Kinetics of T-cell-based assays on cerebrospinal fluid and peripheral blood mononuclear cells in patients with tuberculous meningitis

BACKGROUND/AIMS: The goal of this study was to monitor tuberculosis (TB)-specific T-cell responses in cerebrospinal fluid-mononuclear cells (CSF-MCs) and peripheral blood mononuclear cells (PBMCs) in patients with tuberculous meningitis (TBM) over the course of anti-TB therapy. METHODS: Adult patients (> or = 16 years) with TBM admitted to Asan Medical Center, Seoul, South Korea, were prospectively enrolled between April 2008 and April 2011. Serial blood or CSF samples were collected over the course of the anti-TB therapy, and analyzed using an enzyme-linked immunosorbent spot (ELISPOT) assay. RESULTS: Serial ELISPOT assays were performed on PBMCs from 17 patients (seven definite, four probable, and six possible TBM) and CSF-MC from nine patients (all definite TBM). The median number of interferon-gamma (IFN-gamma)-producing T-cells steadily increased during the first 6 months after commencement of anti-TB therapy in PBMCs. Serial CSF-MC ELISPOT assays revealed significant variability in immune responses during the first 6 weeks of anti-TB therapy, though early increases in CSF-MC ELISPOT results were associated with treatment failure or paradoxical response. CONCLUSIONS: Serial analysis of PBMCs by ELISPOT during the course of treatment was ineffective for predicting clinical response. However, increases in TB-specific IFN-gamma-producing T-cells in CSF-MC during the early phase of anti-TB therapy may be predictive of clinical failure.

Serum and Cerebrospinal Fluid Neuron-Specific Enolase for Diagnosis of Tuberculous Meningitis

PURPOSE: Late diagnosis and treatment lead to high mortality and poor prognosis in tuberculous meningitis (TbM). A rapid and accurate diagnosis is necessary for a good prognosis. Neuron-specific enolase (NSE) has been investigated as a biochemical marker of nervous tissue damage. In the present study, the usefulness of NSE was evaluated, and a cut-off value for the differential diagnosis of TbM was proposed. MATERIALS AND METHODS: Patient charts were reviewed for levels of serum and cerebrospinal fluid (CSF) NSE, obtained from a diagnostic CSF study of samples in age- and gender-matched TbM (n=15), aseptic meningitis (n=28) and control (n=37) patients. RESULTS: CSF/serum NSE ratio was higher in the TbM group than those of the control and aseptic groups (p=0.001). In binary logistic regression, CSF white blood cell count and CSF/serum NSE ratio were significant factors for diagnosis of TbM. When the cut-off value of the CSF/serum NSE ratio was 1.21, the sensitivity was 86.7% and the specificity was 75.4%. CONCLUSION: The CSF/serum NSE ratio could be a useful parameter for the early diagnosis of TbM. In addition, the authors of the present study suggest a cut-off value of 1.21 for CSF/serum NSE ratio.

Detection of antigen and antibody in childhood tuberculous meningitis.

OBJECTIVE: Mycobacterium tuberculosis excretory secretory 31 kDa, a serine protease antigen (M. tb ES-31), prepared from Mycobacterium tuberculosis H37Ra culture medium has been shown to have potential in detecting tuberculosis. Precise diagnosis and management of tuberculous meningitis, in children in particular, is essential to curtail mortality and morbidity. METHODS: In this study, M. tb ES-31 antigen, was used in Indirect ELISA to detect tuberculous IgG antibody, in sera and CSF samples while affinity purified anti ES-31 goat antibody was used in sandwich ELISA for detection of tuberculous antigen. In sixty-five samples each of CSF and sera from cases with neurotuberculosis and control with non-tuberculous diseases were collected from Kasturba Hospital, Sevagram. RESULTS: Among the 20 patients suffering from neurotuberculosis the IgG antibody was detected in 17(85%) of CSF and 16(80%) of sera samples, while antigen was detected in 18 (90%) in CSF and 16 (80%) in sera. Overall specificity of the assay for both IgG antibody and antigen detection in CSF was 96% while in sera it was 94% for IgG antibody and 96% for antigen detection. CONCLUSION: This study showed the usefulness of mycobacterial serine protease antigen and its antibody in detecting neurotuberculosis.

Diagnostic utility of estimation of mycobacterial antigen A60 specific immunoglobulins in serum and CSF in adult neurotuberculosis.

