Amplisensor—聚合酶反应定量检测脑脊液中结核分支杆菌DNA的研究

目的 探讨Amplisensor-聚合酶反应（Amplisensor-PCR)定量检测脑脊液中结核分支杆菌DNA（TB－DNA）对结核性脑膜炎的诊断价值。方法 采用Amplisensor-PCR对117例结核性脑膜炎患者及36例非结核性脑膜炎患者的脑脊液标本进行检测,并与PCR（凝胶电泳后,经溴化乙锭染色）、涂片、培养法比较。结果 Amplisensor-PCR的敏感性显著高于涂片及培养,阳性率分别为57．13％、1．7％、6．7％（P＜0．001）。结论 Amplisensor-PCR可以通过标准曲线划定检出下限,并可换算出标本中原始的靶DNA值,同时具有较高的特异性和敏感性,对结核性脑膜炎的诊断有一定的临床意义。     

TaqMan-PCR技术诊断结核病的临床应用研究

目的　探讨TaqMan-聚合酶链反应 (TaqMan-PCR)技术在结核病快速诊断中的临床价值。方法 对 155例活动性肺结核患者的痰和外周血、130例结核性胸膜炎患者的胸腔积液和外周血以及 61例结核性脑膜炎患者的脑脊液和外周血应用TaqMan -PCR检测结核分支杆菌DNA ,痰、胸腔积液和脑脊液进行抗酸染色涂片检查 ,另外 ,痰标本还进行了BACTEC和改良罗氏培养；同期以52例肺癌患者的痰和外周血、50例恶性胸腔积液患者的胸腔积液以及 33例健康人的外周血作为对照进行TaqMan-PCR检测。 结果 155例活动性肺结核患者的痰和外周血、130例结核性胸膜炎患者的胸腔积液和外周血以及 61例结核性脑膜炎患者的脑脊液和外周血TaqMan-PCR的阳性率分别为 49.0%和 51.6%、45.4%和 38.5%以及 50.8%和 42.6% ,痰、胸腔积液和脑脊液TaqMan-PCR的阳性率显著高于抗酸染色涂片以及BACTEC和罗氏培养 (P＜0.05)。TaqMan-PCR检测痰、胸腔积液和外周血的特异性分别为 96.2%、98%和 96.5%。结论 TaqMan-PCR具有较高的敏感性和特异性 ,对结核病的快速诊断具有一定的价值。     

三种快速检测技术诊断早期结核性脑膜炎的评价

目的 评价3种快速检测技术在早期结核性脑膜炎诊断中的应用价值。方法 采用离心涂片抗酸染色法、改良抗酸染色法、GeneXpert MTB/RIF技术(简称“GeneXpert技术”)对2016年8月至2018年6月河北省胸科医院和石家庄市第五医院收治的根据临床表现、影像学及实验室检查等临床确诊的45例结核性脑膜炎、42例非结核性脑膜炎患者的脑脊液标本进行分枝杆菌检测,并对结果进行分析。采用SPSS 19.0软件进行统计学分析,率的比较采用χ ²检验、校正χ ²检验,以P<0.05为差异有统计学意义。结果 以临床诊断为标准,45例结核性脑膜炎患者脑脊液分别应用离心涂片抗酸染色法、改良抗酸染色法、GeneXpert技术检测分枝杆菌的敏感度分别为4.44%(2/45)、88.89%(40/45)、35.56%(16/45);依次两两比较,差异均有统计学意义(χ ²值分别为64.46、13.16、27.23,P值均=0.000)。42例非结核性脑膜炎患者的脑脊液用离心涂片抗酸染色法、改良抗酸染色法、GeneXpert技术检测分枝杆菌均为阴性。3种检测技术检测分枝杆菌的特异度均为100.00%。结论 改良抗酸染色法较离心涂片法及GeneXpert技术检测分枝杆菌阳性率高,具有较好的确诊结核性脑膜炎的价值。     

