Quantifiable stool-based TB PCR to Improve Diagnostics and Treatment Monitoring

项目总结 结核分枝杆菌(Mtb)是世界上最大的传染病杀手，导致180万人死亡和10.4人死亡 每年新增病例达百万例。尽管最近取得了进展，但40%的病例因诊断缺陷而被遗漏。 最严重的是儿童和艾滋病毒携带者(PLWH)，他们的治疗延误导致结果不佳。 目前的结核病诊断测试对儿童和PLWH缺乏敏感性。在结核病高发人群中，培养通常是无法获得的- 负担设置，结果有很长的延迟，并且对治疗失败和复发的预测很差。Xpert Ultra(超级) 结果在大约2小时内得到结果，但在涂片阴性、培养阳性的成年人中，超敏感度为63%，在 儿童结核病，并依赖于难以采集的呼吸道标本。认识到这些局限性，我们开发了 一种基于粪便的可定量聚合酶链式反应(QPCR)，其初步评估显示：i)检测限相当于 每50毫克粪便96个菌落形成单位的培养，ii)相当于以痰为基础的Xpert的敏感性，以及iii)20- 在Xpert和培养阴性的临床结核病患者中，产量增加了30%。我们现在准备好了 在成人、青少年和感染和不感染艾滋病毒的儿童中验证我们的发现 在我们在斯威士兰、坦桑尼亚和莫桑比克的网站学习。 结核病的治疗反应通过症状缓解、放射学改善、连续显微镜和 文化。由于45%的艾滋病毒相关性结核病和80%的儿童结核病是涂片阴性的，在这些关键地区进行治疗监测 人群仅限于对症状不太敏感的评估。我们的试点工作表明，坚持不懈 在结核病治疗2个月后通过定量聚合酶链式反应检测结核分枝杆菌与增加3倍的患病几率有关。 治疗失败。我们现在已经做好了评估qPCR和 区分治疗失败的参与者和将实现无复发治愈的参与者。 通过最大限度地减少适当治疗的时间来优化结核病的治疗结果。可用的药物 药敏试验(Xpert和Line Probe Assay)因依赖涂片阳性呼吸道而受到限制 样本和测试放置在中心实验室。粪便基因药敏试验(DST) 可为临床医生提供数据，以指导适当的结核病治疗，特别是在痰涂片阴性的情况下 患者，并避免耐药性的发展。在我们基于凳子的平台上，我们将评估 从粪便中提取DNA完成LPA的可行性和检测性能。 目前的痰收集技术很难被患者、父母和医护人员接受。 任何诊断性测试的影响都受到实施的可行性和可接受性的影响。至 全面评估我们基于凳子的平台的潜在影响，我们将收集以下关键数据 可操作性、可接受性和认知度。 快速、负担得起的检测：1)准确诊断结核病；2)有力地识别抗药性；3)指导 利用可获得的非呼吸道样本进行治疗可能会使结核病的护理和控制发生革命性变化。

PROJECT SUMMARY Mycobacterium tuberculosis (Mtb), the world’s leading infectious killer, results in 1.8 million deaths and 10.4 million new cases annually. Despite recent advancements, >40% of cases are missed with diagnostic gaps greatest in children and people living with HIV (PLWH) in whom treatment delay contributes to poor outcomes. Current TB diagnostic tests lack sensitivity in children and PLWH. Culture is often inaccessible in TB high- burden settings, has a long delay to result, and poorly predicts treatment failure and relapse. Xpert Ultra (Ultra) yields results in ~2 hours, but Ultra sensitivity is 63% in smear-negative, culture-positive adults and 67% in child TB, and relies upon difficult to collect respiratory specimens. Recognizing these limitations, we developed a stool based quantifiable PCR (qPCR) whose initial evaluations show i) a limit of detection equivalent to culture at 96 colony forming units per 50mg of stool, ii) sensitivity equivalent to sputum-based Xpert, and iii) 20- 30% increased yield amongst Xpert and culture-negative individuals with clinical TB. We are now poised to validate our findings in adults, adolescents, and children with and without HIV-infection in a multi-centered study at our sites in Swaziland, Tanzania and Mozambique. TB treatment response is monitored by symptom resolution, radiologic improvement, serial microscopy and culture. As 45% of HIV-associated and 80% of child TB is smear-negative, treatment monitoring in these key populations is limited to poorly sensitive symptomatic evaluation. Our pilot work demonstrates that persistent detection of Mtb by qPCR after 2 months of TB treatment was associated with 3-fold increased odds of treatment failure. We are now positioned to assess the treatment monitoring potential of qPCR and discriminate between participants who will fail treatment and participants who will achieve relapse free cure. TB treatment outcomes are optimized by minimizing time to appropriate treatment. Available drug susceptibility tests (Xpert and Line Probe Assay) are limited by reliance on smear-positive respiratory specimens and test placement at central laboratories. Stool-based genotypic drug susceptibility testing (DST) could provide clinicians with data to guide appropriate TB treatment, particularly in sputum smear negative patients, and avert the development of drug resistance. Building on our stool-based platform, we will assess the feasibility and test performance of LPA completed on DNA isolated from stool. Current sputum collection techniques are poorly accepted by patients, parents and health care workers. The impact of any diagnostic test is influenced by feasibility of implementation and acceptability. To comprehensively assess the potential impact of our stool-based platform, we will gather critical data regarding operational characteristics, acceptability and perceptions. Rapid, affordable tests that i) accurately diagnose TB, ii) robustly identify drug-resistance, and iii) guide treatment from accessible non-respiratory specimens could revolutionize TB care and control.