Title of Invention	AN IN-VITRO METHOD OF DISTINGUISHING TUBERCULOSIS MODELS
Abstract	The present invention relates to an in-vitro method for distinguishing samples with freshly infected, relapsed and extra-pulmonary tuberculosis from healthy BCG-vaccinated samples. The instant invention utilizes Iso citrate Dehydrogenase (ICDs) 1 & 2 for distinguishing the samples.

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FIELD OF THE INVENTION The present invention is in relation to distinguishing samples for extra-pulmonary tuberculosis. More particularly, the present invention is in relation to an in-vitro method for distinguishing samples with freshly infected, relapsed and extra-pulmonary tuberculosis from healthy BCG - vaccinated samples. Back ground of the Invention Tuberculosis, caused by Mycobacterium, tuberculosis, remains a ixiajor threat to human population, roughly responsible for 2 - 3 millions deaths every year worldwide (1 - 3). The secret of the pathogen's success is its ability to escape the host immune system and remain undetected in lungs for decades. Only in 10% of the infected people, the number being higher in immuno-compromised patients, TB erupts as a full-blown disease (4). Delay in distinguishing and treatment impedes the downstream management and control of the disease. With the increasing emergence of multi drug resistant strains and co-infection with HIV the problem is getting further compounded (5 - 7). Early distinguishing, therefore, is a matter of utmost concern not just for TB disease management but also for epidemiological investigations (8). Current distinguishing tools for tuberculosis often lack sensitivity and can be time consuming. TB distinguishing in developing countries largely banks upon tuberculin skin test and staining and culture methods. The epidemiological relevance of tuberculin test with purified protein derivative (PPD) is questionable in areas where BCG vaccination is compulsory because PPD is not sensitive enough to distinguish between vaccinated and infected individual (9). Microscopic determination of the bacilli in the sputum samples is a direct way of examining pulmonary tuberculosis (5). This however requires high titers of bacilli (5000 - 10000 / ml) in sputum - a condition seen only in full blown tuberculosis patients. Culture techniques can detect very low titers but are time consuming taking approximately 3-6 weeks (10). The importance of the major extracellular proteins of the pathogen as candidate components of a subunit vaccine has been reported earlier (11). Current discovery of the RD1 locus in the Mtb genome, encoding mainly the proteins actively secreted by mycobacteria into the culture medium, such as CFP-IO and ESAT-6, have further encouraged immunological tests as an adjunct to conventional distinguishing (12-15). Proteins that are released from Mycobacterium tuberculosis during late logarithmic growth phase, such as, superoxide dismutase and isocitrate dehydrogenase are employed as autolysis markers (16). The use of isocitrate dehydrogenase as a potential antigen for serodistinguishing along with malate dehydrogenase has been suggested (17, 18). The Mycobacterium, tuberculosis genome carries two isoforms of isocitrate dehydrogenase, M.tb ICD-I and M.tb ICD-2. Multiple sequence alignment revealed a closer similarity of M.tb ICD-I to eukaryotic NADP+ dependent ICDs, while M.tb ICD-2 groups with bacterial ICDs (manuscript in preparation). We have evaluated the utility of ICDs as immunogenic markers for tuberculosis through detection of anti-Mtb ICD antibody in sera of different well characterized categories of TB patient through enzyme linked immunosorbent assays. We describe the sensitivity and specificity of ICDs to distinguish TB patients from those vaccinated with BCG, and also from those patients infected with non-tuberculous mycobacteria or other pathogens vis-a-vis the