TB-SA基因检测在结核病诊断的临床应用研究

目的　研究结核分枝杆菌特异性基因(Tuberculosis-Specific AntigenTB-SA)在结核病诊断中的应用价值。方法 选择2004年4—9月期间在成都市结核病防治院的住院结核病患者371例。其中肺结核307例;肺外结核64例(结核性胸膜炎47例、结核性腹膜炎7例、结核性脑膜炎10例);非结核病的呼吸系统疾病患者61例(肺炎4例、肺癌9例,急性支气管炎7例、慢性支气管炎14例、哮喘10例,COPD12例、肺间质纤维化1例、支气管扩张3例、矽肺1例);同时选择无结核疾患健康志愿者40例,全部选例均知情同意。入选肺结核病例和健康选择痰液、肺外结核病例选择胸腔积液、腹腔积液、脑脊液等标本进行抗酸杆菌浓缩集菌,结核杆菌培养和TB-SA基因扩增(PCR)。结果 307例肺结核患者中PCR检测TB-SA阳性259例,敏感性为84.4%(259/307);细菌学培养阳性193例,敏感性62.9%(193/307);痰浓缩集菌镜检阳性95例,敏感性30.9%(95/307)。64例肺外结核患者中,TB-SA阳性35例;敏感性54.7%(35/64);核杆菌培养阳性5例,敏感性7.8%(5/64)。61例非结核呼吸系统病患者中,TB-SA阳性2例,特异性96.7%(59/61)痰涂片无阳性病例。40例健康志愿者,TB-SA及痰涂片均无阳性出现,特异性达100%(40/40)。结论 TB-SA基因检测的特异性及敏感性都取得了满意的效果。操作也较为简单。     

超声引导下获取标本行病理学与结核相关检测对结核性胸膜炎的诊断价值

目的 比较分析超声引导下胸膜穿刺获取的病理组织及胸腔积液标本在疑似结核性胸膜炎中的诊断价值。方法 搜集2016年1月至2018年5月在西安市胸科医院初次就诊的298例疑似结核性胸膜炎患者,其中临床确诊结核性胸膜炎患者112例,非结核性胸膜炎患者186例。所有患者均通过超声引导下胸膜穿刺获取病理组织和胸腔积液标本,对活检组织进行病理学切片检查(简称“病理检查”),胸腔积液分别进行结核分枝杆菌培养(浓缩集菌法,采用液体培养BACTEC MGIT 960操作系统,简称“MGIT 960培养”)、全自动医用PCR分析系统(GeneXpert MTB/RIF,简称“GeneXpert检测”)和抗酸杆菌染色涂片检查(简称“涂片检查”)。上述各种方法以临床确诊患者为参考标准,得出各自的诊断敏感度、特异度、阳性预测值、阴性预测值、约登指数,以评价各种检测方法诊断结核性胸膜炎的效能。结果 病理检查、MGIT 960培养、GeneXpert检测、涂片检查以临床确诊结果为参考标准的诊断敏感度分别为41.96%(47/112)、73.21%(82/112)、58.93%(66/112)和2.68%(3/112);特异度分别为99.46%(185/186)、100.00%(186/186)、100.00%(186/186)、100.00%(186/186);约登指数分别为0.41、0.73、0.59、0.02。结论 超声引导下胸膜穿刺获取标本采用各种技术与方法进行检测,结果证明胸腔积液标本采用浓缩集菌法进行MGIT 960培养相对其他检测技术与方法具有较好的敏感度、特异度,约登指数最高,具有良好的临床诊断结核性胸膜炎的价值。     

