Diagnostic Innovations for Pediatric Tuberculosis in Bolivia

儿童结核病(TB)在低收入和中等收入国家继续构成诊断挑战。 由于少菌病对儿童的影响，以及目前的结核病，结核病发病率很高 培养和聚合酶链式反应检测由于成本、可获得性的限制以及 年幼儿童提供的样本敏感性较差。我们来自杜兰的专家团队，约翰斯 霍普金斯大学、佩鲁亚纳·卡耶塔诺·赫雷迪亚大学和ASocial ación Benéfica Prisma面临 所有这些挑战都是通过在秘鲁和玻利维亚超过25年的合作来实现的。我们的目标是直接 通过评估MPI Tony Hu开发的一种新诊断方法来应对儿童结核病的挑战 在杜兰大学使用CRISPR介导的结核病检测(CRISPR-TB)优化以检测循环 结核分枝杆菌无细胞DNA(Mtb-cfDNA)，并用于分析飞行员冷冻保存的血清 来自成人和儿童推定结核病患者的研究，他们的无症状家庭接触者，以及一群 有症状的艾滋病毒携带者(CLHIV)儿童患结核病的风险很高。来自有症状的成人队列的结果 其综合敏感性为93%，特异性为93%，阳性预测值为95%，阴性预测值为95% 价值92%。在CLHIV的有限先导研究中，CRISPR-TBD结果准确地识别了所有确诊的结核病 (13/13)和大多数儿童未确诊结核病(80%；52/65)。我们建议登记200名推定为结核病的患者 病例和两个研究人群(测试人群和 确认人群)通过与Mario Ortiz Suarez博士儿童医院有关的诊所确定 在玻利维亚的圣克鲁斯。我们将在“测试”中确定外周血中cfDNA浓度的分布。 人口“由两个年龄组(2个月至6岁、7至14岁)患有呼吸系统疾病的儿童组成 根据NIH共识病例定义按结核病可能性分组的疾病(确诊结核病，未确诊结核病 结核病和不太可能的结核病)，以及根据潜在结核病感染(LTBI)分组的年龄匹配的对照组， 在接受抗生素治疗的结核病患者中进行连续时间的cfDNA检测。我们还将验证标准 中国儿童结核病或LTBI临床亚组的cfDNA定量范围 独立验证队列。另一个目的是确定定量cfDNA和 基于肺部疾病证据的基于定量成像的结核病评分，肺部是结核病的主要靶器官 疾病，通过(1)胸片，通过使用CAD4TB v7系统的计算机辅助分析测量，以及通过 (2)肺部超声，使用便携式/低成本探头，由机器学习算法辅助进行 自动翻译。这些生物标记物将作为潜在的辅助因子进行测试，可能与 外周血中cfDNA水平，以提高对儿童结核病的检测。这项研究的结果 将是找到一条路径以检测许多“未经确认”的结核病病例的第一步， 理想情况下，使儿科结核病的诊断达到低资源环境，在那里是如此迫切需要它。

Pediatric tuberculosis (TB) continues to pose diagnostic challenges in low- and middle-income countries with high rates of TB disease, due to the well-described impact of paucibacillary disease in children, and current TB culture and polymerase-chain reaction tests are of limited usefulness due to cost, restricted availability, and poor sensitivity in specimens available from younger children. Our team of experts from Tulane, Johns Hopkins University, Universidad Peruana Cayetano Heredia, and Asociación Benéfica Prisma have confronted all of these challenges through more than 25 years of collaboration in Peru and Bolivia. Our goal is to directly address the challenges of TB in children by evaluating a new diagnostic approach developed by MPI Tony Hu at Tulane University using a CRISPR-mediated TB assay (CRISPR-TB) optimized to detect circulating Mycobacterium tuberculosis cell-free DNA (Mtb-cfDNA), and used to analyze cryopreserved serum in pilot studies from adults and children with presumptive TB, their asymptomatic household contacts, and a cohort of symptomatic children living with HIV (CLHIV) at high risk for TB. Results from symptomatic adult cohorts yielded a pooled sensitivity of 93%; specificity of 93%; positive predictive value of 95%; and negative predictive value of 92%. In limited pilot studies in CLHIV CRISPR-TBD results accurately identified all confirmed TB (13/13) and most children with unconfirmed TB (80%; 52/65). We propose to enroll 200 presumptive TB cases and an equal number of well control subjects in each of 2 study populations (test population and validation population) identified through clinics associated with the “Dr. Mario Ortiz Suarez” Children's Hospital in Santa Cruz, Bolivia. We will determine the distribution of cfDNA concentrations in peripheral blood in a “test population” composed of two age-based groups of children (2 months-6 years, 7-14 years) with respiratory disease grouped by likelihood of TB based on the NIH consensus case definitions (confirmed TB, unconfirmed TB, and unlikely TB) and in age-matched controls grouped by presence of latent TB infection (LTBI), with cfDNA measured serially in time among TB cases receiving antibiotic therapy. We will also validate standard ranges of quantitative cfDNA established for clinical subgroups of children with TB disease or LTBI in an independent validation cohort. An additional aim will determine the correlation between quantitative cfDNA and quantitative imaging-based TB scores based on evidence of disease in the lung, the primary target organ in TB disease, by (1) chest radiograph, measured by computer-aided analysis using the CAD4TB v7 system, and by (2) lung ultrasound, performed with a portable/low-cost probe assisted by machine learning algorithms for automatic interpretation. These biomarkers will be tested as potential cofactors that may be combined with cfDNA levels in peripheral blood, to improve the detection of TB disease in children. The results of this study will be the first step in a process to find a path to allow detection of the many “unconfirmed” TB cases and ideally make the diagnosis of pediatric TB in reach for low resource settings where it is so critically needed.