Centre for Clinical Microbiology, Division of Infection and Immunity, University College London, London, United Kingdom.
Imperial College London, Infection and Immunity, Department of Microbiology, Charing Cross Hospital, London, United Kingdom.
Clin Chem. 2024 Apr 3;70(4):642-652. doi: 10.1093/clinchem/hvae013.
Improved monitoring of Mycobacterium tuberculosis response to treatment is urgently required. We previously developed the molecular bacterial load assay (MBLA), but it is challenging to integrate into the clinical diagnostic laboratory due to a labor-intensive protocol required at biosafety level 3 (BSL-3). A modified assay was needed.
The rapid enumeration and diagnostic for tuberculosis (READ-TB) assay was developed. Acetic acid was tested and compared to 4 M guanidine thiocyanate to be simultaneously bactericidal and preserve mycobacterial RNA. The extraction was based on silica column technology and incorporated low-cost reagents: 3 M sodium acetate and ethanol for the RNA extraction to replace phenol-chloroform. READ-TB was fully validated and compared directly to the MBLA using sputa collected from individuals with tuberculosis.
Acetic acid was bactericidal to M. tuberculosis with no significant loss in 16S rRNA or an unprotected mRNA fragment when sputum was stored in acetic acid at 25°C for 2 weeks or -20°C for 1 year. This novel use of acetic acid allows processing of sputum for READ-TB at biosafety level 2 (BSL-2) on sample receipt. READ-TB is semiautomated and rapid. READ-TB correlated with the MBLA when 85 human sputum samples were directly compared (R2 = 0.74).
READ-TB is an improved version of the MBLA and is available to be adopted by clinical microbiology laboratories as a tool for tuberculosis treatment monitoring. READ-TB will have a particular impact in low- and middle-income countries (LMICs) for laboratories with no BSL-3 laboratory and for clinical trials testing new combinations of anti-tuberculosis drugs.
迫切需要改善对结核分枝杆菌治疗反应的监测。我们之前开发了分子细菌负荷检测（MBLA），但由于在 3 级生物安全水平（BSL-3）下需要劳动密集型的方案，因此难以将其整合到临床诊断实验室中。需要一种改进的检测方法。
开发了快速计数和诊断结核病（READ-TB）检测。测试了乙酸，并与 4 M 异硫氰酸胍进行了比较，以达到同时杀菌和保存分枝杆菌 RNA 的效果。提取基于硅胶柱技术，并结合了低成本试剂：3 M 乙酸钠和乙醇用于 RNA 提取，以替代酚-氯仿。READ-TB 经过了全面验证，并直接与 MBLA 进行了比较，使用了从结核病患者收集的痰液。
当痰液在 25°C 的乙酸中储存 2 周或在-20°C 中储存 1 年时，乙酸对结核分枝杆菌具有杀菌作用，并且 16S rRNA 或未受保护的 mRNA 片段没有明显损失。这种新型的乙酸使用允许在收到样本时在生物安全 2 级（BSL-2）下对 READ-TB 进行痰液处理。READ-TB 是半自动和快速的。当直接比较 85 个人类痰液样本时，READ-TB 与 MBLA 相关（R2=0.74）。
READ-TB 是 MBLA 的改进版本，可供临床微生物学实验室采用，作为结核病治疗监测的工具。READ-TB 将对没有 BSL-3 实验室的实验室和临床试验测试新的抗结核药物组合在中低收入国家（LMICs）产生特别影响。
