Neurology India Medknow Publications on behalf of the Neurological Society of India ISSN: 0028-3886 EISSN: 1998-4022 Vol. 51, Num. 1, 2003, pp. 52-54

Neurology India, Vol. 51, No. 1, Jan-Mar, 2003, pp. 52-54

Detection of heat stable mycobacterial antigen in cerebrospinal fluid by Dot-Immunobinding assay

A. Mathai, V. V. Radhakrishnan, C. Sarada,* S. M.George

Departments of Pathology and *Neurology, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram-695011, Kerala, India.
Dr. V. V. Radhakrishnan, Department of Pathology, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram-695011. Kerala, India. E-mail: vvr@sctimst.ker.nic.in

Accepted on 21.01.2002.

Code Number: ni03010

Background: Isolation of Mycobacterium tuberculosis in cerebrospinal fluid (CSF) specimen in patients with tuberculous meningitis (TBM) is infrequent and carries low sensitivity. Thus development of an alternative laboratory diagnostic test is essential for the early diagnosis and treatment of TBM. Objective: A simple, rapid Dot immunobinding assay (Dot-Iba), for the laboratory diagnosis of TBM is devised. This method minimizes the risk of handling infectious material in the laboratory. Method: The Dot-Iba was standardized with heat-inactivated M tuberculosis antigen (PPD). The heat-inactivated CSF from TBM and non-TBM patients was similarly assayed and it can detect antigen upto 1ng/ml in CSF. Result: A positive result was obtained in all the five culture positive patients with TBM and in 20/25 probable TBM. A negative result was obtained in 38/40 CSF from disease control group. The overall sensitivity and specificity of Dot-Iba was 83.3% and 95% respectively. Conclusion: Dot-Iba can be used as an adjunct for the laboratory diagnosis of TBM, particularly in culture negative TBM patients and also in those clinical situations where no laboratory tests are available to distinguish between TBM and partially treated pyogenic meningitis.

Key Words: Tuberculous Meningitis, Mycobacterium tuberculosis, Dot-Iba, Heat stable, antigen, Tuberculin purified protein derivative, Cerebrospinal fluid.

Tuberculous meningitis (TBM) is one of the most common clinical and morphological manifestations of extrapulmonary tuberculosis. The incidence of TBM in developing countries during the past two decades has shown an upward trend especially after the epidemic HIV infection.¹ Early confirmative laboratory diagnosis and institution of effective antituberculous chemotherapy (ATT) is essential in reducing the mortality rate and the neurological sequale. Absolute criteria i.e. the "Gold standard" for establishing the laboratory diagnosis of TBM depends upon the demonstration of M tuberculosis in CSF specimens from patients with TBM. The conventional bacteriological method is time consuming and lacks sensitivity.² The immunological^3-5 and molecular biological⁶ techniques that have been developed over the past decade as an alternative method for the laboratory diagnosis of TBM have evoked considerable interest among clinicians and laboratory investigators. However their application for routine laboratory diagnosis of TBM still remains speculative.

In this report we describe a simple, sensitive, rapid Dot-Iba for the detection of heat stable mycobacterial antigen in CSF specimens from patients with TBM.

