RAPID DIAGNOSTIC PROCEDURES FOR OPPORTUNISTIC PARASITES

弓形虫是一种机会寄生虫， 由于其频繁感染患者， 获得性免疫缺陷综合症（艾滋病）。 电流 诊断人体内这种寄生虫的方法通常依赖于 血清学方法在艾滋病患者中不可靠， 他们的免疫功能紊乱。 相反，对于这些患者， 昂贵的（和风险相关的）替代CT扫描和大脑 使用了biopsy。 在本提案中，我们描述了 最新发展的诊断感染的技术 通过检测寄生虫核酸。 虽然DNA探针 已经用于诊断其它病原体（例如，细菌 粪便中T.可获得的弓形虫 从艾滋病患者的临床标本，是不够的， 简单的检测方案。 我们在这里描述的初步结果 使用“聚合酶链式反应”（最近由 埃利希和同事）用于扩增寄生虫DNA。 使用这个程序，我们可以具体地放大一个已经 T.这样一个离散的 可以从少至两个DNA开始获得信号 寄生虫 我们还证明，PCR方法可以 从根本上提高了其速度，成本和灵敏度， 使用扩增和转录的交替循环（使用 AMV逆转录酶和T7 RNA聚合酶）。 这项申请要求提供资金，以开展这项工作， 这些程序变成常规诊断方法。 为此我们 将优化反应条件，首先在理想的实验室条件下， 使用纯化的DNA，然后在一定条件下 越来越具有代表性的临床标本（脑脊髓 液体和血液）。 除了弓形虫病的诊断， 这种方法可以很容易地适用于其他疾病的诊断。 艾滋病患者的机会性感染，包括那些 目前很难直接诊断，因为 可获得的微生物。

Toxoplasma gondii is an opportunistic parasite which has recently increased a significance due to its frequent infection of patients with Acquired Immunodeficiency Syndrome (AIDS). Current methods to diagnose this parasite in humans generally rely on serological procedures which are unreliable in AIDS patients due to their immune dysfunction. Instead, for such patients, the costly (and risk-associated) alternatives of CT scan and brain biopsy are used. In this proposal, we describe the application of recently developed technology to the diagnosis of infection through detection of parasite nucleic acid. Although DNA probes have been used in the diagnosis of other pathogens (e.g., bacteria in stool samples), the numbers of T. gondii parasites obtainable from clinical specimens from AIDS patients is insufficient for simple detection schemes. We describe here preliminary results using the "Polymerase Chain Reaction" (recently described by Ehrlich and co-workers) for the amplication of parasite DNA. Using this procedure we can specifically amplify an already repetitive gene in the genome of T. gondii such that a discrete signal can be obtained starting with DNA from as few as two parasites. We also demonstrate that the PCR method can be radically improved in its speed, cost and sensitivity through the use of alternating cycles of amplification and transcription (using AMV reverse transcriptase and T7 RNA polymerase, respectively). This application requests funds to pursue this work to develop these procedures into a routine diagnostic method. To do this, we will optimize the reaction conditions, first under ideal laboratory conditions using purified DNA and then under conditions increasingly representative of clinical specimens (cerebrospinal fluid and blood). In addition to the diagnosis of toxoplasmosis, this procedure could be readily adapted to diagnosis of other opportunistic infections of AIDS patients, including those where direct diagnosis is currently difficult due to the small numbers of microorganisms obtainable.