【作者】 刘士甫；

【导师】 何俊瑛；

【作者基本信息】 河北医科大学 ， 神经病学， 2010， 硕士

【摘要】 目的:结核性脑膜炎(tuberculous meningitis,TBM),约占肺外结核的7-10% [1],是由结核杆菌引起的中枢神经系统感染性疾病,致残率和致死率较高,其预后与早期诊断及时治疗密切相关。但结脑的诊断,尤其是早期诊断比较困难,因为结脑患者的脑脊液(CSF)中结核杆菌的数量很少,脑脊液涂片抗酸染色阳性率很低约为10%,而结核菌培养所需时间(4～8周)较长,且阳性率仅20～30%[2],对临床早期诊断价值有限。相关性研究表明结核抗原存在可以作为结核菌存在的证据[3],因而检测脑脊液(cerebrospinal fluid CSF)中的结核抗原对TBM的诊断很有价值。免疫组化法检测脑脊液单核细胞内结核菌素纯蛋白衍生物(protein purified derivative, PPD)实验,对临床诊断TBM具有一定的参考价值,但此抗原尚有一些不足之处。PPD为成分不明确的复合抗原(多克隆),存在一定的假阴性和假阳性,因此寻找一种独特的抗原对TBM的诊断非常必要。早期分泌性抗原靶6 (6Kd early secretory antigenic target ,ESAT-6)为结核分枝杆菌感染早期所分泌的蛋白,具有高度的分布特异性和良好的免疫原性,当结核杆菌在感染的单核巨噬细胞内生长时, ESAT-6抗原表达量明显提高,因此采用单克隆抗体(免疫组化法)检测脑脊液单核细胞内ESAT-6可能会增加诊断结脑的敏感性和特异性。本课题研究的目的是将ESAT-6与PPD对结核性脑膜炎的诊断价值进行比较,为结核性脑膜炎的诊断提供有效的方法。方法:将2009年1月～2009年7月神经内科住院的40例病人分为两组,一组为结脑组:入选标准参考Ahuja[3]的临床诊断标准,共20例,男8例,女12例,年龄2-59岁,中位值年龄24.5岁,其中单纯结脑16例,合并颅外结核者4例。另一组为对照组:共20例,男9例,女11例,年龄1-59岁,中位值年龄26岁。根据第五版神经病学诊断标准入选,其中化脓性脑膜炎5例,新型隐球菌脑膜炎2例,病毒性脑膜炎8例,脑囊虫病1例,脑肿瘤2例(听神经瘤1例,肺癌脑转移1例),脊髓炎1例,布氏杆菌病1例。对所有患者均进行脑脊液常规、生化、细胞学检查(MGG染色)和阿利新兰染色。取脑脊液1ml,用Therm-4 Shandon Cytospin型玻片离心机(购自英国)离心玻片3张,用免疫组化的方法检测单核细胞内的PPD和ESAT-6抗原。将沉淀有细胞的玻片放入中性甲醛中固定10分钟,然后用蒸馏水冲洗,0.01MPBS浸泡5分钟,3%H2O2去离子水孵育10分钟,以消除内源性过氧化物酶活性。滴加封闭用山羊血清工作液室温孵育15分钟倾去。一张玻片滴加适当比例稀释的单克隆鼠抗人ESAT-6抗体(购自美国ADL公司),另一张滴加兔抗结核分枝杆菌多克隆抗体(购自美国Abcom公司),以PBS缓冲液代替一抗为阴性对照。4℃冰箱孵育过夜。PBS冲洗3次每次3分钟。滴加生物素化二抗(购自中杉金桥公司)工作液室温孵育15分钟,PBS冲洗3次,每次3分钟,滴加辣根酶标记的链酶卵白素工作液, 37℃孵育15分钟,PBS冲洗3次每次3分钟,显色剂显色(DAB)5分钟,流水冲洗10分钟。苏木素染色3分钟,自来水冲洗浮色。光学显微镜(OLYMPUS-BX41日本)下观察,脑脊液图文分析仪(MCDS-2020重庆)采图判读结果,单核细胞胞浆呈棕色为阳性结果,单核细胞胞浆无色为阴性结果。结果1临床资料:结脑组患者发热及意识障碍发生率分别为100%, 65%,对照组为70%, 25%,两组之间差异显著,而头痛、恶心呕吐、颅高压、脑膜刺激征、脑局灶体征、脑电图两组无明显差别。2脑脊液常规、生化:结脑组白细胞数3～14000个/mm3,平均为952.5±3086个/mm3,糖0.8～4.1 mmol/L,平均为1.76±0.735mmol/L;蛋白含量0.61～2.39 g/L,平均为1.35±0.53/L;氯化物96～122.4 mmol/L,平均为110.9±8.46mmol/L;对照组细胞数0～2980个/mm3,平均为352±717.02个/mm3;糖0.09～4.7 mmol/L,平均为2.866±1.089 mmol /L;蛋白0.08±4.95 g /L,平均为0.973±1.356 g /L,氯化物由106～142.0 mmol/L,平均为120.96±9.45 mmol/L。结脑组较对照组糖和氯化物明显降低,而白细胞数和蛋白无明显差别。3细胞学表现:结脑组脑脊液细胞学20例全部为混合细胞反应,均未见肿瘤细胞且阿利新兰染色阴性。对照组中混杂细胞学9例,淋巴样细胞反应5例,正常细胞学反应2例,激活单核细胞样细胞学反应2例,中性粒细胞反应2例,隐球菌脑膜炎者细胞学及阿利新兰染色均找到隐球菌。脑囊虫病嗜酸性粒细胞明显增多。肿瘤患者找到肿瘤细胞且激活单核细胞增多。在动态观察中显示结脑病人随治疗好转中性粒细胞逐渐减少,但持续较长时间,同时激活的淋巴细胞、单核细胞逐渐增加,浆细胞出现,这与结脑动态变化相符。4单核细胞内ESAT-6抗原:可在脑脊液中检测到单核细胞内的ESAT-6,大多数单核细胞都有较强的胞浆染色,且多次检测可持续阳性,激活单核细胞较少染色或染色较浅。20例TBM患者中14例ESAT-6抗原阳性,6例阴性,对照组中仅1例病毒性脑膜炎患者阳性,此方法诊断TBM敏感性为70%,特异性为95%,约登指数0.65。在20例TBM病人中,最早发病5天,最晚发病120天可检测出胞内ESAT-6。5单核细胞内PPD抗原: 20例TBM患者中13例PPD抗原阳性,7例阴性,对照组中2例病毒性脑膜炎患者阳性,18例阴性。此方法诊断TBM敏感性为65%,特异性为90%,约登指数0.55。6 ESAT-6及PPD诊断TBM比较:敏感性P>0.05,特异性P>0.05,二者之间敏感性和特异性均无统计学差异。结论1结脑患者临床表现较重,常伴有发热及意识障碍。结脑患者临床表现复杂多样与其他神经系统疾病较难鉴别。2脑脊液生化可为结脑诊断提供一定依据,但脑脊液常规意义较小。3脑脊液细胞学对结脑的诊断和鉴别诊断具有重要参考价值。4结脑患者脑脊液单核细胞内存在ESAT-6抗原。TBM组与对照组比较ESAT-6阳性率差异显著,说明该方法对诊断和鉴别诊断TBM方面有意义。5检测单核细胞内单克隆ESAT-6抗原与检测单核细胞内多克隆PPD抗原相比其敏感性、特异性无显著差别,可能是病例数少所致。但ESAT-6抗原的约登指数高于PPD抗原的检测,两种方法的优劣有待于扩大病例数进一步比较。更多还原

