Innovating anti-tuberculosis drug susceptibility testing with a novel and rapid non-culture based phenotypic test using MPT64 biomarker

使用 MPT64 生物标志物，通过新型、快速的非培养表型测试来创新抗结核药物敏感性测试

• 批准号: 10663034
• 负责人: Midori Kato-Maeda
• 金额: $ 67.68万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-04 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10663034
• 关键词: Address; Antibiotics; Antitubercular Agents; Biological Assay; Biological Markers; Cells; Clinical; Clinical Sensitivity; Collection; Colony-forming units; Country; Culture Media; DNA Sequence Alteration; Data; Detection; Diagnosis; Drug Exposure; Drug resistance; Drug resistance in tuberculosis; Early Diagnosis; Enzyme Immunoassay; Epidemic; Fluoroquinolones; Goals; Growth; Hour; Infrastructure; Intermediate resistance; Isoniazid resistance; Japan; Laboratories; Linezolid; Liquid substance; Measures; Methods; Molecular; Mutation; Mycobacterium tuberculosis; Oral; Patient-Focused Outcomes; Patients; Performance; Pharmaceutical Preparations; Phenotype; Philippines; Predisposition; Protein Secretion; Protocols documentation; Pulmonary Tuberculosis; Regimen; Resistance; Resistance profile; Rifampin; Sampling; Sensitivity and Specificity; Specificity; Speed; Sputum; Testing; Time; Tuberculosis; Validation; World Health Organization; biobank; detection limit; effective therapy; immunogenic; improved; in vitro Assay; innovation; isoniazid; novel; novel therapeutics; prospective; rapid technique; rapid test; tuberculosis drugs; tuberculosis treatment

Summary/Abstract: 30 lines Drug-resistant tuberculosis (TB) threatens our goal of ending the global TB epidemic by 2035. Early diagnosis of drug-resistant TB for timely initiation of appropriate treatment remains a challenge. Phenotypic drug susceptibility testing (DST) requires Mycobacterium tuberculosis (MTB) culture and takes 8-12 weeks. Molecular-based methods are rapid but require previously identified and validated resistance-conferring mutations. Unfortunately, mutations are not completely known for key drugs included in newer all-oral regimens. Our long-term goal is to develop a non-culture-based method for phenotypic DST that provides results in 48-72 hours from sputum collection and that can be used directly on sputum. Our innovative approach is based on MPT64, a protein secreted only during active growth that is highly specific for MTB. We demonstrated that MPT64 secretion in MTB cultures decreases significantly within 48–72 hours of anti-TB drug exposure for drug-susceptible but not drug-resistant MTB isolates. We have previously measured MPT64 directly in sputum samples with a limit of detection comparable with Xpert MTB/RIF Ultra (Cepheid, Sunnyvale, CA, USA). In this study we will validate culture MPT64 DST chemiluminescence-enzyme immunoassay (CLEA) for isoniazid (INH), rifampin (RIF), fluoroquinolones (FQs), bedaquiline (BDQ), linezolid (LZD), and delamanid (DLM). Our proposed approach will provide results within 72 hours of the time the culture is identified as MTB, compared to the 2-3 weeks required to obtain results from conventional DST (Aim 1). We will use the principles of culture MPT64 DST to develop a sputum MPT64 DST CLEIA, which will provide results in 48-72 hours from the time of sputum collection. We will identify protocols for sputum liquefaction, antibiotic exposure times, and CLEIA that result in the lowest limit of detection (LOD) for resistant MTB cells (Aim 2). We will prospectively evaluate sputum MPT64 DST in 860 patients with TB in the Philippines and in the country of Georgia (Aim 3). We hypothesize that both culture MPT64 DST and sputum MPT64 DST will meet or exceed minimum sensitivity (>95%) and specificity (>98%), which are the WHO high priority target product profiles for rapid DST. The innovative MPT64 DST assays will transform TB management by enabling early initiation of effective treatment based on rapid drug susceptibility data, thus improving patient outcomes and curtailing the spread of drug resistant TB that is critical if TB is to be eliminated by 2035.

摘要/摘要：30行 耐药结核病威胁着我们到2035年结束全球结核病流行的目标。 早期诊断耐药结核病以及时开始适当的治疗仍然是一个重要的问题。 挑战.表型药敏试验（DST）需要结核分枝杆菌 (MTB)培养8 - 12周。基于分子的方法是快速的，但需要预先 鉴定并验证了耐药突变。不幸的是，突变并不是 以新的全口服方案中的关键药物而闻名。我们的长期目标是 开发一种基于非培养物的表型DST方法，其结果为48 - 72 从痰液收集开始数小时，并且可以直接用于痰液。我们的创新 一种基于MPT64的方法，MPT64是一种仅在活跃生长期间分泌的高度特异性的蛋白质 关于MTB我们证明了MTB培养物中MPT 64的分泌在一定时间内显著降低， 对药物敏感但不耐药的MTB分离株暴露于抗TB药物48 - 72小时。 我们以前直接在痰液样本中测量过MPT 64，检测限为 与Xpert MTB/RIF Ultra（Cepheid，桑尼维尔，CA，USA）相当。在这项研究中，我们将 验证异烟肼的培养物MPT64 DST荧光-酶免疫测定法（CLEA） (INH)、利福平（RIF）、氟喹诺酮类（FQs）、贝达喹啉（BDQ）、利奈唑胺（LZD）和地拉曼胺 （DLM）中指定的值。我们提出的方法将在培养后72小时内提供结果。 与传统DST获得结果所需的2 - 3周相比， (Aim 1）。我们将利用培养MPT64 DST的原理，开发痰液MPT64 DST CLEIA将在痰液采集后48 - 72小时内提供结果。我们将确定 痰液液化、抗生素暴露时间和CLEIA的方案， 耐药MTB细胞的检测限（LOD）（目的2）。我们将前瞻性评估痰液 在菲律宾和格鲁吉亚国家的860名结核病患者中进行MPT 64 DST（目标3）。我们 假设培养MPT64 DST和痰MPT64 DST都将达到或超过最小值 敏感性（> 95%）和特异性（> 98%），这是世卫组织高度优先的目标产品特征 快速DST创新的MPT64 DST检测将通过以下方式改变结核病管理： 根据快速药物敏感性数据及早启动有效治疗，从而改善患者 结果和遏制耐药结核病的传播，这是至关重要的，如果要消除结核病， 2035.