RNA恒温扩增实时荧光检测技术对结核性胸腔积液的诊断价值

目的 探讨RNA恒温扩增实时荧光检测技术(simultaneous amplification and testing, SAT-TB)对结核性胸腔积液的诊断价值。方法 选取2014年6月至2016年6月聊城市传染病医院收治的有胸腔积液的患者210例作为研究对象,其中143例临床诊断为结核性胸膜炎,作为结核性胸膜炎组;其余67例患者作为对照组。应用涂片法、改良罗氏培养法、SAT-TB法检测研究对象胸腔积液标本,比较各检测方法的检测效能。结果 SAT-TB检测的敏感度、特异度、阳性预测值、阴性预测值分别为36.36%(52/143)、98.51%(66/67)、98.11(52/53)、42.04%(66/157)。SAT-TB检测的敏感度明显高于涂片法[3.50%(5/143)]及罗氏培养法[20.28%(29/143)],差异均有统计学意义(χ ²=19.08,P=0.021;χ ²=9.12,P<0.01)。SAT-TB法检测完成时间为1~2h,罗氏培养法完成时间为6~8周。 结论 SAT-TB法能快速检测胸腔积液中的结核分枝杆菌,敏感度高于涂片法和罗氏培养法。     

联检脑脊液中LAM-IgG和TB-DNA在诊断结核性脑膜炎中的应用

目的　了解脑脊液 (CSF)中阿拉伯糖甘露糖脂IgG抗体 (LAM-IgG)和TB-DNA指标对结脑的诊断价值。方法 以CSF为标本,用酶联免疫吸附试验 (ELISA)检测LAM-IgG,用聚合酶链反应(PCR)检测TB-DNA。结果 102份结脑病人CSF标本,LAM-IgG阳性率51.0% (52102),TB-DNA阳性率81.4% (83102),LAM-IgG阳性及 或TB-DNA阳性共93例 (91.2%)。40份非结核性的中枢神经系统疾病病例的CSF标本,均未检出LAM-IgG和TB-DNA.结论 LAM-IgG和TB-DNA均是诊断结脑的较好的指标,两者联检可进一步提高检测敏感性。     

误诊结核性脑膜炎38例临床分析

目的　提高对结核性脑膜炎鉴别诊断水平。方法 1985年1月至2000年10月住院结核性脑膜炎342例中对非结核性脑膜炎患者误诊为结核性脑膜炎的38例进行回顾分析。结果 总误诊率11.1% (38342)。误诊疾病病毒性脑膜炎19例占误诊病例50.0%,新型隐球菌脑膜炎7例占18.4%,脑囊虫病3例占8.0%,红斑狼疮脑病、急性播散性脑脊髓炎各2例,各占5.3%,脑脓肿、松果体旁囊肿、脑室膜瘤、脑胶质瘤、非特异性大脑导水管狭窄各1例,均占2.6%。误诊时间最短2d,最长360d,平均37d。结论 ①结核病专科医师应熟悉其他脑部疾病临床表现,提高结核性脑膜炎的诊断与鉴别水平。②对临床经过和脑脊液 (CSF)表现不典型、抗结核治疗效果不良的结核性脑膜炎患者,应提高警惕,需进一步排除隐球菌性脑膜炎及脑囊虫病等其他脑部疾患。③对起病急、进展快、早期出现意识障碍、截瘫、括约肌功能障碍,而CSF改变轻微者,应考虑急性播散性脑脊髓炎的可能。④脑部CT(增强)或核磁共振检查对结核性脑膜炎诊断、鉴别诊断有重要作用。     

急性布鲁氏菌病并发中枢神经损害患者临床特征分析

目的 分析布鲁氏菌病中枢神经损害脑脊液特征,探讨其临床诊断价值。方法 选择2007年至2014年黑龙江省农垦总局总医院住院治疗的急性布鲁氏菌病并发中枢神经损害患者20例作为观察对象。临床诊断:并发脑膜炎8例、脑膜脑炎3例、脊髓炎7例和脑脓肿2例。调查其住院病例资料,观察脑脊液常规检查各项指标,分析和评估脑脊液改变特点。结果 脑脊液异常改变,颅内压增高13例,白细胞计数增多18例(90.00%),蛋白增高19例(95.00%),葡萄糖和氯化物降低16例(80.00 %)。结论 布鲁氏菌病并发中枢神经损害患者脑脊液多发生异常改变,颅内压升高,白细胞计数增多,葡萄糖和氯化物降低为特征,诊断需要与结核性脑膜炎相鉴别。     

