Schwartz Lilith A, Brodeth Adriann L, Susilo Cara T, Rodolf Amelia A, Ivanov Tanya, Corrales Esmeralda Mendoza, Kumar Shivani S, Kamariza Mireille
UCLA Department of Chemistry and Biochemistry, Los Angeles, CA, USA.
UCLA Department of Bioengineering, Los Angeles, CA, USA.
bioRxiv. 2025 Jun 23:2025.06.23.661142. doi: 10.1101/2025.06.23.661142.
Tuberculosis (TB) is the most lethal cause of death from a single infectious agent. In 2024, an estimated 10 million people developed TB, nearly half a million of which were infected with drug-resistant tuberculosis (DR-TB). Early detection of infection and drug resistance is critical to controlling DR-TB as this enables rapid engagement into effective care. Currently, bacterial culture and nucleic acid testing remain the primary methods for diagnosing infection, with smear microscopy being phased out. However, these methods present significant limitations for diagnosing drug resistance such as lengthy time-to-result for phenotypic tests, as well as the need for prior knowledge of resistance mutations and prohibitive cost for molecular tests. To address this, we developed a rapid phenotypic TB drug susceptibility test, termed Tre-DST, based on novel trehalose probes, which upon metabolic conversion emit enhanced fluorescence signal, giving them their unique ability to specifically detect live mycobacteria. We used the nonpathogenic and the virulence-attenuated (Mtb) H37Ra or auxotrophic Mtb to demonstrate a strong correlation between cost-effective plate reader results and flow cytometry data, suggesting the fluorescence plate reader is a suitable fluorescence detector for Tre-DST. We determined that adding a one-week incubation step for Mtb allowed samples originally seeded at 10 CFU/mL to become detectable, over two weeks earlier than colony forming unit analysis. Importantly, we found that Tre-DST reports on drug susceptibility in a drug-agnostic manner, demonstrating loss of fluorescence with frontline TB drugs rifampicin (RIF), isoniazid (INH), and ethambutol, as well as the newer drug bedaquiline. Finally, Tre-DST distinguished RIF- and INH-resistant auxotrophs from susceptible controls and accurately reported resistance activity. Ultimately, because Tre-DST is agnostic to mechanisms of drug resistance, this assay is likely compatible with all WHO-recommended DR-TB drugs as well as any future TB drugs as a diagnostic in reference laboratories.
结核病（TB）是单一传染源导致死亡的最主要原因。2024年，估计有1000万人患上结核病，其中近50万人感染了耐多药结核病（DR-TB）。早期检测感染和耐药性对于控制耐多药结核病至关重要，因为这能够迅速开展有效的治疗。目前，细菌培养和核酸检测仍然是诊断感染的主要方法，涂片显微镜检查正在逐步淘汰。然而，这些方法在诊断耐药性方面存在重大局限性，例如表型检测结果报告时间长，以及需要预先了解耐药突变情况，分子检测成本高昂。为了解决这个问题，我们基于新型海藻糖探针开发了一种快速表型结核病药物敏感性试验，称为Tre-DST，该探针在代谢转化后会发出增强的荧光信号，使其具有特异性检测活分枝杆菌的独特能力。我们使用非致病性和毒力减弱的结核分枝杆菌（Mtb）H37Ra或营养缺陷型Mtb来证明具有成本效益的酶标仪结果与流式细胞术数据之间存在很强的相关性，这表明荧光酶标仪是Tre-DST合适的荧光检测器。我们确定，对Mtb添加一周的孵育步骤可以使最初接种浓度为10 CFU/mL的样本变得可检测，比菌落形成单位分析早两周以上。重要的是，我们发现Tre-DST以与药物无关的方式报告药物敏感性，显示一线结核病药物利福平（RIF）、异烟肼（INH）和乙胺丁醇以及新型药物贝达喹啉会导致荧光消失。最后，Tre-DST区分了耐RIF和INH的营养缺陷型菌株与敏感对照，并准确报告了耐药活性。最终，由于Tre-DST与耐药机制无关，该检测方法可能与世界卫生组织推荐的所有耐多药结核病药物以及未来的任何结核病药物兼容，可作为参考实验室的诊断方法。
