SummaryDespite the clear need to control tuberculosis, the diagnosis and prevention of this serious disease are poorly developed and have remained fundamentally unchanged for more than 50 years. Here, we introduce an innovative approach to directly identify Mycobacterium tuberculosis antigens produced in vivo in humans with tuberculosis. We combined reversed phase high performance liquid chromatography and mass spectrometry and categorize four distinct M. tuberculosis proteins produced presumably in lung lesions and excreted in the urine of patients with pulmonary tuberculosis. The genes (MT_1721, MT_1694, MT_2462 and MT_3444) coding for these proteins were cloned and the recombinant molecules were produced in Escherichia coli. The proteins were recognized by immunoglobulin G antibodies from tuberculosis patients but not from non-diseased subjects. In addition, the recombinant proteins were recognized strongly by peripheral blood mononuclear cells from healthy purified protein derivative of tuberculinpositive individuals and to a lesser extent from patients with tuberculosis. These molecules are the only proteins reported to date that are derived directly from bodily fluids of tuberculosis patients, therefore are interesting candidate antigens for the development of vaccine and/or antigen detection assay for accurate diagnosis of active tuberculosis.

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Although the antigen detection assay has the potential to discriminate active tuberculosis from latent infection, development of such a test for the accurate diagnosis of this serious disease has only recently become a matter of interest. Here we present evidence that a Mycobacterium tuberculosis protein (ornithine carboamyltransferase, coded for by MT_1694; Rv1656 [argF]) is an interesting candidate molecule for this test development. The protein was initially discovered by mass spectroscopy in urine of patients with pulmonary tuberculosis and shown by Western blot analysis to be present in M. tuberculosis crude cell extract as well as in the culture supernatant ("secreted" protein). In addition, a recombinant ornithine carboamyltransferase (rMT1694) produced in Escherichia coli was recognized by immunoglobulin G (IgG) antibodies from patients with active tuberculosis but not by IgG from uninfected healthy subjects. Moreover, rMT1694 was strongly recognized by peripheral blood mononuclear cells from both healthy tuberculin purified protein derivative (PPD)-positive individuals and patients with pulmonary tuberculosis. More importantly, a capture enzymelinked immunosorbent assay formatted with rabbit IgG antibodies specific to rMT1694 was able to identify the presence of this antigen in urine samples from 6 of 16 patients with pulmonary tuberculosis and in none of 16 urine samples collected from healthy PPD ؉ controls. These results indicate that an improved antigen detection assay based on M. tuberculosis ornithine carboamyltransferase may represent an important new strategy for the development of a specific and accurate diagnostic test for tuberculosis.Despite the existence of anti-Mycobacterium tuberculosis drugs and the widespread application of the Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine, global tuberculosis (TB) morbidity and mortality remain high and in many parts of the world are increasing due to coinfection with human immunodeficiency virus (6,17,23). It is estimated that one-third of the world's population is infected with M. tuberculosis (18) and that every year 8 million new cases of TB are diagnosed, and up to 2.5 million deaths are attributed to the disease (15). BCG, the only commercially available vaccine, has been in use since the early 1920s. However, while this vaccine protects children from disseminated TB, it does not prevent adult or pulmonary disease (2, 4), the latter being the most common and contagious form of TB. Effective treatment of TB requires multiple medications that must be used over extended periods of time and is complicated by multidrugresistant M. tuberculosis strains already affecting more than 50 million people around the world.Another limitation to control of TB is the lack of a sensitive and reliable diagnostic procedure. Diagnosis of active TB still relies primarily on the direct finding of the tubercle bacilli either in sputum smears or in culture, procedures that are operator dependent and not sensitive enough to detect more than 65 to 70% of the d...