conventional antigen - HSP 60 (19) and purified protein derivative (PPD). Objects of the Present Invention The principal object of the present invention is to develop an in-vitro method for distinguishing samples with freshly infected, relapsed and extra-pulmonary tuberculosis from healthy BCG- vaccinated samples. Another object of the present invention is to use Iso Citrate Dehydrogenase - 1 (ICD-1) and Iso Citrate Dehydrogenase - 2 (ICD-2) to identify immunoreactivity in the samples. Yet another object of the present invention is to distinguish subjects with freshly infected, relapsed and extra-pulmonary tuberculosis from healthy BCG - vaccinated samples. Still another object of the present invention is to identify immunoreactivity using enzyme linked immunosorbent assays (ELISA). Statement of the Invention The present invention is in relation to an in-vitro method of distinguishing samples with freshly infected, relapsed and extra-pulmonary tuberculosis from healthy BCG-vaccinated samples, said method comprising steps of: a) identifying immunoreactivity in the samples using Iso Citrate Dehydrogenase - 1 (ICD-1) and Iso Citrate Dehydrogenase - 2 (ICD-2); and b) distinguishing the samples with freshly infected, relapsed and extra-pulmonary tuberculosis from healthy BCG- vaccinated samples. Brief description of the accompanying drawings Figure 1: Affinity purification of M.tb ICD-I and M.tb ICD-2. Histidine- tagged recombinant protein was purified by nickel column chromatography under native condition and stained with Coomassie Blue following electrophoresis on 10% SDS polyacrylamide gels. The different lanes are: lanes 1 and 2: M.tb ICD-2; lane M: protein molecular size markers (200 kDa, 1 16 kDa, 97 kDa, 66 kDa, 45 kDa, 31 kDa and 21.5 kDa); lanes 3 and 4: M.tb ICD-I. Figure 2: M.tb ICD-I and ICD-2 show high B-cell reactivity to sera from TB infected patients from different groups as opposed to BCG vaccinated healthy controls. The humoral immune responses directed against the recombinant proteins, M.tb ICD-I (2A) and M.tb ICD-2 (2B) and control antigens HSP 60 (2C) and PPD (2D) were compared between different categories of patients and healthy controls. Group 1 : fresh infections, Group 2: relapsed infection and Group 3: extrapulmonary TB. The respective sample numbers and p values are shown. Figure 3: M.tb ICDs are more immunogenic than HSP 60. The ELISA reactivity to M.tb ICD-I, M.tb ICD-2 and control antigen HSP 60 was compared in different patient groups. Horizontal bands represent the mean reactivity or average levels of humoral response in each category. Figure 4: M.tb ICDs could significantly distinguish TB-infected sera from NTMs and non-TB patient sera. Recombinant M.tb ICD-I and M.tb ICD-2 as well as HSP 60 were tested against sera of NTM (4A) and non- tuberculosis patients (4B). The respective humoral responses were compared to TB - infected sera, the p values for which are given in the figures. HSP 60 could not distinguish TB-infected patient from either NTM or non-TB significantly. Horizontal bands represent the mean reactivity in each category. Detailed description of the present invention The present invention is in relation to an in-vitro method of distinguishing samples with freshly infected, relapsed and extra-pulmonary tuberculosis from healthy BCG-vaccinated samples, said method comprising steps of: ♦ identifying immunoreactivity in the samples using Iso Citrate Dehydrogenase - 1 (ICD-1) and Iso Citrate Dehydrogenase - 2 (ICD-2); and ♦ distinguishing the samples with freshly infected, relapsed and extra- pulmonary tuberculosis from healthy BCG- vaccinated samples. In another embodiment of the present invention wherein the immunoreactivity is identified using enzyme linked immunosorbent assays (ELISA). MATERIALS AND METHODS: Cloning, expression and purification of M.tb ICD-I and M.tb ICD-2: The ORFs, corresponding to M.tb