Improving the molecular diagnosis of drug resistant tuberculosis

 描述（由申请方提供）：多重耐药（对异烟肼和利福平耐药）和广泛耐药（XDR，对氟喹诺酮类[FQ]和二线注射剂[SLI]的额外耐药）结核病（TB）的发生率正在增加，导致严重疾病和高死亡率。耐药结核病的快速诊断对于改善患者预后和防止进一步获得对其他药物的耐药性至关重要。快速分子检测通过将诊断时间从3-6周缩短到<1天，彻底改变了耐药结核病的诊断。目前，世界卫生组织已批准商业检测用于诊断耐多药结核病（MDR TB）。然而，这些方法未能鉴定出相当大比例的结核分枝杆菌分离株，这些分离株通过基于培养的表型试验显示对FQ或SLI具有抗性（黄金标准）至少有两个原因：1）目前的分子测定法不足以鉴定携带突变的耐药杆菌的小亚群与敏感亚群的混合（异源抗性），新出现的数据表明至少对于FQ是常见的，和2）存在尚未鉴定的抗性的另外的遗传标记。该提案的总体目标是从根本上推进前XDR和XDR TB的分子诊断。我们将追求平行的研究目标，这是一种补充和系统的方法，以改善耐药结核病的诊断和管理。在目标1中，我们将使用储存的培养分离株和来自MDR、pre-XDR和XDR TB患者的临床样本，对与结核分枝杆菌耐药性相关的所有已知基因区域进行靶向下一代深度测序，这些患者正在菲律宾马尼拉的两家领先MDR TB治疗中心接受评估。结果将用于表征不同抗结核药物的异质耐药频率以及当前分子检测在混合杆菌群存在下鉴定耐药性的能力。我们还将评估异源耐药对治疗结果的影响。在目标2中，我们将对病例（无已知突变的FQ和SLI耐药分离株）和对照（FQ和SLI敏感分离株）进行全基因组下一代测序，以鉴定FQ和SLI耐药的新型遗传标记。我们将开发预测模型，以量化增加新的遗传标记时FQ和SLI抗性诊断准确性的提高，并进行实验以确定哪些新突变直接导致FQ或SLI抗性。当拟定的分析完成时，我们将全面描述FQ和SLI耐药表型和基因型检测之间不一致的原因，以及异源耐药对治疗结果影响的证据。此外，我们将产生一系列新的突变，这些突变可以改善对FQ和SLI耐药的预测并直接导致FQ和SLI耐药。这些发现将有助于改进耐药结核病的诊断和管理策略。

 DESCRIPTION (provided by applicant): The incidence of multidrug-resistant (resistant to isoniazid and rifampin) and extensively drug-resistant (XDR, additional resistance to fluoroquinolones [FQ] and second-line injectables [SLI]) tuberculosis (TB) is increasing, causing serious illness with high mortality. Rapid diagnosis of drug-resistant TB is critical to improving patient outcomes and preventing further acquisition of resistance to other drugs. Rapid molecular tests have revolutionized the diagnosis of drug-resistant TB by shortening the time to diagnosis from 3-6 weeks to <1 day. Commercial tests have now been approved by the World Health Organization to diagnose multidrug resistant TB (MDR TB). However, these methods fail to identify a substantial proportion of Mycobacterium tuberculosis isolates shown to be resistant to FQ or SLI by culture-based phenotypic tests (the gold standard) for at least two reasons: 1) current molecular assays have inadequate resolution to identify small sub-populations of resistant bacilli harboring mutations mixed with susceptible sub-populations (hetero-resistance), which emerging data suggests is common at least for FQ and 2) there are additional genetic markers of resistance that have yet to be identified. The overall goal of this proposal is to fundamentally advance molecular diagnostics for pre-XDR and XDR TB. We will pursue parallel research aims that represent a complementary and systematic approach to improving the diagnosis and management of drug-resistant TB. In Aim 1, we will perform targeted next-generation deep sequencing of all known gene regions associated with Mycobacterium tuberculosis drug resistance using stored culture isolates and clinical samples from patients with MDR, pre-XDR and XDR TB being evaluated at two leading MDR TB treatment centers in Manila, Philippines. Results will be used to characterize the frequency of hetero-resistance for different anti-TB drugs and the ability of current molecular assays to identify resistance in the presence of mixed bacillary populations. We will also assess the impact of hetero-resistance on treatment outcomes. In Aim 2, we will perform whole genome next-generation sequencing in cases (FQ- and SLI-resistant isolates without a known mutation) and controls (FQ- and SLI-susceptible isolates) to identify novel genetic markers of FQ and SLI resistance. We will develop prediction models to quantify improvements in diagnostic accuracy for FQ and SLI resistance when adding the novel genetic markers and perform experiments to identify which novel mutations directly cause FQ or SLI resistance. When the proposed analyses are complete, we will have produced a comprehensive description of reasons for discordance between phenotypic and genotypic tests for resistance to FQ and SLI and evidence on the impact of hetero-resistance on treatment outcomes. In addition, we will have generated a list of novel mutations that improve prediction of and directly cause FQ and SLI resistance. The findings will contribute to improved diagnostics and management strategies for drug-resistant TB.