Multiplex bead-PCR diagnosis for AIDS diarrhea

多重微珠 PCR 诊断艾滋病腹泻

• 批准号: 7120467
• 负责人: ERIC R HOUPT
• 金额: $ 22.21万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-15 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7120467
• 关键词: AIDS; AIDS /HIV diagnosis; Cryptosporidium; clinical research; diagnosis; diarrhea; oligonucleotides

DESCRIPTION (provided by applicant): Chronic diarrhea is a curse for AIDS patients through its association with advanced lymphopenia and mortality. Its incidence is greatest in sub-Saharan Africa where it may undermine absorption of HAART. Diagnosis is flawed by the poor sensitivity of available tests for pathogen detection. We hypothesize that diagnostic failure to detect opportunistic infection accounts for a large proportion of AIDS diarrhea cases given "no diagnosis." In this proposal we will develop a sensitive bead-PCR assay that detects multiple pathogens in single reaction and then test our hypothesis on 401 existing stool specimens from our prior AIDS cohort study from Tanzania, whereby 78% of diarrhea cases revealed no pathogen via microscopy or antigen detection. Based on the practicality of an R21 application, our experience with protozoa, and reviewer's comments, we will limit this project to Cryptosporidium, Giardia, Isospora, and the two major intestinal Microsporidia. The assay involves conventional PCR using biotinylated primers followed by hybridization of amplicon to oligonucleotide probes that are covalently linked to uniquely colored beads. Fluorescence is generated by addition of streptavidin-PE and quantitated per bead color on a Luminex suspension array machine, such that up to 100 pathogen species can be simultaneously resolved. Development of the assay is not trivial and requires attention to the initial multiplex PCR and optimization of amplicon-probe signal-to-noise ratio. We have preliminary data that the procedure is sensitive and specific, as well as the necessary reagents (parasite DNA), equipment (Luminex machine), and real-time PCR assays for Giardia and Cryptosporidium that can be used to validate the new procedure. Through generation of preliminary data from this proposal we will plan to transfer the technique to our ongoing clinical site in Tanzania, where it can be used on a research basis to characterize prevailing pathogens in order to devise appropriate management algorithms. Finally, syndrome-based multiplex diagnosis is the platform of the future for clinical microbiology, and if this assay works on stool it should work on sputum, blood, or spinal fluid.

描述（由申请人提供）：慢性腹泻是艾滋病患者的诅咒，因为它与晚期淋巴细胞减少症和死亡率有关。它在撒哈拉以南非洲的发病率最高，在那里它可能会破坏HAART的吸收。由于可用的病原体检测方法灵敏度较差，诊断存在缺陷。我们推测，在艾滋病腹泻病例中，很大一部分是由于未能发现机会性感染而导致诊断失败。“在这项提案中，我们将开发一种灵敏的珠PCR检测方法，在单一反应中检测多种病原体，然后在我们之前来自坦桑尼亚的艾滋病队列研究的401份现有粪便标本上测试我们的假设，其中78%的腹泻病例通过显微镜或抗原检测没有发现病原体。基于R21应用的实用性，我们对原生动物的经验，以及评审员的评论，我们将把这个项目限制在隐孢子虫，贾第虫，等孢子虫和两种主要的肠道微孢子虫。该测定涉及使用生物素化引物的常规PCR，然后将扩增子与共价连接至独特颜色珠的寡核苷酸探针杂交。通过添加链霉亲和素-PE产生荧光，并在Luminex悬浮阵列机器上对每种珠颜色进行定量，使得可以同时分辨多达100种病原体物种。该检测方法的开发并不简单，需要注意初始多重PCR和扩增子-探针信噪比的优化。我们有初步的数据表明，该程序是敏感和具体的，以及必要的试剂（寄生虫DNA），设备（Luminex机器），贾第虫和隐孢子虫的实时PCR检测，可用于验证新的程序。通过从该提案中生成初步数据，我们将计划将该技术转移到我们在坦桑尼亚正在进行的临床研究中心，在那里它可以用于研究，以表征流行的病原体，以便设计适当的管理算法。最后，基于综合征的多重诊断是未来临床微生物学的平台，如果这种检测方法对粪便有效，那么它应该对痰液、血液或脊髓液有效。