Point-of-care immunoassay for diagnosis of histoplasmosis in HIV/AIDS

描述(由申请人提供)：进行性播散性组织胞浆菌病是美国和拉丁美洲艾滋病毒/艾滋病患者中一种常见的危及生命的真菌感染。艾滋病毒/艾滋病的发病率可能为20%，在资源有限的国家死亡率为30%，在这些国家，组织胞浆菌是地方性流行病。早期诊断和治疗对于降低这一高死亡率至关重要。目前的诊断依赖于培养或组织病理学。这些方法灵敏度低，耗时长，价格昂贵，需要训练有素的人员。另一种方法是免疫测定法来检测血清或尿液中的包膜多糖。目标抗原被认为是细胞壁的半乳甘露聚糖。到目前为止的研究发现，这种检测方法具有很高的灵敏度。然而，组织原体抗原的免疫分析目前只能通过使用参考实验室以ELISA形式提供，ii)不能在资源有限的国家进行商业分发，iii)依赖于多克隆兔抗体(PABS)的使用，以及iv)不服从于护理点(POC)的使用。我们的总体假设是，针对感染过程中释放到血清和尿液中的包膜多糖抗原的高亲和力单抗(MAbs)可以建立用于诊断HIV/AIDS播散性组织胞浆菌病的POC免疫检测方法。这一假设的一个推论是，使用适当的mAb对进行分析构建将能够靶向共同和独特的包膜螺旋体表位，提供对分析特异性的控制，这是PAb所不可能的。第一个目的是制备适合于杂交瘤筛选和单抗特异性评价的多糖类抗原。第二个具体目标是产生一个与组织胞浆多糖抗原上发现的表位谱起反应的单抗的库，该表位被释放到血清和尿液中。如果第一阶段的目标实现，第二阶段将使用第一阶段的单抗构建和评估POC格式的免疫分析。我们首选的检测平台是横向流动免疫层析(试纸)检测。如果成功，这项转化研究项目可以通过更早的诊断和治疗显著降低组织胞浆菌病的死亡率。重要的是，这可以在资源有限的国家以所需的低成本完成。 公共卫生相关性：进行性播散性组织胞浆菌病是美国和拉丁美洲艾滋病毒/艾滋病患者中一种常见的、危及生命的机会性真菌感染。这是一项转化性研究，其目标是在资源有限的国家建立一种用于快速诊断组织胞浆菌病的即时免疫分析方法。如果成功，该项目可以通过早期诊断和治疗大幅降低流行地区的死亡率。

DESCRIPTION (provided by applicant): Progressive disseminated histoplasmosis is a common and life-threatening fungal infection among patients with HIV/AIDS in the United States and Latin America. Incidence rates in HIV/AIDS can be >20%, with mortality rates >30% in resource-limited countries where the fungus, Histoplasma capsulatum, is endemic. Early diagnosis and treatment are essential to reducing this high mortality rate. Diagnosis currently depends on culture or histopathology. These methods have low sensitivity, are time consuming, are expensive, and require trained personnel. An alternative approach is an immunoassay to detect H. capsulatum polysaccharide in serum or urine. The target antigen is believed to be a cell wall galactomannan. Studies to date have found a high sensitivity for such assays. However, immunoassay for Histoplasma antigen i) is currently available only in ELISA format through use of a reference laboratory, ii) is not commercially available for distribution in resource-limited countries, iii) is dependent on the use of polyclonal rabbit antibodies (pAbs), and iv) is not amenable to point-of-care (POC) use. Our overall hypothesis is that an immunoassay for POC diagnosis of disseminated histoplasmosis in HIV/AIDS can be constructed with high-affinity monoclonal antibodies (mAbs) that target the polysaccharide antigen of H. capsulatum that is shed into serum and urine during infection. A corollary to this hypothesis is that the use of appropriate pairs of mAbs for assay construction will enable targeting of common and unique H. capsulatum epitopes, providing a control over assay specificity that is not possible with pAbs. The first Specific Aim is to produce polysaccharide antigens suitable for screening of hybridomas and evaluation of mAb specificity. The second Specific Aim is to produce a library of mAbs reactive with the spectrum of epitopes found on the Histoplasma polysaccharide antigen that is shed into serum and urine. If the goals of this Phase I are achieved, Phase II will use mAbs from Phase I to construct and evaluate an immunoassay in POC format. Our preferred assay platform would be the lateral flow immunochromatographic (dipstick) assay. If successful, this translational research project could dramatically decrease mortality from histoplasmosis through earlier diagnosis and treatment. Importantly, this can be done at the low cost needed in resource-limited countries. PUBLIC HEALTH RELEVANCE: Progressive disseminated histoplasmosis is a common and life-threatening, opportunistic fungal infection among patients with HIV/AIDS in the United States and Latin America. This is a translational research study whose goal is an immunoassay in point-of-care format for rapid diagnosis of histoplasmosis in resource-limited countries. If successful, the project could dramatically reduce mortality in endemic regions through early diagnosis and treatment.