Rapid molecular diagnostics of tuberculosis resistance by targeted stool sequencing.
Rapid molecular diagnostics of tuberculosis resistance by targeted stool sequencing.
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DOI：
10.1186/s13073-022-01054-6
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发表时间：
2022-05-19
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影响因子：
12.3
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中科院分区：
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--
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对于难以提供痰液的人群，如儿童或艾滋病毒感染者，粪便是结核病的重要诊断标本。然而，结核分枝杆菌（M.结核病）复杂菌株表现不佳。这限制了使用该标本进行表型耐药性检测的机会。因此，迫切需要可靠的分子方法对粪便标本进行全面的耐药性检测。我们评估了下一代靶向测序（tNGS，Deeplex® Myc-TB）用于检测与M相关的突变的性能。来自斯威士兰结核病治疗患者前瞻性队列的参与者提供的粪便标本中分离的DNA对结核复合菌耐药性的影响（n = 66; 56例有M.通过实时定量PCR在粪便中检测到结核复合体DNA），以及一个独立的德国验证队列，其中包括培养确诊的结核病参与者（n = 21）。tNGS试验检测M.结核菌复合体DNA阳性率为68%（38/56），完整的结核菌DNA阳性率为74%（28/38）。获得结核复合菌耐药预测报告。与痰液表型药敏结果高度一致（κ = 0.82）。预测耐药性的能力具有浓度依赖性，在粪便PCR浓度阈值> 100毫微克/微升（fg/μl）、1至100 fg/μl和< 1 fg/μl的样本中，分别有7/10（70%）、18/25（72%）和3/21（14%）例成功（p = 0.0004）。德国队列证实了这些结果，并证明粪便tNGS和痰液表型药物敏感性结果之间具有相似的高一致性（κ = 0.84）。tNGS可以从结核病患者提供的粪便中鉴定耐药性。这为难以提供呼吸道标本的结核病患者提供了获得关键诊断信息的机会。在线版本包含补充材料，可通过10.1186/s13073-022-01054-6获得。
Stool is an important diagnostic specimen for tuberculosis in populations who struggle to provide sputum, such as children or people living with HIV. However, the culture of Mycobacterium tuberculosis (M. tuberculosis) complex strains from stool perform poorly. This limits the opportunity for phenotypic drug resistance testing with this specimen. Therefore, reliable molecular methods are urgently needed for comprehensive drug resistance testing on stool specimens. We evaluated the performance of targeted next-generation sequencing (tNGS, Deeplex® Myc-TB) for the detection of mutations associated with M. tuberculosis complex drug resistance on DNA isolated from stool specimens provided by participants from a prospective cohort of patients treated for tuberculosis in Eswatini (n = 66; 56 with and 10 participants without M. tuberculosis complex DNA detected in stool by real-time quantitative PCR), and an independent German validation cohort of participants with culture-confirmed tuberculosis (n = 21). The tNGS assay detected M. tuberculosis complex DNA in 38 of 56 (68%) samples; for 28 of 38 (74%) samples, a full M. tuberculosis complex drug resistance prediction report was obtained. There was a high degree of concordance with sputum phenotypic drug susceptibility results (κ = 0.82). 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 > 100 femtogram per microliter (fg/μl), 1 to 100 fg/μl, and < 1 fg/μl, respectively (p = 0.0004). The German cohort confirmed these results and demonstrated a similarly high concordance between stool tNGS and sputum phenotypic drug susceptibility results (κ = 0.84). tNGS can identify drug resistance from stool provided by tuberculosis patients. This affords the opportunity to obtain critical diagnostic information for tuberculosis patients who struggle to provide respiratory specimens. The online version contains supplementary material available at 10.1186/s13073-022-01054-6.
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