非脊柱结核疾病组形成的SFC数量。
③ELISPOT方法与临床常用诊断方法阳性率比较
在整个研究过程中,PPD皮肤试验的灵敏度、特异度、阳性预测值和阴性预测值分别为61.5%、46.2%、60.4%和47.4%；血清抗结核抗体试验的灵敏度、特异度、阳性预测值和阴性预测值分别为55.8%、61.5%、65.9%和51.1%；CFP-10/EAST6融合抗原-ELISPOT试验的灵敏度、特异度、阳性预测值和阴性预测值分别为82.7%、87.2%、89.6%和79.1%。脊柱结核组中,ELISPOT试验总体阳性率高于PPD皮肤试验和血清抗结核抗体试验(P<0.05)。
④脊柱结核组ELISPOT试验与PPD皮肤试验、抗结核抗体试验和病理学检查的配对比较
经过χ2检验,CFP-10/EAST6融合抗原-ELISPOT试验检测结果与PPD皮肤试验结果比较有统计学意义(P<0.05),与血清抗结核抗体试验结果比较有统计学意义(P<0.05),与病理学检测结果比较无统计学差异(P>0.05)。病理学检查结果与ELISPOT试验结果有着较好的一致性。
⑤脊柱结核组ELISPOT试验与临床特征的相关性
研究显示年龄、营养状况与SFC数量及ELISPOT最终阳性率有着密切联系。
2.探讨T-SPOT.TB试剂盒在脊柱结核辅助诊断中的应用价值
①研究对象及检测标本情况
在纳入的70例患者中,有5例失访被剔除出此项研究。其中确诊为脊柱结核组的有34例,确诊为非脊柱结核疾病组的有31例。
②T-SPOT.TB试剂盒与常规结核检测方法的比较
在整个研究过程中,PPD皮肤试验的灵敏度、特异度、阳性预测值和阴性预测值分别为61.8%、58.1%、61.8%和58.1%；血清抗结核抗体试验的灵敏度、特异度、阳性预测值和阴性预测值分别为58.8%、70.9%、68.9%和61.1%；T-SPOT.TB试验的灵敏度、特异度、阳性预测值和阴性预测值分别为85.3%、77.4%、80.6%和82.8%。脊柱结核组中,T-SPOT.TB试验总体灵敏度高于PPD皮肤试验和血清抗结核抗体试验。
③脊柱结核组T-SPOT.TB试剂盒与PPD皮肤试验、抗结核抗体试验和病理学检查的配对比较
脊柱结核组中,经过χ2检验,ELISPOT试验检测结果与PPD皮肤试验结果比较有统计学意义(P<0.05),与血清抗结核抗体试验结果比较有统计学意义(P<0.05),与病理学检测结果比较无统计学差异(P>0.05)。病理学检查结果与T-SPOT.TB试验结果有着较好的一致性(一致率：93.8%；Kappa值：0.714,P<0.05)。
④Rv2041c-ELISPOT、Rv1419-ELISPOT和Rv0057-1352-ELISPOT技术检测脊柱结核病人反应情况与试验条件的确定
通过对10例脊柱结核患者的预实验观察,并与CFP-10/ESAT-6-ELISPOT的试验结果相比,Rv1419-ELISPOT试验的反应情况较好,而Rv2041c-ELISPOT和Rv0057-1352-ELSPPOT的反应较弱,在以上研究基础上,选取2.0×105个细胞加入96孔培养板,孵育时间分别为20h,结果显示20μg/ml和40μg/ml蛋白浓度斑点强度相似,最终选取20μg/ml为Rv1419-ELISPOT的终浓度。
⑤脊柱结核组中不同抗原ELISPOT诊断灵敏度的比较
CFP-10/ESAT-6-ELISPOT的灵敏度、特异度、阳性预测值和阴性预测值为：82.4%、83.9%、84.8%和71.9%。T-SPOT.TB试验的灵敏度、特异度、阳性预测值和阴性预测值分别为：85.3%、77.4%、74.4%和80.8%,且两种检测结果有着较好的一致性。
3.非典型性脊柱结核的临床特点及T-SPOT.TB试验对其诊断的应用价值
①非典型性脊柱结核患者的临床症状和体征
从研究纳入的29例非典型性脊柱结核患者临床特点中,可以获知腰背痛是最主要的临床主诉(24例,82.8%)。
②非典型性脊柱结核患者的影像学特征
29例患者主要的影像学特征为：(1)软骨终板终板虫蚀样破坏：本组5例,在MRI上见椎体软骨终板虫蚀样破坏,见于椎体型或以椎体型为主的跳跃型脊柱结核；(2)单一椎体的中心性塌陷或者单一椎体轻度压缩改变：本组5例；(3)多发性脊柱结核包括连续性和跳跃性脊柱结核：本组15例；(4)影像学局部破坏重,而临床全身症状轻,即冷脓肿形成,但无明显骨破坏：本组4例。
③T-SPOT.TB试验与常规结核检测方法的比较
非典型性脊柱结核组中病灶抗酸杆菌涂片和结核分枝杆菌培养的阳性率分别为37.5%和78.3%。在整个研究过程中,PPD皮肤试验的阳性率为58.6%,血清抗结核抗体试验的阳性率为62.1%,T-SPOT.TB试验的阳性率为82.8%。
