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(申请人提供)：艾滋病毒和结核分枝杆菌(结核分枝杆菌)的混合感染已被称为“致命的二重奏”，2010年，在880万结核病(TB)病例中，有110万人同时感染艾滋病毒。结核病(TB)仍然是头号细菌杀手，每20秒就有一人死于这种不断被遗忘的大流行。由于与艾滋病毒合并感染、耐多药结核分枝杆菌菌株的增加以及非常稀少的药物供应，结核病的成功治疗变得困难。目前的结核病治疗方案是2个月的利福平、异烟肼、乙胺丁醇和吡嗪酰胺(PZA)，然后是4个月的利福平和异烟肼。这一“短程”方案的特点是存在PZA，这是一种前药物，已被证明在治疗的初始阶段使用时可以改善灭菌效果。PZA在最近的临床试验中也被证明是有益的，将新的结核病药物作为“治疗武器”，包括PZA更快地消毒和缩短整个疗程。然而，对PZA的临床药敏测试一直是不一致的，美国和结核病和艾滋病毒负担沉重的国家的许多实验室甚至不进行经验性的测试和治疗。这已被证明是一场临床噩梦，因为最近来自南非的研究报告了与耐多药结核病病例有关的65%以上的耐药率。这一发现和希望通过临床试验开发新的结核病药物的愿望提高了开发一种快速而准确的分子方法来确定PZA敏感性的紧迫性。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，以及将前药PZA转化为其活性形式吡津酸的酶。PncA基因突变可能与PZA耐药有关。已经提出了一种检测pncA突变的方法来预测PZA的耐药性，类似于检测rpoB突变来预测利福平耐药性的方法。然而，这目前是不可行的，因为已经发现了100多个跨越pncA基因的非同义突变，我们不知道哪些基因变化与耐药性相对应。目前的挑战，也是这项提案的重点，是通过采取一种创新的分子方法来筛选野生型pncA基因作为PZA易感性的预测因子来克服这些障碍。在目标1中，我们将与疾控中心合作，围绕pncA基因建立一个全面的结核分枝杆菌临床菌株集合。在目标2中，我们将使用来自pncA收集的基因分型DNA来开发和严格比较两个分子平台：高分辨率熔融曲线分析和点亮/熄灭探针，以区分野生型pncA基因和具有遗传改变的基因，并在不到2小时内准确地确定PZA的易感性。