Nanoparticle based rapid diagnostics for TB disease

由结核分枝杆菌(M.TB)引起的结核病是全球传染病死亡的三大原因之一。全球约四分之一的人口受到感染，每年新增病例1000万例，死亡140万例。虽然结核病可以通过适当的抗生素组合来治疗，但疾病症状多种多样，很容易与其他疾病混淆，这意味着患有活动性疾病并有能力感染他人的人并不总是能被迅速识别，从而允许继续传播。快速、准确地识别这些患者是实现迅速给予必要的抗生素以防止感染者传播疾病的关键。世界卫生组织(WHO)已将检测结核病的新诊断技术确定为其结束结核病战略的研究重点之一，该战略旨在到2030年将结核病发病率降低80%。这项提案旨在开发和评估工具，以捕获和检测可能患有结核病的患者样本中的结核分枝杆菌：特别是受感染的成年人咳出的痰。我们的长期目标是创建一种能够识别结核病的快速诊断方法，并且能够用于资源匮乏的环境(即结核病负担最重、医疗基础设施有限的低收入和中等收入国家)。我们的方法结合了三项互补技术来捕获和识别结核分枝杆菌：1.产生能够选择性地与结核分枝杆菌细胞紧密结合的特定糖分子；以及它们附着在2.磁性和3.荧光颗粒上。这将创造两种能够结合结核分枝杆菌的材料--一种磁性颗粒，使细菌具有磁性，并使其能够在磁场中捕获；另一种荧光颗粒，使细菌具有荧光，能够用显微镜检测到。结合这些微粒将使我们能够捕获、浓缩和可视化患者样本中的结核分枝杆菌。我们的实验方法分为三个工作包(WPS)：WP1专注于使用一系列创新方法来合成能够与结核分枝杆菌结合的糖；WP2专注于将这些糖连接到磁性和荧光粒子上，并研究结果材料的物理性质(尺寸、形状、磁性和光学特性)；WP3侧重于评估这些材料与细菌的结合。我们将首先在实验室介质中模拟痰的成分测试与减弱的卡介苗(疫苗)结核分枝杆菌菌株的结合，并将其与这些样本中可能存在的其他细菌的结合进行比较，以确定它们对结核分枝杆菌的选择性高于其他物种。我们将把这些实验扩展到研究从患者身上分离出的结核分枝杆菌菌株。最后，我们将对可能存在结核病感染的患者的样本进行评估，将我们的结果与使用传统方法(培养培养法、痰镜检或检测结核分枝杆菌遗传物质的GeneXpert系统)对相同样本进行的平行测试进行比较。结果将决定这种新方法是否能够检测出患者样本中存在的细菌，并能够区分结核分枝杆菌和其他细菌。工作包1和2将在布里斯托尔大学(UOB)、UOB的工作包3和肯尼亚医学研究所(KEMRI)进行，所有患者来源的细菌和样本实验都将在那里进行。肯尼亚是世卫组织结核病高负担国家；KEMRI拥有评估结核病检测方法所需的专门知识和基础设施。我们的建议将新科学(开发选择性地约束结核分枝杆菌的新材料)与支持帖子、基础设施和双向交流访问相结合，这将发展肯尼亚的研究能力。我们研究团队的多样性以及KEMRI、医疗从业者和政策制定者之间的紧密联系将确保在研究界和潜在最终用户中产生影响。

Tuberculosis (TB) caused by the Mycobacterium tuberculosis (M. tb) bacterium is one the three major causes of infectious disease deaths worldwide. Approximately one quarter of the global population is infected, with annually 10 million new cases and 1.4 million deaths. While TB is treatable with suitable antibiotic combinations, disease symptoms are variable and easily confused with other conditions, meaning that individuals with active disease and capable of infecting others are not always quickly identified, allowing continued transmission. Fast, accurate identification of these patients is key to achieving the prompt antibiotic administration necessary to prevent infected individuals spreading disease. The World Health Organisation (WHO) has identified new diagnostic technologies to detect TB disease as one research focus for their End TB Strategy designed to reduce TB incidence by 80 % by 2030. This proposal aims to develop and evaluate tools to capture and detect M. tb in samples from patients with possible TB disease: specifically sputum coughed up by infected adults. The long term goal is to create a rapid diagnostic method capable of identifying TB disease and that is able to be used in resource-poor settings (i.e. the low and middle-income countries where the TB disease burden is greatest and medical infrastructure is limited).Our method combines three complementary technologies to capture and identify M. tb: 1. generation of specific sugar molecules able to bind selectively and tightly to the M. tb cell; and their attachment to 2. magnetic and 3. fluorescent particles. This will create two types of material able to bind M. tb - a magnetic particle, making the bacterium magnetic and enabling its capture in a magnetic field; and a fluorescent particle making the bacterium fluorescent and able to be detected using a microscope. Combining these particles will enable us to capture, concentrate and visualise M. tb from patient samples. Our experimental approach divides into three workpackages (WPs): WP1 focuses on using a range of innovative approaches to synthesise sugars able to bind M. tb; WP2 focuses on attaching these sugars to magnetic and fluorescent particles and investigating the physical properties (size, shape, magnetic and optical characteristics) of the resulting materials; and WP3 on evaluating the binding of these materials to bacteria. We will first test binding to the weakened BCG (vaccine) strain of M. tb in laboratory media mimicking the composition of sputum, comparing this with binding to other bacteria that may be present in these samples to establish their selectivity for M. tb over other species. We will extend these experiments to study M. tb strains isolated from patients. Finally we will evaluate our method on samples from patients presenting with possible TB infection, comparing our results with parallel tests carried out on the same samples using traditional methods (growth in culture, sputum microscopy or the GeneXpert system that detects M. tb genetic material). The results will determine whether this new approach can detect bacteria in the quantities present in patient samples and is able to discriminate between M. tb and other bacteria.Workpackages 1 and 2 will take place at the University of Bristol (UoB), workpackage 3 at UoB and the Kenya Medical Research Institute (KEMRI) where all experiments with patient-derived bacteria and samples will take place. Kenya is a WHO High Burden TB country; KEMRI has the expertise and infrastructure necessary to evaluate TB detection methods. Our proposal combines novel science (development of new materials that selectively bind M. tb) with support for posts, infrastructure and bidirectional exchange visits that will develop research capacity in Kenya. The diversity of our research team and the strong links between KEMRI, healthcare practitioners and policymakers will ensure delivery of impact in both the research community and potential end users.