Detecting pathogen and host factors on extracellular vesicles for pediatric TB diagnosis and management

摘要 结核病是儿科发病率和死亡率的主要原因(120万新发病例和22.4万例 2021年死亡)，大多数死亡发生在未开始治疗的5岁儿童中。这些孩子 通常患有播散性或肺外结核，因为他们的免疫系统可能无法遏制他们的M。 肺结核病(Mtb)感染。由于宿主反应在结核病发展中起着关键作用，因此分析 结核分枝杆菌和宿主来源的结核分枝杆菌可以提高结核病的早期诊断和管理水平。然而，结核病检测仍然是主要的 依靠来自呼吸道样本的微生物学证据，这些样本很难获得，信息也较少， 年幼的孩子。痰培养只能检测出30%-60%的儿童结核病，而用聚合酶链式反应-- 基于Xpert MTB/RIF和Xpert Ultra分析。因此，迫切需要非基于痰的检测，这种检测可以 有效诊断儿童结核病并监测其对治疗的反应。 血清生物标记物检测是诊断儿童结核病的理想方法。我们最初提案中使用的检测方法可以检测到 血清低丰度结核分枝杆菌蛋白可有效诊断儿童结核病 在等待FDA批准的验证研究之前，FDA提供的设备状态。我们已经进一步表明， 我们可以使用一种廉价的便携式设备读取的纳米颗粒增强免疫分析(NEI)来检测 结核分枝杆菌衍生因子细胞外小泡(EVS)用于结核病诊断，一种适合于资源利用的方法-- 有限的设置。我们还表明，简单的修改可以显著提高灵敏度和 再现性，以改进测量。结核分枝杆菌感染的吞噬细胞分泌的EV上的生物标记物 免疫细胞可以反映结核分枝杆菌的负担和免疫系统对其的控制，并提供关键信息。 需要改进儿科结核病的诊断，并允许快速和准确地评估有效的抗结核病 治疗。然而，很少有研究评估基于EV的结核分枝杆菌的诊断和预后表现。 没有人评估宿主来源的EV生物标记物改善检测性能的能力。 我们现在建议修改我们最初的NEI测试，将结核分枝杆菌和宿主衍生的EV标记结合在一起 以提高其在结核病诊断和治疗监测方面的性能。我们将聘用 1)我们将优化其EV生物标记物的特征 使用额外的结核分枝杆菌和宿主衍生的蛋白，我们将从结核病病例和对照的血清EV中识别。2)我们 将使用STARD指南对这一改进的NEI进行分析验证，以确定其特征。 3)建立用于结核病诊断和治疗的NEI阵列的多路性能预测模型 使用纵向血清和来自HIV感染婴儿的回溯性队列数据进行评估 纵向样本的结核病风险(IMPAACT P1041队列)。4)我们将验证NEI阵列的能力 应用纵向血清诊断LTBI和活动性肺结核并评价抗结核治疗疗效 来自多米尼加共和国O&M医院预期登记的第二批年龄较大的儿童。

ABSTRACT Tuberculosis (TB) is a major cause of pediatric morbidity and mortality (1.2 million new cases and 224,000 deaths in 2021), with most deaths occurring in children < 5 years who do not initiate treatment. These children often present with disseminated or extrapulmonary TB as their immune systems may fail to contain their M. tuberculosis (Mtb) infections in their lungs. Since host responses play critical roles in TB development, analysis of Mtb- and host-derived could improve early diagnosis and TB management. However, TB tests still primarily rely on microbiologic evidence from respiratory samples that are difficult to obtain from, and less informative for, young children. Sputum culture detects only 30-60% of pediatric TB, and similar results are obtained with PCR- based Xpert MTB/RIF and Xpert Ultra assays. There is thus an urgent need for non-sputum-based tests that can effectively diagnose pediatric TB and monitor its response to treatment. Serum biomarker assays are ideal for pediatric TB diagnosis. The assay used in our initial proposal detects low abundance Mtb proteins in serum to effectively diagnose pediatric TB and has since received breakthrough device status by the FDA prior to pending validation studies for its FDA clearance. We have further shown that we can employ a nanoparticle-enhanced immunoassay (NEI) read by an inexpensive portable device to detect Mtb-derived factors on extracellular vesicles (EVs) for TB diagnosis, an approach suitable for use in resource- limited settings. We have also shown that a simple modification can markedly increase sensitivity and reproducibility to improve measurements. Biomarkers on EVs secreted by Mtb-infected phagocytes and by immune cells may reflect Mtb burden and its containment by the immune system, and provide critical information to required improve pediatric TB diagnosis and to allow rapid and accurate assessment of effective anti-TB treatment. However, few studies have evaluated the diagnostic and prognostic performance EV-based Mtb assays, and none have evaluated the ability of host-derived EV biomarkers to improve assay performance. We now propose modify our initial NEI test to incorporate both Mtb- and host-derived EV markers associated with TB disease in order to enhance its performance for TB diagnosis and treatment monitoring. We will employ the following aims to develop and characterize this modified assay: 1) We will optimize its EV biomarker signature using additional Mtb- and host-derived proteins we will identify from serum EVs of TB cases and controls. 2) We will conduct an analytical validation of this refined NEI to establish its characteristics using STARD guidelines. 3) We will establish a prediction model of NEI array’s multiplex performance for TB diagnosis and treatment evaluation using longitudinal serum and data from a retrospective cohort of HIV-exposed infants at increased risk for TB (IMPAACT P1041 cohort) with longitudinal samples. 4) We will validate the NEI array’s ability to diagnose LTBI and active TB and evaluate the efficacy of anti-TB treatment responses using longitudinal serum from a second cohort of older children prospectively enrolled at the O&M Hospital in the Dominican Republic.