PDF(Pubmed)
Abstract：
UNASSIGNED: Direct whole genome sequencing (WGS) of Mycobacterium tuberculosis (Mtb) can be used as a tool to study drug resistance, mixed infections, and within-host diversity. However, WGS is challenging to obtain from clinical samples due to low number of bacilli against a high background.
UNASSIGNED: We prospectively collected 34 samples (sputum, n = 17; bronchoalveolar lavage, n = 13; and pus, n = 4) from patients with active tuberculosis (TB). Prior to DNA extraction, we used a ligand-mediated magnetic bead method to enrich Mtb from clinical samples and performed WGS on Illumina platform.
UNASSIGNED: Mtb was definitively identified based on WGS from 88.2% (30/34) of the samples, of which 35.3% (12/34) were smear negative. The overall median genome coverage was 15.2% (interquartile range [IQR], 7.7%-28.2%). There was a positive correlation between load of bacilli on smears and genome coverage (P < .001). We detected 58 genes listed in the World Health Organization mutation catalogue in each positive sample (median coverage, 85% [IQR, 61%-94%]), enabling the identification of mutations missed by routine diagnostics. Mutations causing resistance to rifampicin, isoniazid, streptomycin, and ethambutol were detected in 5 of 34 (14.7%) samples, including the rpoB S441A mutation that confers resistance to rifampicin, which is not covered by Xpert MTB/RIF.
UNASSIGNED: We demonstrate the feasibility of magnetic bead-based enrichment for culture-free WGS of Mtb from clinical specimens, including smear-negative samples. This approach can also be integrated with low-cost sequencing workflows such as targeted sequencing for rapid detection of Mtb and drug resistance.
UNASSIGNED: We prospectively collected 34 samples (sputum, n = 17; bronchoalveolar lavage, n = 13; and pus, n = 4) from patients with active tuberculosis (TB). Prior to DNA extraction, we used a ligand-mediated magnetic bead method to enrich Mtb from clinical samples and performed WGS on Illumina platform.
UNASSIGNED: Mtb was definitively identified based on WGS from 88.2% (30/34) of the samples, of which 35.3% (12/34) were smear negative. The overall median genome coverage was 15.2% (interquartile range [IQR], 7.7%-28.2%). There was a positive correlation between load of bacilli on smears and genome coverage (P < .001). We detected 58 genes listed in the World Health Organization mutation catalogue in each positive sample (median coverage, 85% [IQR, 61%-94%]), enabling the identification of mutations missed by routine diagnostics. Mutations causing resistance to rifampicin, isoniazid, streptomycin, and ethambutol were detected in 5 of 34 (14.7%) samples, including the rpoB S441A mutation that confers resistance to rifampicin, which is not covered by Xpert MTB/RIF.
UNASSIGNED: We demonstrate the feasibility of magnetic bead-based enrichment for culture-free WGS of Mtb from clinical specimens, including smear-negative samples. This approach can also be integrated with low-cost sequencing workflows such as targeted sequencing for rapid detection of Mtb and drug resistance.
摘要：
■结核分枝杆菌（Mtb）的直接全基因组测序（WGS）可用作研究耐药性的工具，混合感染，和宿主内部的多样性。然而,由于在高背景下的低数量的杆菌,从临床样品中获得WGS是具有挑战性的。
■我们前瞻性收集了34个样本（痰，n=17；支气管肺泡灌洗，n=13；和脓液，n=4）来自活动性结核病（TB）患者。在DNA提取之前,我们使用配体介导的磁珠方法从临床样本中富集Mtb,并在Illumina平台上进行了WGS.
■根据WGS从88.2%（30/34）的样品中确定了Mtb，其中35.3%(12/34)为涂片阴性。总体基因组覆盖率中位数为15.2%(四分位数间距[IQR]，7.7%-28.2%）。涂片上的杆菌负荷与基因组覆盖率呈正相关（P<0.001）。我们在每个阳性样本中检测到世界卫生组织突变目录中列出的58个基因（中位数覆盖率，85%[IQR，61%-94%])，能够识别常规诊断遗漏的突变。导致对利福平耐药的突变,异烟肼,链霉素,在34份样品中有5份(14.7%)检测到乙胺丁醇，包括赋予利福平抗性的rpoBS441A突变，XpertMTB/RIF不包括。
■我们证明了基于磁珠的富集用于临床标本中Mtb的无培养WGS的可行性，包括涂片阴性样本.这种方法也可以与低成本测序工作流程整合，例如靶向测序，以快速检测Mtb和耐药性。
■我们前瞻性收集了34个样本（痰，n=17；支气管肺泡灌洗，n=13；和脓液，n=4）来自活动性结核病（TB）患者。在DNA提取之前,我们使用配体介导的磁珠方法从临床样本中富集Mtb,并在Illumina平台上进行了WGS.
■根据WGS从88.2%（30/34）的样品中确定了Mtb，其中35.3%(12/34)为涂片阴性。总体基因组覆盖率中位数为15.2%(四分位数间距[IQR]，7.7%-28.2%）。涂片上的杆菌负荷与基因组覆盖率呈正相关（P<0.001）。我们在每个阳性样本中检测到世界卫生组织突变目录中列出的58个基因（中位数覆盖率，85%[IQR，61%-94%])，能够识别常规诊断遗漏的突变。导致对利福平耐药的突变,异烟肼,链霉素,在34份样品中有5份(14.7%)检测到乙胺丁醇，包括赋予利福平抗性的rpoBS441A突变，XpertMTB/RIF不包括。
■我们证明了基于磁珠的富集用于临床标本中Mtb的无培养WGS的可行性，包括涂片阴性样本.这种方法也可以与低成本测序工作流程整合，例如靶向测序，以快速检测Mtb和耐药性。
