| Objective: Tuberculous meningitis (TBM) is a central nervous system infection disease caused by Mycobacterium tuberculosis, one of the most serious type in tuberculosis, its fatality rate and disability rate is very high.The incidence of TBM was increasing in recent years due to various factors, such as the prevalence of multiple-drug resistance tuberculosis, the infection of human immunodeficiency virus and the poverty. Early diagnosis and early treatment is the key to affect prognosis. But the diagnosis of the TBM, especially early diagnosis is difficult. Cerebrospinal fluid(CSF)detectes mycobacterium tuberculosis can be used as "gold standard" of diagnosis TBM,but the positive of smeared an acid fast stain or cultured tuberculosis mycobacterium is very low,so their value of earlier diagnosis in clinical is limited.At present there is still no a reliable and practical diagnostic method can satisfy the need of clinical diagnosis. Mycobacterium tuberculosis is phagocytized by mononuclear macrophage cell after invading the body, the body expresses mycobacterium tuberculosis specific antigen at first, so the detection of tuberculosis antigen can be used as a direct evidence of mycobacterium tuberculosis. The purpose of this study is to evaluate the significance in diagnosis of tuberculous meningitis by detecting the antigens of PPD and ESAT-6 in the monocytes of CSF, and then seek a more effective method for the diagnosis of tuberculous meningitis.Methods : The case group were 35 cases clinical diagnosis of tuberculous meningitis patients, and the control group were 35 cases of not tuberculous meningitis patients. All of the patients were examined CSF routine, biochemical, cytology(MGG dye) and aricine blue staining, applied immunocytochemical staining and double immunofluorescence staining to detect the ESAT- 6 and PPD antigen in the cytoplasm of cerebrospinal fluid monocytes, Shandon Cytospin4 centrifugal sedimentation instrument to collect cerebrospinal fluid cells. The immunocytochemical staining used SP method and observed in ordinary optical microscope, tan or yellowish-brown particle deposition inside the cytoplasm of monocytes was positive, colorless was negative result. In the fluorochrome of double immunofluorescence staining , FITC (markered goats anti rabbit IgG),Cy5 (markered of goats resistance rat IgG) and DAPI(4',6-diamidino-2-phenylindole)were used to individually tagged anti ESAT - 6 monoclonal antibodies,anti PPD polyclonal antibody and all the cell nucleus DNA,under the laser scanning confocal microscope(LSCM) to observe the result. The ESAT-6 positive monocytes showed blue nucleus and red cytoplasm; the PPD positive monocytes presented the integrated structure of blue nucleus and green cytoplasm; the negative cells only appeared blue nucleus structure.Results:1 Clinical data: Tuberculous meningitis patients might manifest as fever, headache, meningeal stimulation masculine, brain focal signs, consciousness obstacleetc etal, but it lack of specificity, not easy to identify with other diseases of the nervous system. Mergering extracranial tuberculosis supported the diagnosis.2 Cerebrospinal fluid routine,biochemical examination results: Tuberculous meningitis group,the average of the total cells count were 502.60±457.67×106 /L; the average number of white blood cells count were 310.37±292.20×106 /L; the average glucose were 1.87±0.87mmol/L; protein's average were 1.62±0.65g/L; chloride's average were 109.68±9.25mmol/L; the average pressure of lumbar puncture were 277.19±93.56mmH2O. In the control group, the average numerus respectively were total cells count 473.63±1545.95×106 /L;white blood cells 132.37±293.46×106 /L;glucose 2.91±0.95mmol/L;protein 0.76±0.79g/L;chloride 119.25±6.76mmol/L;the pressure of lumbar puncture 225.56±122.38 mmH2O.The white blood cells count and protein content in the TMB group were obviously higher than the control group , P﹤0.05, there was a statistical significance. The content of glucose and chloride in the case group were lower compared with the controls group , P﹤0.05, difference was significant in statistics. The difference of total cell count and pressure of lumbar puncture were not statistically significant.3 The performance of cerebrospinal fluid cytology: in the 35 patients of TBM group, 25 cases had mixed-cell reaction, 8 cases had neutrophil reaction, 2 cases had dominated of lymphocytes response.The control group, 35 patients had 5 cases of mixed-cell reaction, 5 cases with neutrophil reaction in the priority , 18 cases with lymphocytes reaction in the priority , 4 cases of sensitized monocytes sample reaction, 3 cases had normal cerebrospinal fluid cytology.The mixed-cell reaction in the TBM group accounted for about 71.43%, obviously higher than those in the control group 14.29%.The mixed-cell reaction had important meaning to the diagnosis of TBM, to observe the changes of cerebrospinal fluid cytology dynamicly had certain practical value in the therapy and prognosis of TBM .4 The detection result of ESAT - 6 antigens in cerebrospinal fluid monocytes: 35 cases of tuberculous meningitis patients, 29 cases with immunocyto- chemical staining were positive, in the control group, there were 2 cases were positive, its sensitivity was 82.86%, its specificity was 94.29%, the TBM group compared with the controls, P < 0.05, the difference was statistically significant. In the double immunofluorescence staining, there were 28 patients positive in the case group and 1 case positive in the control group, its sensitivity was 80%, and specificity was 97.14%, by statistical analysis P < 0.05, the difference of the two groups was significant. Two methods joint tested ESAT-6 antigen, there were 30 patients positive in TBM group, 2 cases positive in the control group, its sensitivity was 85.71% and specificity was 94.26%.5 The detection result of PPD antigen in cerebrospinal fluid monocytes:using immunocytochemical staining, there were 30 patients positive in the case group and 6 cases positive in the control group, the sensitivity of detection PPD antigen was 85.71% and the specificity was 82.86%, the case group compared with the controls, P < 0.05, the difference was statistically significant. In the double immunofluorescence staining, the cases of positive patients were 29 in the case group and 5 in the control group, PPD's sensitivity was 82.86% and specificity was 85.71%, two groups were compared, P < 0.05, the difference was statistically significant. Two methods joint test PPD antigen, there were 31 cases of positive in case group and 8 cases of positive in the control group, its sensitivity was 88.57% and specificity was 77.14%.6 The comparison of ESAT-6 and PPD antigen to diagnosis TBM: immunocytochemical staining double joint immunofluorescence staining to test ESAT-6 and PPD antigen , compared their sensitivity and specificity by chi-square test, their difference of sensitivity( P>0.05) wasn't statistically significant,but the specificity (P < 0.05) was statistically significant. So the specificity of two methods joint tests ESAT - 6 antigens higher than PPD antigen.Conclusion: The monocytes of cerebrospinal fluid contains tuberculosis antigen in patients with tuberculous meningitis; the detection of tuberculosis antigen can provide a powerful basis for the early diagnosis of TBM. Immunocytochemical staining combined with double immunofluorescence staining can improve the detection rate, the sensitivity of detected ESAT-6 antigen is 85.71%, specificity is 94.26%; the sensitivity of detected PPD antigen is 88.57%, and specificity is 77.14%. By statistics analysis, the sensitivity of two methods joint test ESAT-6 and PPD antigen is not statistically significant, but the specificity of ESAT-6 antigen higher than PPD antigen. The earliest time of tuberculosis antigen can be detected is 3 days and the latest time is 180 days. Laser scanning confocal microscope techniques can put the cells in CSF to be three-dimensional reconstruction and tomography, the cytological study is improved from the surface, single layer, static describing to a new stage of stereo, fault, and dynamic observation.
|
PDF Full Text Request