ICD-I (Rv3339c, 1.230 kb) and M.tb ICD-2 (Rv0066c, 2.238 kb) were PCR amplified from the genomic DNA of H37Rv. BamHl and HmdIII restriction sites were incorporated in the 5f end of forward and reverse primers respectively for both M.tb ICD-I and M.tb ICD-2. The primers and parameters for thermal cycle amplification have been tabulated in table 1. Table X: PCR primers and thermal cycle parameters for amplification of M.tk> ICD-1 and ICD-2 The amplicons carrying the full length M.tb ICD-I and M.tb ICD-2 were cloned at the BamHl and HmdIII sites of the expression vector pRSET-A (Invitrogen, USA) with six histidine sequence tag at N-terminal. The generated constructs 'setAicdl' and tsetAicd2t were further transformed into the BL21 (DE3) strain of E.coli. The clones were confirmed by sequencing using the T7 promoter primer, on an AB1 prism 377 DNA sequencer (PE Biosystems, USA). The genes were over-expressed in the pRSET-A/ E.coli BL-21 (DE3) expression system. The over-expressed his-tagged recombinant protein was purified by Ni2+-nitrilotriacetate affinity chromatography. The cells transformed with the constructs were grown in Terrific Broth (TB) containing ampicillin (lOOng /ml) to an ODpoo of 0.4 to 0.5 at 37°C5 cooled to 27°C, induced with 0. ImM isopropyl /?-D-thiogalactoside and grown overnight at 27°C. The cells were lysed by sonication, followed by centrifuging at 13000 rpm for 30 minutes at 4°C. The clear lysate was loaded onto Ni2+-NTA column, which was then washed with 50mM NaH2PO4, 30OmM NaCll 20mM imidazole, pH 8. The protein was eluted in the same buffer supplemented with 20OmM imidazole. The proteins were 90-95% pure as seen on 10% SDS-PAGE followed by Commasie Blue staining (Figure 1), The purified recombinant proteins were dialyzed against 20mM TrisCl, pH7.5 with lOOmM NaCl and 3% glycerol and quantified using Bradford Reagent (20). The technology of the instant Application is further elaborated with the help of following examples. However, the examples should not be construed to limit the scope of the invention. Examples: Example: 1 Human Sera: The study population (n= 215) comprised of the M.tb infected human sample population reporting to Mahavir Hospital and Research Centre, Hyderabad and Central JALMA Institute for Leprosy, Agra. These were categorized into three groups, namely group 1 (n=42 patients), group 2 (n=32 patients) and group 3 (n^35 patients). In addition to the above 44 clinically healthy donors, 30 NTM cases and 32 non - TB patients who were proven culture negative for acid fast bacteria were also included as controls in this study. Group 1 comprised of patients with fresh infection with no history of TB treatment. Group 2 comprised of patients with relapsed cases, i.e. those who were treated earlier for tuberculosis but the symptoms re-emerged after the completion of the treatment. Group 3 included patients with extra*-1 pulmonary tuberculosis. Group 1 and group 2 patients were distinguishing by sputum examination (acid-fast bacillus smear positive and negative) while the extra-pulmonary cases were confirmed by tissue biopsy. Clinically healthy donors were M. bovis BCG vaccinated and had no symptoms of TB at the time of sera collection. Randomly picked individuals from the population of healthy controls were subjected to PCR test for TB and were found to be PCR negative. Mycobacteria other than M. leprae that are not included in the M. tuberculosis complex are referred to as nontuberculous mycobacteria or NTM (21). However, the group referred to as NTMs in this study included sera collected from patients infected with non-tuberculous mycobacterial species (n= 14), such as, M. avium, M. xenopi, and M fortuitum as well as sera from patients with M leprae infection (n= 16). The non - TB patient category included infected individuals who were tested negative for acid fast bacteria by staining and culture based