An ELISA assay based on mycobacterial antigen A60 (Anda, Biologicals France) was used to detect specific immunoglobulins (IgM, IgA and IgG) in 48 cases of adult neurotuberculosis (24 TBM; 24 Tuberculoma) and in 48 controls (24 diseased controls; 24 healthy controls). Serum was analysed in all the subjects whereas CSF was assayed only in TBM cases and diseased controls. The cut off values used for IgM, IgG and IgA in this study were 1.500 ODI (optical density index) at 1:100 dil, 250 units/ml and 150units/ml respectively in serum; and 1.500 ODI at 1:10 dil, 10 units/ml and 10 units/ml respectively in CSF. The mean titres of all three antibodies were found to be significantly higher in cases as compared to controls. In cases of TBM, in serum, the percentage positivity for IgM, IgG, IgA and combination of IgG or IgA were 41.67, 87.50 87.50 and 95.83 respectively. The corresponding figures in CSF were 62.50, 75.0, 66.67 and 79.16 for IgM, IgG, IgA and 'IgA or IgM' respectively. In tuberculoma cases, in serum, the figures were 37.50, 75.0, 75.0 and 83.33 respectively. Overall, a high sensitivity and specificity were obtained in cases of TBM (Serum: ST = 95.83%: SP = 87.50%; CSF ST = 79.16%. SP = 100%) and Tuberculoma cases (serum: ST = 83.33% SP = 87.50%) employing the combined antibody estimations.

Diagnosis of tuberculous meningitis by detection of antigen and antibodies in CSF and sera.

OBJECTIVE: To evaluate diagnostic potential of three immunological tests, namely, detection of H37Rv antigen of M. Tuberculosis in CSF, detection of antibodies (IgG) against H37Rv in CSF and detection of antibodies (IgG) against H37Rv in serum for diagnosis of tuberculous meningitis in children. SUBJECTS: 50 children diagnosed as patients of tuberculous meningitis were included as cases and 48 children with CNS diseases of nontubercular etiology [pyogenic meningitis (n = 31), encephalitis (n = 10), seizure disorder of unknown etiology (n = 5), brain tumor (n = 2)] served as controls. METHODS: H37Rv antigen of M. tuberculosis was detected in CSF by Dot ELISA, and antibodies (IgG) against H37Rv in CSF and serum were detected by Plate ELISA. RESULTS: Detection of H37Rv antigen in CSF was the most sensitive (90%) and specific (95.83%) with positive and negative predictive values of 95.74% and 90.19%, respectively, followed by detection of antibodies in CSF (sensitivity-74%, specificity-89.58%, positive predictive value-88.10%, negative predictive value-76.78%). Detection of antibodies in serum had low sensitivity (50%), specificity (91.67%), positive predictive value (86.21%) and negative predictive value (63.76%). CONCLUSIONS: Detection of antigen in CSF is a rapid, sensitive and specific test for diagnosis of tuberculous meningitis in children. Detection of antibody in CSF may be useful in some cases but needs further evaluation. Detection of antibody in serum does not appear to be useful for diagnosis of tuberculous meningitis.

Usefulness of ELISA using antigen A60 in serodiagnosis of neurotuberculosis.

Anti-mycobacterial antibody to A60 antigen were quantified in the sera and cerebrospinal fluid (csf) samples of one hundred patients of neurotuberculosis and twenty non-tubercular controls by immunoenzymatic (ELISA) assay. Sixty three patients (63%) had "significant" antibody titres in serum and/or CSF samples. In contrast, none of the samples from the control group showed this level of antibody concentration. The evaluation of this assay in tuberculoma (group A, 79 cases) vis-a-vis tubercular meningitis or TBM (group B, 21 patients) revealed significant antibody levels in the former 46 (58.2%) in comparison to 17 (77.3%) in the latter group. A positive relationship was observed in the titres of anti-mycobacterial antibodies in serum and in CSF both in cases of tuberculoma and TBM. This study indicates the utility of A60 antigen ELISA assay in categorising these patients into tubercular aetiology specially in the absence of bacteriological isolation from CSF which still remains the gold standard diagnostic criterion.

Serum sodium and osmolal changes in tuberculous meningitis.

Twenty children from 2 months to 7 years (mean age 2.74 years +/- 1.62) diagnosed to have tuberculous meningitis (TBM) were evaluated for serial serum sodium levels and osmolality of cerebrospinal fluid (CSF), serum and urine on admission and the results compared with 20 age and nutritionally matched controls, and these investigations repeated on day 3 and day 10. Mean serum sodium levels (130.7 +/- 6.26 mEq/L), and osmolality of CSF (272.0 +/- 7.0 mOsm/kg) and serum (275.5 +/- 6.09 mOsm/kg) were significantly lower (p < 0.001) than in controls. Hyponatremia was detected in 65% of cases on admission, 47% on day 3 and in 30.8% on day 10. All the patients with hyponatremia had biochemical evidence of syndrome of inappropriate secretion of antidiuretic hormone (SIADH) on admission. Incidence of SIADH gradually decreased to 41.2% on day 3 and 15.4% on day 10. In some of the cases serum sodium levels and osmolality of serum and CSF took about 3 weeks to return to normal. CSF osmolality was lower than concomitant serum osmolality in patients as well as in controls. In patients with SIADH, CSF osmolality followed the same trend as serum values and returned to normal in 2-3 weeks. Overall mortality was 25%. Two out of 3 patients who expired during first 3 days had SIADH but in those cases who survived there was no correlation with degree of meningeal inflammatory changes or ultimate outcome. SIADH is commonly associated with TBM and should be diagnosed early in order to modify the fluid therapy in these cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum concentrations of rifampicin and isoniazid in tuberculosis.

