Demonstration of IgG antibodies to 30 Kd protein antigen in CSF for diagnosis of tuberculous meningitis by antibody-capturing ELISA
Objective: A simple and rapid immunological assay method has been developed to demonstrate the presence of IgG antibodies to 30Kd protein antigen (30Kdpa) and culture filtrate protein (CFP) in the CSF of patients with Tuberculous meningitis (TBM). Method: Antibody capturing Enzyme Linked Immunosorbent Assay (ELISA) was standardized with CFP antigen of MTB. The IgG antibodies were assayed in CSF sample from TBM and non-TBM patients against 30 Kdpa. Results: The sensitivity and specificity of IgG antibodies for the diagnosis of suspected patients of TBM using 30 Kdpa was 80% and 91% respectively and the corresponding figures for CFP were 85% and 94% respectively. The sensitivity and specificity in two confirmed cases of TBM was 100%. Conclusion: The presence of this 30Kdpa in the CSF of suspected cases of TBM consistently would indicate that the selected protein band carries the candidate protein marker antigen, which is specific to M. tuberculosis and could be considered as a diagnostic marker for TBM.
Tuberculous meningitis (TBM) remains a major global health problem and even in developed countries there is resurgence of TBM due to the growing number of people infected with human immunodeficiency virus (HIV)., The diagnosis of TBM is difficult to establish because of its pleomorphic clinical presentation., Delayed diagnosis and treatment may be associated with many serious central nervous system (CNS) complications. The most commonly used laboratory method for the definitive diagnosis of TBM is to demonstrate the presence of tubercle bacilli either by smear or culture. However, direct smear methods are insensitive and often negative in the cerebrospinal fluid (CSF) samples and culturing of Mycobacterium tuberculosis (MTB) is time-consuming., Rapid tests like polymerase chain reaction (PCR), though sensitive, are expensive and not readily available in most of the centers. Various immunoassays such as antigen and/or antibody detection in CSF samples have been developed., Several antigens of MTB have been evaluated for their diagnostic potential in the detection of circulating antibodies., In our laboratory studies we have demonstrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) a protein marker 30 Kd protein in the CSF of patients with TBM. The present study evaluates the IgG response to 30 Kd protein antigen (30Kdpa) in the CSF of patients with TBM and compares it with the culture filtrate protein (CFP) antigen of MTB H37RV strain.
The patient population included all the patients admitted to the Neurology wards, Central India Institute of Medical Sciences (CIIMS) between January 2003 and July 2003, in whom a diagnostic lumbar puncture had been performed. CSF examination included total and differential cell counts, biochemistry, and smear for Gram's, India ink, and acid-fast bacilli (AFB) staining. Patients were grouped as follows:
I. Tuberculous Meningitis:
Diagnosis of TBM was based on the clinical features, which included subacute or chronic fever, signs of meningeal irritation with or without other features of CNS involvement and CSF findings. CSF findings suggestive of TBM were increased proteins, decreased glucose (CSF to blood glucose ratio < 0.5), and pleocytosis with lymphocyte predominance. Patients in whom AFB was demonstrated either by smear or culture, were considered "definite" cases of TBM. In the remaining cases response to antituberculous drugs was considered as a criterion for diagnosis and these patients were considered as "suspected/probable" cases of TBM.
II. Pyogenic meningitis:
Pyogenic meningitis was suspected in patients with acute high-grade fever with features of meningitis and often with associated altered sensorium. CSF findings suggestive of pyogenic meningitis included increased proteins, very low sugar (CSF to blood glucose ratio < 0.2) and pleocytosis with polymorphonuclear predominance. However, CSF findings in partially treated cases of pyogenic meningitis can be very similar to TBM. Response to appropriate antibiotics was also considered as one of the diagnostic criteria.
III. Viral meningitis:
Viral meningitis was suspected in patients who presented with acute onset of fever and symptoms and signs of meningeal irritation. In such patients CSF showed mild increase in protein, glucose often normal (CSF to blood glucose ratio > 0.5) and pleocytosis, predominantly lymphocytic. None of them had clinical evidence of extracranial tuberculosis. Those with clinical suspicion of herpes encephalitis were treated with acyclovir and other viral meningitides were treated symptomatically.
IV. Non-infectious neurological disorders:
All other patients who had no clinical features of meningitis and had no evidence of CNS or extra-CNS tuberculosis were grouped in the non-infectious /control group.
About 2-3 ml of CSF was collected in sterile glass tubes in all the patients and was stored at -800C.
Antigens: After the separation of protein from the CSF of patients with TBM by SDS-PAGE, the 30Kd protein bands were sliced from the gels and pre-equilibriated in the elution buffer (0.15M phosphate buffered saline (PBS), pH-7.4). The gel was electroeluted in a whole gel eluter system (BIOTECH, INDIA) for 1 h, at 30 volts, and harvested from the unit and dialyzed against PBS, and the protein content was measured by Bio Lab Kit. Running native PAGE checked the purity of protein. CFP isolated from M. tuberculosis H37Rv Strain, basically are of bacterial origin (Colorado State University, USA). These are the proteins which are filtered after growing the culture of M. tuberculosis bacilli. These proteins (antigens) were used for antibody-capturing Enzyme Linked Immunosorbent Assay (ELISA).
