International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 2(3), 69-75, March (2013) Int. Res. J. Biological Sci. International Science Congress Association 69 Detection of Immune Complexes in CSF of Tuberculous Meningitis cases in Correlation with Antibody ResponseSooraj S. Nath Department of Medical Microbiology, University of Calicut, Kerala, INDIA Available online at: www.isca.in Received 6th February 2013, revised 9th February 2013, accepted 24th February 2013Abstract A total of 800 CSF samples from patients suspected to TB meningitis were analyzed for anti-TB antibodies and mycobacterial immune complexes. About 24.375% of the cases showed anti-TB immune response by ELISA. Some CSF samples were positive only for anti-TB antibodies whereas some others were positive only for mycobacterial immune complexes. It appears that the immune complexes which are found before the free antibody could be detected. Immune complexes are a mixture of both antigen and antibodies. As the disease progresses the patient would have free antibodies and may become negative for immune complexes. It appears from the present study that the detection of both antibody and immune complexes is significant in the immunodiagnosis of the disease. Keywords: Tuberculosis, meningitis, ELISA, CSF. Introduction Meningitis is inflammation of meninges, the membranes that cover the brain and spinal cord. Meningitis is also defined as inflammation of the Leptomeninges and underlying subarachnoid cerebrospinal fluid (CSF). It is mainly caused by bacteria or viruses. Bacterial meningitis is usually serious and is life-threatening if it is not treated promptly. Mycobacterium tuberculosis is the causative agent of tuberculosis (TB) in humans. Humans are the only reservoir for the bacterium. Tuberculous meningitis (TBM) is meningoencephalitis affecting meninges as well as brain parenchyma and vasculature. The primary pathologic event is formation of thick exudate within subarachnoid space, most prominently the base of brain. Accompanying this exudate is inflammation which affects the adjacent blood vessels. Ischemic cerebral infarction is caused from vascular occlusion found around middle cerebral artery (reflecting presence of exudate within sylvian fissure) and striates arteries as they penetrate the base of the brain2,3. TBM is the clinical manifestation of central nervous system (CNS) involvement in TB. Mycobacterium tuberculosis in the meninges is the main feature and the inflammation is concentrated around the base of brain. Infection onsets in lungs and spread to the meninges by various routes; blood-borne spread occurs generally and patients (25 %) with miliary TB have TBM, by crossing the blood-brain barrier; but many patients get TBM from rupture of cortical focus in brain and even from rupture of a bony focus in the spine (Pott’s spine). The diagnosis of TB mainly depends on clinical suspicion and radiographic findings with bacteriological confirmation by smear examination and culture techniques. The method of diagnosis by solid media culture usually takes 3 to 8 weeks to reveal the causative organisms. TB serological techniques based on antibody detection for diagnosing and monitoring tubercular infection with low cost and flexibility to adapt to small laboratories makes it a popular diagnostic method. The main purpose of this study is to reduce the time required for the diagnosis of TB. The average incubation period required for the growth of this organism is estimated to be about 8 weeks. During this time period, the infection might be fatal to the patient and will lead to rise in mortality rate. Even though the gold standard diagnostic tool for the organism is culture, the time duration for incubation masks its specificity. In order to overcome this, a variety of diagnostic tools that have sensitivity rate of culture techniques can be developed. Material and MethodsSamples: CSF samples (800 samples) used for this study were collected from TB patients diagnosed with active disease, subsided disease and also from patients with no history of TB were