Share This Article:

Rapid immunodiagnostic assays for Mycobacterium Tuberculosis infection

Abstract Full-Text HTML Download Download as PDF (Size:238KB) PP. 171-176
DOI: 10.4236/health.2010.23025    5,155 Downloads   10,225 Views   Citations


Purpose: There is a need for a continued effort to develop rapid immunodiagnostic assays for tuberculosis (TB) infection with greater sensitivity and specificity that can be used in the field and in the laboratory and that can be formatted for use with multiple species. This would help to obtain definitive early diagnosis of TB. The present study was developed to determine the role of using early secreted antigenic target-6 (ESAT-6) in immunodiagnosis of Mycobacterium tuberculosis. Methods: Serum samples were obtained from TB infected patients and normal healthy controls. Two rapid immunodiagnostic assays (Enzyme-linked immunosorbent assay (ELISA) and Immunoblotting) were performed. Results: The sensitivity of immunoblotting assay was 100%; however, ESAT-6 antigen was not able to discriminate between patients and normal controls. Application of direct ELISA using ESAT-6 antigen yielded 97.6% sensitivity and 75% specificity for the diagnosis of TB infection. Conclusion: In conclusion, the detection of antibodies against ESAT-6 antigen in the sera of TB patients by direct ELISA could be used as a preliminary assay for diagnosis of human M. tuberculosis infection. A combination of the ELISA with either radiological or microscopic examination is required to overcome the low specificity of the assay for negative results.

Conflicts of Interest

The authors declare no conflicts of interest.

Cite this paper

Talaat, R. , Radwan, G. , Mosaad, A. , Saleh, S. and Bassiouny, K. (2010) Rapid immunodiagnostic assays for Mycobacterium Tuberculosis infection. Health, 2, 171-176. doi: 10.4236/health.2010.23025.


