| Objective:Of all infectious diseases, tuberculosis remained one of the most leading causes of morbidity and mortality in the world. Its diagnosis and treatment were still facing many difficulties. Tuberculous meningitis(TBM) is one of the most severe extrapulmonary manifestations of tuberculosis, and the proportion of TBM is increasing all these years. Early and accurate detection is a formidable obstacle for the diagnosis and treatment of TBM, and is also the key to control TBM and reduce mortality. The plight of the early diagnosis was lack of high sensitivity(Se) and specificity(Sp) detection method. The purpose of this study was to detect the intracellular early secretary antigenic target-6(ESAT- 6) in the cerebrospinal fluid(CSF) by immunofluorescence staining(IF staining), immunocytochemistry DAB staining(IC-DAB staining) and immunocytochemistry AKP staining(IC-AKP staining) method, which will compare the Se and Sp of the diagnosis in the three methods of staining, will provide a new effective diagnositic method for TMB, and will guide and support for the further study. Methods:1. The diagnostic gold standard of definite TBM was recognized as the South African expert consensus. The case group were the patients with clinical symptoms, who were diagnosed as definite TBM was made either by detection of Acid-Fast Bacilli(AFB) or culture. The control group were the patients, who were diagnosed as cryptococcal meningitis, viral meningitis, etc, and all tuberculosis testes such as AFB and culture were negative. We collected CSF specimens, which case group were 144 and the control group were 180. Each CSF specimens were detected the intracellular ESAT-6 by IF staining, IC-DAB staining and IC-AKP staining, respectively. Each slide was examined and the numbers of total cells and the numbers of positive cells were counted. We calculated the percentage of positive cells by each staining. Receiver Operator Characteristic Curve(ROC curve) of the three staining methods were respectively drawn by PASW statistics 18.0 software. The diagnosis of Se, Sp, Youden index(YI index) for each staining methods were concluded in accordance with the principle of cut-off point. The diagnostic Se and Sp of three staining methods were compared to find the most effective staining and diagnostic method.2. 144 CSF specimens of case group were tested the intracellular ESAT-6 by IF staining and IC-DAB staining. Neutrophils, lymphocytes and monocytes were distinguished by the nucleus and cell morphology. Each slide was examined total 200 cells and the numbers of positive cells of each cell type were counted. We calculated the positive cells of each cell type by the two staining methods. The two staining methods of each positive cell type were compared by matched t-test respectively to find the differences.3. 70 cases of CSF specimens were chosen as the research object, which the cases were the initial diagnosis and no application with tuberculosis and antibiotic treatment. According to the nucleus and cell morphology, positive lymphocyte percentage was calculated in each slide after the IF staining. 70 cases were divided into two groups according to CSF lymphocyte percentage of positive cells > 50% and < 50%. After 8 weeks treatment, the curative effect of TBM was scored by modified Rankin score(MRC score). The cured group included the cases of MRC score 0-1, the effective group included the cases of MRC score 2-3, and the sequela group included the cases of MRC score 4-5. CSF lymphocyte percentage of positive cells > 50% and < 50% groups compared therapeutic effects of the cured group, the effective group and the sequela group of recovery ratio by chi-square test. Using the rank conversion of nonparametric test level information of two samples, the curative effect was compared between different groups. Results:1. When the ESAT- 6 express in the CSF cell, by the IF staining, positive staining was defined as the fluorescence microscopic of green fluorescence in the cytoplasm of cells, by the IC-DAB staining, positive staining was defined as visualization of yellow brown granules in the cytoplasm of cells, and by the IC-AKP staining, positive staining was defined as visualization of purple blue granules in the cytoplasm of cells. In the three staining methods, the area under the ROC curve was 0.94, 0.863 and 0.894 respectively. Area of the IF staining under the ROC curve was the largest, and the diagnostic accuracy of the IF staining method was highest. The IC-DAB and the IC-AKP staining methods were medium high diagnostic accuracy. Positive cell percentage of cut-off point in three staining methods was 10.0%, 14.5% and 10.5% respectively. The Se of three staining methods was 88.89%, 83.3% and 88.89% respectively. The Sp of three staining methods was 93.33%, 82.8% and 91.1% respectively. The Se and the Sp of the IF staining were the highest. The Se of the IC-DAB staining was higher than the IC-AKP staining. The Sp of the IC-AKP staining was higher than the IC-DAB staining. Positive predictive value(PV+) of three staining methods was 91.43%, 80.00% and 91.43% respectively. Negative predictive value(PV-) was 91.30%, 86.21% and 84.10% respectively. False positives rate and false negatives rate of the IF staining were both lowest. Positive likelihood ratio(LR+) was 13.33, 4.84 and 8.82 respectively. Negative likelihood ratio(LR-) was 0.12, 0.20 and 0.24 respectively. YI index was 82.22%, 66.1% and 69.6% respectively. LR+ and YI index of the IF staining were highest. LR- of the IF staining was lowest showed that the authenticity of the application of the IF staining technique is the best. The IF staining was the best authenticity taining method in the three staining methods.2. The IF and the IC-DAB staining positive neutrophils comparative difference were statistically significant. ESAT- 6 positive neutrophil were different by the two staining methods. In two methods of staining ESAT- 6 positive monocytes and lymphocytes comparative difference were not statistically significant.3. The therapeutic effects of the cured group, the effective group and the sequela group of recovery ratioes were compared in the CSF positive lymphocyte percentage of positive cells > 50% group and < 50% group. It was concluded that the therapeutic effects of recovery ratioes in the two groups were difference. Comparison different groups of curative effect, it was concluded that the sequela group of recovery ratioes in the lymphocyte percentage of positive cells < 50% group was larger than > 50% group and the cured group of recovery ratioes in the lymphocyte percentage of positive cells < 50% group was smaller than > 50% group. Conclusion:This research is aimed to detect intracellular ESAT-6 expression in CSF through IF staining and the IC-DAB staining and the IC-AKP staining, which might provide accurate diagnostic approach for TBM. ESAT-6 positive cells can be detected in all these staining methods, and the highest accuracy of diagnosis was observed in IF staining with higher Se and Sp.The ESAT-6 positive cells in neutrophil was different between the IF and IC-DAB staining, but in monocytes and lymphocytes remained statistically no significant differences between two groups. All above indicate IF staining method has higher accuracy for the ESAT-6 positive cells in neutrophil. It was also observed that the therapeutic effects of recovery ratioes in the two groups were different, and the patient with more than 50% lymphocyte percentage of positive cells in CSF has better prognosis than the one with less than 50% lymphocyte percentage of positive cells in CSF.The IF staining of intracellular ESAT- 6 in the CSF was an early, effective and fast method of diagnosis for TBM, which will provided more guidance and help with treatment, prognosis and further studies. |
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