结核蛋白芯片对肺结核诊断的临床价值

目的　探讨结核蛋白芯片、痰涂片抗酸染色及结核菌素(PPD)皮试单独和联合检测对肺结核患者临床诊断的意义。方法 对45例肺结核和30例非结核性肺疾病患者,同时进行结核蛋白芯片、痰抗酸杆菌直接涂片、PPD皮试,观察单项和联合指标对诊断肺结核的敏感性与特异性。结果 结核蛋白芯片、PPD及痰涂片的敏感性分别为66.7%、44.4%、22.2%,特异性分别为93.3%、84.6%、100%。结核蛋白芯片联合PPD对肺结核的阳性检出率为86.7%,结核蛋白芯片联合PPD及痰涂片的阳性检出率提高到88.9%,与单项检测的最高阳性检出率相比差异有显著性意义(P<0.05)。三种方法联合检测的特异性为80%,与PPD或结核蛋白芯片单项检测相比无显著性差异(P>0.05)。结论 结核蛋白芯片是诊断结核病较有效方法,具有快速方便等优点,蛋白芯片联合PPD、痰涂片抗酸染色,能显著提高肺结核的阳性检出率,但特异性并没有明显下降。     

4种检测脑脊液结核分枝杆菌法对结核性脑膜炎诊断价值的探讨

目的探讨脑脊液抗酸杆菌涂片、3D、PhaB和PCR检测对结脑的诊断价值。方法检测60例结脑和30例非结脑患者脑脊液涂片、3D、PhaB和PCR阳性率,经统计学处理后,评价4种方法对结脑诊断的灵敏度、特异度。结果结脑组和其他组脑脊液涂片阳性率均为0,3D阳性率分别为23.3%和0,PhaB的阳性率分别是23.3%和26.7%,PCR的阳性率分别是6%和0,4种方法差异无统计学意义。脑脊液3D灵敏度为23.3%,特异度为100%,PhaB灵敏度为23.3%,特异度为73.3%,PCR的灵敏度10%,特异度为100%。结论脑脊液结核分枝杆菌涂片、3D、PhaB和PCR4种检测方法灵敏度低,积极寻找脑脊液结核分枝杆菌的检测方法和提高阳性率是实验室亟需解决的问题。     

Rapid diagnosis of tuberculous meningitis by ligase chain reaction amplification

A total of 87 cerebrospinal fluid (CSF) samples obtained from 85 patients with suspected meningitis were examined for the presence of Mycobacterium tuberculosis by means of ligase chain reaction amplification (LCx; Abbott Laboratories). The results were compared with direct smear and culture results. Of 61 patients with pathological CSF, 9 (14.8%) were scheduled to receive treatment for tuberculous meningitis. The sensitivity of the smear and culture tests was 11.1 and 33.3%, respectively. The sensitivity, specificity, positive predictive value and negative predictive value of the LCx assay were 55.5, 100, 100 and 92.9%, respectively. The results reveal that amplification by ligase chain reaction is valuable for the diagnosis of tuberculous meningitis.     

