Nanotechnology-Based Detection of Mycoplasma pneumoniae

描述（由申请方提供）：肺炎支原体是一种重要的人类呼吸道病原体，可引起支气管炎和非典型肺炎或“行走性”肺炎。M.肺炎占所有社区获得性肺炎的20%，是年龄较大的儿童和年轻人肺炎的主要原因。由于直接培养带来的重大挑战，血清学检测是诊断的主要方法，但存在严重的局限性，包括需要在单独的医生就诊时获得配对血清，因此对于快速诊断是不切实际的。PCR可以表现出高灵敏度和更快地产生阳性检测，但容易出现来自反应抑制剂的假阴性。无法提供快速和 明确诊断会延迟适当治疗的开始，增加发病率，并增加继续传播、继发感染和长期后遗症（包括与COPD和哮喘相关的慢性肺病）的可能性。因此，缺乏一种简单、可靠、快速的诊断测试是改善M控制的关键障碍。肺炎疾病。纳米技术在生物传感器开发中的应用产生了直接、快速和灵敏的模式识别方法，用于检测感染因子。我们已经表明，纳米纤维掠射角气相沉积产生银纳米棒阵列（NA）表现出极高的电磁场增强的表面增强拉曼光谱（Sers）。与化学计量学分析相结合，该平台可以快速检测和区分病毒和支原体的拉曼光谱，具有出色的灵敏度和特异性，并显示出极大的潜力，以提高支原体的诊断。肺炎感染。我们的总体目标是推进NA-SERS在临床标本中快速灵敏检测支原体的应用，建立在证明其检测支原体能力的关键已发表数据的基础上。在模拟和真实临床咽拭子样本中，肺炎克雷伯氏菌具有临床相关灵敏度。我们的应用有两个具体目标，将该项目从概念验证转向临床应用：（1）使用来自更广泛队列的咽拭子扩展加标和对照样本的分析，包括与qPCR的直接比较;分析支原体的光谱特征并评估其对M.肺炎检测;并评估Sers在与其它细菌病原体包括肺炎衣原体的混合感染中的区分能力;和（2）通过NA-SERS测试，先前确定为M阳性或阴性的真实临床咽拭子样品。pneumoniae通过PCR和培养，在非盲和盲分析中;并表征M.在全球范围内获得的肺炎克雷伯氏菌分离株，以确定菌株多样性是否影响加标咽拭子样本中NA-SERS检测。 公共卫生相关性：肺炎支原体是一种重要的人类呼吸道病原体，占所有社区获得性肺炎的20- 30%，是年龄较大儿童和年轻人肺炎的主要原因。无法提供快速和明确的诊断会延迟适当治疗的开始，增加发病率，并增加持续传播和长期并发症（包括COPD和哮喘）的可能性。因此，缺乏一种简单可靠的快速诊断测试是改善M控制的关键障碍。我们的目标是推进基于纳米技术的平台的开发，用于敏感和方便地检测肺炎支原体。临床标本中的肺炎。

DESCRIPTION (provided by applicant): Mycoplasma pneumoniae is a significant human respiratory tract pathogen, causing bronchitis and atypical or "walking" pneumonia. M. pneumoniae accounts for 20% of all community-acquired pneumonia and is the leading cause of pneumonia in older children and young adults. Serologic testing is the primary method for diagnosis due to the significant challenges posed by direct culture but suffers from severe limitations, including the need for paired sera obtained at separate physician visits, and is thus impractical for rapid diagnosis. PCR can exhibit high sensitivity and yield positive detection sooner but is prone to false-negatives from reaction inhibitors. The inability to provide rapid and definitive diagnosis delays initiation of appropriate treatment, prolongs morbidity, and increases the likelihood of continued transmission, secondary infections, and long-term sequelae, including chronic lung disease associated with COPD and asthma. Lack of a simple, reliable, rapid diagnostic test is thus a critical barrier to improved control of M. pneumoniae disease. Application of nanotechnology to biosensor development is yielding direct, rapid, and sensitive pattern-recognition approaches for detection of infectious agents. We have shown that nanofabrication by glancing angle vapor deposition produces Ag nanorod arrays (NA) exhibiting extremely high electromagnetic field enhancements for surface-enhanced Raman spectroscopy (SERS). Paired with chemometric analysis this platform can rapidly detect and distinguish with outstanding sensitivity and specificity the Raman spectra of viruses and mycoplasmas, and shows great promise in its potential to improve diagnosis of M. pneumoniae infections. Our overall goal is to advance the application of NA-SERS to rapid and sensitive mycoplasma detection in clinical specimens, building upon critical published data that demonstrate its capacity to detect M. pneumoniae at clinically relevant sensitivity in simulated and true clinical throat swab samples. Our application has two specific aims, shifting the project from proof-of-concept toward clinical application: (1) expand analysis of spiked and control samples using throat swabs from a broader cohort, including direct comparison to qPCR; analyze spectral signatures of commensal mycoplasmas and assess their impact on M. pneumoniae detection; and assess NA- SERS discriminatory ability in mixed infections with other bacterial pathogens including Chlamydophila pneumoniae; and (2) test by NA-SERS, true clinical throat swab samples previously established as positive or negative for M. pneumoniae by PCR and culture, in unblinded and blinded analyses; and characterize M. pneumoniae isolates obtained globally to determine whether strain diversity impacts detection by NA-SERS in spiked throat swab samples. PUBLIC HEALTH RELEVANCE: Mycoplasma pneumoniae is a significant human respiratory tract pathogen, accounting for 20- 30% of all community-acquired pneumonia and the leading cause of pneumonia in older children and young adults. The inability to provide rapid and definitive diagnosis delays initiation of appropriate treatment, prolongs morbidity, and increases the likelihood of continued transmission and long-term complications, including COPD and asthma. Lack of a simple and reliable rapid diagnostic test is thus a critical barrier to improved control of M. pneumoniae disease, and our goal is to advance the development of a nanotechnology-based platform for sensitive and expedient detection of M. pneumoniae in clinical specimens.