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）： HIV 和结核分枝杆菌 (Mtb) 的双重感染被称为“致命的二重奏”，2010 年，在 880 万例结核病 (TB) 病例中，有 110 万例与结核分枝杆菌 (Mtb) 双重感染。艾滋病病毒。结核病 (TB) 仍然是头号细菌杀手，每 20 秒就有一个人死于这种不断被遗忘的流行病。由于艾滋病毒合并感染、耐多药结核分枝杆菌菌株的增加以及药物管道非常稀少，结核病的成功治疗已成为问题。目前治疗结核病的方案是利福平、异烟肼、乙胺丁醇和吡嗪酰胺（PZA）2个月，随后利福平和异烟肼4个月。这种“短程”方案的标志是 PZA 的存在，PZA 是一种前药，已被证明在治疗的初始阶段使用时可以改善灭菌效果。在最近的临床试验中，PZA 也被证明是有益的，新的结核病药物作为“治疗武器”，其中包括 PZA 可以更快地消毒并缩短整个治疗过程。然而，针对PZA的临床药敏试验一直不一致，美国以及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试验和治疗。南非最近的研究表明，这已被证明是一场临床噩梦，并报告了与耐多药结核病病例相关的耐药率超过 65%。这一发现以及通过临床试验推动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。现在人们普遍认为，PZA 抗性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嗪酸的酶。据信 pncA 突变与 PZA 抗性相关。已提出一种鉴定 pncA 突变的测定法来预测 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。然而，目前这是不可行的，因为已识别出超过 100 个跨越 pncA 基因的非同义突变，而且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库。在目标 2 中，我们将使用 pncA 集合中的基因分型 DNA 来开发和严格比较两个分子平台：高分辨率熔解曲线分析和开灯/关灯探针，以区分野生型 pncA 基因与具有遗传改变的基因在 2 小时内准确测定 PZA 敏感性。