techniques. These patients were also negative for HIV and HBV, These randomly picked patients were suffering from either pneumonia, lower respiratory infections, septicemia, urinary tract infections, gastrointestinal infections, cirrhosis or fever of unknown origin. The study population had no sex or age bias. This study was approved by the Institutional Ethics Committee. Example: 2 Immunosorbent assays: Enzyme linked immunosorbent assays (ELISA) were performed to check the B cell immune response in human to the M.tb ICD-I and ICD-2 proteins and control antigen HSP 60 and PPD. HSP 60 used was M. tb HSP65/GroEL. In brief, the 96 well microtitre plates (Corning, Costar, USA) were coated with ~500ng of either control antigens or recombinant M.tb ICD-I and M.tb ICD-2. The plates were incubated overnight at 4°C, washed thrice with phosphate buffer saline (PBS) and blocked with 100 jal of blocking buffer (2% BS A in PBS) for 2 hour at 37°C, The plates were then washed thrice with wash buffer PBST (0.05% Tween 20 in 1 X PBS). The M. tuberculosis infected human sera belonging to different clinical groups were diluted 200 times in blocking buffer (1% BSA in PBS) . 50 (al of sera were added to antigen coated wells followed by incubation for lhr at 37°C. The plates were thoroughly washed with PBST and further incubated with anti-human IgG-horseradish peroxidase (HRP) (Sigma, USA) at 37°C for lhr. HRP activity was detected using a chromogenic substance o-phenylenediamine tetrahydrochloride (Sigma, USA) in citrate-phosphate buffer (pH 5.4) and H2O2 (Qualigens, India) as 1 |al/ml. The reactions were terminated using IN H2SO4, and the absorbance values were measured at 492 nm in an ELISA reader (BioRad, USA). Each ELISA was repeated at least twice with some randomly picked sera samples tested thrice for confirmation, with and without replicates for each sample within individual ELISA. Data analysis: t-test was performed to compare the means of two variable groups, healthy and infected classes, using the online scientific calculator of GraphPad (http://www.graphpad.com/quickcalcs/ttestl.cfm) to calculate means, standard error of means (SEM) and p values. Example: 3 RESULTS Expression and purification of M.tb ICD-I and M.tb ICD-2 The over-expressed N-terminal His-tagged M.tb ICD-I was purified to 95% homogeneity on a Nickel affinity column (Figure 1). The molecular size of the recombinant ICD-I was determined to be 49.2 kDa. The purification was carried out under native conditions from soluble fraction with an yield of 3.25 mg protein per 500 ml of start culture. Similarly M.tb ICD-2, a 83 kDa protein, was purified to 90-95% homogeneity (Figure 1) with an yield of about 20.4 mg per 1000 ml of start culture. Example: 4 M.tb ICD-I and M.tb ICD-2 show high reactivity to patient sera as opposed to BCG-vaccinated healthy controls Humoral immune responses directed against the M.tb ICD-I and M.tb ICD-2 were compared between patients- with tuberculosis and BCG-vaccinated healthy controls (Figure 2A and 2B) . The recombinant proteins were used to screen the infected and the healthy sera by ELISA1 using anti-human IgG-HRP as conjugates. The sera were also tested against M.tb HSO 60 and the purified protein derivative (PPD) (Figure 2 C and 2D). The immunoreactivity of ICD-I, ICD-2, HSP 60 and PPD were statistically analysed and compared with respect to both infected and healthy sera. These data demonstrate that sera of all the infected patients mounted a statistically significant (p A correlation between reactivity against M.tb ICD-I and M.tb ICD-2 in patient sera with the state of disease, fresh or relapse, was attempted by comparing the antibody responses to M.tb ICD-.L and M.tb ICD-2 between various clinical categories (Figure 2A and 2B respectively). M.tb ICD-I failed to discriminate between fresh, relapsed and extra-pulmonary TB cases as no significant differences in