A rapid molecular approach to determine PZA susceptibility

确定 PZA 敏感性的快速分子方法

• 批准号: 8709716
• 负责人: BARRY Neal KREISWIRTH
• 金额: $ 22.75万
• 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/8709716
• 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 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）的联合感染被称为“致命的二重唱”，2010年，在880万例结核病（TB）病例中，110万例合并感染了艾滋病毒。结核病仍然是头号细菌杀手，因为每20秒就有一人死于这种不断被遗忘的流行病。由于合并感染艾滋病毒，耐多药结核菌株的增加以及药物渠道非常稀少，成功治疗结核病已成为问题。目前的结核病治疗方案是利福平、异烟肼、乙胺丁醇和吡嗪酰胺（PZA）治疗2个月，然后是利福平和异烟肼治疗4个月。这种“短期”治疗方案的特点是使用PZA，这是一种前药，在治疗的初始阶段使用时已被证明可以改善消毒。在最近的结核病新药临床试验中，PZA也被证明是有益的，作为包括PZA在内的“治疗武器”，PZA可以更快地消毒并缩短整个治疗过程。然而，对PZA的临床敏感性检测一直不一致，美国和结核病和艾滋病毒高负担国家的许多实验室甚至没有进行经验性检测和治疗。事实证明，这是一场临床噩梦，因为最近来自南非的研究报告称，耐多药结核病病例的耐药率超过65%。这一发现以及推动结核病新药通过临床试验的愿望提高了开发一种快速准确的分子方法来确定PZA易感性的紧迫性。现在人们普遍认为PZA耐药的靶标是编码吡嗪酰胺酶的基因pncA，该酶可将PZA前药转化为活性形式吡嗪酸。人们认为pncA突变与PZA耐药有关。已经提出了一种检测pncA突变来预测PZA耐药性的方法，类似于检测rpoB突变来预测利福平耐药性的方法。然而，这目前还不可行，因为有超过100个跨pncA基因的非同义突变被发现，我们不知道哪些遗传变化与抗性相对应。目前的挑战，也是本提案的重点，是通过采用创新的分子方法来筛选野生型pncA基因作为PZA易感性的预测因子，从而克服这些障碍。在目标1中，我们将与CDC合作，围绕pncA基因型建立一个全面的结核分枝杆菌临床菌株收集。在目标2中，我们将使用来自pncA收集的基因型DNA来开发和严格比较两种分子平台：高分辨率熔融曲线分析和开灯/关灯探针，以区分野生型pncA基因和具有遗传改变的基因，并在不到2小时内准确确定PZA易感性。