Blood Transcriptional Profiles in Human Active and Latent Tuberculosis

This series regroups different datasets (training set, test set, validation set, longitudinal set, separated cell set) to identify and characterise a specific transcriptional signature for patients with active TB, distinct from patients with latent TB and healthy controls. The training set dataset was used to identify a whole blood transcriptional signature for active TB patients in London, across a range of ethnicity. This signature was then validated in an independent cohort of patients, also recruited in London (the test set), and then further confirmed in an additional independent cohort recruited in Cape Town, South Africa (validation set), in order to confirm that the defined signature was present in both high (Cape Town, South Africa) and medium incidence regions (London, UK). The longitudinal dataset was then used to explore how successful TB treatment modifies this transcriptional signature. The separated cell set compares the transcriptional profiles in purified cell subsets (neutrophils, monocytes and T cells) to assess which cell types are contributing to the whole blood signature, and in what way. These studies may ultimately help to improve the diagnosis of active tuberculosis which normally relies on culture of the bacilli, which can take up to 6 weeks, and sometimes the bacilli cannot be obtained from sputum thus requiring invasive techniques such as bronchoalveolar lavage (BAL). In some cases (30%) the bacill cannot be grown from sputum or BAL. Any diagnostic tool would need to be valid across a range of ethnicities, and be valid in both high and low incidence countries. A further aim was to determine whether latent TB patients have a distinct homogeneous or heterogeneous signature, since it is not currently possible to determine using present tests (Tuberculin skin test - TST - or MTb antigen responsiveness of blood cells to produce IFN-gamma - IGRA assay) whether the mycobacteria have been cleared, are still present but are controlled by an active immune response, or to predict which patients will develop active TB. Defining heterogeneity in the latent TB patients would be an important step in developing diagnostics which could detect those most at risk of developing active TB, and thus enable targeted preventive therapy. The latter situation may be determined if Latent patients have a blood transcriptional signature similar to that in Active patients. The transcriptional signature in whole blood and cell subsets from Active TB patients may also provide information as to the factors leading to immunopathogenesis, thus possibly identifying therapeutic targets. The transcriptional profile in latent TB may give information regarding protective factors controlling the infection, important for vaccine development. Finally, definition of a transcriptional signature which responds to therapy could facilitate the development of surrogate biomarkers for drug or vaccine studies. Since any active TB signature may reflect common inflammatory responses evoked during many diseases, we also performed analysis of significance, comparing transcriptional profiles from patients with TB to those from patients with other bacterial and inflammatory diseases to identify a TB specific signature. The resulting signature was then tested against patients normalized to their own controls from 7 independent datasets: TB (Training and Validation Sets), Staphylococcus infection, Group A Streptococcus infection, Still's disease, and adult and pediatric SLE. This SuperSeries is composed of the SubSeries listed below. Overall design: Active Pulmonary TB: PTB - All patients were confirmed by isolation of Mycobacterium Tuberculosis on culture of sputum or bronchoalvelolar lavage fluid. Latent TB: LTB - All patients were screened at a tuberculosis clinic, being either new entrants to the UK from endemic countries or being household contacts of infectious cases, or in the case of the validation set recruited in South Africa, were residents of a high incidence country. All UK patients were positive by tuberculin skin test (>14mm if BCG vaccinated, >5mm if not vaccinated) and were also positive by Interferon-Gamma Release assay(IGRA); specifically Quantiferon Gold In-Tube Assay (Cellestis, Australia). The South African latent TB patients were all positive by Interferon-Gamma Release assay (IGRA); specifically Quantiferon Gold In-Tube Assay. Latent patients had no clinical, radiological or microbiological evidence of active infection and were asymptomatic. Healthy controls - these were volunteers without exposure to TB who were negative by both tuberculin skin test (48,000 probes, Illumina Inc, San Diego, CA, USA), washed, blocked, stained and scanned on an Illumina BeadStation 500 following the manufacturer's protocols. Illumina's BeadStudio version 2 software was used to generate signal intensity values from the scans, substract background, and scale each microarray to the median average intensity for all samples (per-chip normalisation). This normalised data was used for all subsequent data analysis.