immunoreactivity in different patient groups were observed (Figure 2A). Yet as compared to BCG- vaccinated healthy controls, each category yielded p values less than 0.0001 indicating that M.tb ICD-I can differentiate substantially between BCG-vaccinated healthy population and any category of M.tb infected patients, pulmonary or non-pulmonary. M.tb ICD-2, on the other hand, could also discriminate relapsed cases from both fresh infections (p distinguish the extrapulmonary infections from BCG vaccinated healthy controls (p= 0.2177). These results demonstrate that (i) recombinant M.tb ICD-I and ICD-2 proteins could differentiate sera from TB infected patients vis-a-vis healthy BCG vaccinated controls, (ii) the extrapulmonary infections that could not be distinguished from healthy controls by HSP 60, could be significantly identified and categorized by M.tb ICDs and (iii) M.tb ICD-2 mounted a stronger antibody response in relapsed cases and could significantly discriminate them from Group 1 and Group 3 categories. These proteins, which have an apparently important metabolic role, are thus able to elicit a strong B-cell response as a function of the TB infection. Example: 5 Immunodominace of ICDs over HSP 60 We compared the immunogenicity of ICDs over HSP 60. Humoral response to HSP 60 in all the three categories of TB patients was tested and compared with those to ICDs (Figure 3). The data clearly indicate that the mean reactivity (represented by the horizontal bands in Figure 3) of HSP 60 in all the classes of patient sera was much lower than either ICD-I or ICD-2 (Figure 3). Thus ICDs are immunodominant and serologically more sensitive than HSP 60. The mean values for ICD-I in the Groups 1, 2 and 3 were 0.481, 0.565 and 0.457 respectively, while those for ICD-2 were 0.165, 0.362 and 0.188 respectively. It is therefore apparent that ICD-I elicited a stronger response in all the three categories of patients tested than ICD-2. The data also confirm the discriminatory power of ICD-2 for relapsed case as compared to other categories. Example: 6 Immunospecificity of M.tb ICDs The potential of M.tb ICDs to specifically distinguish between TB, NTMs and non-TB patient sera (those essentially culture negative for acid fast bacteria but harboring other pathogens) was tested by examining the cross-reactivity of the recombinant proteins with NTMs and non-TB patient sera. Thirty NTMs and thirty two non-TB patient sera were tested for their immunogenic response against M.tb ICD-I, M.tb ICD-2 and HSP 60. The data were statistically analyzed to check if ICDs could significantly distinguish between TB infected patients and NTMs or non- TB patients. Figure 4A and 4B show that ICDs could significantly distinguish TB-infected sera from NTMs (p Example: 7 DISCUSSION: The main objective of our study was to evaluate M.tb ICD-I and M.tb ICD-2 in terms of their immune features as compared to the control antigens HSP 60 and PPD that are ferquently used for distinguishing of tuberculosis. ICDs serve as marker of autolysis (16, 17) and are amongst the secretory proteins released during late logrithmic phase. While earlier efforts have pointed to the antigenic potential of M.tb ICDs (17), the present study is the first systematic investigation of their potential as an immune marker. The cases of tuberculosis were identified and enrolled based on their history of treatment as fresh infections, relapsed cases and extrapulmonary infections. The categorization of patients largely depended upon the treatment history dictated by the patients or their family members. As evident from Figure 2, there is little doubt about the ability of either M.tb ICD-I or M.tb ICD-2 to elicit a strong B-cell response, irrespective of patient category. When compared to M.tb ICD-2, M.tb ICD-I was more antigenic (Figure 2 and 3). It would be interesting to explore this disparity. A comparative analysis