Comprehensive molecular drug resistance profiles derived from stool-based targeted sequencing of Mycobacterium tuberculosis complex: a cross-sectional observational study

BackgroundStool is an important diagnostic specimen for tuberculosis in populations who struggle to provide sputum, such as children or people living with HIV. However, as culture of M tuberculosis complex strains from stool performs poorly, drug resistance testing is currently limited.MethodsWe evaluated the performance of targeted next-generation sequencing (NGS, Deeplex® Myc-TB) for the detection of mutations associated with M tuberculosis complex drug resistance on DNA isolated from stool specimens provided by patients with tuberculosis from a prospective cohort in Eswatini, and an independent German validation cohort. FindingsAnalysis of the Eswatini cohort included specimens from 56 unique participants with and 10 participants without M tuberculosis complex DNA detected in stool by real-time quantitative PCR. The Deeplex® Myc-TB assay detected M tuberculosis complex DNA in 38 of 56 (68%) samples, and for 28 of 38 (74%) samples, a full M tuberculosis complex drug resistance prediction report was obtained. The ability to predict resistance was concentration dependent; and successful in 7/10 (70%), 18/25 (72%) and 3/21 (14%) of samples with stool PCR concentration thresholds of < 1 femtogram per microliter (fg/?l), 1 to 100 fg/?l, and > 100 fg/?l, respectively (p = 0.0004). The German cohort confirmed these results and demonstrated a high concordance between stool NGS and sputum phenotypic drug susceptibility results (? = 0.84). InterpretationThe Deeplex® Myc-TB assay can identify drug resistance via targeted NGS from stool provided by tuberculosis patients. This discovery affords the opportunity to obtain critical diagnostic information for tuberculosis patients who struggle to provide respiratory specimens.

【背景】粪便对于难以留取痰液的人群（如儿童或人类免疫缺陷病毒感染者）而言，是结核病重要的诊断标本。然而，从粪便中分离培养结核分枝杆菌复合群（Mycobacterium tuberculosis complex）菌株的效果不佳，因此当前药物敏感性检测手段十分有限。
【方法】本研究针对斯威士兰一项前瞻性队列中的结核病患者粪便标本分离得到的DNA，以及独立的德国验证队列标本，评估靶向二代测序（next-generation sequencing，NGS，Deeplex® Myc-TB）检测结核分枝杆菌复合群耐药相关突变的性能。
【研究结果】对斯威士兰队列的分析共纳入56例粪便实时定量聚合酶链反应（real-time quantitative PCR）检测出结核分枝杆菌复合群DNA的受试者，以及10例未检出的受试者。Deeplex® Myc-TB检测在56份样本中检出38份（68%）结核分枝杆菌复合群DNA，其中38份阳性样本中有28份（74%）获得了完整的结核分枝杆菌复合群耐药预测报告。耐药预测成功率与粪便标本的DNA浓度相关：当粪便PCR检测浓度阈值分别为<1飞克每微升（fg/μL）、1~100 fg/μL和>100 fg/μL时，检测成功率分别为7/10（70%）、18/25（72%）和3/21（14%）（p=0.0004）。德国验证队列验证了上述结果，且粪便NGS检测与痰液表型药物敏感性检测结果具有高度一致性（κ=0.84）。
【结论】Deeplex® Myc-TB检测可通过靶向NGS技术，从结核病患者的粪便标本中鉴定结核分枝杆菌复合群的耐药性。这一发现为难于留取呼吸道标本的结核病患者提供了获取关键诊断信息的途径。