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 (TB) 威胁到我们到 2035 年结束全球结核病流行的目标。 早期诊断耐药结核病并及时开始适当的治疗仍然是一个难题 表型药物敏感性测试（DST）需要结核分枝杆菌。 (MTB) 培养需要 8-12 周，基于分子的方法速度很快，但需要提前进行。 不幸的是，突变并非如此。 因新的全口服疗法中包含的关键药物而闻名，我们的长期目标是 开发一种基于非培养的表型 DST 方法，可提供 48-72 的结果 距离痰液采集仅数小时，可直接用于痰液。 该方法基于 MPT64，这是一种仅在活跃生长期间分泌的高度特异性蛋白质 对于 MTB，我们证明 MTB 培养物中的 MPT64 分泌在体内显着减少。 对于药物敏感但不耐药的 MTB 分离株，暴露 48-72 小时的抗结核药物。 我们之前直接在痰样本中测量了 MPT64，但检测限有限 在这项研究中，我们将与 Xpert MTB/RIF Ultra（Cepheid，桑尼维尔，加利福尼亚州，美国）进行比较。 验证异烟肼培养物 MPT64 DST 化学发光-酶免疫分析 (CLEA) (INH)、利福平 (RIF)、氟喹诺酮类 (FQ)、贝达喹啉 (BDQ)、利奈唑胺 (LZD) 和德拉马尼 (DLM)。我们提出的方法将在培养后 72 小时内提供结果。 与传统 DST 需要 2-3 周才能获得结果相比，被确定为 MTB （目标1）我们将利用MPT64 DST培养原理来开发痰MPT64 DST。 CLEIA，将在采集痰液后 48-72 小时内提供结果，我们将进行鉴定。 痰液液化、抗生素暴露时间和 CLEIA 方案可实现最低 耐药 MTB 细胞的检测限 (LOD)（目标 2）。 对菲律宾和格鲁吉亚的 860 名结核病患者进行 MPT64 药敏试验（目标 3）。 坚持认为培养物 MPT64 DST 和痰 MPT64 DST 将达到或超过最低要求 敏感性 (>95%) 和特异性 (>98%)，这是 WHO 高度优先的目标产品概况 创新的 MPT64 DST 检测将通过启用来改变结核病管理。 根据快速药敏数据及早开始有效治疗，从而改善患者病情 如果要通过以下途径消除结核病，这一点至关重要： 2035.