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. 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 (<15mm if BCG vaccinated, <6mm if unvaccinated); and IGRA (as described above). Experimental variables : Patient group: Active PTB; Latent TB, Healthy controls (BCG vaccinated and unvaccinated). Ethnicity - a wide range of ethnic groups is represented. The active PTB group incorporates a range of smear positive and smear negative disease and a spectrum of disease extent/severity. Experimental methods: Whole blood was collected into Tempus tubes (Applied Biosystems, Foster City, CA, USA) and stored between -20degrees Celsius and -80 degrees Celsius before RNA extraction. For the training set cohort, and the active TB patients baseline samples in the longitudinal cohort, total RNA was isolated from whole blood using the PerfectPure RNA Blood kit (5 PRIME Inc, Gaithersburg, MD, USA). For the separated cell samples, total RNA was isolated using the Qiagen RNeasy Mini Kit. For all other cohorts Total RNA was isolated from whole blood using the MagMAX 96 well RNA isolation kit (Applied Biosystems, Foster City, CA, USA). Isolated total RNA was then globin reduced using the GLOBINclear 96-well format kit (Ambion, Austin, TX, USA) according to the manufacturer's instructions. Total and globin-reduced RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA). Biotinylated, amplified RNA targets (cRNA) were then prepared from the globin-reduced RNA using the Illumina CustomPrep RNA amplification kit (Ambion, Austin, TX, USA). Labeled cRNA was hybridized overnight to Sentrix HT12 V3 BeadChip arrays (>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.

本数据集系列整合了多组数据集（训练集、测试集、验证集、纵向队列数据集、分离细胞亚群数据集），用于识别并表征活动性结核病（TB）患者特有的转录特征，该特征区别于潜伏性结核患者与健康对照人群。

训练集数据集用于在伦敦地区、覆盖多族裔人群中，识别活动性结核患者的全血转录特征。该特征随后在另一组同样招募自伦敦的独立患者队列（测试集）中得到验证，并进一步在南非开普敦招募的另一独立队列（验证集）中得以确认，以证实所定义的特征在高发病区域（南非开普敦）与中等发病区域（英国伦敦）中均存在。

纵向队列数据集用于探究成功的结核病治疗如何改变该转录特征。分离细胞亚群数据集则比对纯化细胞亚群（中性粒细胞、单核细胞与T细胞）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方式。

此类研究最终或可助力改善活动性肺结核的诊断现状——当前活动性结核诊断通常依赖结核分枝杆菌培养，该过程耗时可达6周，且有时无法从痰液中分离到病原菌，需借助支气管肺泡灌洗（BAL）等有创技术。另有30%的病例无法从痰液或BAL样本中培养出病原菌。理想的诊断工具需在多族裔人群中均具备有效性，且可在高、低发病国家中通用。

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质性或异质性转录特征，因为当前现有检测手段（结核菌素皮肤试验（TST），或血液细胞针对结核分枝杆菌抗原产生γ干扰素的IGRA检测）无法区分以下几种情况：结核分枝杆菌已被清除、病原菌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，是开发可识别活动性结核高风险人群的诊断工具的重要一步，从而可开展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，则可通过该特征判断后者的发病风险。

活动性结核患者全血及细胞亚群的转录特征，还可用于揭示导致免疫病理发生的相关因素，从而有望确定治疗靶点。潜伏性结核患者的转录谱或可提供控制感染的保护性因素相关信息，这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，可助力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的转录特征可能反映多种疾病引发的共性炎症应答，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性及炎症性疾病患者的转录谱进行比对，以识别结核特异性的转录特征。所得特征随后在7组独立数据集的标准化对照样本中进行测试：包括结核数据集（训练集与验证集）、葡萄球菌感染、A组链球菌感染、斯蒂尔病，以及成人与儿童系统性红斑狼疮（SLE）。

本超级数据集由以下子数据集构成。

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而确诊。

潜伏性结核（Latent TB, LTB）：所有患者均在结核病门诊筛查，要么为来自结核流行国家的英国新入境人员，要么为传染性病例的家庭接触者；而南非招募的验证集队列中的患者，则均为高发病国家的居民。所有英国患者的结核菌素皮肤试验结果均为阳性（接种卡介苗者硬结直径>14mm，未接种者>5mm），且γ干扰素释放试验（IGRA，具体为Quantiferon Gold In-Tube检测试剂盒，澳大利亚Cellestis公司产品）结果亦为阳性。南非的潜伏性结核患者均通过γ干扰素释放试验（IGRA，具体为Quantiferon Gold In-Tube检测试剂盒）确认阳性。潜伏性结核患者无活动性感染的临床、影像学或微生物学证据，且无相关症状。

健康对照：为无结核暴露史的志愿者，结核菌素皮肤试验结果均为阴性（接种卡介苗者硬结直径<15mm，未接种者<6mm），且IGRA检测结果亦为阴性（检测方法同上）。

实验变量：患者分组：活动性肺结核、潜伏性结核、健康对照（包括卡介苗接种与未接种者）。族裔：覆盖广泛的族裔群体。活动性肺结核组纳入了痰涂片阳性与痰涂片阴性的病例，且涵盖不同疾病程度与严重程度的谱系。

实验方法：全血样本采集至Tempus管（美国应用生物系统公司，加州福斯特城），在-20℃至-80℃条件下保存，随后进行RNA提取。对于训练集队列及纵向队列中的活动性结核患者基线样本，采用PerfectPure RNA Blood试剂盒（美国5 PRIME公司，马里兰州盖瑟斯堡）从全血中分离总RNA。对于分离细胞亚群样本，采用Qiagen RNeasy Mini试剂盒分离总RNA。其余所有队列的总RNA均采用MagMAX 96孔板RNA分离试剂盒（美国应用生物系统公司，加州福斯特城）从全血中分离。

分离得到的总RNA随后按照制造商说明书，采用GLOBINclear 96孔板试剂盒（美国Ambion公司，德克萨斯州奥斯汀）进行珠蛋白RNA去除处理。

总RNA及经珠蛋白去除处理的RNA的完整性，采用Agilent 2100生物分析仪（美国Agilent公司，加州帕洛阿尔托）进行评估。

采用Illumina CustomPrep RNA扩增试剂盒（美国Ambion公司，德克萨斯州奥斯汀），从经珠蛋白去除处理的RNA制备生物素标记的扩增RNA靶标（cRNA）。

将标记后的cRNA与Sentrix HT12 V3 BeadChip芯片（>48000个探针，美国Illumina公司，加州圣地亚哥）进行过夜杂交，随后按照制造商操作流程，在Illumina BeadStation 500上进行洗涤、封闭、染色与扫描。

采用Illumina BeadStudio v2软件从扫描结果中生成信号强度值，扣除背景信号，并将每张微阵列芯片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）。该标准化后的数据用于所有后续数据分析。