Delamanid susceptibility testing of Mycobacterium tuberculosis using the resazurin microtitre assay and the BACTEC™ MGIT™ 960 system
Delamanid susceptibility testing of Mycobacterium tuberculosis using the resazurin microtitre assay and the BACTEC™ MGIT™ 960 system
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DOI：
10.1093/jac/dkw044
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发表时间：
2016-06-01
影响因子：
5.2
通讯作者：
Cirillo, Daniela Maria
中科院分区：
文献类型：
--
作者：
Schena, Elisa;Nedialkova, Lubov;Cirillo, Daniela Maria
目的：本研究的目的是通过建立准确区分结核分枝杆菌对delamanid的敏感和耐药的断点，通过比色瑞祖林微滴法（REMA）和半自动BACTEC (TM) MGIT (TM) 960系统（MGIT），制定快速delamanid药敏试验（DST）的标准化方案。方法：采用MGIT法、REMA法和固体琼脂法对19株预鉴定菌株进行mic测定。然后建立了delamanid的MIC分布，用于一组从未接触过该药物的临床菌株，并以不同的地理来源和易感模式为特征。WGS研究了参与细胞内delamanid激活的5个基因（ddn、fgd1、fbiA、fbiB和fbiC）的遗传多态性。结果：REMA和MGIT均可快速、准确测定delamanid MIC，与固体琼脂参比法一致性好，重现性和重复性高。我们建议REMA和MGIT的暂定断点为0.125 mg/L，以便可靠地区分结核分枝杆菌对delamanid的易感和耐药。ddn （Trp-88 -> Stop）和fbiA （Lys-250 -> Stop）的停止密码子突变仅在耐delamanid菌株中观察到。结论：我们建立了mdit和REMA中delamanid的DST方案，确认了它们在常规结核病诊断中的可行性，两种方法使用相同的判别浓度。此外，利用WGS分析，我们确定了两个参与delamanid激活的基因中可能与抗性相关的多态性。
Objectives: The objective of this study was to develop standardized protocols for rapid delamanid drug susceptibility testing (DST) using the colorimetric resazurin microtitre assay (REMA) and semi-automated BACTEC (TM) MGIT (TM) 960 system (MGIT) by establishing breakpoints that accurately discriminate between susceptibility and resistance of Mycobacterium tuberculosis to delamanid.Methods: MICs of delamanid were determined by the MGIT, the REMA and the solid agar method for 19 pre-characterized strains. The MIC distribution of delamanid was then established for a panel of clinical strains never exposed to the drug and characterized by different geographical origins and susceptibility patterns. WGS was used to investigate genetic polymorphisms in five genes (ddn, fgd1, fbiA, fbiB and fbiC) involved in intracellular delamanid activation.Results: We demonstrated that the REMA and MGIT can both be used for the rapid and accurate determination of delamanid MIC, showing excellent concordance with the solid agar reference method, as well as high reproducibility and repeatability. We propose the tentative breakpoint of 0.125 mg/L for the REMA and MGIT, allowing reliable discrimination between M. tuberculosis susceptible and resistant to delamanid. Stop codon mutations in ddn (Trp-88 -> STOP) and fbiA (Lys-250 -> STOP) have only been observed in strains resistant to delamanid.Conclusions: We established protocols for DST of delamanid in the MGIT and REMA, confirming their feasibility in routine TB diagnostics, utilizing the same discriminative concentration for both methods. Moreover, taking advantage of WGS analysis, we identified polymorphisms potentially associated with resistance in two genes involved in delamanid activation.