obtained from neighboring diagnostic laboratory as well as hospitals. Staining for AFB (acid-fast bacteria): Preliminary and initial detection of TB was done by Ziehl-Neelsen (ZN) method of staining and graded as per RNTCP (Revised National Tuberculosis Control Programme) guidelines. Culturing on Lowenstein-Jensen (LJ) medium: Readymade LJ medium slants were inoculated with 0.1 ml of each of the samples and incubated at 37 °C for 8 weeks. The LJ slants were inspected weekly for the growth of the mycobacterial colonies on the LJ slants i.e., rough, tough and buff-colored, were International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(3), 69-75, March (2013) Int. Res. J. Biological Sci. International Science Congress Association 70 subjected to ZN staining to confirm the presence of acid-fast bacilli. Samples that show no growth in LJ medium after 8 weeks of incubation at 37 °C were considered as negative. Culturing in modified Middlebrook 7H9 broth: Middlebrook 7H9 broth with indicator and cocktail of antibiotics (PANTA) (Himedia, Mumbai, India) was inoculated with 0.1 ml of each of the samples. The indicator in the medium makes it possible to visualize growth within 10–14 days. Antibiotic cocktail present in the medium retards the growth of contaminating flora. Coating of MTSE antigen and blocking of MTSE plates: 5 ml of 20x PBS solution was taken and volume was made into 1x solution with distilled water. From this 5 ml was taken and 10 l of MTSE antigen was added into it. 50 l of this solution was transferred into the wells of ELISA (Microtitre) plates. The plate was kept in the refrigerator for overnight. The next day the MTSE plate was washed with PBST solution for 3 times. 5 ml of 20x PBS solution was taken and its volume was made into 1x solution with distilled water. To this 50 ml of solution 1 g of skimmed milk powder was added. 150 l of 2 % PBST milk was added into antigen coated ELISA plate. Then the plate was incubated at 37 ºC for 2 hrs. The plates were blot dried and were stored in the refrigerator. Coating of BCG antigen and blocking of immune complexes plates: 5 ml of 20x PBS solution was taken and its volume was made into 1x solution with distilled water. From this 30 ml was taken and 9 l of anti-BCG antigen was added into it. 50 l of this solution was transferred into ELISA (Microtitre) plates which were marked as IcM and IcG. These plates were kept in the refrigerator for overnight. The next day the BCG coated plates were washed with PBST solution for 3 times. 5 ml of 20x PBS solution was taken and its volume was made into 1x solution with distilled water. To this 50 ml of solution 1 g of skimmed milk powder was added. 150 l of 2% PBS milk was added into Ag coated microtitre ELISA plates. Then the plates were incubated at 37 ºC for 2 hrs. The plates were blot dried and were stored in the refrigerator. Procedure for ELISA of MTSE: 1% PBST Milk was prepared (0.2 g of skimmed milk powder was added to 20 ml of PBST solution. This is the sample diluent). 400 l of PBST milk was added into each dilution tube. 100 l of sample was added to it. These 1:5 dilutions were mixed well. The microtitre plate was marked as MTSE. The first well is the blank and to the second well MTSE Positive control was added. From the dilution tubes 50 l was transferred to the ELISA plate wells in duplicates and plate was incubated at 37ºC for 1 hr and 30 min. After the incubation was over the plate was washed for six times with PBST solution and it was blot dried. The conjugate IgG HRP and IgM HRP of 5 l were added to 15 ml and 5 ml of PBST milk (diluents). The IgG conjugate was added to the plate (1:3000 dilution). The plate was then incubated at 37ºC for 1 hr. After the incubation was over the plate was washed with the wash buffer (PBST) for 8 times and it was blot dried. 75l of the substrate was added in the ELISA plate into each well. The plate was then kept in a dark place for 10 min for colour development. The colour change was noted after stopping the reaction by using the stop solution 1N sulphuric acid (50 l) into each well of ELISA microtitre plate. The plate was read using the ELISA reader (492 nm) 8. Procedure for ELISA of immune complexes: 1% PBST Milk was prepared (0.2 g of skimmed milk powder was added to 20 ml of PBST solution (sample diluent). 