[1] Brock, I., Weldingh, K., Leyten, E.M.S., Arend, S.M., Ravn, P. and Andersen, P. (2004) Specific T-Cell epitopes for immunoassay-based diagnosis of mycobacterium tuberculosis Infection. Journal of Clinical Microbiology, 42(6), 2379-2387.
[2] World Health Organization (WHO) (2007) Global tuberculosis control: Surveillance, planning, ?nancing. WHO report. Geneva: World Health Organization. WHO/ HTM/ TB/2007.376.
[3] Di Perri, G., Cruciani, M., Danzi, M.C., Luzzati, R., De Checchi, G., Malena, M., Pizzighella, S., Mazzi, R., Solbiati, M., Concia, E., et al. (1989) Nosocomial epidemic of active tuberculosis among HIV infected patients. Lancet, 2, 1502-1504.
[4] Lucas, S. and Nelson, A.M. (1994) Pathogenesis of tuberculosis in human immunodeficiency virus-infected people. American Society for Microbiology Press, Washington, D.C., 29.
[5] Dye, C., Watt, C.J., Bleed, D.M., Hosseini, S.M. and Raviglione, M.C. (2005) Evolution of tuberculosis control and prospects for reducing tuberculosis incidence, pre- valence, and deaths globally. Journal of the American Medical Association, 293, 2767-2775.
[6] Jasmer, R.M., Nahid, P. and Hopewell, P.C. (2002) Latent tuberculosis infection. New England Journal of Medicine, 347, 1860-1866.
[7] Bruchfeld, J., Aderaye, G., Palme, I.B., Bjorvatn, B., Britton, S., Feleke, Y., Kallenius, G. and Lindquist, L. (2002) Evaluation of outpatients with suspected pulmonary tuberculosis in a high HIV prevalence setting in Ethiopia: Clinical, diagnostic and epidemiological characteristics. Scandinavian Journal of Infectious Diseases, 34, 331-337.
[8] Anderson, D., Anderson, V., Pentland, L., Sawyer, S., Starr, M. and Johnson, P.D. (2000) Attention function in secondary school students receiving isoniazid prophylaxis for tuberculosis infection. Epidemiology and Infection, 124(1), 97- 101.
[9] Sarmiento, O.L., Weigle, K.A., Alexander, J., Weber, D.J. and Miller, W.C. (2003) Assessment by meta-analysis of PCR for diagnosis of smear-negative pulmonary tuber- culosis. Journal of Clinical Microbiology, 41, 3233-3240.
[10] Chaparas, S.D., Maloney, C.J. and Hedrick, S.R. (1970) Specificity of tuberculins and antigens from various species of mycobacteria. American Review of Respiratory Diseases, 101, 74-83.
[11] Harboe, M. (1981) Antigens of PPD, old tuberculin, and autoclaved Mycobacterium bovis BCG studied by crossed immunoelectrophoresis. American Review of Respiratory Diseases, 124, 80-87.
[12] Huebner, R.E., Schein M.F. and Bass Jr.J. (1993) The tuberculin skin test. Clinical Infectious Diseases, 17, 968-975.
[13] Huebner, R.E., Schein, M.F., Hall, C.A. and Barnes, S.A. (1994) Delayed type hypersensitivity energy in human immunodeficiency virus-infected persons screened for infection with Mycobacterium tuberculosis. Clinical Infectious Diseases, 19, 26-32.
[14] Converse, P.J., Jones, S.L., Astemborski, J., Vlahov, D. and Graham, N.M. (1997) Comparison of a tuberculin interferon-gamma assay with the tuberculin skin test in high-risk adults: Effect of human immunodeficiency virus infection. Journal of Infectious Disease, 176, 144-150.
[15] Stenger, S. and Modlin, R.L. (1999) T-cell mediated immunity to Mycobacterium tuberculosis. Current Opinion in microbiology, 2, 89-93.
[16] Flynn, J.L. and Chan, J. (2001) Immunology of tuberculosis. Annual Review of Immunology, 19, 93-129.
[17] Delgado, J.C., Tsai, E.Y., Thim, S., Baena, A., Boussiotis, V.A., Reynes, J.M., Sath, S., Grosjean, P., Yunis, E.J. and Goldfeld, A.E. (2002) Antigen-specific and persistent tuberculin energy in a cohort of pulmonary tuberculosis patients from rural Cambodia. Proceedings of the National Academy of Sciences, 99, 7576-7581.
[18] Imaz, M.S., Schmelling, M.F., Kaempfer, S., Spallek, R. and Singh, M. (2008) Serodiagnosis of tuberculosis: Specific detection of free and complex-dissociated antibodies antimycobacterium tuberculosis recombinant antigens. Brazilian Journal of Infectious Diseases, 12(3), 234-244.