of ELISA reactivities amongst different categories of patients for M.tb ICDs (Figure 2 A and 2B) revealed higher reactivity in the Group 2 as compared to fresh (Group 1) and extrapulmonary (Group 3) infections. More specifically, the antigenic response in this category of patients to ICD 2 was significantly higher than that in Group 1 and Group 3. Since, these patients had undergone treatment earlier, high number of autolysed infected macrophages and autolysed pathogens could possibly explain the comparative high antibody response against M.tb ICDs in this category. Drugs, like isoniazid, are known to affect the cell envelope architecture of mycobacteria and hence the increase in the production of the secreted proteins (22). Comparative immunoreactivity of M.tb ICD-I , M.tb ICD-2 and HSP 60 clearly indicates that the antigenic distinction between healthy and tuberculosis patients is statistically significant for both M.tb ICD-1 and M.tb ICD-2 (p infect mammalian hosts. These are referred to as nontuberculous mycobacteria (±JTM). NTMs are omnipresent in the environment and most species are either non-pathogenic for humans or are rarely associated with disease, except a few like M. avium, that are opportunistic pathogens, more frequently associated with immunocompromised patients (23, 24). The clinical significance of many NTM remains unclear, however it is important to check the crossreactivity of M.tb antigens with this group of mycobacteria. Our experiments could establish that M.tb ICDs do not cross-react with either NTMs or non-TB patient sera (Figure 4A and 4B). The existing distinguishing tests for tuberculosis, even to this day, largely depends on tuberculin skin test and staining and culture techniques. These methods are slow, cumbersome and lack sensitivity and specificity in BCG vaccinated cases. As more and more recombinant antigens are being tested (25 - 31) serological methods are likely to be favoured over others. ELISA per se is unlikely to replace the current tuberculosis distinguishing, however in combination or parallel with other rapid PCR based distinguishing techniques, ELISA can largely improve the accuracy and rapidity of tuberculosis distinguishing for an effective disease management. Our data, for the first time, reveal the antigenic potential of recombinant M.tb ICD-I and also present a systematic study on immunogenicity of recombinant M.tb ICD-2. M.tb ICD-I and M.tb ICD-2 can be further analyzed for their pathogen specific antigenic epitopes. Given their important role in the energy cycle, we are currently evaluating these two enzymes of M.tb as possible drug targets. That such important enzymes can also have strong antigenic attributes which enable them to significantly discriminate between BCG-vaccinated healthy controls and TB patients and at the same time TB from other pathogenic infections is a very exciting and novel proposition possibly pointing to their immunomodulatory function. ACKNOWLEDGEMENTS This project was supported by research grants from the Council of Scientific and Industrial Research (CS1R) and Department of Biotechnology, Government of India to SEH. SB thanks the CSIR for Senior Research Fellowship. We thank Dr. Shekhar Mande for providing purified recombinant M. tb HSP 65/GroEL. REFERENCES 1. Dye, C, Scheele, S., Dolin, P., Pathania, V., & Raviglione, M. C. (1999) JAMA. 282, 677-686. 2. Bloom, B. R., & Murray, C. J. L. (1992) Science. 257, 1055-1064. 3. Chakhaiyar, P. & Hasnain, S., E. (2004) Medical Principles and Practice. (In Press). 4. Helmuth, L. (2000) Science. 289, 1123-1125. 5. Dye, C, Espinal, M. A., Watt, C. J., Mbiaga, C, & Williams, B. G. (2002) J. Infect. Dis. 185, 1197-1202. 6. Siddiqi, N., Shamim, M., Hussain, S., Choudhary, R. K., Ahmed, N. , Prachee, Banerjee, S., Savithri, G. R., Alam, M., Pathak, N. , et al. (2002) Anf)microb. Agents Chemother. 46, 443-450. 