Cabrera Adriana, Lee Tracy, Kolehmainen Kathleen, Hird Trevor, Jorgensen Danielle, Lo Calvin Ka-Fung, Hamze Hasan, O'Dwyer Alan, Fornika Dan, KhunKhun Rupinder Kaur, Rodrigues Mabel, Prystajecky Natalie, Tyson John, Zlosnik James E A, Sekirov Inna
British Columbia Centre for Disease Control Public Health Laboratory, Vancouver, BC V5Z 4R4, Canada.
Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of British Columbia, Vancouver, BC V5Z 3N9, Canada.
Trop Med Infect Dis. 2025 Jul 10;10(7):194. doi: 10.3390/tropicalmed10070194.
Treatment of requires multi-drug regimens, and resistance to any individual antibiotic can compromise outcomes. For slow-growing organisms like , rapid detection of resistance-conferring mutations enables timely initiation of effective therapy. Conversely, confirming wild-type status in resistance-associated genes supports confidence in standard regimens. We developed an amplicon-based next generation sequencing (amplicon tNGS) assay on the Illumina platform targeting eight genes linked to resistance to isoniazid, rifampin, ethambutol, pyrazinamide, and fluoroquinolones. Sequencing results were analyzed using a custom bioinformatics pipeline. Forty-seven samples were used for assay development, and 37 additional samples underwent post-implementation clinical validation. Compared to whole genome sequencing (WGS), amplicon tNGS demonstrated 97.7% sensitivity, 98.9% specificity, and 98.7% overall accuracy for variant detection in targeted regions. Resistance prediction showed 79.3% concordance with WGS; discrepancies were primarily due to mutations outside of target regions. Among post-implementation samples, 27/37 passed quality metrics for all targets, with 95.7% concordance between amplicon tNGS results and final susceptibility results. This assay is now in use in our laboratory and offers significantly faster turnaround than both WGS and phenotypic methods on cultured isolates, enabling more rapid, informed treatment decisions for tuberculosis patients.
[疾病名称]的治疗需要多药联合方案，对任何一种抗生素产生耐药性都可能影响治疗结果。对于像[病原体名称]这样生长缓慢的微生物，快速检测赋予耐药性的突变能够及时启动有效的治疗。相反，确认耐药相关基因中的野生型状态有助于对标准治疗方案充满信心。我们在Illumina平台上开发了一种基于扩增子的下一代测序（扩增子tNGS）检测方法，该方法针对与对异烟肼、利福平、乙胺丁醇、吡嗪酰胺和氟喹诺酮类药物耐药相关的八个基因。使用定制的生物信息学流程分析测序结果。47个样本用于检测方法开发，另外37个样本在实施后进行临床验证。与全基因组测序（WGS）相比，扩增子tNGS在目标区域的变异检测中显示出97.7%的灵敏度、98.9%的特异性和98.7%的总体准确率。耐药性预测与WGS的一致性为79.3%；差异主要是由于目标区域以外的突变。在实施后的样本中，37个样本中有27个通过了所有目标的质量指标，扩增子tNGS结果与最终药敏结果的一致性为95.7%。该检测方法目前已在我们实验室使用，与WGS和对培养分离株的表型检测方法相比，周转时间明显更快，能够为结核病患者做出更快速、明智的治疗决策。