【Abstract】 Objective:Tuberculous meningitis(TBM), 7～10% of tuberculosis, is a central nervous system infection disease caused by Mycobacterium tuberculosis. The early diagonosis and appropriate treatment were strongerly associated with the patient’s outcom .Early diagnosis and appropiate treatment in a patient with TBM will lead to complete neurological recovery while a delay in diagnosis and treatment often leads to irreversible neurological sequelea or death.But the diagnosis of the TBM is difficulty, because the quantity of M.tuberculosis in CSF is very few. The acid-fast bacilli are seldom demonstrated in CSF by Ziehl-Neelsen staining,about 10%.The culture of is not only time-consuming, about 4～8 weeks,but also less sensitivity ,the sensitivity is only 20～30%. So their value of earlier diagnosis in clinical is limited.Search for a method of earlier diagnosis is a highly anticipated lesson. Correlation studies have shown that Mycobacterium tuberculosis antigens can be used as the evidence of M.tuberculosis existence. For this reason ,detecte the intracellular mycobacterial antigens in monocytes (macrophages) in CSF is valuable. Immunohistochemical detection of cerebrospinal fluid mononuclear cells of tuberculin purified protein derivative (protein purified derivative, PPD) test for diagnosis of TBM has a certain reference value, but this antigen has a number of shortcomings. PPD as a component of the complex is not clear antigen (polyclonal), there is a certain degree of false negative and false positive, so look for a unique antigen for the diagnosis of TBM was necessary. Early secretory antigen target 6 (ESAT-6) is a protein secreted in the early stage of Mycobacterium tuberculosis infection. It has a high degree specific distribution and good immunogenicity , When M. tuberculosis in infected monocyte-macrophage cells is growing, ESAT-6 antigen expression will significantly increased. Therefore, using monoclonal antibodies (immunohist -ochemistry) to detect ESAT-6 in the mononuclear cells of cerebrospinal fluid may increase the diagnostic of tuberculous meningitis sensitivity and specificity .The purpose of this study is to evaluate the significance in diagnosis of tuberculous meningitis by detecting antigens of PPD and ESAT-6 in the monocytes of CSF,and then provide an effective method for the diagnosis of tuberculous meningitis.Methods:All patients are from depatment of nuerology the second affliliated hospital of Hebei Medical University during the period 2009 january～2009 july.These 40 patients was divided into two groups.One group is tuberculous meningitis ,the diagnosis criteria is according to Ahuja’s clinical diagnosis criteria.Totally 20 patients,8 are male, 12 are female, the median age is 24.5 years.Among them 16 patients are pure TBM, 4 patients combine tuberculosis outside of skull.Another is control group,totally 20 patients,9 are male, 11 are female, the median age is 26 years .Diagnosis of every patient was according to the standard of 5th neurology textbook.Among them 5 had purulent meningitis, 2 had cryptococcal meningitis, 8 had viral meningitis, 1 had cerebral cysticercosis , 2 had brain tumor (acoustic neuroma in 