Development of a dual-iDDS probe multiplex test for detecting endemic fungal agents

开发用于检测地方性真菌因子的双 iDDS 探针多重测试

• 批准号: 10591947
• 负责人: DAVID A SHAFER
• 金额: $ 20.7万
• 依托单位: GENETAG TECHNOLOGY, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-06 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10591947
• 关键词: Acute Respiratory Distress Syndrome; Adult; Amphotericin B; Anatomy; Aspergillus; Biological Assay; Blastomyces; Blastomyces dermatitidis; Blastomycosis; CLIA certified; Candida; Certification; Cessation of life; Characteristics; Child; Chronic; Clinical; Clinical Pathology; Coccidioides; Coccidioides immitis; Coccidioides posadasii; Coccidioidomycosis; Communicable Diseases; Cryptococcus; DNA; Detection; Development; Diagnosis; Diagnostic; Diagnostic tests; Differential Diagnosis; Disease; Doctor of Philosophy; Drug resistance; Ensure; FDA approved; Fluconazole; Fluorescence; Freeze Drying; Future; Health Benefit; Healthcare; Histoplasma; Histoplasma capsulatum; Histoplasmosis; Hospitals; Immunocompetent; Immunocompromised Host; Individual; Infection; Intervention; Itraconazole; Label; Laboratories; Life; Liquid substance; Lung diseases; Lymphoma; Malignant Neoplasms; Malignant neoplasm of lung; Manufacturer; Marketing; Medical; Meningeal; Methods; Molecular; Molecular Diagnostic Testing; Monitor; Morbidity - disease rate; Mycoses; Pathway interactions; Patients; Performance; Pericarditis; Phase; Physicians; Pneumonia; Public Health; Pulmonary Coin Lesion; Rationalization; Reflex action; Reporting; Research Contracts; Resistance; Routine Diagnostic Tests; Sampling; Sarcoidosis; Serology; Site; Specialist; Specificity; Specimen; Symptoms; System; Technology; Test Result; Testing; Thoracic Radiography; Time; Tube; Validation; accurate diagnosis; color detection; commercialization; cost; cross reactivity; desert fever; design; detection limit; diagnostic assay; effective therapy; experience; flu; instrument; locked nucleic acid; mortality; multiplex diagnostics; pathogen; patient subsets; prevent; resistance mutation; respiratory; success

Development of a dual-iDDS probe multiplex test for detecting endemic fungal agents Confidential PI: Shafer, David A., PhD PROJECT SUMMARY In this study, we propose developing a simple, lyophilized molecular test for detecting the causative agents of Valley fever (coccidioidomycosis), histoplasmosis, or blastomycosis, based on our proprietary internal DNA- Detection Switch (iDDS) probe technology. Timely diagnosis of these fungal infections is key for selecting effective treatments and preventing severe morbidity or death. Over 500,000 such cases are thought to occur in the US annually, although only one FDA-approved molecular test exists for diagnosing Valley fever, and no FDA- approved molecular tests exist for diagnosing histoplasmosis or blastomycosis. The symptoms of coccidioidomycosis, histoplasmosis, and blastomycosis are frequently vague and chest X-rays do not distinguish between these and other lung diseases. Thus, differential diagnosis between these diseases and other that present with similar symptoms (acute respiratory distress syndrome, lung cancer, lymphoma, pericarditis, sarcoidosis, or solitary pulmonary nodules) is key for proactive healthcare interventions. Moreover, diagnosis using culture- or serology-based methods may have a turnaround time of weeks or months, or show false-positive results, and the only related FDA-approved test (the GeneSTAT.MDx Coccidioides Assay) is only approved for use on the manufacturer’s instrument, which dissuades from use at reference labs. GeneTAG Technology (www.genetagtech.com) specializes in developing sensitive and reliable qPCR probe systems. Our error-resistant iDDS probe system employs a fluorescently labeled target specific probe and a slightly mismatched quencher-labeled antiprobe. In the absence of the intended target, the antiprobe hybridizes to the probe, quenching its fluorescence and preventing off-target detection. We have also developed dual-iDDS probe assays for increased specificity and automatic confirmation of the test results. This added layer of specificity is key for accurate diagnosis, especially with low levels of fungal infection. Redundant detection at multiple target sites greatly reduces the need for reflex/confirmatory testing. Our Specific Aims are (1) to develop a 3-tube dual-iDDS probe assay for C. immitis, C. posadasii, H. capsulatum, and B. dermatitidis, and (2) to evaluate key performance characteristics of the multiplex test for endemic fungal agents in lyophilized format. Specifically, in Aim 1, we will (i) develop locked nucleic acid (LNA)-enhanced dual-iDDS probes for diagnostic sequence-specific targets in each pathogen, (ii) convert the dual-iDDS probes to lyophilized format, and (iii) conduct cross-reactivity testing with the lyophilized assays to assess false-positive detection of other pathogens commonly found in bronchoalveolar fluid. In Aim 2, we will determine the (i) linearity, (ii) limit of detection (LoD), (iii) precision, (iv) effects of interfering substances (if any), and (v) stability of the dual-iDDS probe assays. Success in this endeavor will justify expanded testing with clinical specimens in Phase II through a BSL III- certified contract research organization.

