Multiplex bead-PCR diagnosis for AIDS diarrhea

多重微珠 PCR 诊断艾滋病腹泻

• 批准号: 7273893
• 负责人: ERIC R HOUPT
• 金额: $ 18.39万
• 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 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7273893
• 关键词: AIDS diagnosis; Accounting; Acquired Immunodeficiency Syndrome; Africa South of the Sahara; Algorithms; Antigens; Attention; Biological Assay; Blood; Cerebrospinal Fluid; Chronic; Clinical Microbiology; Cohort Studies; Color; Cryptosporidium; DNA; Data; Detection; Diagnosis; Diagnostic; Diarrhea; Equipment; Failure; Feces; Fluorescence; Future; Generations; Giardia; Giardia lamblia; Highly Active Antiretroviral Therapy; Incidence; Intestines; Isospora; Isospora belli; Link; Lymphopenia; Measures; Microscopy; Microsporidia; Noise; Oligonucleotide Probes; Opportunistic Infections; Parasites; Pathogen detection; Patients; Polymerase Chain Reaction; Procedures; Protozoa; Published Comment; Reaction; Reagent; Research; Research Personnel; Screening procedure; Signal Transduction; Specimen; Sputum; Streptavidin; Suspension substance; Suspensions; Syndrome; Tanzania; Techniques; Testing; Time; Work; absorption; assay development; base; clinical research site; design; experience; internal control; mortality; pathogen; programs; rRNA Genes

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）：慢性腹泻与晚期淋巴细胞减少症和死亡率相关，对艾滋病患者来说是一种诅咒。其发病率在撒哈拉以南非洲地区最为严重，可能会破坏高效抗逆转录病毒疗法的吸收。由于现有病原体检测测试的敏感性较差，诊断存在缺陷。我们假设“未诊断”的艾滋病腹泻病例中很大一部分是由于诊断失败而未能发现机会性感染。在本提案中，我们将开发一种灵敏的珠子 PCR 检测方法，可在单次反应中检测多种病原体，然后用来自坦桑尼亚的 401 份现有粪便样本检验我们的假设，其中 78% 的腹泻病例通过显微镜或抗原检测未发现病原体。基于 R21 应用的实用性、我们对原生动物的经验以及审稿人的意见，我们将这个项目限制为隐孢​​子虫、贾第鞭毛虫、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到独特颜色珠子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个珠子颜色进行定量，从而可以同时解析多达 100 种病原体。该检测方法的开发并非易事，需要关注初始多重 PCR 和扩增子探针信噪比的优化。我们有初步数据表明该程序敏感且特异，以及必要的试剂（寄生虫 DNA）、设备（Luminex 机器）以及贾第鞭毛虫和隐孢子虫的实时 PCR 检测，可用于验证新程序。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站点，在那里可以在研究基础上使用它来描述流行病原体的特征，以便设计适当的管理算法。最后，基于综合征的多重诊断是临床微生物学未来的平台，如果这种检测方法适用于粪便，那么它也适用于痰液、血液或脊髓液。