Materials and Methods

CSF specimens were collected from 30 patients with a clinical diagnosis of TBM admitted to the neurology unit of the Sree Chitra Tirunal Institute for Medical Sciences and Technology_a tertiary care referral centre for neurological diseases. At the time of admission, none of the patients had associated diseases like diabetes, and human immunodeficiency virus infection. These patients had neither manifestations of pulmonary tuberculosis nor had received chemotherapy for tuberculosis in the recent past. The diagnosis of TBM in all 30 patients was based on clinical features, such as neck rigidity, positive Kernig's sign and compatible CSF biochemical parameters, viz., elevated protein levels (60-400 mg% mean 98 mg%), low glucose concentration (8-30 mg% mean 23 mg%) and pleocytosis (30-700 cells/cm³) in their CSF specimens. The CSF specimens were collected from all patients under aseptic conditions and were centrifuged at 5000 x g for 30 min. The deposits were examined by Ziehl-Neelsen staining. The remainder was innoculated into a Lowenstein-Jenson medium for culturing M tuberculosis. The supernatant CSF specimens were coded and stored at -20ºC until used. After 8 weeks, the culture results showed the presence of M tuberculosis in the CSF specimens in 5 patients and these patients were regarded as having "confirmed" TBM. For the remaining 25 patients repeated bacteriological investigations were negative for fungi (Aspergillus) and bacteria (pneumococci meningiococci and haemophilus). India-Ink preparations of the CSF specimens were also negative for Cryptococcus neoformans. Routine chest X-ray did not demonstrate any active pulmonary tuberculous lesions. However, magnetic resonance imaging for all patients showed varying degree of exudates in the base of the brain. Since the clinical and neurological features were suggestive of TBM, these 25 patients were categorized as having "probable" TBM. Patients with "confirmed" or "probable" TBM after CSF studies were given ATT daily for 6 weeks during the hospital stay. These patients were advised to continue the ATT for 3 months. All the patients were followed in the infectious diseases clinic for the assessment of their clinical response to ATT. CSF specimens from 40 patients with non-tuberculous neurological disease were selected as controls. The diagnosis of the control group of patients were (i) bacterial meningitis due to Hemophilus influenza (n = 3) and Neissesia meningitis (n = 2) (ii) Partially treated pyogenic meningitis (n = 15) Japanese B virus encephalitis (n=10) and Chronic meningitis (n = 10).

Tuberculin PPD antigen was prepared from H₃₇Ra strain of M tuberculosis in this laboratory. This strain was obtained from Tuberculosis Research Centre Madras. Pellicle cultures from 10-14 day old "seed cultures" were grown on Proskauer and Beck medium (Hi-media-Bombay) for 8 10 weeks. At optimum growth, culture was filtered through Whatman filter paper IV and 0.45mµ pore-sized millipore membrane. 1000 ml cell-free of culture filtrate was autoclaved at 15 Ibs for 30 min. Ammonium sulphate was added (297 gm/l of filtrate) to bring the saturation to 50%. The precipitate was recovered by centrifugation and reconstituted to 10 ml in 0.15 M phosphate buffered saline (PBS pH 7.4). The reconstituted material was dialysed against PBS for 48 h at 4°. The protein content was measured by Lowry's method⁷ and preserved in aliquots (1 mg/ml) at -20°.

Polyvalent antibody to H₃₇Ra strain of M tuberculosis was raised in adult rabbits⁸ by the repetitive (weekly once) subcutaneous and intramuscular administration of 2 mg of culture filtrate antigen, mixed thoroughly with 2.5 mg of dried autoclaved bacillary sediment of H₃₇Ra strain of M tuberculosis and 1 ml of incomplete Freund's adjuvant. On immunoelectrophoresis this polyvalent antibody demonstrated multiple precipitation arcs against the culture filtrate antigen. A persistent and high titre of antisera could be raised following the fifth immunization. The rabbit immune serum to M tuberculosis was dispersed in aliquots and stored at -20°.

500 µl of CSF from TBM group both confirmed, probable and CSF of control group were autoclaved at 15 Ibs for 30 min and kept at 4° until the assay.

Prior to patient sampling the Dot-Iba was standardized with different concentration with Tuberculin PPD (1 ng, 10 ng, 100 ng and 1000 ng) in a nitrocellulose membrane (NCM). Circular NCM discs (dia 1 cm) were placed in each well of a flat bottom polystyrene plate (Nunc; Roskilde; Denmark). Five microlitre of the Tuberculin PPD at each concentration was spotted onto the NCM disc and the discs were incubated at 37° for 1 h. The NCM discs were thoroughly washed with 0.15M PBS (pH 7.4). The unbound sites in the NCM discs were quenched with 2.5% skimmed milk in PBS at 40° for 1 h. Subsequently, the NCM discs were treated with rabbit polyvalent antibody to M tuberculosis (1:40 dilution) in 2.5% skimmed milk in PBS for 2 h at 40°. The NCM discs were washed three times with 0.15M PBS. The NCM discs were subsequently incubated with 1:600 anti-rabbit IgG biotin conjugate and 1:500 Extr-avidin HRP (Sigma Chemicals, St Louis MO) for 1 h respectively at 40° and washed repeatedly with PBS. The NCM discs were then treated with the substrate for 10 min (3 mg of 4-chloro-1-napthal in 1 ml of cold methanol. For routine use, 4 ml of PBS pH7.4 added 1 ml 4-chloro-1-napthol solution and 3 µl 30% hydrogen peroxide. The reaction was stopped by `pouring off' the substrate and followed by thorough washing in PBS. A positive reaction was indicated by the development of insoluble blue colour in NCM discs. The standardization of Dot-Iba gave positive results for those NCM discs that contained 1ng of the Tuberculin PPD antigen per ml and above.

