Metabolomics-based discovery of small molecule biomarkers for noninvasive dengue

描述(由申请人提供)：流行性登革热(DF)和登革出血热(DHF/DSS)已在整个热带世界出现，对公共卫生造成毁灭性后果。过去十年拉丁美洲严重登革热(DEN)的急剧增加令人严重关切；目前有30%的病例被诊断为严重登革热病毒感染。在许多发展中国家，DEN在临床护理方面的公共卫生能力令人难以承受。这项提案的总体目标是为DENV感染的诊断和预后(D&P)提供一个重大变化。代谢组学方法将被用来识别出现在血清和非侵入性临床标本(尿液和唾液)中的候选代谢物小分子生物标记物(SMB)，这些标记物可以诊断DENV感染并预测病情进展。通过对DEN患者急性期标本的初步研究，利用基于LC-MS的代谢组学方法，在血清、唾液和尿液中确定了DF和DHF/SS的一些分子特征和候选SMB。在这个项目的R21阶段，我们将使用代谢指纹方法来确认现有的SMB，并在尼加拉瓜高质量的儿科DEN医院和队列研究中找到新的候选SMB样本，将开始描述所涉及的SMB和代谢途径，将在医院研究中调查这些SMB在预期收集的血清、唾液和尿样中对D&P的有效性，并将确定区分DEN、严重DEN和非DEN疾病的最重要的分子特征组合。我们将开发包括SMB、临床体征和症状以及临床实验室结果在内的算法，用于DENV感染的D&P。在项目的R33阶段，从医院研究和以社区为基础的队列研究中预期收集的血清、唾液和尿样将通过使用LC-MS/MS(LC-MS/MS)的代谢谱进行分析，以确定候选的SMB。尼加拉瓜的诊断方案将包括对选定中小企业的第一代EIA检测。将确定候选中小企业和“第一代”中小企业的诊断和预后敏感性和特异性，以及基于中小企业的诊断的首选临床标本。总体而言，这些研究将确定一组SMB(例如5-10个)，用于制定用于临床和医院DEN D&P的快速护理点(POC)测试的目标产品概况(TPP)。检测唾液和尿液中可预测严重DEN的SMB是创新的，并通过使用廉价、容易获得、非侵入性的临床标本为DEN的D&P提供真正的诊断范式转变的机会。 公共卫生相关性：这项研究将利用液相色谱-串联质谱仪来识别DEN患者血清、唾液和尿液中的小分子生物标记物(SMB)，以预测和诊断DEN感染。这将对发展中国家的医生和公共卫生工作者具有巨大的价值，以识别最有可能出现严重DEN的患者，进行支持性护理和早期治疗干预，非侵入性样本的使用将为DEN诊断和患者护理提供真正的范式转变。

DESCRIPTION (provided by applicant): Epidemic dengue fever (DF) and dengue hemorrhagic fever (DHF/DSS) have emerged throughout the tropical world with devastating public health consequences. A dramatic increase in severe dengue disease (DEN) in Latin America in the last decade is of grave concern; 30% of cases are now diagnosed as severe dengue virus (DENV) infections. DEN is overwhelming public health capacity for clinical care in much of the developing world. The overall goal of this proposal is to provide a major change in the diagnosis and prognosis (D&P) of DENV infections. A metabolomics approach will be used to identify candidate metabolite small molecule biomarkers (SMBs) that occur both in serum and in non-invasive clinical specimens (urine and saliva) that diagnose DENV infection and predict progression to severe disease. Preliminary studies using acute phase specimens from DEN patients have identified a number of molecular features and candidate SMBs of DF and DHF/SS in serum, saliva, and urine using liquid chromatography-mass spectrometry (LC-MS)-based metabolomics. In the R21 phase of this project, we will use a metabolic fingerprinting approach to confirm existing and identify new candidate SMBs in retrospectively collected serum specimens available from the high-quality pediatric DEN hospital-based and cohort studies in Nicaragua, will begin to characterize the SMBs and metabolic pathways involved, will investigate the efficacy for D&P of these SMBs in prospectively collected serum, saliva, and urine specimens in the hospital study, and will identify a portfolio of the most significant molecular features that differentiate DEN, severe DEN, and non-DEN disease. We will develop algorithms including SMBs, clinical signs and symptoms, and clinical laboratory results for the D&P of DENV infections. In the R33 phase of the project, prospectively collected serum, saliva, and urine samples from both the hospital study and a community-based cohort study will be analyzed by metabolic profiling using LC-tandem MS (LC-MS/MS) to identify candidate SMBs. First generation EIA tests for selected SMBs will be included in the diagnostic regimen in Nicaragua The diagnostic and prognostic sensitivity and specificity of the candidate SMBs and "first generation" SMB tests will be determined as will the preferred clinical specimen for SMB-based diagnoses. Overall these studies will identify a panel of SMBs (e.g. 5-10), which will be used to formulate the Target Product Profiles (TPP) for rapid point-of-care (POC) tests for use in clinics and hospitals for DEN D&P. Detection of SMBs in saliva and urine that are predictive of severe DEN is innovative and provides the opportunity for a true paradigm shift in diagnosis by using inexpensive, easily procured, non-invasive clinical specimens for D&P of DEN. PUBLIC HEALTH RELEVANCE: The studies will exploit liquid chromatography-tandem mass spectrometry to identify small molecule biomarkers (SMBs) in serum, saliva, and urine of DEN patients for both prognosis and diagnosis of DEN infections. This will be of enormous value to physicians and public health workers in the developing world to identify patients at greatest risk for severe DEN for supportive care and early therapeutic intervention, and the use of non-invasive samples will provide a true paradigm shift in DEN diagnosis and patient care.