Rapid Diagnosis of CNS Tuberculosis by a T-Cell Interferon-γ Release Assay on Cerebrospinal Fluid Mononuclear Cells

Central nervous system tuberculosis remains a clinical diagnostic challenge. The ex vivo Mycobacterium tuberculosis-specific enzyme-linked immunospot assay (ELISPOT) is a novel assay for the rapid detection of M. tuberculosis-specific T-lymphocytes in the peripheral blood. However, when performed on peripheral blood, this assay cannot distinguish between active tuberculosis or latent tuberculosis infection. On the assumption that M. tuberculosis-specific T-lymphocytes migrate to sites of infection, we were able to demonstrate high levels of M. tuberculosis-specific cells by ELISPOT in the cerebrospinal fluid of a patient with tuberculous meningitis and intracerebral tuberculoma four weeks before cerebrospinal fluid culture became positive for M. tuberculosis by culture.     

Rapid diagnosis of Mycobacterium tuberculosis meningitis by enumeration of cerebrospinal fluid antigen-specific T-cells.

SETTING: Hospital in-patients with suspected tuberculous meningitis (TBM), predominantly in India. OBJECTIVE: To determine whether interferon-gamma (IFN-gamma) secreting Mycobacterium tuberculosis antigen-specific T-cells are present in the cerebrospinal fluid (CSF) of patients with TBM and to evaluate the feasibility of CSF enzyme-linked immunospot (ELISpot) for the diagnosis of active TBM. DESIGN: Prospective blinded hospital-based study. RESULTS: The overnight ELISpot assay detected M. tuberculosis antigen-specific IFN-gamma secreting T-cells in CSF from nine of 10 prospectively recruited patients with TBM, and zero of seven control patients with meningitis of other aetiology. This corresponds to a diagnostic sensitivity of 90% (95%CI 56-100) and specificity of 100% (95%CI 59-100). CONCLUSION: This pilot study demonstrates proof-of-principle for a new T-cell-based diagnostic test for TBM which is rapid, sensitive and specific.     

TB PCR in the early diagnosis of tuberculous meningitis: evaluation of the Roche semi-automated COBAS Amplicor MTB test with reference to the manual Amplicor MTB PCR test. .

SETTING: Cecilia Makiwane Hospital, Mdantsane, Eastern Cape, Republic of South Africa. OBJECTIVE: To assess the role of the semi-automated Roche COBAS AMPLICOR(TM)Mycobacterium tuberculosis PCR test in the diagnosis of tuberculous meningitis (TBM). DESIGN: Eighty-three specimens of cerebrospinal fluid (CSF) were collected prospectively from 69 patients with suspected TBM. The COBAS AMPLICOR TB PCR test was compared with the manual AMPLICOR(TM)TB PCR test, clinical and cerebrospinal fluid (CSF) findings, direct ZN smear and radiometric TB culture. RESULTS: CSF from 7/40 (17.5%) patients treated for TBM were positive by TB COBAS AMPLICOR(TM). The sensitivity of the test was not significantly different (p=0.375) from the manual TB AMPLICOR(TM)PCR test. The comparative sensitivities of the TB COBAS AMPLICOR(TM)PCR and the manual AMPLICOR PCR for detecting cases of definite and probable TBM from CSF collected within 9 days of commencing antituberculosis treatment were 40% and 60% respectively. All 29 patients not treated for TBM were negative by COBAS AMPLICOR(TM), giving a specificity of 100%. CONCLUSION: The COBAS AMPLICOR(TM)TB PCR test is a rapid and highly specific diagnostic test for TBM. However, there was a non-significant trend favouring slightly greater sensitivity using the manual AMPLICOR(TM)TB PCR test.     

A comparative study of the polymerase chain reaction and conventional procedures for the diagnosis of tuberculous pleural effusion.

