Rapid Diagnosis of Early TB in HIV+ Patients

描述（由申请人提供）：印度的结核病诊断依赖于临床症状、痰涂片的显微镜检查和/或胸部x光片（CXR）。HIV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）结核病和肺外结核（EPTB）的发病率增加，因此结核病的微生物学确认存在问题。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，导致结核病的诊断严重不足，而结核病的进展迅速，往往是致命的。一种简单、快速的检测方法可以识别HIV+患者的痰涂阳（SP）和SN症状或无症状结核病，这将对结核病相关的发病率和死亡率产生重大影响。已经确定了结核分枝杆菌的五种免疫原性蛋白，它们是设计一种简单的HIV+患者结核病诊断测试的潜在候选者。这些是MS， MPT51, PPE55， ESAT6和CFP10。这些抗原的抗体（Abs）已被证明存在于所有水平的免疫抑制中，无论HIV+TB+患者的肺部表现和结核病部位如何，并且在随后2-6个月内进展为症状性结核病的无症状HIV+患者的血清中也可检测到。在最终未发展为结核病的CD4 T细胞匹配的HIV+患者的回顾性血清中缺乏针对这些抗原的抗体，这表明在HIV+患者中，无论是否存在结核病临床症状，在细菌负荷达到直接显微镜检测的阈值之前，这些抗体反应都是活动性结核病的信号。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有症状/无症状的HIV+TB+患者的潜力，并绘制它们在印度患者中被抗体识别的显性表位。为此，我们将招募约200名ART-na HIV+患者，并使用直接显微镜以及去污/浓缩痰涂片、固体培养基培养和痰、血和尿NAAT评估对他们进行结核病调查（目标1）；评估来自招募个体的血清标本中重组纯化MS、MPT51、ESAT6、CFP10和PPE55抗体的存在，并将抗体的存在与结核病的细菌学证实联系起来（目的2），并在肽芯片上鉴定来自印度患者的血清抗体识别的5种抗原上的免疫优势表位，以确定可用于设计基于肽的印度HIV+患者结核病快速检测的肽。

DESCRIPTION (provided by applicant): 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-na¿ve 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. PUBLIC HEALTH RELEVANCE: 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 lack of appropriate diagnostic tools results in identification of less than half of the HIV+TB+ patients being diagnosed before death in TB-endemic countries. More sensitive diagnostic tests like microscopy with decontaminated and concentrated smears, culture of M. tb and PCR-based nucleic acid-amplification tests (NAAT) significantly enhance the identification of SN patients. However, routine implementation of these advanced diagnostic techniques is not economically feasible in high-burden countries. Studies over the last 15 years have led to the identification, cloning, and evaluation of a panel of well-defined highly immunogenic proteins of M. tb that are potential candidates for devising a simple diagnostic test for TB in HIV+ patients whether or not clinical symptoms of TB are present and before the bacterial burden reaches the threshold of detection by direct microscopy. The goal of the current studies is to evaluate the ability of Abs to these antigens to detect SN and SP symptomatic/asymptomatic HIV+TB+ patients in prospective studies. We will also map the dominant epitopes of these proteins that are recognized by Abs in Indian patients that may be used to devise a peptide-based rapid diagnostic test for HIV+TB+ patients.