Advanced Diagnostics Laboratory, National Jewish Health, Denver, Colorado, USA.
Department of Medicine, National Jewish Health, Denver, Colorado, USA.
Microbiol Spectr. 2024 Oct 3;12(10):e0163824. doi: 10.1128/spectrum.01638-24. Epub 2024 Aug 27.
Complete identification methods are critical for evaluating nontuberculous mycobacteria (NTM). Here, we describe a novel diagnostic method for identification of eight NTM, complex, and three drug resistance markers using PCR/matrix-assisted, laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) from cultured organisms. With this technology, a multiplex end-point PCR is performed for targets of interest. Detection probes that are extended in the presence of a target are added. The extended probes have greater molecular weight and can be detected by MALDI-TOF MS. An AFB Primary Panel was designed to differentiate ; subsp. ; complex (other); subsp. , , and ; , and complex. This design should cover 90% (3,483/3,691) of mycobacteria seen onsite. A development set of unblinded isolates ( = 217) was used to develop PCR primers, detection probes, and probe barcodes. It demonstrated 99.1% (215/217) agreement with reference methods. An evaluation set using blinded isolates ( = 320) showed an overall sensitivity of 94.3% (range by target: 90.0-100%). Overall specificity from negative media, non-target mycobacteria, and bacteria was 99.1% (108/109; range by target: 94.4-100%). Three drug resistance markers (41), and demonstrated 100%, 91%, and 100% sensitivity, respectively, and >99% specificity. Limit of detection per target ranged from 2.2 × 10 to 9.9 × 10 CFU/mL. The AFB Primary Panel allows for mycobacterial speciation, subspeciation, and resistance mutation detection, which is essential for diagnosis, appropriate therapy, identifying outbreaks, and managing treatment-refractory disease. It can perform with high-throughput and high specificity and sensitivity from isolates.IMPORTANCEEven closely related mycobacteria can have unique treatment patterns, but differentiating these organisms is a challenge. Here, we tested an innovative platform that combines two commonly used technologies and creates something new: matrix-assisted, laser-desorption ionization time-of flight mass spectrometry was performed on PCR amplicons instead of on proteins. This created a robust system with the advantages of PCR (high discriminatory power, high throughput, detection of resistance) with the advantages of mass spectrometry (more targets, lower operational cost) in order to identify closely related mycobacterial organisms.
完整的鉴定方法对于评估非结核分枝杆菌（NTM）至关重要。在这里，我们描述了一种使用聚合酶链反应/基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）从培养物中鉴定八种 NTM、复合和三种耐药标记物的新型诊断方法。使用这项技术，可以对感兴趣的靶标进行多重终点 PCR。在存在靶标的情况下添加延伸的检测探针。延伸的探针具有更大的分子量，可以通过 MALDI-TOF MS 检测。设计了 AFB 初级面板以区分；复合亚种（其他）；亚种、、和；和复合。这个设计应该可以覆盖现场所见的 90%（3483/3691）分枝杆菌。一组未盲的分离株（=217）被用于开发 PCR 引物、检测探针和探针条码。它与参考方法的一致性达到 99.1%（215/217）。使用盲法分离株（=320）进行的评估集显示总体灵敏度为 94.3%（按靶标范围：90.0-100%）。来自阴性培养基、非靶标分枝杆菌和细菌的总体特异性为 99.1%（108/109；按靶标范围：94.4-100%）。三种耐药标记物（41）、和分别显示出 100%、91%和 100%的灵敏度，特异性均>99%。每个靶标的检测限范围为 2.2×10 至 9.9×10 CFU/mL。AFB 初级面板允许进行分枝杆菌种属鉴定、亚种鉴定和耐药突变检测，这对于诊断、适当的治疗、识别暴发和治疗难治性疾病的管理至关重要。它可以从分离物中进行高通量、高特异性和高灵敏度的检测。
即使是密切相关的分枝杆菌也可能有独特的治疗模式，但区分这些生物体是一项挑战。在这里，我们测试了一种创新的平台，该平台结合了两种常用技术并创造了新的方法：基质辅助激光解吸电离飞行时间质谱在 PCR 扩增子上而不是在蛋白质上进行。这创建了一个强大的系统，具有 PCR（高鉴别力、高通量、耐药检测）的优势，以及质谱（更多靶标、更低的运营成本）的优势，以鉴定密切相关的分枝杆菌。