400 l of PBST milk was added into each dilution tube. 100 l of sample was added to it. These 1:5 dilutions were mixed well. The microtitre plate was marked as IcM, IcG. The first well is the blank and to the second well IgM Positive control was added to the second well of IgM plate. The first well is the blank and to the second well IgG Positive control was added to the second well of IgG plate. From the dilution tubes 50 l of the diluted samples was transferred to the ELISA plate in duplicate and it was incubated at 37 ºC for 1 hr and 30 min. After the incubation was over the plate was washed for six times with PBST solution and it was blot dried. 5 l of the conjugate IgG HRP and IgM HRP was added to 15 ml and 5 ml of PBST milk (diluents). The IgG conjugate was added to the IcG plate (1:3000 dilution). The IgM conjugate was added to the IcM plate (1:1000 dilution). The plates were then incubated at 37 ºC for 1 hr. After the incubation was over the plates were washed with the wash buffer (PBST) for 8 times and it was blot dried. 75 l of the substrate was added in the ELISA plate into each well. The plates were then kept in a dark place for 10 min for colour development. The colour change was noted after stopping the reaction by using the stop solution 1N sulphuric acid (50 l) into each well of ELISA microtitre plate. The plates were read using the ELISA reader (492 nm). Results and Discussion The control of TB depends mainly on early detection of infection and effective treatment. Various attempts have been made in improvement of diagnosis of TB by implementing clinical and laboratory methods10, 11. Laboratory diagnosis have limitations: microscopic examination gives a low sensitivity, whereas culture techniques require more time and sometimes fail to detect the disease early 12. The development of the ELISA technique13 and dot-immunobinding assay14 using mycobacterial antigens has revolutionized serological tests for the diagnosis of mycobacterial infections. A total of 800 CSF samples from patients suspected to TBM were analyzed for anti-TB antibodies and mycobacterial immune complexes. It is observed that 195 samples were found to be positive for either anti-TB antibodies or mycobacterial immune complexes. The percentage of positivity is 24.375%. The aim of this present study is to detect the presence of immune complexes IcG and IcM and MTSE antibody in CSF samples from TBM cases by ELISA. International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(3), 69-75, March (2013) Int. Res. J. Biological Sci. International Science Congress Association 71 Figure-1 A microtitre plate module with positive results Figure-2 Bar graph representation of total immune response positive cases out of 800 samples tested for tuberculous meningitis immune complexes by ELISA IMMUNE RESPONSE505530606.256.8753.757.524.37510203040506070MTSEIgGIgMIgG+IgMOVERALL %NUMBER OF CASES AND PERCENTAGE NUMBER OF CASES PERCENTAGE International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(3), 69-75, March (2013) Int. Res. J. Biological Sci. International Science Congress Association 72 Figure-3 Bar graph representation of analysis of anti tuberculosis immune response in relation to age Figure-4 Bar graph representation of anti tuberculosis immune response analysis gender wise 2021273135283310.25610.76913.84615.89717.94914.35916.92310152025303540 10 yrs11 - 20yrs21 - 30yrs31 - 40yrs41 - 50yrs51 - 60yrs&#x-3.2;Ö” 60 yrsNUMBER OF CASES AND PERCENTAGE NUMBER OF CASES PERCENTAGE 59.12540.87525.3722.93610203040506070MALEFEMALEPERCENTAGE TOTAL PERCENTAGE OFSUSPECTED CASES PERCENTAGE OF POSITIVECASES International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(3), 69-75, March (2013) Int. Res. J. Biological Sci. International Science Congress Association 73 Figure-5 Bar graph representation of antibody response in relation to cell