Detection of Prions

朊病毒检测

• 批准号: 9161647
• 负责人: BYRON CAUGHEY
• 金额: $ 94.47万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9161647
• 关键词: Affect; Agriculture; Animals; Biological Assay; Bovine Spongiform Encephalopathy; Cattle; Cerebrospinal Fluid; Chronic Wasting Disease; Clinic; Collaborations; Conversion disorder; Creutzfeldt-Jakob Syndrome; Detection; Development; Diagnosis; Diagnostic; Diagnostic Sensitivity; Discrimination; Disease; Early Diagnosis; Generations; Human; Infection; Life; Microtus; Neurodegenerative Disorders; Nose; Olfactory Mucosa; Patients; Peptide Hydrolases; Performance; Prion Diseases; Prions; Recombinants; Relative (related person); Reporting; Research; Resistance; Rodent; Sampling; Scrapie; Seeds; Sheep; Source; Specificity; Specimen; Speed; Testing; Time; Variant; base; brain tissue; cervid; disease diagnosis; improved; polymerization; prion seeds; protein misfolding cyclic amplification; rapid detection

1) We have furthered the development of a test for Creutzfeldt-Jakob disease (CJD) using nasal brushings with Gianluigi Zanusso and other collaborators because definite diagnosis of sporadic CJD in living patients remains a challenge. In our previous study The RT-QuIC assays seeded with nasal brushings were positive in 30 of 31 patients with CJD but were negative in 43 of 43 patients without CJD, indicating a sensitivity of 97% and specificity of 100% for the detection of CJD. By comparison, testing of cerebrospinal fluid (CSF) samples from the same group of patients had a sensitivity of 77% and a specificity of 100%. Since our initial report, we have collected these two specimen types simultaneously from 12 additional patients, with a sensitivity of 100% from olfactory mucosa (12 of 12 samples) and 83% from cerebrospinal fluid (10 of 12 samples). Thus, our current overall diagnostic performance for RT-QuIC of nasal brushings is 42 positive out of 43 CJD patients (98%) diagnostic sensitivity and 0 positive out of 43 non-CJD patients (100%) specificity. 2) In collaborations with Italian, Canadian and US colleagues, we have also improved our CSF-based testing for CJD using RT-QuIC. Previous RT-QuIC testing of human CSF has required 2.5 to 5 days. Our improved RT-QuIC assay identifies positive CSF samples within 4 to 14 h with better analytical sensitivity. Moreover, analysis of 11 CJD patients demonstrated that while 7 were RT-QuIC positive using the previous conditions, 10 were positive using the new assay. In these and further analyses, a total of 46 of 48 CSF samples from sporadic CJD patients were positive, while all 39 non-CJD patients were negative, giving 95.8% diagnostic sensitivity and 100% specificity. This second-generation RT-QuIC assay markedly improves the speed and sensitivity of detecting prion seeds in CSF specimens from CJD patients. This should enhance prospects for rapid and accurate ante mortem CJD diagnosis. 3) Statutory surveillance of bovine spongiform encephalopathy (BSE) indicates that cattle are susceptible to both classical BSE (C-BSE) and atypical forms of BSE. Atypical forms of BSE appear to be sporadic and thus may never be eradicated. A major challenge for prion surveillance is the lack of sufficiently practical and sensitive tests for routine BSE detection and strain discrimination. As noted above, the RT-QuIC test was known to be highly specific and sensitive for the detection of multiple human and animal prion diseases but not BSE. Collaborating with the Cristina Casalone and others, we have now tested brain tissue from cattle affected by C-BSE and atypical L-type bovine spongiform encephalopathy (L-type BSE or L-BSE) with the RT-QuIC assay and found that both BSE forms can be detected and distinguished using particular rPrPSen substrates. Specifically, L-BSE was detected using multiple rPrPSen substrates, while C-BSE was much more selective. This substrate-based approach suggests a diagnostic strategy for specific, sensitive, and rapid detection and discrimination of at least some BSE forms. 4) Because prions propagate as multiple strains in a wide variety of mammalian species, the detection of all such strains by a single ultrasensitive assay such as RT-QuIC would facilitate prion disease diagnosis, surveillance and research. In a collaboration with Romolo Nonno, Wenquan Zou, Bernadino Ghetti and Pierluigi Gambetti, we have found that bacterially expressed recombinant bank vole prion protein (residues 23-230) is an effective substrate for the sensitive RT-QuIC detection of all of the different prion types that we have tested so far--a total of 28 from humans, cattle, sheep, cervids and rodents, including several that have previously been undetectable by RT-QuIC or Protein Misfolding Cyclic Amplification. Furthermore, comparison of the relative abilities of different prions to seed positive RT-QuIC reactions with bank vole and not other recombinant prion proteins allowed discrimination of prion strains such as classical and atypical L-type bovine spongiform encephalopathy, classical and atypical Nor98 scrapie in sheep, and sporadic and variant Creutzfeldt-Jakob disease in humans. Comparison of protease-resistant RT-QuIC conversion products also aided strain discrimination and suggested the existence of several distinct classes of prion templates among the many strains tested.