Ninety-four patients, 1-13 years of age suffering from different types of tuberculosis were investigated for serum rifampicin (RIF) and isoniazid (INH) concentrations using microbiological and fluorimetric methods, respectively. Of these, 64 (68.1%) had pulmonary primary complex (PPC); 20 (21.3%) progressive primary disease (PPD) and 10 (10.6%) tuberculous meningitis (TBM). Patients with PPC, PPD and TBM were given two-drug (6HR), three drug (2HRZ, 4HR) and four drug (2SHRZ, 4HRE, 3HE) regimens, respectively. RIF and INH were administered in a dose of 12 and 10 mg/kg/day, respectively. After 10-12 days of continuous therapy, their serum concentrations were estimated at 0, 2, 4, 6, 8 hours for RIF and 0, 1, 3, 5, 7 hours for INH. For RIF, the time to achieve maximum concentrations (Tmax) was 2 hours, range of mean of maximum concentration (Cmax) 3.38 to 3.88 micrograms/ml, terminal half life elimination (T1/2) 3.03 to 3.81 hours and area under serum concentration curve (AUC) 0-8 hours 24.7 to 28.3 micrograms/ml hours in different forms of tuberculosis. INH had a Tmax of 1 h, Cmax 4.38 to 8.17 micrograms/ml, T1/2 4.0 to 4.98 hours and AUC 0-7 hours 34.1 to 57.5 micrograms/ml hours. The concentrations achieved at 7-8 hours with these dosages were much above those required for therapeutic efficacy (minimum inhibitory concentration), being 50 to 250 times for RIF and 35-60 times for INH. We recommend pharmacokinetic studies with lower doses of RIF and INH for less toxic, equally effective and cheaper antitubercular chemotherapy.

Cerebrospinal fluid N-acetyl neuraminic acid estimation for early diagnosis and differentiation of bacterial meningitis.

Cerebrospinal fluid (CSF) analysis for free, bound and total N-Acetyl Neuraminic Acid (NANA) as well as serum NANA was done in 68 patients of bacterial meningitis, of which 37 cases were of pyrogenic meningitis and 31 of tuberculous meningitis. Ten patients were included in the control group. The free NANA levels were increased in only pyogenic meningitis, independent of protein levels but the bound form increased with the increase in CSF proteins. The increase of free NANA in CSF of pyogenic meningitis patients was not related to the cell count or sugar content in CSF or to the duration or severity of illness. This finding can be of great help in differentiating cases of pyogenic meningitis, particularly partially treated patients, who may have ambiguous pictures of CSF analysis, from the cases of tuberculous meningitis.

Acute phase proteins in tuberculous patients.

The serum concentrations of some acute phase proteins were determined on admission, during treatment, at the end of treatment and at 12 months after stopping treatment in 20 patients with pulmonary tuberculosis. Measurements were also made, on admission and at the end of treatment, in 19 patients with abdominal tuberculosis, and 11 children with tuberculous meningitis. All 20 patients with pulmonary TB had quiescent disease by the end of treatment and none had a bacteriological relapse during the follow-up period of 12 months. The response to treatment was considered favourable in 18 of the 19 patients with abdominal TB, and the CSF findings had returned to normal in 9 of 11 patients with TB meningitis. There was a significant decrease with treatment in the concentrations of C-reactive protein, ceruloplasmin, haptoglobin and alpha-1-acid glycoprotein in all 3 groups of patients. While there was an increase in the concentrations of transferrin in patients with pulmonary and abdominal TB, there was a significant decrease in those with TB meningitis; alpha 2-macroglobulin did not appear to function as an acute phase reactant in any of the 3 groups. Amalgamating the findings in all 3 groups of tuberculous patients, the proportions of patients with abnormal values on admission and at the end of treatment were 62% and 14% for C-reactive protein, 78% and 50% for ceruloplasmin, 86% and 26% for haptoglobin and 92% and 6% for alpha 1-acid glycoprotein, respectively.