| Abstract|| |
Background: The aim of this study was to investigate the significance of positive expression of Mycobacterium tuberculosis, (MTB) antigen in the cerebrospinal fluid (CSF) monocytes in diagnosing tuberculous meningitis (TBM). Materials and Methods: A total of 50 inpatients of TBM, 30 viral meningitis and 20 healthy controls were studied at the 1 st , 2 nd , and 4 th week during their treatment course. Immunohistochemical assay were used to detect early secreted antigenic target 6 (ESAT-6) positive cells, and positive cases were also observed. Results: The percentage of positive cases and positive cells of ESAT-6 in CSF monocytes were all higher in the 1 st and 2 nd week than in the 4 th week in TBM patients (P < 0.01); and percentage of positive cases and positive cells of MTB antigen in CSF monocytes were higher in TBM patients than in viral meningitis and health control in the 1 st and 2 nd week (P < 0.01). The sensitivity was 90% and the specificity was 92% in the early stage (within 2 weeks) of TBM. Conclusion: The positive expression of ESAT-6 in CSF monocytes is helpful for the early diagnosis of TBM.
Keywords: Diagnosis, early secreted antigenic target 6, Mycobacterium antigen, tuberculosis, tuberculous meningitis
|How to cite this article:|
Song FX, Sun XW, Wang XT, Nai Y, Liu ZL. Significance of Mycobacterium tuberculosis antigen expression in cerebrospinal fluid monocytes in diagnosing tuberculous meningitis. Indian J Pathol Microbiol 2014;57:265-8
|How to cite this URL:|
Song FX, Sun XW, Wang XT, Nai Y, Liu ZL. Significance of Mycobacterium tuberculosis antigen expression in cerebrospinal fluid monocytes in diagnosing tuberculous meningitis. Indian J Pathol Microbiol [serial online] 2014 [cited 2023 Sep 26];57:265-8. Available from: https://www.ijpmonline.org/text.asp?2014/57/2/265/134705
| Introduction|| |
The diagnosis of tuberculous meningitis (TBM) remains a challenge to date. Nowadays laboratory techniques for the diagnosis of tuberculosis (TB) primarily involve the fields of bacteriology, immunology, and molecular biology. In bacteriology, although acid-fast bacillus detection on cerebrospinal fluid (CSF) smears or Mycobacterium tuberculosis (MTB) isolation through CSF culture can serve as the gold standard for the diagnosis of TBM, both methods have a low positive rate (meanwhile, CSF MTB culture needs 4 weeks to 6 weeks of time); therefore, neither has great clinical significance.  Molecular biological methods such as nucleic acid amplification (NAA) can only be used in laboratories with suitable conditions; meanwhile, they have high false positive rates and low specificity. ,,,, NAA has only been applied to sputum smear-positive MTBs for further diagnostic confirmation, and it may get a negative result from patients with typical clinical manifestations. , These drawbacks greatly limit the clinical application of NAA. Compared with bacteriological and molecular biological techniques, immunological diagnostic techniques have the virtues of easier operation, lower costs, higher specificity, and easier popularization, for which they have attracted more and more attention. Now-a-days, MTB antigen detection is presumed to possess higher sensitivity and specificity than antibody detection.  Antibody detection is likely to result in false negativity for patients at early TBM stage, with hypoimmunity, or after glucocorticoid treatment. Furthermore, antibody detection cannot differentiate acute TBM and previous TBM from each other. Due to these shortcomings of antibody detection, detection of MTB antigens in CSF may become one of the important auxiliary methods for TBM diagnosis. However, studies on the immunohistochemical marker specific for MTB antigens in CSF are rare.
Early secreted antigenic target 6 (ESAT-6) is an early secreted and low-molecular-weight protein of MTB, which mainly exists in pathogenic mycobacteria, and not in all Mycobaterium bovis bacillus Calmette-Guerin (BCG) strains, nor in most environmental mycobacterials examined so far.  In the previous studies, it is demonstrated that most TB patients (35-92%) can recognize ESAT-6, While healthy unrelated controls did not. ,,,, Consequently, the possible use of ESAT-6 as a marker of MTB infection has been proposed in the regions of low TB endemicity. 
In the current study, to explore the significance of ESAT-6 positive expression in CSF monocytes for the early diagnosis of TBM, ESAT-6 in CSF monocytes were immunohistochemically marked based on conventional CSF cytology.