1 case, 1 had lung cancer with brain metastasis ), Myelitis 1 case, 1 cases of brucellosis .All patients were investigated of CSF include the routing test, biochemical, cytology (MGG dye), and Alixin dye. Immunohistochemistry demonstration of ESAT-6 and PPD in the cytoplasm of monocytes was carried out at the same time.Take CSF 1 ml, centrifug 3 smears with Therm-4 Shandon Cytospin type centrifugal sedimentation instrument and prepare for Immunohistochemistry staining.First the smears were fixed in neutral formaldehyde for 10 minutes at 4℃, then washed with 0.01 M PBS,( pH 7.4 ) 3 times, every time is 5 minutes.After that droping 3% H2O2 deionized water, incubated for 10 minutes to eliminate endogenous peroxidase activity. move it into PBS which contained 3% normal goat serum for 15 minutes, then incubated with rabbit anti- tuberculousis antibody IgG and ESAT-6 antibody IgG , The negtive comperation was substituted the primery antibody with PBS buffer.4℃overnight.Take out the smears and washed with the PBS buffer 3 times.Subsequently adding the biotinylated secondary antibody (purchased from the cedar Jinqiao Company) and then incubated for 15 minutes at room temperature. Washed by PBS buffer 3 times. Dropping horseradish enzyme labeled avidin-chain working solution, 37℃incubation15 minutes .PBS washed 3 times every 3 minutes, the color reagent (DAB), running water for 10 minutes. Hematoxylin 3 minutes, tap water rinse floating color.,and visualized under the optical microscope ( OLYMPUS-BX41Japan ) . cerebrospinal fluid Graphic Analyzer (MCDS-2020 Chongqing) to adopt map interpretation of results, Mononuclear cells of brown as a positive result.Mononuclear cells of colorless as negative results.Results1 Clinical data : Tuberculous meningitis patients the incidence of fever and consciousness disturbance were 100%, 65% vs 70%, 25%, significant difference between the two groups. The headache, nausea, vomiting, cranial pressure, meningeal irritation, focal cerebral signs, EEG was no significant difference between the two groups.2 Cerebrospinal fluid routine and biochemical examination: Sugar and chloride in tuberculous meningitis group was lower than in the control group, while the white blood cell count and protein, no significant difference between the two groups.3 CSF Cytology:TBM group all patients show a mixted-cell response in CSF. The Alixin and Indian ink staining of patients with TBM are negtive. All the patients no tumor cells.5 patients in control group is dominate of lymphoid-cell response , 9 patients show a mixted-cell response . Cryptococcal was detected in CSF with cryptococcal meningitis ,and the Alixin are all positive.Neurocysticercosis disease show eosinophilosis. At the dynamic observation , neutral cell gradually reduced when the condition of TBM patients become better after the treatment. But neutral cell will last a long time, the monocyte and lymphoid cell increase gradually at the same time, plasmcell emergence, this agree with the TBM’s CSF feature. 