Tuberculosis is the leading cause of death among infectious diseases worldwide. Detection of Mycobacterium tuberculosis (Mtb), using routine culture-based methods is time consuming resulting in delayed diagnosis and poor treatment outcomes. Currently available molecular tests provide faster diagnosis but are able to screen only limited hot-spot mutations. Whole genome sequencing from direct sputum offers a potential solution, however, due to the presence of other microbes and host DNA its use in diagnostic testing remains challenging. In this study, we present a targeted Mtb-enrichment assay for lineage-4 coupled with an improved analysis pipeline that uses 1657 bacterial taxa as background for reducing non-Mtb genome from sputum DNA. This method drastically improved the recovery of Mtb DNA from sputum (Mtb alignment increased from 3% to >65%) as compared to non-enrichment-based sequencing. We obtained >99% Mtb genome coverage as compared to 49% in non-enriched sputum sequencing. We were able to identify Mtb positive samples from controls with 100% accuracy using Mpt64 gene coverage. Our method not only achieved 100% sensitivity to resistance variants profiled by line probe assay (LPA), but also outperformed LPA in determining drug resistance based on phenotypic drug susceptibility tests for 6 anti-tuberculosis drugs (accuracy of 97.7% and 92.8% by enriched WGS and LPA, respectively).

中文翻译：

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全基因组富集方法通过直接痰测序快速检测结核分枝杆菌和耐药相关突变

结核病是全世界传染病的主要死因。使用基于培养的常规方法检测结核分枝杆菌 (Mtb) 非常耗时，导致诊断延迟和治疗效果不佳。目前可用的分子测试提供更快的诊断，但只能筛选有限的热点突变。来自直接痰液的全基因组测序提供了一种潜在的解决方案，然而，由于存在其他微生物和宿主 DNA，其在诊断测试中的应用仍然具有挑战性。在这项研究中，我们提出了一种针对谱系 4 的靶向 Mtb 富集测定，以及改进的分析管道，该分析管道使用 1657 个细菌分类群作为背景，从痰 DNA 中减少非 Mtb 基因组。这种方法极大地提高了痰中 Mtb DNA 的回收率（Mtb 比对从 3% 增加到 > 65%) 与非基于富集的测序相比。我们获得了 >99% 的 Mtb 基因组覆盖率，而在非富集痰测序中为 49%。我们能够使用 Mpt64 基因覆盖率以 100% 的准确度从对照中鉴定 Mtb 阳性样本。我们的方法不仅对线性探针分析 (LPA) 分析的耐药变异实现了 100% 的敏感性，而且在基于 6 种抗结核药物的表型药物敏感性测试确定耐药性方面也优于 LPA（通过富集的准确度分别为 97.7% 和 92.8%）。分别为 WGS 和 LPA）。