Tuberculosis Diagnostics: Towards More Precise Testing in Children

描述（由申请人提供）：这是一份修订后的R03申请，旨在开发一种改进的细胞介导免疫体外试验，以测量用于诊断儿童结核病的细胞因子产生。具有百年历史的结核菌素皮肤试验至今仍是诊断潜伏性结核（TB）感染的标准方法，常作为临床结核诊断的辅助试验。皮肤测试需要与医疗保健提供者进行两次单独的接触，而且有相当一部分人，特别是处于不利地位的移民人口，很难返回进行皮肤测试。此外，测试需要仔细应用测试剂量，并仔细阅读才能可靠，而且往往做得不好。显然，我们需要更好的测试。一种基于PPD抗原刺激致敏淋巴细胞释放干扰素的全血测试（Cellestis Ltd.的QuantiFERON-TB测试）。南墨尔本，澳大利亚)之前已被FDA批准。最近，一个更新的版本（QuantiFERON-TB黄金测试）也被批准，但这两种测试都不是针对儿科人群的。使用最近获得的高特异性ESAT-6和CFP-10肽（包括在QuantiFERON-TB金试验中），这些肽在所有卡介苗菌株和大多数非结核分枝杆菌（除了堪萨斯分枝杆菌、苏盖分枝杆菌、海洋分枝杆菌、麻风分枝杆菌、牛分枝杆菌和非洲分枝杆菌）中都不存在，应该有助于结核病的诊断，并消除大多数非结核分枝杆菌感染或以前接种过卡介苗的个体的交叉反应性问题。在目前的研究中，我们将使用QuantiFERON-TB金测试，该测试包含ESAT-6和CFP-10新获得的重叠肽池作为单独的成分。我们还将使用两者的鸡尾酒加上TB7.7 (p4)，以及Dr. D. Goletti提供的ESAT-6和CFP-10的RD1选择肽。将在临床状况良好的儿童（主要是移民人群）中进行一组基于这种改进试验的试点研究，以评估其与传统结核菌素皮肤试验（TST）的相关性，并在短期内确定其预测临床结核病的能力。将特别强调较年幼的婴儿（1至5岁）人口，因为他们的疾病严重程度和需求都很大。此外，由于在疑似临床结核病患者中，由于抑制细胞因子和其他与成熟不成熟相关的机制，可能会出现“假阴性”皮肤试验，这可能会掩盖对MTB的真正细胞介导免疫，因此还将包括研究，以确定最佳剂量-反应关系（对儿童尤其重要），这可能允许在实验室中克服这种“假阴性”。因此，这些研究将探索区分假阴性和假阳性反应的技术，并有望允许更可靠的体外测试，以确定对MTB的真细胞介导免疫，并可能区分潜伏性结核病和活动性结核病。

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.