RAPID DIAGNOSTIC PROCEDURES FOR OPPORTUNISTIC PARASITES

弓形虫是一种机会性寄生虫，近年来 由于其对患者的频繁感染而增加了意义 患有获得性免疫缺陷综合症(艾滋病)。当前 诊断人类这种寄生虫的方法通常依赖于 对艾滋病患者进行的不可靠的血清学检查 他们的免疫功能障碍。相反，对于这样的患者， 昂贵的(且与风险相关的)CT扫描和脑扫描替代品 采用活组织检查。在本提案中，我们描述了 最近发展起来的诊断感染的技术 通过检测寄生虫核酸。虽然DNA探针 已用于其他病原体(例如细菌)的诊断 在粪便样本中)，可获得的弓形虫寄生虫数量 从艾滋病患者的临床样本中提取的样本不足以 简单的检测方案。我们在这里描述初步的结果 使用“聚合酶链式反应”(最近由 Ehrlich和同事)用于寄生虫DNA的扩增。 使用此程序，我们可以明确地放大已经存在的 弓形虫基因组中的重复基因 信号可以从DNA开始，从两个信号开始 寄生虫。我们还证明了该PCR方法可以 通过以下方式从根本上提高了速度、成本和灵敏度 使用交替的扩增和转录循环(使用 AMV逆转录酶和T7RNA聚合酶)。 此应用程序请求资金来继续这项工作以开发 将这些程序转化为常规诊断方法。为了做到这一点，我们 将优化反应条件，首先在理想实验室下进行 使用纯化的DNA，然后在条件下 越来越有代表性的临床标本(脑脊液 液体和血液)。除了弓形虫病的诊断外， 这一过程可以很容易地适应于其他疾病的诊断 艾滋病患者的机会性感染，包括 目前直接诊断很困难，因为数量很少 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.