Tuberculosis Diagnostics: Towards More Precise Testing in Children

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DESCRIPTION (provided by applicant): This is a revised R03 application to develop an improved in vitro assay of cell mediated immunity to measure cytokine production for the diagnosis of tuberculosis in children. The century-old tuberculin skin test remains the standard way of diagnosis of latent tuberculosis (TB) infection, and is often used as an ancillary test in the diagnosis of clinical TB. The skin test requires two separate encounters with a health-care provider, and there is a sizable proportion of persons, particularly in disadvantaged, immigrant populations, who find it difficult to return to have their skin tests read. In addition, the test requires careful application of the test dose, and careful reading to be reliable, and is often done poorly. Clearly, better tests are needed. A whole blood test based upon release of interferon-gamma from sensitized lymphocytes stimulated with PPD antigen (the QuantiFERON-TB test, from Cellestis Ltd. South Melbourne, Australia) had been previously approved by the FDA. Recently a newer version (the QuantiFERON-TB Gold test) has also been approved, but neither test for the pediatric population. The use of the recently available, highly specific pools of ESAT-6 and CFP-10 peptides (included in the QuantiFERON-TB Gold test) which are absent from all BCG strains and from most non-tuberculosis mycobacteria (with the exception of M. kansasii, M. szulgai, M. marinum, M. leprae, M. bovis and M. africanum) should aid diagnosis of tuberculosis and eliminate the problem of cross- reactivity in individuals infected with most non-tuberculous mycobacteria or previously immunized with BCG. In the present studies we shall be using the QuantiFERON-TB Gold Test which contains as individual components the newly available pools of overlapping peptides of ESAT-6 and CFP-10. We shall also employ cocktails of the two plus TB7.7 (p4), as well as RD1- selected peptides of ESAT-6 and CFP-10 provided by Dr. D. Goletti. A set of pilot studies based upon the use of this improved test will be performed in a clinically well pediatric, mostly immigrant population to evaluate its correlation with conventional tuberculin skin testing (TST), and, on a short-term basis, to determine its ability to predict clinical TB. A particular emphasis will be placed on the younger infant-child (1 to 5 year-old) population where both the severity of disease and the need is great. Moreover, since in patients with suspected clinical TB, there may be "false-negative" skin tests due to suppressor cytokines and other mechanisms related to maturational immaturity that may obscure true cell-mediated immunity to MTB, studies will be also included to determine optimal dose- response relationships (particularly important in children) which may permit overcoming this "false- negativity" in the laboratory. Thus, these studies will explore techniques for differentiating false negative and false positive reactions, and hopefully permit more reliable In vitro testing for the determination of true cell- mediated immunity to MTB and possible differentiating latent from active TB disease.