A rapid molecular approach to determine PZA susceptibility

确定 PZA 敏感性的快速分子方法

• 批准号: 8667400
• 负责人: BARRY Neal KREISWIRTH
• 金额: $ 20.2万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8667400
• 关键词: Air; Antitubercular Agents; Area; Biological Assay; Cataloging; Catalogs; Centers for Disease Control and Prevention (U.S.); Clinical; Clinical Trials; Codon Nucleotides; Collaborations; Collection; Communities; Complex; Country; DNA; DNA Sequence; Diagnosis; Disease; Drug Resistant Tuberculosis; Enrollment; Enzymes; Epidemiology; Ethambutol; Extreme drug resistant tuberculosis; Fluorescence; Gene Targeting; Genes; Genotype; HIV; HIV Infections; HIV therapy; Hour; Human; India; Infection; Laboratories; Length; Methodology; Molecular; Multi-Drug Resistance; Multidrug-Resistant Tuberculosis; Mutation; Mycobacterium tuberculosis; Nightmare; Patient Care; Patients; Pharmaceutical Preparations; Phase; Phase II Clinical Trials; Phase III Clinical Trials; Population; Predisposition; Prodrugs; Protease Inhibitor; Proteins; Protocols documentation; Pyrazinamide; Pyrazinamide resistance; Reporting; Reproducibility; Research; Research Institute; Resistance; Resolution; Resources; Reverse Transcriptase Polymerase Chain Reaction; Rifampicin resistance; Rifampin; Role; Running; South Africa; Speed; Sterilization; Technology; Temperature; Testing; Treatment Protocols; Tuberculosis; Work; arm; cost; design; disorder prevention; drug discovery; enzyme activity; forgetting; improved; innovation; isoniazid; melting; mutant; novel; pandemic disease; pathogen; public health relevance; public health research; pyrazinamide deamidase; pyrazinoic acid; repository; research clinical testing; resistant strain; success; tool; tuberculosis drugs; tuberculosis treatment

DESCRIPTION (provided by applicant): The co-infection of HIV and Mycobacterium tuberculosis (Mtb) has been termed the "deadly duet" and in 2010, among the 8.8 million incident cases of tuberculosis (TB), 1.1 million were co-infected with HIV. Tuberculosis (TB) remains the number one bacterial killer as every 20 seconds a human dies of this continually forgotten pandemic. As a result of the co-infection with HIV, the rise in multidrug resistant Mtb strains and the very sparse drug pipeline, successful treatment of TB has become problematic. The current TB treatment regimen for TB is 2 months of rifampin, isoniazid, ethambutol and pyrazinamide (PZA), followed by 4 months of rifampin and isoniazid. The hallmark of this "short-course" protocol is the presence of PZA, a pro-drug that has been shown to improve sterilization when used in the initial phase of treatment. PZA has also proven beneficial in recent clinical trials with new TB drugs as "treatment arms" that include PZA sterilize more quickly and shorten the overall course of therapy. However, the clinical susceptibility testing against PZA has always been inconsistent and many laboratories in the US and in countries with a high burden of TB and HIV do not even test and treat empirically. This has proven to be a clinical nightmare as recent studies from South Africa and have reported over 65% resistance rates associated with MDR tuberculosis cases. This finding and the desire to move new TB drugs through clinical trials have elevated the urgency of developing a rapid and accurate molecular approach to determine PZA susceptibility. It is now well accepted that the target for PZA resistance is the gene pncA which encodes for pyrazinamidase, and enzyme that converts the pro- drug PZA to its active form, pyrazinoic acid. It is believed that mutation in pncA will correlate with PZA resistance. An assay to identify the pncA mutations has been proposed to predict PZA resistance, similar to the approach being used to detect rpoB mutations to predict rifampin resistance. However, this is currently not feasible, as there are over 100 non-synonymous mutations identified that span the pncA gene and we do not know which genetic changes correspond with resistance. The present challenge, and the focus of this proposal, is to overcome these hurdles by taking an innovative molecular approach to screen for the wild type pncA gene as a predictor of PZA susceptibility. In Aim 1, in collaboration with the CDC, we will build a comprehensive M. tuberculosis clinical strain collection curated around the pncA genotype. In Aim 2, we will use genotyped DNA from the pncA collection to both develop and rigorously compare two molecular platforms: high resolution melt curve analysis and lights-on/lights-off probes to distinguish the wild type pncA gene from those with genetic alterations and accurately determine PZA susceptibility in less than 2 hours.

描述（由申请人提供）：艾滋病毒和结核分枝杆菌（MTB）的共同感染已被称为“致命二重奏”，在2010年，在880万例结核病事件（TB）中，有110万例与HIV共同感染。结核病（TB）仍然是第一个细菌杀手，因为每20秒就会死于这种不断被遗忘的大流行。由于与HIV的共同感染，多药抗MTB菌株的增加和非常稀疏的药物管道，成功的结核病治疗已成为问题。 TB的当前结核病治疗方案是利福平，异烟肼，乙酰胺醇和吡嗪酰胺（PZA）的2个月，其后为4个月的利福平和异尼氏酶。该“短道”方案的标志是PZA的存在，PZA的存在，这是一种在治疗初始阶段使用时已证明可以改善灭菌的情况。 PZA在最近使用新结核病药物的临床试验中证明了有益的“治疗组”，其中包括PZA对PZA进行更快的量并缩短整体治疗过程。但是，针对PZA的临床敏感性测试一直是不一致的，在美国和TB和HIV负担很高的国家中，许多实验室甚至都无法进行经验测试和治疗。事实证明，这是南非最近研究的临床噩梦，并且报告了与MDR结核病病例相关的65％阻力率。这一发现以及通过临床试验转移新结核病药物的愿望提高了开发快速准确的分子方法来确定PZA敏感性的紧迫性。现在，PZA耐药性的靶标是编码吡嗪酰胺酶的基因PNCA，以及将促PZA转化为其活性形式的吡唑酸的基因PNCA。据认为，PNCA中的突变将与PZA耐药性相关。已经提出了一种鉴定PNCA突变的测定方法来预测PZA耐药性，类似于用于检测RPOB突变以预测利福平耐药性的方法。但是，目前这是不可行的，因为跨越PNCA基因的100多个非同义突变，我们不知道哪些遗传变化与抗性相对应。目前的挑战以及该提案的重点是通过采用创新的分子方法来筛选野生型PNCA基因作为PZA敏感性的预测指标来克服这些障碍。在AIM 1中，与CDC合作，我们将建立一个围绕PNCA基因型策划的全面结核分枝杆菌临床收集。在AIM 2中，我们将使用基因分型DNA从PNCA集合中进行开发和严格比较两个分子平台：高分辨率熔体曲线分析和点亮/照明探针，以将野生型PNCA基因与具有遗传变化的野生型PNCA基因区分开来，并在不到2个小时内准确地确定PZA易感性。