| Objective: Tuberculous meningitis (TBM) is an infection disease ofcentral nervous system caused by Mycobacterium tuberculosis. Accounts forapproximately7～10%of all TB cases. TBM is a non suppurativeinflammation with meningeal and the brain. Spinal cord and spinal meningescan also affected. It is difficult to diagnose as clinical manifestations arecomplicated and atypical. Also there were no specific features in the earlyimaging inspection. Definitive diagnosis of tuberculous meningitis majordepends upon the detection of cerebrospinal fluid (CSF) biochemical andcytology. It can not be used differential diagnosis of TBM as the result mayoverlap with other forms of intracranial infection. Mycobacteriumtuberculosis is an intracellular bacterial parasite.MTB was first phagocytosed by mononuclear phagocyte aftertuberculosis infection. So immunological methods to detect tuberculosisspecific antigen can be a simple, sensitive and high specificity way indiagnosis of TBM. Early secretory antigen target6(ESAT-6) is a secretedlow molecular weight protein purified in the short period culture filtrate of Mycobacterium tuberculosis. ESAT-6antigen expression significantlyincreased when Mycobacterium tuberculosis is growing in early stageinfected monocyte-macrophage cells. We supposed that ESAT-6in themonocyte-macrophage cells has a high sensitivity in the diagosis of TBM. Inthe first part of this experiment the early secretion antigen of mycobacteriumtuberculosis ESAT-6and PPD were chosed as test object. Immunefluorescence cell chemical staining were used to observe the existence ofESAT-6.We tested ESAT-6in the monocyte-macrophage cells of PB and CSFand meanwhile scanned the cells with layering scanning method by laserscanning confocal microscope (LSCM) among some TBM patients.Three-dimensional picture was constructed and Fluorescence intensity valuewas detected to analyzed the qualitive, ration, location and dynamic changesof the tuberculosis antigen in the monocyte-macrophage cells. To furtherinvestigate the mechanisms of TBM.Methods: All60cases of the clinical diagnosed TBM patients wereselected from neurology department of Ningxia Medical UniversityAffiliated Hospital and Ningxia Tuberculosis Hospital during December2009to February2012. Totally60patients,32are male,28are female, theage of onset is between16and76years. The median age is28.5years. Thediagnosis criteria is according to clinical diagnosis criteria suggested by MMThwaites. All patients were investigated of CSF routine test, biochemical, cytology (MGG dye), and Alixin dye. Immune fluorescence cell chemicalstaining were used to label the PPD and ESAT-6antigen in the monocytes ofPB and CSF and examined with fluorescence microscopy. Some smearswere observed by LSCM and detected the fluorescent contents.Results: All TBM patients before treatment showed a mixed-cellresponse in CSF. Dominated by lymphocyte and activated lymphocyte.15days after treatment neutral cell slightly reduced and activated lymphocyteratio obviously increased.30days after treatment MGG staining showed amixed-cell response dominated by lymphocyte in the CSF, aging neutrophil,more activated lymphocyte still remained and activated monocyte can beseen.The positive rate of PPD in the PB and CSF were63.3%(38/60) and83.33%(50/60) separately in the TBM group,and the positive rate of PPD inthe non-TBM group were6.45%(4/62)and4.84%(3/62)separately. Thepositive rate of ESAT-6in the PB and CSF were53.33%(32/60) and76.67%(46/60) separately in the TBM group, and The positive rate of ESAT-6in thenon-TBM group both were3.23%(2/62). The positive rate of PPD andESAT-6were statistically significantly higher in the TBM group comparedwith non-TBM group.ESAT-6antigen had better specificity compared with PPD. PB and CSFcell smears from20EAST-6positive TBM patients and20EAST-6negativecontrolled patients were observed by LSCM after double immunofluorescence staining. The ESAT-6was observed uniformlydistributed in the plasma and cytomembrane of the monocytes of CSF.Positive monocyte is big, round or orbicularovate in shape. Cell plasma wasloose, expression of green fluorescent. While in the control group showedfew green fluorescent. All the nuclei showed blue fluorescent. The averagefluorescent value of ESAT-6in the PB and CSF were (22.45±13.35；26.55±11.34) in the TBM group, showed statistically significantly highercompared with the control group(2.45±0.89；2.22±0.37).20TBM patientswere selected to detect the average fluorescent value again after treatmentfor3weeks. The result showed the average fluorescent value werestatistically significantly decreased.Conclusion:1. Immune fluorescence cell chemical method to detect antigen in theTBM monocyte was established preliminarily.2. The ESAT-6in the monocytes of CSF was significantly higher thanthe control group. Indicating that it can be an important index in thediagnosis of TBM.3. We can detect the qualitive, ration, location and dynamic changes ofthe tuberculosis antigen in the CSF monocytes with LSCM to furtherunderstand the mechanisms of TBM. |
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