4. ELISPOT试验对脊柱结核手术前后疗效监测的作用
①患者基本情况分析
本次研究共纳入12例脊柱结核患者,其中有3例脊柱结核患者行单纯结核病灶清除术,5例脊柱结核患者行一期前路病灶清除植骨融合内固定术,4例脊柱结核患者行一期前路病灶清除椎间植骨并后路椎弓根螺钉系统固定术。
②脊柱结核患者入院时,手术前后SFC数目的比较分析
12例脊柱结核患者入院时,手术前后对应ESAT-6的SFC平均数目分别为35.0±22.4、36.7±23.8、30.1±16.2；对应CFP-10的SFC平均数目分别为55.1±68.5、57.4±68.3、44.1±47.9。经过配对样本t检验,患者入院时与手术前、手术前与手术后ESAT-6和CFP-10刺激形成的SFC数量比较均无统计学差异(P>0.05)。
结论
1.以CFP-10/ESAT-6融合蛋白作为抗原的ELISPOT法应用于脊柱结核诊断有着良好的灵敏度和特异度,能为脊柱结核感染患者提供有效的辅助诊断。
2. T-SPOT.TB试验在脊柱结核的诊断中有着较高的灵敏度(尤其是非典型性脊柱结核的诊断),可能成为脊柱结核感染早期诊断的一项重要的筛查工具,并辅助脊柱结核病的诊断。
3.结核分枝杆菌Rv1419融合抗原具有较高的脊柱结核免疫学诊断价值。
Backgroud
Tuberculosis (TB) is one of the most important infectious disease in the world. TB is a disease that can result in very high mortality, especially in developing countries. The incidence of TB has become increasingly serious because of several reasons: the biological characteristics of tuberculosis, the floating of the population and an epidemic of Multi-drug resistant and the AIDS. What's more, delayed diagnosis and treatment can increase the risk for dissemination of M. tuberculosis and decrease survival for some subgroups of TB patients. TB has been an important public health problem, which will become a priority of disease control. According to the World Health Organization (WHO) surveillance study, there were about8.8million new TB cases around the world in2010. According to recent statistics, China is one of the leading countries with a high incidence of TB. Due to of people's living conditions, social and economic development, the differences of the health habits, the morbidity of tuberculosis is high. TB prevention and control work in Guangdong province has made considerable progress and achievements, but also very difficult task. The number of the TB patients always stands in the top list of A or B class infectious diseases in the whole province. According to recent statistics,46thousand people were infected by TB in Guangdong province2010, with about2000reported multidrug resistant. The first group is farmer, occupied70%of TB patients.
