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Abstract：
Targeted next-generation sequencing (tNGS) can be used to perform Mycobacterium tuberculosis (MTB) complex-specific amplification or target capture directly from sputum samples, yielding simultaneous coverage of many genes and DNA regions associated with antimicrobial resistance (AMR). Performance comparisons of tNGS and another molecular testing tool, Xpert MTB/rifampicin (RIF), have been empirical. Here, using a dilution series of a RIF-resistant clinical isolate of MTB, we found that tNGS had a slightly lower limit of bacterial detection (102 CFU/mL) compared with Xpert MTB/RIF (103 CFU/mL) in culture medium. However, the minimum detection limit of the rpoB S450L mutation in this isolate was significantly lower with tNGS (102 CFU/mL) than with Xpert MTB/RIF (106 CFU/mL). Sputum samples collected from 129 suspected pulmonary tuberculosis patients were also prospectively studied with the clinical diagnosis as a reference, revealing that the sensitivity of tNGS (48.6%) was higher than those of culture (46.8%), Xpert MTB/RIF (39.4%), and smear microscopy (34.9%) testing. Notably, AMR analysis of 56 MTB-positive samples as determined by tNGS revealed high mutation frequencies of 96.4%, 35.7%, 26.8%, and 19.6% in the following AMR-associated genes: rrs, rpoB, katG, and pncA, respectively. The findings of this study provide theoretical support for the differential clinical application of tNGS and Xpert MTB/RIF and suggest that tNGS has greater application value in tuberculosis drug resistance monitoring and prevention.IMPORTANCETargeted next-generation sequencing (tNGS) can be used to perform Mycobacterium tuberculosis (MTB) complex-specific amplification or target capture directly from sputum samples, yielding simultaneous coverage of genes and DNA regions associated with antimicrobial resistance (AMR). Performance comparisons of tNGS and Xpert MTB/rifampicin (RIF) have been empirical. The Xpert MTB/RIF assay is a commercial system that uses the nucleic acid amplification detection method for rapid (2 hours) diagnosis of tuberculosis (TB). The cost of the tNGS and Xpert MTB/RIF assays in this study was similar, at USD 98 and USD 70-104 per sample, respectively, but the time required for tNGS (3 days) was much longer than that required for the Xpert MTB/RIF assay. However, tNGS yielded more accurate results and a larger number of AMR-associated gene mutations, which compensated for the extra time and highlighted the greater application value of tNGS in TB drug resistance monitoring and prevention.
摘要：
靶向下一代测序（tNGS）可用于直接从痰液样本中进行结核分枝杆菌（MTB）复合物特异性扩增或靶标捕获，同时覆盖与抗菌素耐药性（AMR）相关的许多基因和DNA区域。tNGS和另一种分子测试工具的性能比较，XpertMTB/利福平（RIF），一直是经验性的。这里,使用耐RIF的MTB临床分离株的稀释系列，我们发现,与XpertMTB/RIF(103CFU/mL)相比,tNGS的细菌检测限(102CFU/mL)略低.然而,该分离株中rpoBS450L突变的最低检测限用tNGS(102CFU/mL)显著低于XpertMTB/RIF(106CFU/mL).并以临床诊断为参考,对129例疑似肺结核患者的痰液样本进行前瞻性研究,显示tNGS的敏感性(48.6%)高于培养物的敏感性(46.8%)，XpertMTB/RIF（39.4%），和涂片镜检(34.9%)。值得注意的是,通过tNGS确定的56个MTB阳性样品的AMR分析显示，高突变频率为96.4％，35.7%,26.8%,在以下AMR相关基因中占19.6%：rrs，rpoB,katG,和pncA，分别。本研究结果为tNGS和XpertMTB/RIF的差异临床应用提供了理论支持，提示tNGS在结核病耐药监测和预防中具有较大的应用价值。IMPORTANCETargeted下一代测序（tNGS）可用于进行结核分枝杆菌（MTB）复合物特异性扩增或直接从痰液样本中捕获靶标，同时覆盖与抗菌素耐药性（AMR）相关的基因和DNA区域。tNGS和XpertMTB/利福平（RIF）的性能比较是经验性的。XpertMTB/RIF测定是使用核酸扩增检测方法快速(2小时)诊断结核病(TB)的商业系统。在这项研究中，tNGS和XpertMTB/RIF测定的成本相似，每个样本98美元和70-104美元，分别,但tNGS所需的时间(3天)比XpertMTB/RIF测定所需的时间长得多.然而,tNGS产生更准确的结果和更多的AMR相关基因突变，弥补了额外的时间，凸显了tNGS在结核病耐药性监测和预防中的更大应用价值。