| Materials and Methods|| |
This study was conducted in accordance with the declaration of Helsinki. This study was conducted after obtaining approval from the Ethics Committee of Hospital. Written informed consent was taken from all participants. A total of 50 TBM patients hospitalized between 2009 and 2011 were recruited. Of the patients, 29 were males and 21 were females. Their ages ranged from 13 to 68 years with an average of 41.7 years. All patients had an acute onset according to the Thwaites diagnostic criteria  and visited the doctors' office within 1 week after disease occurrence. Their manifestations included fever, headache, positive meningeal irritation sign, and diplopia. 22 patients had hyperpyrexia, 4 of whom were complicated with active TB; 3 were complicated with cerebral infarction during the course of disease, 6 had intracranial tuberculoma, 1 had the complications of spinal cord lesion and intestinal obstruction, and 4 had the complication of epilepsy. All patients had CSF white blood cell count of 185 × 10 6 /L to 630 × 10 6 /L (the normal range: 0-5 ng 10 6 /L) and protein quantity of 1844.7 g/L to 2911.8 g/L (the normal range: 120-600 mg/L). 50 patients had reduced glucose and chloride quantity; 32 were observed with abnormal signals using cranial magnetic resonance imaging (MRI) that were manifested by basal cistern exudates, meningeal enhancement, tuberculoma, hydrocephalus, cerebral infarction, encephaledema, and so on.
A total of 30 patients with viral meningitis, including 14 males and 16 females, were also enrolled. Their ages ranged from 13 to 42 years with an average of 33.4 years. All these patients had an acute onset with the manifestations of fever, headache, emotional disturbance, seizure disorder, and positive meningeal irritation sign, according to the diagnostic criteria for viral meningitis.  They received treatment within 1 week after disease occurrence. First lumbar puncture showed CSF white blood cell count of 0 × 10 6 /L to 220 × 10 6 /L and protein quantity of 343.7 g/L to 891.6 g/L. In addition, 20 healthy persons comprised the control group.
Fresh CSF at 4 mL was, respectively taken from both experimental groups at 1, 2, and 4 weeks after disease occurrence for CSF routine, protein quantitation, and bacteriological examinations. Remnant CSF was treated using the natural precipitation method: 0.5 mL of CSF was left in each precipitator and then placed at 4°C overnight for cytological examination and ESAT-6 detection by immunohistostaining was performed on the 2 nd day. For each glass slide, a field where cells were evenly distributed was selected and 100 monocytes were counted under a 200 power microscope. Positive cell percentages were calculated and positive case numbers were recorded. Cells with buffy-stained deposited particles in plasma were positive cells, and patients with positive cells in CSF were considered as positive cases.
The CSF sample was stained by May-Gruwald-Giemsa method; cell types were classified and calculated by light microscope.
The CSF sample was fixed with acetone and then incubated with mouse anti-human ESAT-6 monoclonal antibody (primary antibody; Abcam, UK) overnight at 4°C. After phosphate-buffered saline (PBS) washing, it was incubated with biotinylated goat anti-mouse secondary antibody (Abcam, UK) for 30 min at room temperature. After PBS washing, the sample was colored with DAB solution, counterstained with hematoxylin, washed, dehydrated, vitrified by dimethylbenzene, and then mounted.
Data were presented by means ± standard error of means (x- ± s). Statistical analysis was carried out by using SPSS 10.0 software(SPSS Company, Chicago,USA) using χ2 test for comparisons among the MTB positive case numbers at 1, 2, and 4 weeks, ANOVA for comparisons of the MTB positive cell percentages among groups, and q test for comparisons between groups. T-test was used for comparison between the TBM group and the viral meningitis group. P < 0.05 was considered to be statistically significant.
| Results|| |
Cytological examination of cerebrospinal fluid
In the TBM group, neutrophilic granulocytes occupied a high percentage at early stage; as the disease proceeded, the percentages of lymphocytes, activated lymphocytes, activated monocytes, and plasmocytes increased, and the coexistence of neutrophilic granulocytes, activated lymphocytes, monocytes, activated monocytes, and plasmocytes (i.e., mixed cell reaction) commonly appeared; at late stage, neutrophilic granulocytes disappeared, whereas lymphocytes and monocytes became dominant. By contrast, in the viral meningitis group, activated lymphocyte reaction was presented; at late stage, monocytes, and activated monocytes appeared.
In the TBM group, the respective ESAT-6 positive case numbers and positive cell percentages at 1, 2, and 4 weeks were 45 (90%) and 23.2 ± 2.45%, 45 (90%) and 22.67 ± 2.88%, and 9 (30%) and 9.8 ± 1.97%. In the viral meningitis group, the positive case number and positive cell percentage at 1 week were 3 (10%) and 5.0 ± 1.41%; at 2 and 4 weeks, negative results were obtained. In the control group, the positive number and positive cell percentage at 1 week were 1 (5%) and 4%; negative results were obtained at 2 and 4 weeks [Table 1].