According to traditional chinese medicine, osteoarticular TB was called "Gulao".Osteoarticular TB usually presents in a slowly indolent manner with nonspecific clinical presentations, so the diagnosis of tuberculous arthritis is often difficult and delayed, and remains a great challenge for clinicians. Osteoarticular TB accounts for3-5%of all forms of TB and approximately35-50%of cases with extrapulmonary TB. Spinal TB is found in1%to3%of all TB cases and in50%to60%cases of osteoarticular TB. The typical clinical presentations of spinal TB includes:lower back stiffness, night sweats, anorexia, hot flushes and weakness. At physical examination of the patients, kyphosis and neurological deficit was also seen in the late change of the spinal TB. Actually, spinal TB are a great challenge to physicians because of the nonspecific and wide spectrum of clinical presentations that result in delay of diagnosis and the risk of significant potential morbidity and mortality due to several complications. Early diagnosis and treatment is the key to avoiding this long-term disability.
As we know, if the doctor can make an early diagnosis and treatment for spinal TB, they could cure the TB patients without surgery and help the country save a lot of medical costs. According to recent statistics, the morbidity of atypical spinal TB was approximately2.1-12%of cases with spinal TB. The misdiagnosis rate of this disease was32.2%. Definite diagnosis is generally considered to require microbiological confirmation; however, the bacteriological gold standard result, including solid media-based techniques using Lowenstein-Jensen slants and Middlebrook7H10/7H11agar and liquid media-based methods, such as with the BACTEC960MGIT system, usually takes several weeks to be available. These may lead to delay in diagnosis and initiation of treatment. Therefore, we urgently need a faster, more sensitive, and specific test for the diagnosis of spinal TB in clinical practice.
Recently, an enzyme-linked immunospot (ELISPOT) assay using pools of early secretory antigenic target6(ESAT-6) and culture filtrate protein10(CFP-10) peptides was manufactured and commercialized. It is based on the detection of interferon-gamma (IFN-y) released by activated T lymphocytes. The ELISPOT assay has been demonstrated to be useful for the diagnosis of pulmonary TB. However, few studies have investigated the sensitivity and specificity of this assay for diagnosing atypical spinal TB.
Objectives
1.To assess performance of a laboratory-developed recombinant CFP-10/ESAT-6fusion protein (rCFP-10/ESAT-6)-based ELISPOT assay in diagnosis of spinal tuberculosis.
2.To assess clinical value of an enzyme-linked immunospot assay (T-SPOT. TB) for the diagnosis of spinal tuberculosis.
3.To analyze the clinical characteristics of the atypical spinal TB and assess the diagnostic value of an enzyme-linked immunospot (T-SPOT.TB) assay in clinically suspected cases of atypical spinal TB.
4. To assess clinical value of an enzyme-linked immunospot assay (T-SPOT. TB) for the monitoring efficacy of surgical treatment of spinal TB.
Methods
1.To assess performance of a laboratory-developed recombinant CFP-10/ESAT-6fusion protein (rCFP-10/ESAT-6)-based ELISPOT assay in diagnosis of spinal tuberculosis.
Suspected spinal TB patients were prospectively recruited recruited in two hospitals (Nanfang Hospital, Southern Medical University; Guangzhou thoracic hospital) from May2011to July2012. Data on clinical characteristics of the patients and conventional laboratory results were collected. The specific data were collected on age, sex, underlying diseases, radiographic image examination, lymphocyte count, pathology, microbiological results, and follow-up observations.4-5ml of venous blood samples from the volunteers were taken in heparinized glass tubes, and peripheral blood mononuclear cells (PBMC) were isolated and quantified the second day after the patients went into hospital. This study aimed to assess performance of a laboratory-developed rCFP-10/ESAT-6-ELISPOT assay in diagnosis of spinal TB.
2. To assess clinical value of an enzyme-linked immunospot assay (T-SPOT.TB) for the diagnosis of spinal TB.
Suspected spinal TB patients were prospectively recruited recruited in two hospitals (Nanfang Hospital, Southern Medical University; Guangzhou thoracic hospital) from May2011to August2012. Data on clinical characteristics of the patients and conventional laboratory results were collected. The specific data were collected on age, sex, underlying diseases, radiographic image examination, lymphocyte count, pathology, microbiological results, and follow-up observations. In addition to conventional tests for TB, we used ELISPOT assays to measure the IFN-y response to ESAT-6, CFP-10, rCFP-10/ESAT-6, Rv2041c, Rv1419and Rv0057-1352in T-cells in samples of peripheral blood mononuclear cells (PBMC).