count Figure-6 Bar graph representation of immune response and immune status of positive cases 1730152040732.1253.751.8752.59.12510203040506070802050100150200.42;É€200NUMBER OF CASES AND PERCENTAGE NUMBER OF CASES PERCENTAGE 1952017524.3752.521.87550100150200250TOTAL HIVNON-HIVNUMBER OF CASES AND PERCENTAGE NUMBER OF CASES PERCENTAGE International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(3), 69-75, March (2013) Int. Res. J. Biological Sci. International Science Congress Association 74 It was observed that the total immune complexes are more when compared with total MTSE antibody. Out of 195 positive cases 60 (30.769 %) were total immune complexes. Then the IgG positive cases were 55 (28.205 %), IgM positive cases were 30 (15.385 %) and MTSE antibody positive cases were 50 (25.641 %). The study shows increased amount of antibodies and immune complexes in the age group between 41 and 50 (17.949 %). In other words the age group between 41 and 50 are mostly affected by TB when compared with other age groups according to the results obtained from this table. When compared to gender’s males (25.37 %) showed higher positivity to both antibody and immune complexes than females (22.936 %). Thus this table suggests that when compared to gender’s males showed higher positivity to both antibody and immune complexes than females. Whenever the cell count is high the positivity is also high and whenever the cell count is low the positivity is also low. Hence this means that when the cell count is low the severity of the disease might be less Thus immune response and cell count might be interlinked with each other. Earlier studies demonstrated that the detectable anti-TB antibody was found in cerebrospinal fluid (CSF)1517. The detection of antibodies correlated well with the disease and was positive in 68 to 80% of the cases. Scientists have shown that antigen or antibody detection is useful in the diagnosis of pulmonary, meningeal, pleural and abdominal TB by radioimmunoassay15,18,19. In a limited study of 10 CSF samples from patients with TBM, the potential usefulness of ELISA was demonstrated in the detection of antigen20. Studies reported that in a cross sectional study on changes in IgG and IgA and IgM levels along with other serum proteins in pulmonary TB during therapy, serum IgG and IgA levels have mostly reported to be increased while most of the authors have reported unchanged IgM levels21. Measurements of different classes of immunoglobulins using antigenic preparations have shown that IgM antibody levels have been found to be so low that their reliable measurement has been difficult. In the present study, IcM is found to be positive in less number when compared with IcG and MTSE antibody. Overall the presence of immune complexes is marginally more predominant than mycobacterial antibody (MTSE). It appears that the immune complexes which are found before the free-antibody could be detected, as immune complex is a mixture of both antigen and antibodies. Conclusion TBM is meningoencephalitis affecting meninges as well as brain parenchyma and vasculature. The primary pathologic event is formation of thick exudate within subarachnoid space, most prominently the base of brain. A total of 800 samples suspected from TBM were analyzed for anti-TB antibodies and mycobacterial immune complexes by using ELISA. It is observed that 195 samples were found to be positive for either anti-TB antibodies or mycobacterial immune complexes. The percentage of positivity is 24.375%. The positive results were further analyzed for total immune response, immune response in relation to cell count, age wise analysis of immune response, gender wise analysis of immune response and immune status of the patients. Some CSF samples were positive only for anti-TB antibodies whereas some others were positive only for mycobacterial immune complexes. It appears that the immune complexes which are found before the free antibody could be detected. Immune complexes are a mixture of both antigen and antibodies. As the disease progresses the patient would have free