Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain.
Servicio de Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañóngrid.410526.4, Madrid, Spain.
mSphere. 2021 Dec 22;6(6):e0074421. doi: 10.1128/mSphere.00744-21. Epub 2021 Dec 15.
Detection of mixed Mycobacterium tuberculosis (MTB) infections is essential, particularly when resistance mutations are present in minority bacterial populations that may affect patients' disease evolution and treatment. Whole-genome sequencing (WGS) has extended the amount of key information available for the diagnosis of MTB infection, including the identification of mixed infections. Having genomic information at diagnosis for early intervention requires carrying out WGS directly on the clinical samples. However, few studies have been successful with this approach due to the low representation of MTB DNA in sputa. In this study, we evaluated the ability of a strategy based on specific MTB DNA enrichment by using a newly designed capture platform (MycoCap) to detect minority variants and mixed infections by WGS on controlled mixtures of MTB DNAs in a simulated sputum genetic background. A pilot study was carried out with 12 samples containing 98% of a DNA pool from sputa of patients without MTB infection and 2% of MTB DNA mixtures at different proportions. Our strategy allowed us to generate sequences with a quality equivalent to those obtained from culture: 62.5× depth coverage and 95% breadth coverage (for at least 20× reads). Assessment of minority variant detection was carried out by manual analysis and allowed us to identify heterozygous positions up to a 95:5 ratio. The strategy also automatically distinguished mixed infections up to a 90:10 proportion. Our strategy efficiently captures MTB DNA in a nonspecific genetic background, allows detection of minority variants and mixed infections, and is a promising tool for performing WGS directly on clinical samples. We present a new strategy to identify mixed infections and minority variants in Mycobacterium tuberculosis by whole-genome sequencing. The objective of the strategy is the direct detection in patient sputum; in this way, minority populations of resistant strains can be identified at the time of diagnosis, facilitating identification of the most appropriate treatment for the patient from the first moment. For this, a platform for capturing M. tuberculosis-specific DNA was designed to enrich the clinical sample and obtain quality sequences.
检测混合分枝杆菌（MTB）感染至关重要，特别是在少数细菌群体中存在耐药突变的情况下，这可能会影响患者的疾病演变和治疗。全基因组测序（WGS）扩展了可用于诊断 MTB 感染的关键信息，包括鉴定混合感染。在诊断时获得基因组信息以进行早期干预需要直接对临床样本进行 WGS。然而，由于痰液中 MTB DNA 的代表性较低，因此很少有研究成功采用这种方法。在这项研究中，我们评估了一种基于使用新设计的捕获平台（MycoCap）特异性 MTB DNA 富集的策略的能力，该策略用于在模拟的痰液遗传背景下检测 MTB DNA 混合物中少数变体和混合感染。进行了一项试点研究，该研究使用 12 个样本，这些样本包含 98%来自无 MTB 感染患者痰液的 DNA 池和 2%的 MTB DNA 混合物，比例不同。我们的策略允许我们生成与从培养物获得的质量相当的序列：62.5×深度覆盖率和 95%的广度覆盖率（至少 20×的读取）。通过手动分析进行少数变体检测的评估使我们能够识别高达 95:5 比例的杂合位置。该策略还自动区分高达 90:10 比例的混合感染。我们的策略可以有效地在非特异性遗传背景下捕获 MTB DNA，允许检测少数变体和混合感染，并且是直接在临床样本上进行 WGS 的有前途的工具。我们提出了一种通过全基因组测序鉴定分枝杆菌属混合感染和少数变体的新策略。该策略的目的是直接在患者的痰液中检测；通过这种方式，可以在诊断时识别耐药菌株的少数群体，从而从第一时间为患者确定最合适的治疗方法。为此，设计了一种用于捕获 MTB 特异性 DNA 的平台，以富集临床样本并获得高质量的序列。
