Determining the persister populations in sputum during tuberculosis therapy. A supplementary study to the RIFASHORT trial.

2014年，近1000万人患上结核病，150万人死亡。尽管耐药性是一个严重的问题，但绝大多数感染都可以用标准抗生素治疗，这种抗生素对结核杆菌（Mtb）有效，导致这种毁灭性的疾病。结核病的治疗目前需要患者服用抗生素6个月。即使在此之后，疾病在高达5%的个体中复发，通常在停止治疗后一年内。开发持续时间更短、疾病复发率更低的治疗方法是全球结核病控制工作的一个主要目标。测试新的治疗方法非常昂贵，需要数百名患者，并需要多年时间。我们的项目旨在减少这些测试障碍，并缩短治疗时间。需要长期治疗和复发的频率被认为是由于大量处于改变状态的Mtb细菌在暴露于抗生素后存活;改变的细菌被称为持久性。虽然Mtb的生长细胞在常规实验室测试中被迅速杀死，但处于非生长持续状态的细胞不会。我们的团队多年来一直在研究结核病中的持续存在现象。在过去的十年中，我们在新诊断患者的痰（咳出的痰）中发现了结核分枝杆菌的特征，令人惊讶的是，这些特征表明临床实验室通常分离的细菌中存在非生长的持久性。这些特征以3种方式识别，显微镜检查检测Mtb细胞内的脂肪滴，在生长刺激剂Rpf存在下的实验室培养，以及遗传测试显示该细菌表达的基因（称为转录组）。因为所有三种都表明痰中存在非生长的结核分枝杆菌，并且因为存在的水平因患者而异，所以我们想知道这些“肥胖和懒惰”细菌是否与持久性有关。如果是这样的话，那么不同患者中具有这些特性的结核分枝杆菌的数量可能会告诉我们，他们对治疗的反应有多快，以及一旦接受治疗，持续存在的结核分枝杆菌被消除的速度有多快。在初步研究中，我们观察了结核病患者中脂肪结核分枝杆菌细胞的频率，并将这些与患者对治疗的反应联系起来。虽然数量很少，但我们发现脂肪结核杆菌数量较多的患者反应较慢。然后，与利物浦和马拉维的同事一起，我们询问痰液样本中的脂肪Mtb细胞计数是否可以告诉我们哪些患者尽管感染了抗生素敏感的细菌，但结核病治疗失败。当我们比较了9名治疗失败和29名治愈患者的脂肪结核分枝杆菌水平时，我们发现，失败者平均有两倍的这些“持久性样”细胞的频率。在这里，我们的目标是通过研究我们的测试预测治疗失败的能力，为测试缩短结核病治疗的临床试验增加价值。RIFASHORT TB治疗试验为我们提供了一个特殊的机会，可以测试足够的痰液样本的这些特性，以实现3个目标：1）重新测试一个月时计数脂肪Mtb细胞的预测能力; 2）确定痰液样本中脂肪、Rpf和转录组特征之间的相互关系;以及3）确定在治疗的前2个月内应用的哪种测试与治疗失败最密切相关。本研究的结果将具有明确的潜力，1）能够快速评估新的抗结核治疗方法，包括有效对抗耐药结核分枝杆菌的新药，以及2）确定哪些患者可以在短至3个月的时间内安全地停止治疗。如果我们能够在2个月而不是18个月内可靠地比较新的治疗方法，我们将能够测试更多的替代方案，并以比目前所需的少得多的资源选择最佳方案。如果我们能够缩短结核病治疗时间，将挽救许多生命，减轻许多痛苦。

Nearly ten million people developed TB in 2014 and 1.5 million died. Although drug resistance is a serious problem, the vast majority of infections are treatable with standard antibiotics effective against the TB bacterium, known as Mtb, which causes this devastating disease. Treatment of TB currently requires patients to take antibiotics for 6 months. Even after this the disease returns in up to 5% of individuals, usually within a year after stopping therapy. Developing treatments with shorter duration and lower disease return (or relapse) rates is a major goal of global TB control efforts. Testing new treatments is very expensive, requires hundreds of patients and takes many years. Our project aims to reduce these barriers to testing and enable shortened treatment. The need for prolonged therapy and the frequency of relapse are thought to be due to significant numbers of Mtb bacteria in an altered state that survive exposure to antibiotics; the altered bacteria are known as persisters. While growing cells of Mtb are rapidly killed in conventional lab tests, cells in the non-growing persister state are not. Our team has been studying the persister phenomenon in TB for many years. In the past decade we discovered features of Mtb in the sputum (coughed up phlegm) of newly diagnosed patients that, surprisingly, indicated the presence of non-growing persisters amongst the bacteria that clinical labs normally isolate. These features are recognised in 3 ways, microscopy to detect fatty droplets inside the Mtb cells, lab culture in the presence of a growth stimulator known as Rpf and a genetic test to show which genes the bug is expressing (known as the transcriptome). Because all three indicate the present of non-growing Mtb in sputum and because the levels present varied from patient to patient we wondered whether these "Fat and Lazy" bacteria were related to the persisters. If this is so, then the numbers of Mtb with these properties in different patients might tell us how quickly they will respond to treatment and, once on therapy, how rapidly the persisters were being eliminated.In preliminary studies we looked at the frequency of fatty Mtb cells in TB patients and related these to how patients responded to treatment. Although numbers were small, we found that patients with higher numbers of fatty Mtb responded more slowly. Then, with colleagues in Liverpool and Malawi, we asked whether counting fatty Mtb cells in sputum samples could tell us which patients were going to fail TB treatment in spite of being infected with an antibiotic sensitive bug. When we compared the levels of fatty Mtb between 9 treatment failures and 29 cured patients we found that the failures had, on average, double the frequency of these "persister-like" cells.Here we aim to add value to a clinical trial testing a shortened treatment of TB by studying the ability of our tests to predict treatment failure. The RIFASHORT TB treatment trial provides an exceptional opportunity for us to test sufficient sputum samples for these properties to achieve 3 objectives: 1) re-testing the predictive capacity of counting fatty Mtb cells at one month; 2) determining the interrelationships between the fatty, Rpf and transcriptome features in sputum samples; and 3) determining of which test, applied within the first 2 months of treatment, is most strongly linked to treatment failure.The results of this study will have clear potential to, 1) enable rapid assessment of new anti-TB treatments including new drugs effective against resistant Mtb, and 2) determine which patients might safely stop treatment after periods as short as 3 months. If we can reliably compare new treatments in 2 rather than 18 months, we will be able to test more alternatives and select the best with far less resource than currently required. If we can shorten TB treatment, many lives will be saved and much suffering alleviated.