[19] Mahairas, G.G., Sabo, P.J., Hickey, M.J., Singh, D.C. and Stover, C.K. (1996) Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. Journal of Bacteriology, 178, 1274-1282.
[20] Behr, M.A., Wilson, M.A., Gill, W.P., Salamon, H., Schoolnik, G.K., Rane, S. and Small, P.M. (1999) Com- parative genomics of BCG vaccines by whole genome DNA microarray. Science, 284, 1520-1523.
[21] Arend, S.M., van Meijgaarden, K.E., de Boer, K., de Palou, E.C., van Soolingen, D., Ottenhoff, T.H. and van Dissel, J.T. (2002) Tuberculin skin testing and in vitro T cell responses to ESAT-6 and culture filtrate protein 10 after infection with mycobacterium marinum or M. kansasii. Journal of Infectious Disease, 186, 1797-1807.
[22] Sorensen, A.L., Nagai, S., Houen, G., Andersen, P. and Andersen, A.B. (1995). Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis. Infection and Immunity, 63, 1710-1717.
[23] Berthet, F.X., Rasmussen, P.B., Rosenkrands, I., Andersen, P. and Gicquel, B. (1998) A mycobacterium tuberculosis operon encoding ESAT-6 and a novel low-molecular-mass culture filtrate protein (CFP-10). Microbiology, 144 (Pt. 11), 3195-3203.
[24] Arend, S.M., Andersen, P., van Meijgaarden, K.E., Skjot, R.L., Subronto, Y.W., van Dissel, J.T. and Ottenhoff, T.H. (2000) Detection of active tuberculosis infection by T cell responses to early-secreted antigenic target 6-kDa protein and culture filtrate protein 10. Journal of Infectious Disease, May, 181(5), 1850-1854.
[25] Ewer, K., Deeks, J., Alvarez, L., Bryant, G., Waller, S., Andersen, P., Monk, P. and Lalvani, A. (2003) Comparison of T-cell-based assay with tuberculin skin test for diagnosis of Mycobacterium tuberculosis infection in a school tuberculosis outbreak. Lancet, 361, 1168-1173.
[26] Crookham, J. and Dapson, R. (1991) Hazardous chemi- cals in the histopathology laboratory, 2nd ED, Anatech.
[27] Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities ?of protein utilizing the principle of protein dye binding. Analytical Biochemistry, 72, 248-254.?
[28] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the ?head of bactriophage T4. Nature, 277, 680-685.?
[29] Towbin, M., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of ?proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and ?applications. Proceedings of the National Academy of Sciences, 76, 4350-4354.?
[30] Burnette, W.N. (1981) “Western Blotting”: Electrophoretic transfer of proteins from ?SDS-PAGE to unmodified nitrocellulose and radiographic detection with antibody ?and radioiodinated protein. Analytical Biochemistry, 112, 195-203.?
[31] Storla, D.G., Yimer, S. and Bjune, G.A. (2008) A systematic review of delay in the diagnosis and treatment of tuberculosis. BMC Public Health, 8, 15.
[32] World Health Organization (WHO) (2005) Global tuberculosis control: Surveillance, planning, ?nancing. WHO report. Geneva: World Health Organization. WHO/HTM/ TB/2005.349.
[33] Bjune, G. (2005) Tuberculosis in the 21st century: An emerging pandemic? Norsk Epidemiologi, 15(2), 133-139.
[34] Bothamley, G.H. (1995) Serological diagnosis of tuber- culosis. European Respiratory Journal (Supplement 20), 676s-688s.
[35] Lodha, R. and Kabra, S.K. (2004) Newer diagnostic modalities for tuberculosis. Indian Journal of Pediatrics, 71(3), 221- 227.Links.
[36] Orme, I.M. (2006) Preclinical testing of new vaccines for tuberculosis: A comprehensive review. Vaccine, 9;24(1), 2-19.
[37] Pollock, J.M. and Andersen, P. (1997) The potential of the ESAT-6 antigen secreted by virulent mycobacteria for specific diagnosis of tuberculosis. Journal of Infectious Disease, 175, 1251-1254.
[38] Harboe, M., Oettinger T., Wiker H.G., Rosenkrands I. and Andersen P. (1996) Evidence for occurrence of the ESAT-6 protein in Mycobacterium tuberculosis and virulent Mycobacterium bovis and for its absence in Mycobacterium bovis BCG. Infection and Immunity, 64, 16-22.