Antibodies: A polyvalent antibody to 30Kd protein was raised in rabbits [1:1000] (Bangalore Genei, Bangalore), and H37Rv Culture filtrate protein antibodies [1:5000] (rabbit polyclonal sera C-193) were obtained from Colorado State University, USA.
Antibody-capturing ELISA described by Voller et al was employed with minor modification. The assay was first standardized by reacting 30Kdpa or CFP antigen with respective antibodies with different dilutions prior to analysis of patients' samples. The flat-bottom microliter wells were coated with 100 µl of 30Kdpa (15µgm/ml) or CFP antigen (10µgm/ml) and incubated overnight at 40C. The wells were then washed with PBS solution, pH 7.4 and then non-specific sites of coated antigen were blocked by the addition of 100 µl of 0.5% BSA/PBS at 370C for 1 hour. The blocking solution was then removed from the wells. 100 µl of diluted CSF 1:10 in PBS was added to the wells and incubated at 370C for 60 minutes. The wells were again washed with the PBS. 100 µl of affinity-purified horseradish peroxides conjugated anti-human IgG (Bangalore Genei, India) diluted 1:10,000 in PBS was added into the wells and incubated at 370C for 60 min. After another washing with PBS, 100µl of Tetramethyl benzindine/hydrogen peroxide substrate solution was added into the wells and incubated at room temperature for about 20 min. The reaction was then stopped with 100 µl of 2.5 N sulphuric acid.
The clinical profiles of 20 patients with TBM are presented in [Table - 1]. Among the 20 patients who fulfilled the inclusion criteria for TBM, 2 were definite cases of TBM and 18 were suspected/probable cases of TBM. Two of the probable cases of TBM had active pulmonary TB on chest X-ray. CT findings suggestive of CNS tuberculosis such as tuberculoma, hydrocephalus, and vasculitis with infarcts were noted in 6 patients.
The absorbance value of IgG to 30 Kdpa and CFP antigen in CSF are presented in [Figure - 1]. The cut-off values (OD=450nm) for positivity to 30Kdpa and CFP antigen in the control CSF were 0.046 and 0.150 respectively. In the patients with definite, probable or suspected TBM, the CSF positivity for 30 Kdpa was 80% (16/20) and for CFP it was 85% (17/20) [Table - 2]. While the CSF positivity for patients with pyogenic meningitis was 12.5% (1/8) for 30 Kdpa and 25% (2/8) for CFP. In the control group, 2 patients had CSF positivity for CFP and none for 30 Kdpa.
The sensitivity and specificity of IgG antibodies for the diagnosis of suspected patients of TBM using 30 Kdpa was 80% and 91% respectively. However, the sensitivity and specificity in the two confirmed cases of TBM was 100%. The sensitivity and specificity of the IgG antibodies using CFP antigen was 85% and 94% respectively. No significant difference was observed in the positivity to 30 Kdpa and CFP antigen in the CSF of TBM patients.
In the absence of any definite rapid diagnostic test, the diagnosis of TBM with certainty remains difficult. A biochemical or immunological marker is required to substantiate the clinical diagnosis of TBM. We have demonstrated in our earlier work a 30Kd protein marker by SDS-PAGE in the CSF of 90% of the suspected and definite cases of TBM.  This protein band is absent in the control group as well as in patients with pyogenic meningitis. In the present study we evalu ated the response of IgG antibodies against 30 Kdpa and compared it with the standard CFP antigen of M. tuberculosis of H37RV strain by antibody-capturing ELISA. The high sensitivity and specificity of IgG antibodies for the diagnosis of suspected patients of TBM using 30 Kdpa would indicate that the selected protein band carries the candidate protein marker antigen which is specific to M. tuberculosis and could be considered a diagnostic marker for TBM. The major limitation of this study is a lack of definite diagnosis of TBM by demonstration of M. tuberculosis in the majority of the cases. However, the consistent presence of IgG antibodies against 30 Kdpa in CSF in clinically suspected cases of TBM gives a confidence to the treating physicians to start antituberculous drugs before a final confirmation of TBM. The other advantage of this test is that it can be made available in most parts of the country on account of its low cost.
In this study antibody-capturing ELISA has been used to detect 30Kdpa antibodies in the CSF of TBM and control patients. Antibody-capturing ELISA is most commonly used for antibody detection in TBM because it is technically very simple with high sensitivity and specificity. Hernandz et al by using antibody-capturing ELISA demonstrated either IgG or/and IgM antibodies to BCG in CSF from all the patients with TBM and none in the control subjects, indicating that both the sensitivity and specificity of antibody detection by capturing ELISA were 100%. This data strongly suggests that capturing ELISA of CSF IgG antibodies is a promising tool for the diagnosis of TBM
Earlier many workers have attempted the use of various MTB antigen markers such as lipoarabinomannan, purified protein derivatives, heat shock protein of 62 Kd and 14Kd, GroE, Ag 85 complex, 38Kd antigen, etc,, which have now been recognized as potential markers for the diagnosis of TBM and pulmonary tuberculosis. However, their presence remains questionable and many of these antigens are reported in the blood only but not in the CSF and thus question the veracity of the various diagnostic techniques currently under use for the diagnosis of TBM.
We would like to acknowledge the help of Colorado State University, USA and NIH, NIAID Contract No.1 AI-75320, entitled 'Tuberculosis Research Materials and Vaccine Testing'.