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，及时开始适当治疗仍是一项重要任务 挑战。表型药敏试验(DST)需要结核分枝杆菌 (MTB)培养，需要8-12周。基于分子的方法是快速的，但以前需要 识别和验证导致抗药性的突变。不幸的是，突变并不是 完全以包括在较新的全口服疗法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的表型DST方法，在48-72年中提供结果 从收集痰的几个小时开始，可以直接用于痰。我们的创新 该方法基于MPT64，一种只有在活跃的生长过程中才分泌的高度特异的蛋白质 对于MTB。我们证明在结核分枝杆菌培养中MPT64的分泌在 对药物敏感但不耐药的结核分枝杆菌分离株进行48-72小时的抗结核药物暴露。 我们以前曾在痰样本中直接检测MPT64，检测限为 可与Xpert MTB/RIF Ultra(美国加利福尼亚州森尼维尔Cepheid)进行比较。在这项研究中，我们将 异烟肼培养MPT64 DST化学发光-酶免疫分析(CLEA)方法的验证 (INH)、利福平(RIF)、氟喹诺酮(FQS)、贝达奎兰(BDQ)、利奈唑胺(LZD)和地拉曼 (DLm)。我们建议的方法将在培养时间的72小时内提供结果 确认为结核分枝杆菌，而从常规DST获得结果需要2-3周 (目标1)。我们将利用培养MPT64 DST的原理来开发一种痰MPT64 DST CLEIA将在48-72小时内提供结果，从收集痰的时间起算。我们将确定 痰液化、抗生素暴露时间和CLEIA的方案导致最低 耐药结核杆菌细胞的检测限(LOD)(目标2)。我们将前瞻性地评估痰 菲律宾和格鲁吉亚860名结核病患者的MPT64 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(&gt；95%)和特异性(&gt；98%)，这是世卫组织高度优先的目标产品配置文件 用于快速DST。创新的MPT64 DST检测将通过以下方式改变结核病管理 根据快速药敏数据及早开始有效治疗，从而改善患者状况 结果和遏制耐药结核病的传播，如果要通过以下方式消除结核病，这是至关重要的 2035年。