| 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. |
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