Tuberculosis Diagnostics: Towards More Precise Testing in Children

描述(由申请人提供)：这是一个修订的R03申请，以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的产生。已有百年历史的结核菌素皮肤试验仍然是诊断潜伏性结核病(TB)感染的标准方法，并经常被用作临床结核病诊断的辅助试验。皮试需要两次单独的与医疗保健提供者的接触，有相当大比例的人，特别是在处境不利的移民人口中，发现很难回来接受皮试。此外，测试需要仔细应用测试剂量，仔细阅读才能可靠，而且往往做得很差。显然，需要更好的测试。一种基于PPD抗原刺激的致敏淋巴细胞释放干扰素-伽马的全血测试(Quantiferon-TB测试，来自澳大利亚南墨尔本的Cellestis Ltd.)先前已被FDA批准。最近，一种较新的版本(Quantiferon-TB Gold测试)也获得了批准，但这两种测试都不适用于儿科人群。使用最近可用的高度特异的ESAT-6和CFP-10多肽池(包括在Quantiferon-TB Gold测试中)，所有卡介苗菌株和大多数非结核分枝杆菌(除堪萨斯分枝杆菌、斯祖尔盖分枝杆菌、马丁氏分枝杆菌、麻风分枝杆菌、牛分枝杆菌和非洲分枝杆菌外)都不存在ESAT-6和CFP-10多肽，这应有助于结核病的诊断，并消除感染大多数非结核分枝杆菌或以前接种过卡介苗的人的交叉反应问题。在目前的研究中，我们将使用Quantiferon-TB Gold Test，它包含新获得的ESAT-6和CFP-10重叠多肽库作为单独成分。我们还将使用两个加TB7.7(P4)的鸡尾酒，以及D.Goletti博士提供的Rd1选择的ESAT-6和CFP-10多肽。将在临床良好的儿科人群(主要是移民)中进行一系列基于使用这种改进的测试的试点研究，以评估其与传统的结核菌素皮肤测试(TST)的相关性，并在短期基础上确定其预测临床结核病的能力。将特别重视年龄较小的婴幼儿(1至5岁)人群，这些人群的疾病严重程度和需求都很大。此外，在疑似临床结核病的患者中，由于抑制细胞因子和其他与成熟不成熟有关的机制，可能会出现“假阴性”皮肤测试，这可能会掩盖对结核杆菌的真正细胞介导免疫，因此还将进行研究，以确定最佳剂量-反应关系(对儿童尤其重要)，从而可以在实验室克服这种“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，并有望实现更可靠的体外测试，以确定对结核分枝杆菌的真实细胞介导免疫，并可能区分潜伏性和活动性结核病。

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.