|Table 1: Tuberculosis antigen positive case numbers and positive cell percentages of the experimental and control groups at 1, 2, and 4 weeks|
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In the TBM group, both ESAT-6 positive case numbers at 1 and 2 weeks were significantly larger than that at 4 weeks (χ2 = 35.04, P = 0.0000), but no significant difference was observed between the numbers at 1 and 2 weeks (q = 1.3736, P > 0.05); the ESAT-6 positive cell percentages at 1, 2, and 4 weeks also showed significant differences (F = 166.01, P = 0.0000), and the percentages at both 1 and 2 weeks were noticeably higher than that at 4 weeks (q = 24.5561, P < 0.01; q = 23.5849, P < 0.01). At 1 and 2 weeks, the TBM group presented larger MTB antigen positive case numbers (χ2 = 64.03, P = 0.0000) and noticeably increased positive cell percentages (t = 19.18, P = 0.0000; t = 16.06, P = 0.0000) compared with the control group. MTB antigen marking in CSF monocytes had sensitivity of 90% and specificity of 92% for the early diagnosis of TBM. The results are summarized in [Table 1].
| Discussion|| |
Tuberculosis infection remains a major health problem in the 21 st century. Nowadays about one-third of the population of the world are suffering from this infection, and approximately 8,000,000 new TB cases occur worldwide yearly. Of the patients with TB, those with TBM account for 5-10%.  Even after active regular anti-tubercular treatment, TBM still leads to an early mortality rate as high as 19%, and around 50% of the survivals show varying severity degrees of sequelae. , Diagnosis and treatment time are directly correlated with the prognosis of TBM; early diagnosis and timely anti-TB treatment can improve the prognosis of the disease.  Frequently, central nervous system TB has a poor prognosis, which can be chiefly attributed to delayed diagnosis; if a patient with such a disease begins to receive an anti-TB treatment 5 days later after disease occurrence, the risk of severe complications will increase by 3.7 times in him.  Therefore, early diagnosis plays a vital role in the treatment of TBM.
Mycobacterium tuberculosis is intracellular bacteria, and mononuclear phagocytes are their parasitic place. When an organism is invaded by MTBs and suffered with TBM, MTBs enter into CSF monocytes; MTB specific antigens such as ESAT-6 appear first in CSF monocytes. Therefore, MTB antigen detection in CSF monocytes is feasible for the diagnosis of TBM.
In this study, immunohistochemical assay were adopted to detect ESAT-6 in CSF monocytes. Patients with TBM were strictly selected according to the diagnostic criteria; the clinical diagnoses were further confirmed based on CSF cytological characteristics. Although, the clinical and radiological manifestations of meningitis syndromes (including TBM, viral meningitis, and so on) vary, abnormality in CSF can always be a reliable basis for the diagnosis of TBM.  CSF cytological mixed cell reaction in TBM patients lasts as long as more than 4 weeks; this is the most important CSF cytological characteristic of TBM, as well as a distinctive feature which differentiates TBM from other types of meningitis.  In this study, all patients presented this typical CSF cytological characteristic, and then the clinical diagnoses were further confirmed, which successfully guaranteed the reliability of the experimental results; in addition, in order to deeply explore the correlation between TB antigen expression in CSF monocytes and the evolution of patients' condition, changes in TB antigens in CSF at 1 week after disease occurrence as well as after treatment were dynamically monitored. The results show that marking ESAT-6 in CSF monocytes using immunohistochemistry is reliable for the early diagnosis of TBM. In this study, the TBM group had 45 positive cases at 1 week that were confirmed positive by repeated detection at 2 weeks and positive cell percentages above 20% at different times. By contrast, at 1 week, the respective case numbers in the viral meningitis and control groups were 3 and 1, and both groups had a noticeably lower positive cell percentage than the TBM group; at 2 and 4 weeks, all the cases in these two groups were detected negative. Considering that the clinical manifestations and CSF cytological characteristics in these cases were not in line with those of TBM, they were presumed to be false positive. Now-a-days, the immune effect of BCG remains controversial.  Moreover approximately one-third of the population of the world are suffering from TB. Based on this consideration, the cases with positive cells, but low positive percentages in the viral meningitis and control groups may be subject to in apparent infection. In the TBM group, the TB antigen positive cell percentages at 1 and 2 weeks did not show a significant difference, whereas that at 4 weeks decreased, and so did the positive rate. This indicates that MTB antigens exist at early stage of tubercle bacillus infection; with the prolongation of the course of disease, as well as the application of anti-tubercular drugs, these antigens are degraded and eliminated by mononuclear phagocytes. It also indicates pathogenic condition improvement after anti-tubercular treatment. Although five patients in this group had negative MTB antigen expression after repeated detection, their clinical manifestations and CSF cytological characteristics met the diagnostic criteria of TBM. This phenomenon is presumably associated with the titer of the applied primary antibody. In addition, the possibility that these patients were infected with different MTB lines should also not be excluded.
Early secreted antigenic target-six positive expression has sensitivity of 90% and specificity of 92% for the early diagnosis of TBM and therefore is of great significance. The limitations of this method are certain degrees of false positive and negative rates, which entail larger sample sizes and specific high-titer MTB antibodies to further improve the specificity of immunologic examination.
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Department of Neurology, Yantai Yuhuangding Hospital, No. 20 Yudong Road, Zhifu, Yantai - 264 000, Shandong Province
Source of Support: None, Conflict of Interest: None