本超级数据集系列整合了多组独立数据集（训练集（training set）、测试集（test set）、验证集（validation set）、纵向数据集（longitudinal dataset）、分离细胞亚群数据集（separated cell set）），旨在识别并表征活动性结核病（active TB）患者特异性转录特征（transcriptional signature），该特征可与潜伏性结核（latent TB）患者及健康对照（healthy controls）相区分。
训练集用于识别伦敦地区不同种族背景下活动性结核患者的全血（whole blood）转录特征。该特征随后在另一组同样招募自伦敦的独立患者队列（cohort）（测试集）中得到验证，并进一步在南非开普敦（Cape Town, South Africa）招募的额外独立队列（验证集）中确认，以验证所定义的特征在高发病区（南非开普敦）与中等发病区（英国伦敦）均存在。
纵向数据集用于探究成功的结核治疗如何改变该转录特征。分离细胞亚群数据集则对比纯化细胞亚群（中性粒细胞（neutrophils）、单核细胞（monocytes）及T细胞（T cells））的转录谱，以明确哪些细胞类型参与构成全血转录特征及其具体作用机制。
当前活动性结核病的诊断通常依赖于分枝杆菌（mycobacteria）培养，耗时可达6周，且部分患者无法从痰液（sputum）中分离到致病菌，需借助支气管肺泡灌洗（bronchoalveolar lavage, BAL）等侵入性操作，甚至有30%的病例无法通过痰液或BAL培养获得致病菌。本系列研究有望助力改善活动性结核的诊断现状。理想的诊断工具需适用于不同种族人群，且在高、低发病国家均具备有效性。
本研究的另一目标是明确潜伏性结核患者是否存在独特的均一化或异质性转录特征。当前现有检测手段（如结核菌素皮肤试验（Tuberculin skin test, TST）或血液细胞针对分枝杆菌抗原产生γ干扰素的γ干扰素释放试验（Interferon-Gamma Release assay, IGRA assay））无法区分以下三种情况：分枝杆菌已被清除、仍存在但被活跃免疫应答所控制，或预测哪些患者会进展为活动性结核。明确潜伏性结核患者的转录异质性，将有助于开发可识别活动性结核高风险人群的诊断工具，从而实现针对性的预防性治疗。若潜伏性结核患者的血液转录特征与活动性结核患者相似，则可进一步验证上述假设。
活动性结核患者全血及细胞亚群的转录特征，还可为阐明免疫致病机制提供线索，进而助力治疗靶点的筛选。潜伏性结核的转录谱则可揭示控制感染的保护性因素，对疫苗开发具有重要价值。此外，明确可响应治疗的转录特征，将有助于开发用于药物或疫苗研究的替代生物标志物。
由于任何活动性结核相关转录特征可能反映多种疾病引发的共同炎症应答，本研究还开展了显著性分析：将结核患者的转录谱与其他细菌感染及炎症性疾病患者的转录谱进行对比，以筛选结核特异性转录特征。所得特征随后通过7组独立数据集的自身对照标准化样本进行验证，这些数据集包括：结核数据集（训练集与验证集）、葡萄球菌感染数据集、A组链球菌感染数据集、斯蒂尔病数据集，以及成人与儿童系统性红斑狼疮（SLE）数据集。
本超级数据集系列由以下子数据集系列构成。整体实验设计：
1. 活动性肺结核（Active Pulmonary TB, PTB）：所有患者均经痰液或支气管肺泡灌洗液培养分离到结核分枝杆菌（Mycobacterium Tuberculosis）得以确诊。
2. 潜伏性结核（Latent TB, LTB）：所有患者均在结核病门诊筛查，招募来源包括来自结核病流行国家的英国新入境人员、传染性病例的家庭密切接触者；南非招募的验证集队列则均为高发病区居民。所有英国籍患者的结核菌素皮肤试验结果均为阳性（接种卡介苗（BCG）者硬结直径>14mm，未接种者>5mm），且γ干扰素释放试验（IGRA assay）结果亦为阳性，具体采用澳大利亚Cellestis公司的定量结核菌素金标试管试验（Quantiferon Gold In-Tube Assay）。南非籍潜伏性结核患者的IGRA检测结果均为阳性，同样采用上述定量试验。所有潜伏性结核患者均无临床、影像学或微生物学证据提示活动性感染，且无相关症状。
3. 健康对照（Healthy controls）：招募无结核暴露史的志愿者，其结核菌素皮肤试验结果均为阴性。
本研究采用Illumina公司（美国加利福尼亚州圣地亚哥）的包含48000个探针的微阵列（microarray）芯片，按照厂商说明书进行样本洗涤、封闭、染色及扫描，扫描操作在Illumina BeadStation 500平台完成。使用Illumina BeadStudio version 2软件从扫描图像中生成信号强度值，扣除背景噪音，并将每个微阵列的信号强度标准化至所有样本的中位数平均强度（芯片内标准化（per-chip normalisation））。经上述标准化处理的数据用于后续所有数据分析。