Preliminary reports by ourselves and others suggest that amplification of mycobacterial DNA by the polymerase chain reaction (PCR) is a sensitive and rapid diagnostic test for tuberculosis. We recently described a PCR assay with a 336 bp repetitive sequence specific for Mycobacterium tuberculosis as the DNA target, which gave encouraging results in culture-positive smear-negative clinical specimens. In the present prospective study of patients with pleural effusions we compared PCR of the pleural fluid with conventional procedures. 84 adult patients with pleural effusions were divided into 4 groups. In group A (44 patients), M. tuberculosis was detected by culture of pleural fluid, pleural biopsy or extrapleural source. In group B (6 patients), tuberculous infection was confirmed by histology (group A excluded). Group C (3 patients) had clinical evidence of tuberculosis. Group D (31 patients) had no evidence of active M. tuberculosis infection. Analysis of the pleural fluid confirmed a sensitivity for PCR of 81%. The sensitivity of pleural fluid culture, culture of pleural biopsy, and histology of biopsy was 52.8%, 69.8% and 77.3% respectively. There were however 7 PCR positive results within group D; 6 of these were in patients with malignant effusions. We conclude that for the diagnosis of M. tuberculosis PCR is more sensitive than laboratory culture as determined by the analysis of pleural fluids. Positive PCR results among patients with malignant effusions may be false-positives or the result of latent tuberculous infections. PCR should remain an investigational procedure until prospective studies in high and low prevalence areas have critically evaluated the specificity of the assay.     

Correlation between culture of Mycobacterium tuberculosis and IgG antibody to Mycobacterium tuberculosis antigen-5 in the cerebrospinal fluid of patients with tuberculous meningitis

A retrospective study was made of the correlation between culture of Mycobacterium tuberculosis and detection of IgG antibody to M. tuberculosis antigen-5 in cerebrospinal fluid (CSF) by means of an enzyme linked immunosorbent assay (ELISA). Mycobacterium tuberculosis was cultured from the CSF in 14 of 70 patients with a clinical diagnosis of tuberculous meningitis (TBM). IgG antibody to M. tuberculosis antigen-5 was demonstrated in significant titres (80-640) in all 14 culture-positive patients. Thus, positive correlation was observed between culture of M. tuberculosis and detection of IgG antibody in the CSF. As a result of this observation, the CSF from 56 culture-negative patients with a clinical diagnosis TBM was specifically investigated for the detection of IgG antibody to M. tuberculosis antigen-5 and the findings were correlated with those of culture-positive patients. The assay was positive in 34 of 56 patients, the antibody titre ranging between 80 and 640. In the CSF of 70 patients with non-tuberculous neurological diseases, the assay was negative at a dilution of 1 in 80. Thus, detection of IgG antibody to M. tuberculosis antigen-5 by indirect ELISA carried 100% specificity and 60.7% sensitivity for a tuberculous aetiology in culture-negative patients with TBM. The results of this study suggest that indirect ELISA for IgG antibody to M. tuberculosis antigen-5 in CSF holds definite promise in diagnosis of TBM, particularly when repeated cultures of CSF are negative for M. tuberculosis.     

Familial outbreak of disseminated multidrug-resistant tuberculosis and meningitis.

Rapidly progressive multidrug-resistant tuberculosis (MDR-TB) is well documented in human immunodeficiency virus (HIV) positive subjects, but it is not fully recognised in HIV-negative subjects in the familial environment. We report three cases of MDR-TB in three young HIV-negative subjects from the same family. All the patients showed signs of meningitis during the course of their disease, and in two cases a resistant strain of Mycobacterium tuberculosis was isolated in cerebrospinal fluid. Two of the three subjects died from neurological complications; the other was successful treated utilising both systemic and intrathecal therapy for tuberculous meningitis. By a retrospective analysis of DNA obtained from Lowenstein-Jensen cultures, the strains were confirmed as M. tuberculosis resistant to rifampicin and isoniazid, and were closely related in the two cases where specimens were available for analysis. The resistance was acquired in two patients initially infected with a susceptible strain; in the other patient, the resistance was present on the first sensitivity test for which results were available. This report demonstrates the high risk of fatality from MDR-TB for HIV-negative subjects in the absence of reliable early diagnostic and preventive tools. It also reinforces the concept that genetic susceptibility to M. tuberculosis may be an important factor in the clinical presentation and outcome of MDR-TB.