开发用于检测地方性真菌因子的双 iDDS 探针多重测试机密 PI：Shafer，David A. 博士 项目概要 在这项研究中，我们建议开发一种简单的冻干分子测试来检测病原体 山谷热（球孢子菌病）、组织胞浆菌病或芽生菌病，基于我们专有的内部 DNA- 检测开关 (iDDS) 探头技术。及时诊断这些真菌感染是选择的关键 有效的治疗并预防严重的发病或死亡。据认为，超过 500,000 起此类案件发生在 美国每年都会进行一次，尽管只有一项经 FDA 批准的用于诊断谷热的分子测试，并且没有 FDA- 已批准的分子检测可用于诊断组织胞浆菌病或芽生菌病。 球孢子菌病、组织胞浆菌病和芽生菌病的症状通常不明确，胸部 X 线检查 不区分这些和其他肺部疾病。因此，这些疾病之间的鉴别诊断 以及其他表现出类似症状的疾病（急性呼吸窘迫综合征、肺癌、淋巴瘤、 心包炎、结节病或孤立性肺结节）是主动医疗干预的关键。而且， 使用基于培养或血清学的方法进行诊断可能需要数周或数月的周转时间，或者显示 假阳性结果，并且 FDA 批准的唯一相关测试（GeneSTAT.MDx Coccidioides Assay）仅 批准在制造商的仪器上使用，这会阻止在参考实验室中使用。 GeneTAG Technology (www.genetagtech.com) 专注于开发灵敏可靠的 qPCR 探针 系统。我们的防错 iDDS 探针系统采用荧光标记的目标特异性探针和 稍微不匹配的猝灭剂标记的反探针。在没有预期目标的情况下，反探针杂交 到探针上，猝灭其荧光并防止脱靶检测。我们还开发了双iDDS 探针测定可提高特异性并自动确认测试结果。这增加了一层 特异性是准确诊断的关键，尤其是在真菌感染水平较低的情况下。冗余检测 多个目标位点大大减少了反射/确认测试的需要。我们的具体目标是 (1) 发展 对 C. immitis、C. posadasii、H. capsulatum 和 B. dermatitidis 进行 3 管双 iDDS 探针测定，以及 (2) 评估冻干形式地方性真菌剂多重测试的关键性能特征。 具体来说，在目标 1 中，我们将 (i) 开发用于诊断的锁核酸 (LNA) 增强型双 iDDS 探针 每种病原体中的序列特异性靶标，(ii) 将双 iDDS 探针转化为冻干形式，以及 (iii) 使用冻干检测进行交叉反应测试，以评估其他病原体的假阳性检测 常见于支气管肺泡液中。在目标 2 中，我们将确定 (i) 线性，(ii) 检测限 (LoD)， (iii) 精度，(iv) 干扰物质的影响（如果有），以及 (v) 双 iDDS 探针检测的稳定性。 这一努力的成功将证明通过 BSL III 在第二阶段扩大临床样本测试是合理的。 经认证的合同研究组织。