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

项目摘要 结核分枝杆菌（Mtb）是世界上主要的传染性杀手，导致180万人死亡，1040万人死亡。 每年新增病例100万例。尽管最近取得了进展，但仍有超过40%的病例因诊断缺口而被遗漏 在儿童和艾滋病毒感染者（PLWH）中最严重，延迟治疗会导致不良结果。 目前的结核病诊断测试对儿童和艾滋病毒携带者缺乏敏感性。在结核病高发地区， 负担设置，具有长时间的结果延迟，并且很难预测治疗失败和复发。Xpert Ultra（Ultra） 在约2小时内得出结果，但在涂片阴性、培养阳性的成人中，Ultra灵敏度为63%， 儿童结核病，并依赖于难以收集的呼吸道标本。认识到这些局限性，我们开发了 基于粪便的可定量PCR（qPCR），其初始评价显示i）检测限相当于 每50 mg粪便培养96个菌落形成单位，ii）灵敏度等同于基于大肠杆菌的Xpert，iii）20- Xpert和培养阴性临床TB患者的产量增加30%。我们现在准备 验证我们的研究结果，在成人，青少年，和儿童有和没有艾滋病毒感染，在一个多中心的 在我们位于斯威士兰、坦桑尼亚和莫桑比克的研究中心学习。 结核病治疗反应通过症状缓解、放射学改善、连续显微镜检查和 文化由于45%的艾滋病毒相关结核病和80%的儿童结核病是涂片阴性， 人群仅限于敏感性差的症状评估。我们的试点工作表明， 结核病治疗2个月后通过qPCR检测Mtb与结核病发生的几率增加3倍相关。 治疗失败。我们现在可以评估qPCR的治疗监测潜力， 区分治疗失败的参与者和实现无复发治愈的参与者。 通过最大限度地缩短适当治疗的时间来优化结核病治疗结果。获得的药物 敏感性检测（Xpert和Line Probe Assay）因依赖于痰涂片阳性呼吸道感染而受到限制， 中心实验室的样本和检测位置。粪便基因型药敏试验（DST） 可以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 患者，并避免耐药性的发展。基于我们基于凳子的平台，我们将评估 LPA在从粪便中分离的DNA上完成的可行性和测试性能。 目前的痰液收集技术不被患者、父母和卫生保健工作者接受。 任何诊断测试的影响都受到实施的可行性和可接受性的影响。到 为了全面评估我们基于凳子的平台的潜在影响，我们将收集有关以下方面的关键数据 操作特性、可接受性和感知。 快速、负担得起的检测，i）准确诊断结核病，ii）可靠地识别耐药性，iii）指导 利用可获得的非呼吸道标本进行治疗可能会彻底改变结核病的护理和控制。

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.