antibodies and may become negative for immune complexes. It appears from the present study that the detection of both antibody and immune complexes is significant in the immunodiagnosis of the disease. References 1.Horner P.J. and Moss F.M., Tuberculosis in HIV infection, Int. J. STD AIDS, , 162-167 (1991) 2.Selwyn P.A., Tuberculosis in the AIDS era: A new threat from an old disease, NY State J. Med.,91, 233-235 (1991) 3.Katti M.K., Assessment of antibody responses to antigens of Mycobacterium tuberculosis and cysticercus cellulosae in cerebrospinal fluid of chronic meningitis patients for definitive diagnosis as TBM/NCC by passive hemagglutination and immunoblot assays, FEMS Immunol. Med. Microbiol., 33, 57-61 (2002) 4.Kashyap R.S., Agarwal N., Chandak N.C., Taori G.M., Biswas S.K. and Purohit H.J., Application of mancini technique as a diagnostic test in CSF of tuberculous meningitis patients, Med. Sci. Monit., 6, 95-98 (2002) 5.Kennedy D.H. and Fallon R.J., Tuberculous meningitis, JAMA, 241, 264-268 (1979) 6.Thwaites G., Chau T.T., Mai N.T., Drobniewski F. and McAdam K., Tuberculous meningitis, J. Neurol. Neurosurg. Psychiatry, 68, 289-299 (2000) 7.Chakraborty N., Mukherjee A., Santra S., Sarkar R.N., Banerjee D., Guha S.K., Chakraborty S. and Bhattacharyya S.K., Current trends of opportunistic infections among HIV seropositive patients from Eastern India, Jpn. J. Infect. Dis.,, 49-53 (2008) 8.Sooraj S.N., Nathiya K., Dhanabalan R., Angayarkanni J. and Palaniswamy M., Detection of mycobacterial antibodies in serum samples by enzyme linked immunosorbent assay, Afr. J. Biotechnol., 10, 16012-16015 2011) 9.Sooraj S.N., Nathiya K. and Palaniswamy M., Vaccine turned to diagnostic tool, Asian J. Pharm. Clin. Res., , 54-56 (2012) 10.Frieden T.R., Sterling T.R., Munsiff S.S., Watt C.J. and Dye C., Tuberculosis, Lancet, 362, 887–899 (2003) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(3), 69-75, March (2013) Int. Res. J. Biological Sci. International Science Congress Association 75 11.Grzybowski S., Barnett G.D. and Styblo K., Contacts of cases of active pulmonary tuberculosis, Bull Int. Union Tuberc.,50, 90-106 (1975) 12.Lowell A.M., Tuberculosis: its social and economic impact and some thoughts on epidemiology. P. Kubica and L. G. Wayne (ed.), The mycobacteria. Part B. Marcel Dekker, New York., 1021-1056 (1984) 13.Daniel T.M. and Debanne S.M., The serodiagnosis of tuberculosis and other mycobacterial diseases by enzyme linked immunosorbent assay, Am. Rev. Respir. Dis.,135,1137-1151 (1987) 14.Van Vooren J.P., Turneer M., Yernault J.C., De Bruyn J., Burton E., Legros F. and Farber C.M., A multidot immunobinding assay for the serodiagnosis of tuberculosis, comparison with an enzyme linked immunosorbent assay, J. Immunol. Methods, 113, 45-49 (1988) 15.Samuel A.M., Kadival G.V., Irani S., Pandya S.K. and Ganatra R.D., A sensitive and specific method for the diagnosis of tuberculous meningitis, Ind. J. Med. Res., 77, 752-757 (1983) 16.Kalish S.B., Radin R.C., Leitz 0 Zeiss C.K. and Phair J.P., The enzymelinked immunosorbent assay method for IgG antibody to purified protein derivative in cerebrospinal fluid of patients with tuberculous meningitis, Ann. Int. Med., 99, 630 (1983) 17.Chandramukhi A., Bothamley G.H., Brennan P.J. and Evanyi J., Levels of antibodies to defined antigens of Mycobacterium tuberculosis in tuberculous meningitis, J. Clin. Microbiol., 27, 821-825 (1989) 18.Kadival G.V., Samuel A.M., Virdi S.S., Kale R.N. and Ganatra R.D., Radioimmunoassay of tuberculous antigen, Indian J. Med. Res.,75, 765-770 (1982) 19.Samuel A.M., Kadival G.V., Ashtekar M.D. and Ganantra R.D., Evaluation of tubercular antigen and anti tubercular antibodies in pleural and ascitic effusions, Ind. J. Med. Res., 80, 563-565 (1984) 20.Sada E., Ruiz-Palacios G.M., Lopezvidal Y. and Ponce De Leon S., Detection of mycobacterial antigens in CSF of patients with tuberculous meningitis by ELISA, Lancet, , 651-652 (1984) 21.Bhatia A.S., Kumar S. and Harinath B.C., Immunodiagnosis of Tuberculosis: An update, Ind. J. Clin. Biochem., 16(1), 132-135 (2001)