[39] Geluk, A., van Meijgaarden, K.E., Franken, K.L., Subronto, Y.W., Wieles, B., Arend, S.M., Sampaio, E.P., de Boer, T., Faber, W.R., Naafs, B. and Ottenhoff, T.H. (2002) Identification and characterization of the ESAT-6 homologue of Mycobacterium leprae and T-cell cross-reactivity with Mycobacterium tuberculosis. Infection and Immunity, 70, 2544-2548.
[40] Andersen, P., Andersen, A.B., Sorensen, A.L. and Nagai, S. (1995) Recall of long-lived immunity to Mycobacterium tuberculosis infection in mice. Journal of Immunology, 154, 3359-3372.
[41] Brandt, L., Oettinger, T., Holm, A. and Andersen, P. (1996) Key epitopes on the ESAT-6 antigen recognized in mice during the recall of protective immunity to Mycobacterium tuberculosis. Journal of Immunology, 157, 3527- 3533.
[42] Ulrichs, T., Munk, M. E., Mollenkopf, H., Behr-Perst, S., Colangeli, R., Gennaro, M. L. and Kaufmann, S.H. (1998) Differential T cell responses to Mycobacterium tuberculosis ESAT-6 in tuberculosis patients and healthy donors. European Journal of Immunology, 28, 3949-3958.
[43] Ravn, P., Demissie, A., Eguale, T., Wondwosson, H., Lein, D., Amoudy, H.A., Mustafa, A.S., Jensen, A.K., Holm, A., Rosenkrands, I., Oftung, F., Olobo, J., von Reyn, F. and Andersen, P. (1999) Human T cell responses to the ESAT-6 antigen from Mycobacterium tuberculosis. Journal of Infectious Disease, 179(3), 637-645.
[44] Laurens, A., Van Pinxteren, H., Ravn, P., Agger, E.M., Pollock, J. and Andersen, P. (2000) Diagnosis of tuberculosis based on the two specific antigens ESAT-6 and CFP10. Clinical and Diagnostic Laboratory Immunology, 7(2), 155-160.
[45] Lalvani, A., Pathan, A.A., Durkan, H., Wilkinson, K.A., Whelan, A., Deeks, J.J., Reece, W.H., Latif, M., Pasvol, G. and Hill, A.V. (2001a) Enhanced contact tracing and spatial tracking of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells. Lancet, 357, 2017-2021.
[46] Lalvani, A., Nagvenkar, P., Udwadia, Z., Pathan, A.A., Wilkinson, K.A., Shastri, J.S., Ewer, K., Hill, A.V., Mehta, A. and Rodrigues, C. (2001b) Enumeration of T cells specific for RD1-encoded antigens suggests a high prevalence of latent Mycobacterium tuberculosis infection in healthy urban Indians. Journal of Bacteriology, 183, 469-477.
[47] Lalvani, A., Pathan, A.A., McShane, H., Wilkinson, R.J., Latif, M., Conlon, C.P., Pasvol, G. and Hill, A.V. (2001c) Rapid detection of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells. American Journal of Respiratory and Critical Care Medicine, 163, 824-828.
[48] Pathan, A.A., Wilkinson, K.A., Klenerman, P., McShane, H., Davidson, R.N., Pasvol, G., Hill, A.V. and Lalvani, A. (2001) Direct ex vivo analysis of antigen-specific IFN-γ-secreting CD4 T cells in Mycobacterium tuberculosis-infected individuals: Association with clinical disease state and effect of treatment. Journal of Immunology, 167, 5217-5225.
[49] Chapman, A.L., Munkanta, M., Wilkinson, K.A., Pathan, A.A., Ewer, K., Ayles, H., Reece, W.H., Mwinga, A., Godfrey-Faussett, P. and Lalvani, A. (2002) Rapid detection of active and latent tuberculosis infection in HIV-positive individuals by enumeration of Mycobacterium tuberculosis-specific T cells. AIDS, 16, 2285-2293.
[50] Vincenti, D., Carrara, S., De Mori, P., Pucillo, L.P., Petrosillo, N., Palmieri, F., Armignacco, O., Ippolito, G., Girardi, E., Amicosante, M. and Goletti1, D. (2003) Identification of early secretory antigen target-6 epitopes for the immunodiagnosis of active tuberculosis. Molecular Mmedicine, 9(3), 105-111.
[51] Lyashchenko, K., Colangeli, R., Houde, M., Al Jahdali, H., Menzies, D. and Gennaro, M.L. (1998) Heterogeneous antibody responses in tuberculosis. Infection and Immunology, 66(8), 3936-3940.
[52] Teixeira, H.C., Abramo, C. and Munk, M.E. (2007) Immunological diagnosis of tuberculosis: Problems and strategies for success. Jornal Brasileiro de Pneumologia, 33(3), 323-334.

comments powered by Disqus

Copyright © 2018 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.