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

描述（由申请人提供）：迫切需要为NIAID A类、B类和C类生物威胁病原体开发快速样本到答案的诊所和现场可部署的诊断方法，以保护军事和平民群体。该应用的目标是推进和验证一种领先的候选诊断-NetBio-to最近开发的一种新型微流体核酸检测和测序诊断平台，以识别导致危及生命的呼吸道疾病的B类病原体鹦鹉热衣原体（Cps），该病原体在历史上一直是生物武器开发的焦点，也是一种研究不足的病原体。我们还将检测新出现的Cps病原体和非生物威胁病原体，这些病原体是非典型肺炎的主要原因，在临床表现中经常与生物威胁因子混淆：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，我们对国家的生物防御RFA-AI-11-014做出了回应，“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具有高度优先级。“目前在美国很少或没有针对这些病原体的商业诊断。NetBio平台由一个坚固耐用的分析仪器组成，该仪器可接受生物芯片组（BCS）-初步研究显示，可从临床样本中纯化基因组DNA，扩增，分离和激光检测以及Ct类型。整个系统从DNA纯化到检测需要约1小时。其目的是：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; 3）采用由Read博士开发的引物选择管道，根据可用基因组的比较基因组学和Aim 1中测序的基因组来识别引物，以区分生物威胁和非生物威胁。对非典型呼吸道病原体进行生物威胁，并开发用于DNA扩增的多重检测方法，以区分每种病原体;和4）与Ct、Cpn、Mp和Lp的可用商业核酸扩增测试相比，使用首先掺入的样品，然后使用临床NP拭子和痰样品，评估NetBio测定的灵敏度、特异性以及阳性和阴性预测值（不存在Cps），并与FDA批准的临床试验之前的高灵敏度内部RT-PCR测定进行比较。未来的目标是将该检测方法扩展到其他生物威胁呼吸道病原体。我们设想，NetBio样本到答案测定将用于急诊室，医学博士办公室，诊所，军事设施，医院和外地，以促进我们的理解 非典型呼吸道疾病的流行病学和最佳治疗战略，这将为生物威胁防备提供信息。一种广泛使用的诊断方法将检测出常见但也具有生物威胁感染的患者，从而能够早期识别攻击并快速治疗受感染的军事和平民人口。 公共卫生相关性：鹦鹉热衣原体是一种B类生物防御因子，很难与肺炎衣原体、沙眼衣原体、肺炎支原体和嗜肺军团菌的临床表现区分开来;所有这些都是呼吸道病原体，是当今世界非典型肺炎的主要原因。对于这些人类病原体中的任何一种，都没有可靠的商业诊断方法。我们计划开发一种易于使用，廉价，一小时的诊断，将检测和区分每种病原体，用于急诊室，诊所，医院，军事设施和现场，以促进我们对非典型呼吸道疾病流行病学和最佳治疗策略的理解，这将为生物威胁准备提供信息。广泛使用的诊断将识别患有常见但也具有生物威胁感染的患者，从而能够在早期阶段识别攻击，并协助卫生保健人员快速治疗受感染的军人和平民，以挽救生命。

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. PUBLIC HEALTH RELEVANCE: Chlamydia psittaci is a Category B Biodefense agent that is difficult to distinguish from the clinical presentation of Chlamydia pneumoniae, Chlamydia trachomatis, Mycoplasma pneumoniae and Legionella pneumophila; all are respiratory pathogens and represent the major causes of atypical pneumonia in the world today. There are no reliable commercial diagnostics available for any of these human pathogens. We plan to develop an easy to use, cheap, one-hour diagnostic that will detect and differentiate each pathogen for use in ERs, clinics, hospitals, military facilities and the field to advance our understanding of the epidemiology of atypical respiratory diseases and the best therapeutic strategies, which will inform biothreat preparedness. A broadly-used diagnostic will identify patients with common but also with biothreat infections, enabling the identification of an attack i the early stages and assisting health care personnel in rapidly treating infected military and civilian populations to save lives.