3. To analyze the clinical characteristics of the atypical spinal TB and assess the diagnostic value of an enzyme-linked immunospot (T-SPOT.TB) assay in clinically suspected cases of atypical spinal TB.
From March2011to September2012, a total of29patients with atypical spinal TB were enrolled. Data on clinical characteristics of the patients and conventional laboratory results were collected, and blood samples were obtained for T-SPOT.TB assay. The aim of this study was to assess the diagnostic value of T-SPOT.TB assay in clinically atypical spinal TB.
4. To assess clinical value of an enzyme-linked immunospot assay (T-SPOT. TB) for the monitoring efficacy of surgical treatment of spinal TB.
A sequential subgroup of spinal TB patients was enrolled for evaluation of responses of T-SPOT.TB assay on admission, two days before and after surgery. All these spinal TB patients underwent focal cleaning or one-stage anterior debridement, bone graft plus anterior or posterior instrumentation.
Results
1. The performance of a laboratory-developed rCFP-10/ESAT-6-based ELISPOT assay in diagnosis of spinal TB.
①Patient characteristics
A total of102patients with suspected spinal TB were prospectively recruited during the study period.11patients were excluded from the study, among which5did not complete the rCFP-10/ESAT-6-ELISPOT assay and6had no final diagnosis. The remaining91patients were ultimately included for ELISPOT analyses. The spinal TB group contained52cases. The non-TB disease group contained39subjects.47patients with spinal TB had available biopsy or surgical specimens for histopathological examination.42patients with spinal TB had AFB staining and40patients had cultures for M. tuberculosis, respectively.
②Spread of SFCs above the negative control/2.5×105peripheral blood mononuclear cells (PBMCs) in response to recombinant CFP-10/ESAT-6fusion protein in spinal TB patients and non-spinal TB patients.
The mean number of SFC in spinal TB patients was significantly higher than that of non-spinal TB patients.
③Clinical diagnostic value of the ELISPOT assay for spinal TB
Among the spinal TB patients and non-TB disease patients, the overall sensitivity, specificity, positive predictive value, and negative predictive value for spinal TB diagnosis by the PPD skin test were61.5%,46.2%,60.4%and47.4%, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value for spinal TB diagnosis by the Antibody detection were55.8%,61.5%,65.9%and51.1%. By comparison, the sensitivity, specificity, positive predictive value and negative predictive value for spinal TB diagnosis by the ELISPOT assay were82.7%,87.2%,89.6%and79.1%, respectively. The overall sensitivity of ELISPOT is higher than that of PPD skin test and antibody detection test (P<0.05).
④Comparsion of spinal tuberculosis patients detected by PPD skin test, TB antiboby test, Histopathology diagnosis and ELISPOT
When we assessed the concordance between the histopathology detection and the ELISPOT assay using the κ coefficient, we found moderate agreement between the two tests.
⑤Clinical characteristics associated with the ELISPOT results in spinal TB patients
In this study, we found that age and emaciation were associated with the number of SFC or positive rate of ELISPOT in spinal TB patients.
2. Clinical value of an enzyme-linked immunospot assay (T-SPOT.TB) for the diagnosis of spinal tuberculosis.
①Patient characteristics
A total of70patients with suspected spinal TB were prospectively recruited during the study period.5patients were excluded from the study.
The remaining65patients were ultimately included for T-SPOT.TB analyses. The spinal TB group contained34cases. The non-TB disease group contained31subjects.
