Field evaluation of nanopore targeted next-generation sequencing to predict drug-resistant tuberculosis from native sputum in South Africa and Zambia.

IF 6.1 2区 医学 Q1 MICROBIOLOGY
Journal of Clinical Microbiology Pub Date : 2025-03-12 Epub Date: 2025-02-12 DOI:10.1128/jcm.01390-24
Tiana C Schwab, Lavania Joseph, Andrew Moono, Pauline C Göller, Mamello Motsei, Guy Muula, Denise Evans, Stefan Neuenschwander, Gunar Günther, Carolyn Bolton, Peter M Keller, Alban Ramette, Matthias Egger, Shaheed V Omar, Lukas Fenner
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引用次数: 0

Abstract

Rapid and comprehensive drug susceptibility testing (DST) is essential for diagnosing and treating drug-resistant tuberculosis effectively, and next-generation sequencing can be an effective genotypic DST method. We implemented and evaluated the performance of a nanopore targeted sequencing assay, called the Tuberculosis Drug Resistance Test (TBDR, Oxford Nanopore Diagnostics, Ltd., United Kingdom), which predicts drug resistance to 16 TB drugs, at a South African reference laboratory and a district diagnostic laboratory in Zambia. We compared the sequencing success rates between unprocessed and decontaminated sputum samples and determined the diagnostic accuracy against local DST (Xpert MTB/RIF Ultra, Xpert MTB/XDR, and BD BACTEC MGIT phenotypic DST). We prospectively sequenced 236 samples and have 148 samples with sequencing results from unprocessed and decontaminated sputum. We obtained successful sequencing results from 66.4% (94/148) unprocessed sputum samples and 75% (111/148) decontaminated samples. Sequencing success rates at the two sites differed, with 50.7% (36/71) successful sequencing results from unprocessed sputum in Zambia and 75.3% (58/77) in South Africa. Samples with "low" bacterial load, measured by Xpert MTB/RIF Ultra, tended to produce fewer successful sequencing results. TBDR sequencing predicted resistances in 48 samples, detecting resistance for rifampicin (n = 41) and isoniazid (n = 20), as well as 10 second-line drugs (n = 15). Sensitivity was variable compared to phenotypic DST, ranging from 33 (ethionamide) to 94% (rifampicin), while specificity remained above 90% for all drugs, except clofazimine. The TBDR assay can provide rapid, comprehensive genotypic DST. Technical and operational challenges need to be addressed for its broader implementation in high tuberculosis-burden settings.IMPORTANCEThis study illustrates the use of the Tuberculosis Drug Resistance Test (TBDR, Oxford Nanopore Diagnostics, Ltd., United Kingdom) as a rapid drug susceptibility testing (DST) approach for diagnosing drug-resistant TB in the high TB-burden countries of South Africa and Zambia. The TBDR assay predicts resistance to 16 TB drugs, including first- and second-line treatments. By implementing the TBDR assay in a national reference laboratory in South Africa and a district diagnostic laboratory in Zambia, we demonstrate how this technology can provide faster diagnostic results (days) compared to traditional phenotypic DST methods (~2 months), with adequate sensitivity. Missed resistances compared to phenotypic DST indicate that technical improvements are needed. Successful sequencing from unprocessed and decontaminated sputum samples at different sites suggests feasibility in diverse settings, though operational challenges remain. Implementing this rapid, comprehensive DST approach could enhance drug-resistant tuberculosis diagnosis and treatment, ultimately improving patient outcomes and helping to combat tuberculosis in high-burden regions.

南非和赞比亚纳米孔靶向下一代测序预测本地痰液耐药结核病的现场评估。
快速、全面的药敏试验(DST)是有效诊断和治疗耐药结核病的必要手段,新一代测序可作为一种有效的基因型药敏试验方法。我们在南非的一个参考实验室和赞比亚的一个地区诊断实验室实施并评估了一种名为结核耐药试验(TBDR, Oxford nanopore Diagnostics, Ltd.,英国)的纳米孔靶向测序测定方法的性能,该方法预测了对16种结核药物的耐药性。我们比较了未处理和净化的痰样本的测序成功率,并确定了对当地DST (Xpert MTB/RIF Ultra, Xpert MTB/XDR和BD BACTEC MGIT表型DST)的诊断准确性。我们对236份样本进行了前瞻性测序,其中148份样本的测序结果来自未处理和去污染的痰。66.4%(94/148)未处理的痰液样本和75%(111/148)净化的痰液样本测序成功。两个地点的测序成功率不同,赞比亚未处理痰液的测序成功率为50.7%(36/71),南非为75.3%(58/77)。通过Xpert MTB/RIF Ultra测量的“低”细菌负荷样品往往产生较少的成功测序结果。TBDR测序预测了48份样本的耐药性,检测到利福平(n = 41)和异烟肼(n = 20)的耐药性,以及10种二线药物(n = 15)的耐药性。与表型DST相比,敏感性是可变的,范围从33%(乙硫酰胺)到94%(利福平),而除氯法齐明外,所有药物的特异性都保持在90%以上。TBDR法可以提供快速、全面的基因型DST。为了在结核病高负担环境中更广泛地实施,需要解决技术和操作方面的挑战。这项研究表明,在南非和赞比亚等结核病高负担国家,结核病耐药试验(TBDR, Oxford Nanopore Diagnostics, Ltd., uk)作为一种快速药敏试验(DST)方法用于诊断耐药结核病。TBDR试验预测对16种结核病药物的耐药性,包括一线和二线治疗。通过在南非的一个国家参比实验室和赞比亚的一个地区诊断实验室实施TBDR检测,我们展示了与传统表型DST方法(~2个月)相比,该技术如何能够提供更快的诊断结果(天),并具有足够的灵敏度。与表型DST相比,遗漏的抗性表明需要技术改进。从不同地点的未处理和净化的痰样本成功测序表明,在不同环境下是可行的,尽管操作上的挑战仍然存在。实施这种快速、全面的DST方法可以加强耐药结核病的诊断和治疗,最终改善患者的治疗结果,并帮助在高负担地区防治结核病。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Journal of Clinical Microbiology
Journal of Clinical Microbiology 医学-微生物学
CiteScore
17.10
自引率
4.30%
发文量
347
审稿时长
3 months
期刊介绍: The Journal of Clinical Microbiology® disseminates the latest research concerning the laboratory diagnosis of human and animal infections, along with the laboratory's role in epidemiology and the management of infectious diseases.
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