Background

Drug-resistant tuberculosis remains a major obstacle in ending the global tuberculosis epidemic. Deployment of molecular tools for comprehensive drug resistance profiling is imperative for successful detection and characterisation of tuberculosis drug resistance. We aimed to assess the diagnostic accuracy of a new class of molecular diagnostics for drug-resistant tuberculosis.

Methods

We conducted a prospective, cross-sectional, multicentre clinical evaluation of the performance of two targeted next-generation sequencing (tNGS) assays for drug-resistant tuberculosis at reference laboratories in three countries (Georgia, India, and South Africa) to assess diagnostic accuracy and index test failure rates. Eligible participants were aged 18 years or older, with molecularly confirmed pulmonary tuberculosis, and at risk for rifampicin-resistant tuberculosis. Sensitivity and specificity for both tNGS index tests (GenoScreen Deeplex Myc-TB and Oxford Nanopore Technologies [ONT] Tuberculosis Drug Resistance Test) were calculated for rifampicin, isoniazid, fluoroquinolones (moxifloxacin, levofloxacin), second line-injectables (amikacin, kanamycin, capreomycin), pyrazinamide, bedaquiline, linezolid, clofazimine, ethambutol, and streptomycin against a composite reference standard of phenotypic drug susceptibility testing and whole-genome sequencing.

Findings

Between April 1, 2021, and June 30, 2022, 832 individuals were invited to participate in the study, of whom 720 were included in the final analysis (212, 376, and 132 participants in Georgia, India, and South Africa, respectively). Of 720 clinical sediment samples evaluated, 658 (91%) and 684 (95%) produced complete or partial results on the GenoScreen and ONT tNGS workflows, respectively, with 593 (96%) and 603 (98%) of 616 smear-positive samples producing tNGS sequence data. Both workflows had sensitivities and specificities of more than 95% for rifampicin and isoniazid, and high accuracy for fluoroquinolones (sensitivity approximately ≥94%) and second line-injectables (sensitivity 80%) compared with the composite reference standard. Importantly, these assays also detected mutations associated with resistance to critical new and repurposed drugs (bedaquiline, linezolid) not currently detectable by any other WHO-recommended rapid diagnostics on the market. We note that the current format of assays have low sensitivity (≤50%) for linezolid and more work on mutations associated with drug resistance is needed.

Interpretation

This multicentre evaluation demonstrates that culture-free tNGS can provide accurate sequencing results for detection and characterisation of drug resistance from Mycobacterium tuberculosis clinical sediment samples for timely, comprehensive profiling of drug-resistant tuberculosis.

Funding

Unitaid.

中文翻译：

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评估无培养靶向下一代测序诊断耐药结核病：两个端到端商业工作流程的多中心临床研究

 背景

耐药结核病仍然是终结全球结核病流行的主要障碍。部署分子工具进行全面的耐药性分析对于成功检测和表征结核病耐药性至关重要。我们旨在评估一类新的耐药结核病分子诊断的准确性。

 方法

我们在三个国家 （格鲁吉亚、印度和南非） 的参考实验室对两种靶向下一代测序 （tNGS） 检测耐药结核病的性能进行了前瞻性、横断面、多中心临床评估，以评估诊断准确性和指数检测失败率。符合条件的受试者年龄在 18 岁或以上，分子证实患有肺结核，并且有患利福平耐药结核病的风险。根据表型药物敏感性检测和全基因组测序的复合参考标准，计算利福平、异烟肼、氟喹诺酮类药物（莫西沙星、左氧氟沙星）、二线注射剂（阿米卡星、卡那霉素、卷曲霉素）、吡嗪酰胺、贝达喹啉、利奈唑胺、氯法齐明、乙胺丁醇和链霉素的 tNGS 指数检测（GenoScreen Deeplex Myc-TB 和 Oxford Nanopore Technologies [ONT] 结核病耐药性试验）的敏感性和特异性。

 发现

在 2021 年 4 月 1 日至 2022 年 6 月 30 日期间，共邀请了 832 人参加该研究，其中 720 人被纳入最终分析（格鲁吉亚、印度和南非分别为 212、376 和 132 名参与者）。在评估的 720 个临床沉积物样本中，658 个 （91%） 和 684 个 （95%） 分别在 GenoScreen 和 ONT tNGS 工作流程中产生了完整或部分结果，其中 616 个弥散条带阳性样本中有 593 个 （96%） 和 603 个 （98%） 产生了 tNGS 序列数据。与复合参考标准品相比，两种工作流程对利福平和异烟肼的敏感性和特异性均超过 95%，对氟喹诺酮类药物（敏感性约为 ≥94%）和二线注射剂（敏感性 80%）的准确度较高。重要的是，这些检测还检测到了与关键新药和再利用药物（贝达喹啉、利奈唑胺）耐药性相关的突变，这些药物目前在市场上任何其他 WHO 推荐的快速诊断都无法检测到。我们注意到目前的检测形式对利奈唑胺的敏感性较低 （≤50%），需要对与耐药性相关的突变进行更多工作。

 解释

这项多中心评估表明，免培养 tNGS 可以提供准确的测序结果，用于检测和表征结核分枝杆菌临床沉积物样本的耐药性，以便及时、全面地分析耐药结核病。

 资金

 国际药品采购机制。