Multiplex diagnostic for biothreat C. psittaci & non-threat respiratory pathogens

生物威胁鹦鹉热衣原体的多重诊断

• 批准号: 8481514
• 负责人: DEBORAH Anne DEAN
• 金额: $ 103.09万
• 依托单位: CHILDREN'S HOSPITAL & RES CTR AT OAKLAND
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-06 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8481514
• 关键词: Adenoviruses; Antibiotic Resistance; Antibiotics; Bacillus anthracis; Biological; Biological Assay; Burkholderia pseudomallei; Categories; Cervical; Chlamydia; Chlamydia trachomatis; Chlamydophila pneumoniae; Chlamydophila psittaci; Clinic; Clinical; Clinical Research; Clinical Trials; Clinics and Hospitals; Collection; Coxiella burnetii; DNA; DNA amplification; DNA purification; Data; Detection; Development; Diagnostic; Disease; Drug resistance; Early Diagnosis; Early identification; Epidemiology; Evaluation; Fluorescence; Francisella tularensis; Future; Genome; Genomics; Goals; Health Personnel; Hospitals; Hour; Housing; Human; Individual; Infection; Influenza; Lasers; Lead; Legionella pneumophila; Life; Location; Lung diseases; Measles; Medical; Methods; Microfluidics; Military Personnel; Mycoplasma pneumoniae; Names; National Institute of Allergy and Infectious Disease; New Agents; Nucleic Acid Amplification Tests; Organism; Parainfluenza; Pathogenesis; Patients; Performance; Population; Predictive Value; Prevalence; Procedures; Readiness; Reading; Respiratory syncytial virus; Reverse Transcriptase Polymerase Chain Reaction; Sampling; Sensitivity and Specificity; Sexually Transmitted Diseases; Source; Specificity; Sputum; Staging; Swab; Symptoms; System; Technical Expertise; Technology; Testing; Therapeutic; United States National Institutes of Health; Urethra; Vagina; Virulence; Virulent; Work; Yersinia pestis; age group; atypical pneumonia; base; biodefense; biothreat; comparative genomics; design; genome sequencing; human DNA; innovation; instrument; microbial; novel; nucleic acid detection; pathogen; respiratory; treatment strategy

DESCRIPTION (provided by applicant): There is an urgent need to develop rapid sample-to-answer clinic and field deployable diagnostics for NIAID Category A, B and C biothreat pathogens to protect both military and civilian populations. The goal of this application is to advance and validate a lead candidate diagnostic - a novel microfluidics nucleic acid detection and sequencing diagnostic platform recently developed by NetBio-to identify Category B pathogen Chlamydia psittaci (Cps) that causes life-threatening respiratory diseases and has historically been a focus of bioweapons development as well as being a vastly understudied pathogen. We will also detect emerging Cps pathogens and non-biothreat pathogens that are the leading cause of atypical pneumonia and often confused with biothreat agents in clinical presentation: Chlamydia trachomatis (Ct), Chlamydia pneumoniae (Cpn), Mycoplasma pneumoniae (Mp) and Legionella pneumophila (Lp). Indeed, the Biodefense RFA-AI-11-014 we are responsive to states, "Medical diagnostics that use platforms to rapidly distinguish whether an individual is infected with a biological threat agent or a common infection with similar, generalized symptoms are of high priority." There are currently few or no commercial diagnostics for these pathogens in the US. The NetBio platform consists of a ruggedized, analytic instrument that accepts a biochipset (BCS)-shown in Preliminary Studies to purify genomic DNA from clinical samples, amplify, electrophoretically separate and laser detect and type Ct. The entire system from DNA purification to detection takes ~1 hour. The Aims are to: 1) genome sequence representative Cps biothreat and non- biothreat atypical respiratory pathogens for robust primer selection; 2) modify the BCS to purify genomic DNA from human clinical nasopharyngeal (NP) swab and sputum samples; 3) employ the primer selection pipeline developed by Dr. Read to identify primers based on comparative genomics of available genomes and those sequenced in Aim 1 for differentiating biothreat and non-biothreat atypical respiratory pathogens, and develop a multiplexed assay for DNA amplification to distinguish each pathogen; and 4) evaluate the sensitivity, specificity and positive and negative predictive value of the NetBio assay using first spiked samples and then clinical NP swabs and sputum samples compared to the available commercial nucleic acid amplification tests for Ct, Cpn, Mp and Lp (none exist for Cps), and compared to highly sensitive in-house RT-PCR assays prior to clinical trials for FDA approval. A future goal will be to expand the assay to other biothreat respiratory pathogens. We envision that the NetBio sample-to-answer assay will be used in ERs, MD offices, clinics, military facilities, hospitals and the field to advance our understanding of the epidemiology of atypical respiratory diseases and best treatment strategies, which will inform biothreat preparedness. A broadly used diagnostic will detect patients with common but also biothreat infection, enabling early identification of an attack and rapid treatment of infecte military and civilian populations.

描述（由申请人提供）：迫切需要开发针对 NIAID A、B 和 C 类生物威胁病原体的快速从样本到答案的临床和现场可部署诊断方法，以保护军事和平民群体。该应用的目标是推进和验证主要候选诊断——NetBio 最近开发的一种新型微流控核酸检测和测序诊断平台，以识别 B 类病原体鹦鹉热衣原体 (Cps)，这种病原体会导致危及生命的呼吸道疾病，并且历史上曾出现过这种疾病。一直是生物武器开发的焦点，也是一种未被充分研究的病原体。我们还将检测新出现的 Cps 病原体和非生物威胁病原体，它们是非典型肺炎的主要原因，并且在临床表现中经常与生物威胁病原体混淆：沙眼衣原体 (Ct)、肺炎衣原体 (Cpn)、肺炎支原体 (Mp) 和嗜肺军团菌（LP）。事实上，我们响应的 Biodefense RFA-AI-11-014 指出，“使用平台快速区分个体是否感染了生物威胁因子或具有类似普遍症状的常见感染的医疗诊断是高度优先的。 ”目前在美国很少或根本没有针对这些病原体的商业诊断方法。 NetBio 平台由坚固耐用的分析仪器组成，可接受初步研究中所示的生物芯片组 (BCS)，用于从临床样本中纯化基因组 DNA、扩增、电泳分离、激光检测和 Ct 分型。整个系统从DNA纯化到检测大约需要1小时。目标是： 1) 代表 Cps 生物威胁和非生物威胁非典型呼吸道病原体的基因组序列，用于稳健的引物选择； 2）修改BCS以从人类临床鼻咽（NP）拭子和痰样本中纯化基因组DNA； 3) 采用 Read 博士开发的引物选择流程，根据可用基因组的比较基因组学和目标 1 中测序的引物来识别引物，以区分生物威胁和非生物威胁非典型呼吸道病原体，并开发用于 DNA 扩增的多重检测，以区分每种病原体病原; 4) 与现有的 Ct、Cpn、Mp 和 Lp 商业核酸扩增测试（不存在Cps），并在获得 FDA 批准的临床试验之前与高度敏感的内部 RT-PCR 检测进行比较。未来的目标是将检测扩展到其他生物威胁呼吸道病原体。我们预计 NetBio 样本到答案检测将用于急诊室、医学博士办公室、诊所、军事设施、医院和现场，以增进我们的理解 非典型呼吸道疾病的流行病学和最佳治疗策略，这将为生物威胁防范提供信息。广泛使用的诊断将检测常见感染和生物威胁感染的患者，从而能够及早识别攻击并快速治疗受感染的军事和平民群体。