Rapid Diagnosis of Early TB in HIV+ Patients

在印度，结核病的诊断依赖于临床症状、直接从痰和/或胸部X光(CXR)获得的涂片的显微镜检查。艾滋病毒感染引起的免疫功能障碍改变了结核病的临床和放射学表现，由于HIV+TB+患者涂片阴性(SN)和肺外结核病(EPTB)的频率增加，结核病的微生物确认存在问题。尽管现有药物完全可以治疗，但在艾滋病毒阳性患者中，不敏感的诊断测试表现较差，导致在结核病进展迅速且往往致命的人群中严重低估结核病。一种简单、快速的测试来识别HIV+患者中的涂阳(SP)和SN症状或无症状的结核病，将对结核病相关的发病率和死亡率产生重大影响。 已经确定了五种结核分枝杆菌免疫原蛋白，它们可能是设计一种简单的HIV+患者结核病诊断试验的候选者。它们是MS、MPT51、PPE55、ESAT6和CFP10。这些抗原的抗体已经在所有水平的免疫抑制中被证明，无论HIV+TB+患者的肺部表现和结核的部位，也可以在随后2-6个月发展为有症状的TB的无症状HIV+患者的血清中检测到。在CD4T细胞相合的HIV+患者中，这些抗原的追溯血清中没有针对这些抗原的抗体，这表明在HIV+患者中，这些抗体反应向活动性结核病发出信号，无论是否有结核病的临床症状，并且在细菌负荷达到直接显微镜检测的阈值之前。在目前的研究中，我们建议评估这些抗体在前瞻性研究中识别SN和SP症状/无症状HIV+TB+患者的潜力，并绘制其在印度患者中被Abs识别的优势表位。为此，我们将招募约200名初诊HIV+患者，通过直接显微镜以及去污染/浓缩痰涂片、固体培养和痰、血、尿NAAT评估来调查他们是否患有结核病(目标1)；评估从招募的受试者血清标本中是否存在重组纯化的MS、MPT51、ESAT6、CFP10和PPE55抗体，并将抗体的存在与结核病的细菌学确认相关联(目标2)，并鉴定由印度患者血清中的抗体识别的5种抗原上的主要免疫表位，以确定可用于印度HIV+患者结核病的多肽快速检测方法。

TB diagnosis in India relies on clinical symptoms, microscopic examination of smears made directly from sputum and/or chest X-rays (CXR). The immune dysfunction caused by HIV-infection alters clinical and radiological presentation of TB, and microbiological confirmation of TB is problematic since HIV+TB+ patients have increased frequency of smear-negative (SN) TB and extrapulmonary TB (EPTB). Despite being totally treatable with the existing drugs, the poorer performance of insensitive diagnostic tests in HIV+ patients leads to significant under-diagnosis of TB in the very population where TB progression is rapid and often fatal. A simple, rapid test to identify smear-positive (SP) and SN symptomatic or asymptomatic TB in HIV+ patients would have significant impact on TB-related morbidity and mortality. Five immunogenic proteins of M. tuberculosis that are potential candidates for devising a simple diagnostic test for TB in HIV+ patients have been identified. These are MS, MPT51, PPE55, ESAT6 and CFP10. Antibodies (Abs) to these antigens have been demonstrated at all levels of immune-suppression, regardless of pulmonary manifestations and site of TB in HIV+TB+ patients, and are also detectable in sera of asymptomatic HIV+ patients who progressed to symptomatic TB in the subsequent 2-6 months. Absence of Abs to these antigens in retrospective sera from CD4 T cell-matched HIV+ patients who did not eventually develop TB suggests that in HIV+ patients, these Ab responses signal active TB whether or not clinical symptoms of TB are present and before the bacterial burden reaches the threshold of detection by direct microscopy. In the current studies we propose to evaluate the potential of these Abs to identify SN and SP symptomatic/asymptomatic HIV+TB+ patients in prospective studies and to map their dominant epitopes that are recognized by Abs in Indian patients. For this, we will recruit a cohort of ~200 ART-naive HIV+ patients and investigate them for TB using direct microscopy as well as with decontaminated/concentrated sputum smears, cultures on solid media and NAAT evaluation of sputum, blood and urine (Aim 1); evaluate the presence of Abs to recombinant purified MS, MPT51, ESAT6, CFP10 and PPE55 in serum specimens from the recruited individuals and correlate the presence of Abs with bacteriological confirmation of TB (Aim 2) and identify the immunodominant epitopes on the 5 antigens recognized by serum Abs from Indian patients on peptide microarrays to define peptides that can be used to devise a peptide-based rapid test for TB in Indian HIV+ patients.