CSF samples from patients with confirmed cases of TBM, probable cases of TBM and control groups were similarly assayed as described in the standardized Dot-Iba. The CSF from test and control groups were performed in batches of 10 at a time. PPD antigen (5 ng/ml) as positive and heat-inactivated pneumococci (5 ng/ml) as negative control were used in the assay. All the coded CSF samples were tested on two different occasions to evaluate the inter-observation variation as well as the reproducibility of the assay.

Results and Discussion

Standardization of the Dot-Iba indicated that the sensitivity of the assay is 1ng of Tuberculin PPD antigen per ml. Dot-Iba gave positive results for all 5 confirmed TBM. Thus giving a sensitivity of 100%. 20 of 25 CSF specimens from patients with probable TBM were positive by Dot-Iba and the sensitivity of the assay was 80%. None of the CSF from confirmed and probable TBM gave positive reaction when incubated with pneumococcal antigen in the assay.

For the 40 CSF samples from patients with non-tuberculous neurological disease the assay gave negative reaction in 38 cases. Thus specificity of the assay was 95%. Two patients with chronic meningitis gave positive results. In these two patients no microbial agents were isolated to account for the cause of chronic meningitis. Thus etiology of chronic meningitis in these two patients remained undetermined. However, these two patients were given ATT and at three months of follow-up both the patients showed partial clinical response.

The data of the study highlight three observations. Firstly, autoclaving of the CSF will eliminate the risk of handling the infective material in the laboratory. Secondly, disposal of infectious material is less cumbersome and there is no risk of environmental pollution. Thirdly, heat-inactivation of the CSF specimen eliminates the non-specific cross-reacting mycobacterial antigen in the CSF, particularly in patients with meningitis due to non-tuberculous etiology.

The immunoassays described in the literature for the detection of mycobacterial antigen in the CSF in patients with TBM include Latex particle agglutination test with anti-plasma membrane antibody,³ a sandwich ELISA with anti-M bovis antibody⁴ and inhibition ELISA with polyvalent antibody against M tuberculosis.⁵ Mastroianni et al⁹ applied a Dot-Iba method in the study of 38 patients with TBM. They used anti M. bovis BCG antibody in their study and the assay detected mycobacterial antigen at a concentration of 100 ng/ml. The assay detected the presence of antigen in all culture positive patients of TBM. Two of the 25 patients with non-tuberculous meningitis gave false positive results in their study. In that study the physico-chemical properties of the antigen detected in the CSF was not highlighted. We have isoload a 14 kDa mycobacterial antigen using an immunoabsorbant affinity chromatography.^10,11 This antigen was used in Dot-Iba. However, isolation of the 14 kDa antigen from culture filtrate is cumbersome and requires technical expertise.

Dot-Iba as described in the study is reproducible and there is no inter-observation variation when the assay is performed in the same CSF on two different occasions. More importantly, it can be readily performed in any routine clinical laboratory and does not require expensive equipments like ELISA reader. The results can be interpreted by visual examination of NCM discs. The entire procedure requires only 6 h after the receipt of CSF specimen in the laboratory. Although recent molecular biological techniques have highlighted the usefulness of PCR for the diagnosis of TB, the precise application PCR for the routine laboratory diagnosis of TBM SC II remains to be determined. The technical aspects of Dot-Iba described in this study can be performed very easily and a large number of CSF specimens can be easily handled in a single laboratory. We consider that a positive Dot-Iba has definite diagnostic value. The reagents used in the assay have a shelf life of more than six months. We therefore consider that this approach is most suited to laboratories in developing countries where TBM is still prevalent.

Acknowledgment

The authors are grateful to Director, SCTIMST, Trivandrum, India, for providing all facilities to undertake this study and kind permission to publish this paper. The authors are also indebted to Miss. P. Sobha Kumari for excellent secretarial assistance.

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