②Clinical diagnostic value of the T-SPOT.TB assay for spinal TB
Among the spinal TB patients and non-TB disease patients, the overall sensitivity, specificity, positive predictive value, and negative predictive value for spinal TB diagnosis by the PPD skin test were61.8%,58.1%,61.8%and58.1%, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value for spinal TB diagnosis by the Antibody detection were58.8%,70.9%,68.9%,61.1%. By comparison, the sensitivity, specificity, positive predictive value and negative predictive value for spinal TB diagnosis by the T-SPOT.TB assay were85.3%,77.4%,80.6%and82.8%, respectively. The overall sensitivity of T-SPOT.TB is higher than that of PPD skin test and antibody detection test (P<0.05).
③Comparsion of spinal tuberculosis patients detected by PPD skin test, TB antiboby test, Histopathology diagnosis and T-SPOT.TB
When we assessed the concordance between the histopathology detection and the T-SPOT.TB assay using the κ coefficient, we found moderate agreement between the two tests.
④The determination of test condition about Rv2041c-ELISPOT, Rv1419-ELI SPOT and Rv0057-1352-ELISPOT
To change the condition of experiment, and find that the best numbers of cell is2×105, the best incubated time is20hour and the best density of proteinum is20μg/ml.
④Clinical diagnostic value of different antige response to the ELISPOT assay for spinal TB
The sensitivity, specificity, positive predictive value and negative predictive value for spinal TB diagnosis by the CFP-10/ESAT-6-ELISPOT assay were82.4%,83.9%,84.8%and71.9%, respectively. The sensitivity, specificity, positive predictive value and negative predictive value for spinal TB diagnosis by the T-SPOT.TB were85.3%,77.4%,74.4%and80.8%.When we assessed the concordance between these two tests using the κ coefficient, we found moderate agreement between the two tests.
3. To analyze the clinical characteristics of the atypical spinal TB and assess the diagnostic value of an enzyme-linked immunospot (T-SPOT.TB) assay in clinically suspected cases of atypical spinal TB.
①Clinical presentation
Back pain was the most common clinical complaint (24patients,82.8%).
②Imaging study findings
Through the imaging study, worm-eaten destruction of vertebral endplate was seen in5patients. Destruction of centricity of the single vertebral body or concentric collapse of single vertebral body were seen in5patients.15patients presented contiguous or skipped vertebral body destruction. Tuberculous abscess with no identifiable osseous lesion were visible by CT and MRI in4patients
③Clinical diagnostic value of the T-SPOT.TB assay for atypical spinal TB
We found that37.5%,78.3%of patients were positive for AFB smear and cultures for M. tuberculosis, respectively. The overall sensitivity, specificity, positive predictive value, and negative predictive value for atypical spinal TB diagnosis by the PPD skin test were58.6%,59.4%,56.7%and61.3%. By comparison, the sensitivity, specificity, positive predictive value and negative predictive value for atypical spinal TB diagnosis by the T-SPOT.TB assay were82.8%,81.3%,80.0%and83.9%, respectively. The overall sensitivity of the T-SPOT.TB assay was higher than that of PPD skin test (P<0.05).
4. To assess clinical value of an enzyme-linked immunospot assay (T-SPOT. TB) for the monitoring efficacy of surgical treatment of spinal TB.
①Patient characteristics
Additional responses were evaluated in a consecutive subset of12spinal TB patients on two days before and after surgery.3spinal TB patients only underwent focal cleaning. The remaining9spinal TB patients underwent one-stage anterior debridement, bone graft plus anterior or posterior instrumentation.
②Responses to ESAT-6, CFP-10before and after Surgery
The median ESAT-6(30spots per250,000PBMCs) and CFP-10(44spots per250,000PBMCs) response were slightly lower after surgery than those before surgery (ESAT-6:36spots per250,000PBMCs; CFP-10:57spots per250,000PBMCs), but those failed to reach statistical significance (P>0.05).
Conclusions
1. A laboratory-developed rCFP-10/ESAT-6-based ELISPOT assay is a useful adjunct to current tests for diagnosis of spinal TB.
2. The T-SPOT.TB assay is a promising tool for the clinical evaluation of spinal TB and merits additional assessment in a diagnostic trial.
3. The recombinant Rv1419protein of mycobacterium tuberculosis has relatively high value in the immunological diagnosis of tuberculosis.
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