Quantitative diagnosis of TB/HIV co-infection using pathogen-specific exosomes in blood

摘要 同时感染艾滋病毒和结核病(TB)的患者表现出这两种疾病进展加快。A错过了 艾滋病毒阳性患者的结核病诊断，反之亦然，可能会产生严重的后果，因为死亡率是 艾滋病毒/结核病混合感染患者的比例非常高。因此，世界卫生组织建议对结核病进行艾滋病毒检测 对感染艾滋病毒的患者和结核病患者进行筛查。HIV/TB混合感染的早期诊断和适当 定时干预可以通过减缓疾病进展和降低死亡率来改善健康结果 这些感染的传播。鉴于对艾滋病毒和结核病的早期诊断的迫切需要，我们建议开发和 验证一种新的快速直接诊断艾滋病毒和结核病感染的方法，使用一种敏感的 纳米等离子增强散射(NPEs)平台检测病原体特异性(Ps)外切体(PsExo) 在患者血清样本中携带结核和/或艾滋病毒衍生抗原。PS抗原通常很难被检测到 循环，由于低浓度和非特异性蛋白质相互作用或降解性的潜在掩蔽 酶，特别是在感染早期，当抗原浓度较低时。体液中含有丰富的外切体， 外切体是多种Ps抗原的丰富来源。外切体富集有效地浓缩Ps- 抗原，并保护它们免受循环水解酶的影响，从而增强和改进检测。边使用边使用 PsExo作为诊断特定传染病的生物标志物是新的，显示出巨大的前景和意义 技术挑战，包括目前缺乏针对特定疾病的有效PxExo标记物，以及简单和 PsExo分析的快速分析方法已经存在。我们最近展示了我们可以使用我们的NPEs检测平台 对癌症来源的外切体进行量化，并从疾病对照中诊断早期胰腺肿瘤。我们会 在本提案中，采用这种方法来检测艾滋病毒和结核病PsExo。目标1将通过以下方式解决第一个问题： 感染细胞外切体特异或优势表达的致病蛋白及PsExo检测 创新基金会对感染患者进行定量蛋白质组分析的候选对象 诊断(查找)样本库。AIM 2将使用AIM 1 PsExo生物标记物建立优化的NPE阵列， 应用机器学习开发可有效诊断HIV、TB和HIV/TB患者的NPE阵列 基于NPE的结果，并在独立的发现队列中验证这些发现。这种方法不会 需要单独的外切体分离步骤，并且只使用微量血清体积。建议的化验方法将 有益于不能产生痰的混合感染患者，经常需要 用于诊断的侵入性活检，以及HIV抗体仍为阴性的急性HIV感染患者。

ABSTRACT Patients co-infected with HIV and tuberculosis (TB) exhibit accelerated progression of both diseases. A missed TB diagnosis in a HIV-positive patient, and vice versa, can have severe consequences, since mortality rates are very high in HIV/TB co-infected patients. The World Health Organization thus recommends HIV testing for TB patients and TB screening for HIV-affected patients. Early diagnosis of HIV/TB co-infections and appropriately timed interventions can improve health outcomes by slowing disease progression and reducing mortality and the spread of these infections. Given this urgent need for early diagnosis of HIV and TB, we propose to develop and validate a novel assay that rapidly and directly diagnoses both HIV and TB infections, using a sensitive nanoplasmon-enhanced scattering (nPES) platform to detect pathogen-specific (Ps) exosomes (PsExo) that carry TB- and/or HIV-derived antigens in patient serum samples. Ps antigens are often difficult to detect in circulation, due to low concentrations and potential masking by non-specific protein interactions or degradative enzymes, especially early in infection when antigen concentrations are low. Body fluids are rich in exosomes, and exosomes are rich sources of multiple Ps-antigens. Exosome enrichment effectively concentrates Ps- antigens and protects them from circulating hydrolases, thereby enhancing and improving detection. While use of PsExo as biomarkers for diagnosing specific infectious diseases is new and shows great promise, significant technical challenges, including the current lack of validated PxExo markers for specific diseases and simple and rapid assays for PsExo analysis, exist. We have recently demonstrated that we can use our nPES assay platform to quantify cancer-derived exosomes and diagnose early-stage pancreatic tumors from disease controls. We will adapt this approach to detect HIV and TB PsExo in this proposal. Aim 1 will address the first issue by identifying pathogenic proteins specifically or predominantly expressed on exosomes of infected cells and evaluating PsExo candidates by quantitative proteomics analysis of infected patients from Foundation for Innovative New Diagnostics (FIND) sample bank. Aim 2 will establish an optimized nPES array using Aim 1 PsExo biomarkers, apply machine learning to develop an nPES array that can effectively diagnose HIV, TB and HIV/TB patients based on nPES results, and validate these findings in an independent FIND cohort. This approach does not require a separate exosome isolation step and uses only microsample serum volumes. The proposed assay will benefit co-infected patients who are unable to produce sputum, extrapulmonary TB patients who often require invasive biopsies for diagnosis, and patients with acute HIV infections who are still HIV antibody negative.