Improving the molecular diagnosis of drug resistant tuberculosis

 描述(申请人提供)：耐多药(对异烟肼和利福平耐药)和广泛耐药(广泛耐药，对氟喹诺酮[FQ]和二线注射剂[SLI])耐药的结核病(TB)的发病率正在上升，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。通过将诊断时间从3-6周缩短到1天，快速分子检测使耐药结核病的诊断发生了革命性的变化。世界卫生组织现已批准商业试验来诊断耐多药结核病(MDR TB)。然而，这些方法未能通过基于培养的表型试验(金标准)确定相当大比例的结核分枝杆菌耐药株，原因至少有两个：1)目前的分子检测没有足够的分辨率来确定含有突变和敏感亚群(异种耐药)的耐药杆菌亚群，新数据表明这至少在FQ是常见的；2)还有其他耐药的遗传标记尚未确定。这项提案的总体目标是从根本上推进对广泛耐药前和广泛耐药结核病的分子诊断。我们将追求并行的研究目标，以补充和系统的方法来改进耐药结核病的诊断和管理。在目标1中，我们将使用菲律宾马尼拉两个领先的耐多药结核病治疗中心正在评估的MDR、Pre-XDR和XDR TB患者的存储培养分离物和临床样本，对所有与结核分枝杆菌耐药相关的已知基因区域进行有针对性的下一代深度测序。结果将被用来表征不同抗结核药物的异种耐药性的频率，以及在存在混合细菌种群的情况下，当前分子分析识别耐药性的能力。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(耐FQ和SLI但没有已知突变的菌株)和对照(FQ和SLI敏感菌株)进行下一代全基因组测序，以确定FQ和SLI抗性的新遗传标记。我们将开发预测模型，以量化在添加新的遗传标记时对FQ和SLI耐药性诊断准确性的改善，并进行实验以确定哪些新突变直接导致FQ或SLI耐药性。当拟议的分析完成后，我们将对FQ和SLI抗性表型和基因测试之间不一致的原因进行全面描述，并提供异质耐药对治疗结果影响的证据。此外，我们将生成一个新的突变列表，这些突变可以改善对FQ和SLI耐药性的预测，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.