Wadsworth Center, New York State Department of Health, Albany, NY, United States.
Front Public Health. 2023 Jun 29;11:1206056. doi: 10.3389/fpubh.2023.1206056. eCollection 2023.
complex (MTBC) infections are treated with combinations of antibiotics; however, these regimens are not as efficacious against multidrug and extensively drug resistant MTBC. Phenotypic (growth-based) drug susceptibility testing on slow growing bacteria like MTBC requires many weeks to months to complete, whereas sequencing-based approaches can predict drug resistance (DR) with reduced turnaround time. We sought to develop a multiplexed, targeted next generation sequencing (tNGS) assay that can predict DR and can be performed directly on clinical respiratory specimens. A multiplex PCR was designed to amplify a group of thirteen full-length genes and promoter regions with mutations known to be involved in resistance to first- and second-line MTBC drugs. Long-read amplicon libraries were sequenced with Oxford Nanopore Technologies platforms and high-confidence resistance mutations were identified in real-time using an in-house developed bioinformatics pipeline. Sensitivity, specificity, reproducibility, and accuracy of the tNGS assay was assessed as part of a clinical validation study. In total, tNGS was performed on 72 primary specimens and 55 MTBC-positive cultures and results were compared to clinical whole genome sequencing (WGS) performed on paired patient cultures. Complete or partial susceptibility profiles were generated from 82% of smear positive primary specimens and the resistance mutations identified by tNGS were 100% concordant with WGS. In addition to performing tNGS on primary clinical samples, this assay can be used to sequence MTBC cultures mixed with other mycobacterial species that would not yield WGS results. The assay can be effectively implemented in a clinical/diagnostic laboratory with a two to three day turnaround time and, even if batched weekly, tNGS results are available on average 15 days earlier than culture-derived WGS results. This study demonstrates that tNGS can reliably predict MTBC drug resistance directly from clinical specimens or cultures and provide critical information in a timely manner for the appropriate treatment of patients with DR tuberculosis.
复杂（MTBC）感染用抗生素联合治疗；然而，这些方案对多药和广泛耐药的 MTBC 疗效不佳。像 MTBC 这样生长缓慢的细菌的表型（基于生长）药物敏感性测试需要数周到数月才能完成，而基于测序的方法可以在缩短周转时间的情况下预测药物耐药性（DR）。我们试图开发一种多重、靶向的下一代测序（tNGS）检测方法，该方法可以预测 DR，并且可以直接在临床呼吸道标本上进行。设计了一种多重 PCR，以扩增一组十三个全长基因和启动子区域，这些区域的突变已知与对一线和二线 MTBC 药物的耐药性有关。使用 Oxford Nanopore Technologies 平台对长读长扩增文库进行测序，并使用内部开发的生物信息学管道实时鉴定高可信度的耐药突变。该 tNGS 检测的敏感性、特异性、重现性和准确性作为临床验证研究的一部分进行了评估。总共对 72 个原发性标本和 55 个 MTBC 阳性培养物进行了 tNGS 检测，并将结果与对配对患者培养物进行的临床全基因组测序（WGS）进行了比较。82%的涂片阳性原发性标本生成了完整或部分药敏谱，tNGS 鉴定的耐药突变与 WGS 完全一致。除了对原发性临床样本进行 tNGS 检测外，该检测还可用于对与其他分枝杆菌混合的 MTBC 培养物进行测序，这些培养物无法产生 WGS 结果。该检测可在临床/诊断实验室中有效实施，周转时间为 2-3 天，即使每周分批进行，tNGS 结果也比培养衍生的 WGS 结果平均提前 15 天提供。本研究表明，tNGS 可以直接从临床标本或培养物中可靠地预测 MTBC 耐药性，并及时提供关键信息，为 DR 结核患者的适当治疗提供依据。