1)我们已经与Gianluigi Zanusso和其他合作者进一步开发了一种使用鼻刷的克雅氏病（CJD）检测方法，因为在活体患者中明确诊断散发性CJD仍然是一个挑战。在我们之前的研究中，用鼻刷接种的RT-QuIC检测在31名CJD患者中有30名阳性，但在43名非CJD患者中有43名阴性，表明检测CJD的灵敏度为97%，特异性为100%。相比之下，对同一组患者的脑脊液（CSF）样本进行检测的敏感性为77%，特异性为100%。自我们的首次报告以来，我们从另外12名患者中同时收集了这两种标本类型，嗅粘膜（12/12份样本）的敏感性为100%，脑脊液（10/12份样本）的敏感性为83%。因此，我们目前对鼻刷的RT-QuIC的总体诊断性能是43名CJD患者中的42名阳性（98%）诊断灵敏度和43名非CJD患者中的0名阳性（100%）特异性。 2)在与意大利、加拿大和美国同事的合作中，我们还使用RT-QuIC改进了基于CSF的CJD检测。 以前的人CSF RT-QuIC测试需要2.5至5天。我们改进的RT-QuIC测定在4至14小时内鉴定阳性CSF样品，具有更好的分析灵敏度。此外，对11名CJD患者的分析表明，虽然7名使用以前的条件为RT-QuIC阳性，但10名使用新的检测方法为阳性。在这些和进一步的分析中，48例散发性CJD患者的CSF样本中共有46例为阳性，而所有39例非CJD患者均为阴性，诊断灵敏度为95.8%，特异性为100%。这种第二代RT-QuIC检测显著提高了检测CJD患者CSF标本中朊病毒种子的速度和灵敏度。这将提高快速和准确的死前诊断克雅氏病的前景。 3)牛海绵状脑病（BSE）的统计学监测表明，牛对经典BSE（C-BSE）和非典型BSE均易感。非典型形式的疯牛病似乎是零星的，因此可能永远不会被根除。朊病毒监测的一个主要挑战是缺乏足够实用和敏感的常规BSE检测和菌株鉴别试验。如上所述，已知RT-QuIC检测对于多种人类和动物朊病毒疾病的检测具有高度特异性和灵敏度，但对于BSE则没有。与Cristina Casalone等人合作，我们现在已经用RT-QuIC检测试剂盒检测了受C-BSE和非典型L型牛海绵状脑病（L型BSE或L-BSE）影响的牛的脑组织，发现使用特定的rPrPSen底物可以检测和区分这两种BSE形式。具体而言，使用多种rPrPSen底物检测L-BSE，而C-BSE更具选择性。这种基于底物的方法提出了一种特异、灵敏和快速检测和区分至少某些BSE形式的诊断策略。 4)由于朊病毒在多种哺乳动物物种中作为多个菌株传播，因此通过单一超灵敏测定如RT-QuIC检测所有此类菌株将有助于朊病毒疾病的诊断、监测和研究。在与Romolo Nonno，Wenquan Zou，Bernadino Ghetti和Pierluigi Gambetti的合作中，我们发现细菌表达的重组库田鼠朊蛋白（残基23-230）是我们迄今为止测试的所有不同朊病毒类型的灵敏RT-QuIC检测的有效底物-总共28种来自人、牛、羊、鹿和啮齿动物，包括先前通过RT-QuIC或蛋白质错误折叠循环扩增检测不到的几种。此外，不同朊病毒与库田鼠而不是其他重组朊病毒蛋白的种子阳性RT-QuIC反应的相对能力的比较允许区分朊病毒株，例如经典和非典型L型牛海绵状脑病，绵羊中的经典和非典型Nor 98瘙痒症，以及人类中的散发性和变异性克雅氏病。蛋白酶抗性RT-QuIC转化产物的比较也有助于菌株鉴别，并表明在许多测试的菌株中存在几种不同类型的朊病毒模板。