4 Result of ESAT-6 antigens demostration in monocytes:At every stages of disease the antigens of ESAT-6 can be detected in the cytoplasm of monocyes in the CSF, and most monocytes have stronger staining.The positive staining will keep for a long time with continue test.The actived monocytes staining are shallow or no stain. In 20 TBM ,14 patients show positive immunostaining, 6 patients show negative, while the control group only one showed positive.The positive rate of ESAT-6 in experimental group was significantly higher than the control group. its sensitivity is 70%,and specificity is 95%, youdens index is 0.65. In the 20 cases of TBM patients, the earliest incidence of 5 days, no later than 120 days can be detected in the incidence of intracellular ESAT-6.5 Result of PPD antigens demostration in monocytes: 20 cases of TBM patients, 13 cases of PPD antigen-positive and 7 cases negative, the control group 2 cases of viral meningitis in patients with positive and negative in 18 cases.The positive rate of PPD in experimental group was significantly higher than the control group its sensitivity is 65%,and specificity is 90%, youdens index is 0.55.6 ESAT-6 and PPD comparison: sensitivity and specificity between the two there was no statistical difference.Conclusion1 The clinical manifestations of tuberculous meningitis in patients serious, often accompanied by fever and disturbance of consciousness. Junction complex and diverse clinical manifestations in patients with brain and other nervous system diseases difficult to identify.2 CSF biochemistry can provide a basis for diagnosis of tuberculous meningitis, but routine examination of cerebrospinal fluid have small significance .3 The value. cerebrospinal fluid cytology in the diagnosis of tuberculous meningitis and differential diagnosis is great.4 The ESAT-6 in the plasma of the monocytes of CSF. TBM group and the control group ESAT-6 positive rate significantly different, indicating that the method in the diagnosis and differential diagnosis of TBM is meaningful . The positive rate of ESAT-6 in experimental group was significantly higher than the control group ,its sensitivity is 75%,and specificity is 90%, youdens index is 0.65. Observation in TBM patients, the earliest detection of the ESAT-6 antigens in CSF is at 5th day . the latest detection of the ESAT-6 antigens in CSF is at 120th day . So we can say that this method applies not only to early stage, is applicable for the whole course of the disease.Detection the ESAT-6 in the monocytes of CSF is a simple and specific method in diagnosis of tuberculous meningitis.Detection of mononuclear cells of monoclonal ESAT-6 5 antigen compared with polyclonal PPD antigen, the sensitivity and specificity was no significant difference could be due to a small number of cases. ESAT-6 antigen Youden index higher than the PPD antigen detection, advantages and disadvantages of both approaches need to be to expand the number of cases for further comparison.更多还原