Rapid Susceptibility Testing of MDR M. tuberculosis- Phase II

描述（由申请人提供）：本项目的长期目标是开发一种基于高效液相色谱（HPLC）的系统，用于快速准确地检测结核分枝杆菌（MTB）的抗生素敏感性。暴露于抗MTB抗生素的MTB的敏感菌株和抗性菌株在分枝菌酸含量方面表现出显著差异。初步研究将侧重于使基于HPLC的敏感性测试适应当前市售的生长/敏感性系统。Mycobacterium Sherlock（r）Mycobacteria Identification System是一种基于HPLC的系统，已获得FDA 510（k）许可，可用于鉴定MTB。将修改MTBSherlock软件以允许自动计算来自“对照”或来自含分枝杆菌的培养基的MTB菌株的分枝菌酸含量的比率。尽管异烟肼导致分枝菌酸损失35-40%，但在敏感菌株中，暴露于抑制剂仅20分钟，利福平、乙胺丁醇和吡嗪酰胺可能需要长达72小时才能进行最佳测定。这与其他自动化敏感性测试系统所需的典型6-9天形成对比。由于在分析中观察到MTB分枝菌酸的高度特异性模式，因此非分枝杆菌物种的污染不会导致错误的耐药性评估，从而解决了其他自动化系统中的严重问题。将评估“混合培养”算法在识别非结核分枝杆菌污染方面的有效性。初步研究将使用市售培养基和抗MTB抗生素，通过分枝菌酸含量分析确定耐药性或敏感性。这将促进具有基于生长的系统的实验室采用分枝菌酸测试。尽管对项目的成功并不重要，但也将评价用于结核分枝杆菌鉴定和药敏试验的直接痰液分析。

DESCRIPTION (provided by applicant): The long-term objective of this project is the development of a system based on high performance liquid chromatography (HPLC) for rapid and accurate antibiotic susceptibility testing of Mycobacterium tuberculosis (MTB). Susceptible and resistant strains of MTB, exposed to anti-MTB antibiotics, exhibit pronounced differences in mycolic acid content. Initial research will focus on adapting the HPLC-based susceptibility testing to current commercially available growth/susceptibility systems. The MIDI Sherlock(r) Mycobacteria Identification System is an HPLC-based system that has FDA 510(k) clearance for identification of MTB. The MIDI Sherlock software will be modified to allow for automated calculation of ratios of mycolic acid content of MTB strains from "control" or from antibiotic-containing media. Although isoniazid causes 35-40% loss of mycolic acids, in susceptible strains, with as little as 20 minutes exposure to the inhibitor, rifampin, ethambutol and pyrazinamide may require up to 72 h for optimal determination. This is in contrast to the typical 6-9 days required for other automated susceptibility test systems. As the highly specific pattern of mycolic acids of MTB is seen in the analysis, contamination by non-mycobacterial species do not cause false resistance evaluations, thus solving a serious problem in other automated systems. A "mixed culture" algorithm will be evaluated for effectiveness in identifying contamination by non-TB mycobacteria. Initial research will use commercially available media and anti-MTB antibiotics to determine resistance or susceptibility by mycolic acid content analysis. This will facilitate adoption of the mycolic acid test by laboratories having the growth-based systems. Although not crucial to the success of the project, direct-from-sputum analysis for MTB identification and susceptibility testing will also be evaluated.