Fast detection of Mycobacterium tuberculosis in culture-positive sputum samples by nitrate reductase activity

ABSTRACT Microscopy and bacterial culture are the main tools in the diagnosis of tuberculosis. Since the slow growth of Mycobacterium tuberculosis impairs rapid diagnosis strategies, especially in countries where the latter are the only available resources, the ongoing development of new and inexpensive tools based on mycobacterial metabolism optimizing growth detection with preliminary identification is greatly welcome. When compared to the other species from the M. tuberculosis complex, M. tuberculosis is a strong nitrate reducer. Current assay compares the nitrate reductase activity of M. tuberculosis from pulmonary specimens cultivated in nitrate-supplemented media. Fifty-five sputum samples were decontaminated and inoculated in conventional (Middlebrook 7H9, Ogawa Kudoh-OK) and in nitrate-supplemented media (Middlebrook 7H9-N, Ogawa Kudoh-N). An aliquot from the media directly reacted with Griess reagent (7H9-N and OK-N) every five days, or transferred to a nitrate substrate solution (7H9, OK). Nitrate to nitrite reduction was considered positive, revealed by the pink color, indicating bacterial growth. As reference method, the Mycobacteria Growth Indicator Tube (MGIT) was used for sensitivity calculations and statistical analysis. 7H9-N and OK-N assays proved to perform better in detecting M. tuberculosis than conventional assays (7H9 and OK). Indeed, broth nitrate-supplemented medium (7H9-N) was comparable to MGIT to detect M. tuberculosis, except in growth detection time. Results show that 7H9-N may be used as an alternative tool particularly in low-income countries since it is a simple and cheap technique, and does not restrict diagnosis to single-source products.

摘要：显微镜检查法与细菌培养是结核病诊断的两大核心手段。由于结核分枝杆菌（Mycobacterium tuberculosis）生长缓慢，掣肘了快速诊断策略的落地，尤其在仅能依赖此类手段的国家中，基于分枝杆菌代谢、优化生长检测并兼顾初步鉴定的新型低成本检测工具的持续研发，广受推崇。相较于结核分枝杆菌复合群（Mycobacterium tuberculosis complex）的其他菌种，结核分枝杆菌具备极强的硝酸盐还原能力。
本研究采用的检测方法旨在对比呼吸道标本来源的结核分枝杆菌在添加硝酸盐培养基中的硝酸盐还原酶活性。本次研究共收集55份痰液样本，经去污染处理后，分别接种于常规培养基（Middlebrook 7H9、Ogawa Kudoh-OK）与添加硝酸盐培养基（Middlebrook 7H9-N、Ogawa Kudoh-N）中。每间隔5日，取添加硝酸盐培养基（7H9-N与OK-N）的等分培养液直接与格里斯试剂（Griess reagent）反应；而常规培养基（7H9、OK）的培养液则需先转移至硝酸盐底物溶液后再进行检测。当硝酸盐还原为亚硝酸盐的反应呈现粉红色时，判定为阳性结果，提示存在细菌生长。
本研究以分枝杆菌生长指示管（Mycobacteria Growth Indicator Tube，MGIT）作为参考方法，用于敏感性计算与统计学分析。实验结果显示，添加硝酸盐的培养基检测体系（7H9-N与OK-N）在结核分枝杆菌检测效能上优于常规培养基体系（7H9与OK）。具体而言，液体添加硝酸盐培养基（7H9-N）的结核分枝杆菌检测效能与MGIT相当，仅在生长检测时长上存在差异。研究结果表明，7H9-N体系可作为结核病诊断的替代方案，尤其适用于低收入国家：该技术操作简便、成本低廉，且无需局限于单一来源的检测产品。