7. Ahmed, N., Caviedes, L., Alam, M., Rao, K. R., Sangal, V., Sheen, P. , Gilman, R. H., & Hasnain, S. E. (2003) J. Clin. Microbiol 41, 1712-1716. 8. Ahmed, N., Alam, M., RajenderRao, K., Kauser, F., Ashok Kumar, N. , Qazi, N., N., Sangal, V., Sharma, V., D., Das, R., Katoch, V., M., et al (2004). J. Clin. Microbiol. (In Press). 9. Roche, P. W., Triccas, J .A., Avery, D. T., Fifis, T., Billman-Jacobe, H., & Britton, W. J. (1994) J. Infect. Dis. 170, 1326-1330. 10. Laidlaw, M. (1989) In Practical Medical Microbiology, eds. Colle, J. G., Duguid, J. P., Fraser, A. G., & Marimon, B, P. (New York, Churchill Livingstone), pp. 399-416. 11. Horwitz, M. A., Lee, B. W., Dillon, B. J., & Harth, G. (1995) Proc. Natl. Acad. Sd. USA 92, 1530-1534. 12. Mustafa, A. S. (2002) MoZ. Immunol. 39, 113-119. 13. Louise, R., Skjot, V., Agger, E. M., & Andersen, P.(2001) Scand. J. Infect. Dis. 33, 643-647. 14. Trajkovic, V., Natarajan, K., & Sharaia, P. (2004) Microbes Infect, p, 513-519. 15. Mori, T., Sakatani, M., Yamagishi, F., Takashima, T., Kawabe, Y.3 Nagao, K., Shigeto, E., Harada, N., Mitarai, S., Okada, M., et al (2004) Am. J. Respir. Crit. Care Med. 0, 200402179-0 (In Press). 16. Anderson, P., Askgaard. D., Ljungqvist, L., Bennedsen, J., & Heron I. ( 1991) Infect. Immun. 59, 1905-1910. 17. Ohman, R., & Ridell, M. (1996) Tuber. Lung Dis. 77, 454-461. 18. Florio, W., Bottai, D., Batoni, G., Esin, S., Pardini, M. , Maisetta, G., & Campa, M. (2002) Clin. Diagn. Lab. Immunol. 9, 846-851. 19. Perschinka, H., Mayr, M., Millonig, G., Mayerl, C, van der Zee, R., Morrison, S.G., Morrison, R.P., Xu, Q., & Wick, G. (2003) Artefioscler Thromh Vase Biol. 23, 1060-1065. 20. Bradford, M. M. (1976) Analyt. Biochem. 72, 248-252. 21. Saiman, L. (2004) Paediatr. Respir. Rev. 221-3. 22. Bardou, F., Quemard, A., Dupont, M. A., Horn, C, Marchal, G., & Daffe, M. (1996) Antimicrob. Agents Chemother, 40, 2459-2467. 23. Shiratsuch, H., 86 Basson, M. D. (2003) Am. J. Surg. 186, 547-551. 24. Hadad, D. J., Palaci, M., Pignatari, A. C, Lewi, D. S., Machado, M. A., Telles, M. A., Martins, M. C, Ueki, S. Y., Vasconcelos, G.M., Palhares, M. C. (2004) Epidemiol Infect. 132,151-155. 25. Choudhary, R. K.. Mukhopadhyay, S., Chakhaiyar, P., Sharma, N., Murthy, K. J. R., Katoch, V. M., & Hasnain, S. E. (2003) Infection Immunity. 71, 6338-6343. 26. Ramalingam, B., UmaDevi, K. R., & Raja, A. (2003) Scand. J. Infect. Dis. 35, 234- 239. 27. Brusasca, P. N., Peters, R. L., Motzel, S. L., Klein, H. J., & Gennaro M. L. (2003) Comp. Med. 53, 165- 172. 28. Maekura, R., Kohno, H., Hirotani, A., Okuda, Y., Ito, M., Ogura, T., & Yano, I. (2003) J. Clin. Microbiol. 41, 1322-1325. 29. Perkins, M. D., Conde, M. B., Martins, M., & Kritski, A. L. (2003) Chest 123, 107- 112. 30. Maekura, R., Okuda, Y., Nakagawa, M., Hiraga, T., Yokota, S., Ito, M., Yano, I., Kohno, H., Wada M., Abe, C, et dl. (2001) J. Clin. Microbiol. 39, 3603-3608. 31. Chakhaiyar, P., Nagalakshmi, Y., Aruna, B., Murthy, K., J., R,, Katoch, V., M., & Hasnain, S., E. (2004) J. Infect Dis. (In Press). LEGENDS We claim: 1) An in-vitro method of distinguishing samples with freshly infected, relapsed and extra-pulmonary tuberculosis from healthy BCG- vaccinated samples, said method comprising steps of: a) identifying immunoreactivity in the samples using Iso Citrate Dehydrogenase - 1 (ICD-1) and Iso Citrate Dehydrogenase - 2 (ICD-2); and b) distinguishing the samples with freshly infected, relapsed and extra- pulmonary tuberculosis from healthy BCG- vaccinated samples. 2) The in-vitro method as claimed in claim 1, wherein the immunoreactivity is identified using enzyme linked immunosorbent assays (ELISA). 3) An in-vitro method of distinguishing samples with freshly infected, relapsed and extra-pulmonary tuberculosis from healthy BCG- vaccinated samples, substantially as herein described along with accompanied examples.

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