Optimization and evaluation of a PCR assay for detecting toxoplasmic encephalitis in patients with AIDS
Optimization and evaluation of a PCR assay for detecting toxoplasmic encephalitis in patients with AIDS
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DOI：
10.1128/jcm.40.12.4499-4503.2002
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发表时间：
2002-12-01
影响因子：
9.4
通讯作者：
Evans, CA
中科院分区：
文献类型：
--
作者：
Joseph, P;Calderón, MM;Evans, CA
弓形虫是一种常见的危及生命的机会性感染。我们使用实验性鼠弓形虫感染来优化诊断用途的 PCR，确定其敏感性，并表征实验性弓形虫病的时间过程和组织分布。调整 PCR 条件，直到检测可靠地检测到源自少于单一寄生虫的 DNA 量。 42 只小鼠腹膜内接种弓形虫速殖子，并在 6 至 72 小时后处死。通过 PCR 和组织学检查组织，发现感染从血液进展到肺、心脏、肝脏和大脑，PCR 始终比显微镜检查更早检测出寄生虫，并且没有假阳性结果。然后我们评估了这种 PCR 检测对人类患者的诊断价值。我们研究了 12 名艾滋病患者和确诊的弓形虫脑炎患者（定义为小鼠接种阳性和/或所有疾病控制中心临床诊断标准）、12 名既没有 CDC 诊断标准也没有小鼠接种阳性的人类免疫缺陷病毒感染疑似脑弓形体病患者、26 名患有其他机会性感染且无感染迹象的人类免疫缺陷病毒感染患者的脑脊液和血清样本。脑弓形体病和免疫功能正常的神经囊尾蚴病患者 18 例。 12 名确诊弓形体病患者中，有 11 名血液或脑脊液样本 PCR 结果呈阳性（9 份血液样本中有 6 份，12 份脑脊液样本中有 8 份）。对照患者的所有样本均为阴性。这项研究证明了 PCR 在资源匮乏的环境中诊断弓形虫脑炎的高敏感性、特异性和临床实用性。
Toxoplasma gondii is a common life-threatening opportunistic infection. We used experimental murine T. gondii infection to optimize the PCR for diagnostic use, define its sensitivity, and characterize the time course and tissue distribution of experimental toxoplasmosis. PCR conditions were adjusted until the assay reliably detected quantities of DNA derived from less than a single parasite. Forty-two mice were inoculated intraperitoneally with T. gondii tachyzoites and sacrificed from 6 to 72 h later. Examination of tissues with PCR and histology revealed progression of infection from blood to lung, heart, liver, and brain, with PCR consistently detecting parasites earlier than microscopy and with no false-positive results. We then evaluated the diagnostic value of this PCR assay in human patients. We studied cerebrospinal fluid and serum samples from 12 patients with AIDS and confirmed toxoplasmic encephalitis (defined as positive mouse inoculation and/or all of the Centers for Disease Control clinical diagnostic criteria), 12 human immunodeficiency virus-infected patients with suspected cerebral toxoplasmosis who had neither CDC diagnostic criteria nor positive mouse inoculation, 26 human immunodeficiency virus-infected patients with other opportunistic infections and no signs of cerebral toxoplasmosis, and 18 immunocompetent patients with neurocysticercosis. Eleven of the 12 patients with confirmed toxoplasmosis had positive PCR results in either blood or cerebrospinal fluid samples (6 of 9 blood samples and 8 of 12 cerebrospinal fluid samples). All samples from control patients were negative. This study demonstrates the high sensitivity, specificity, and clinical utility of PCR in the diagnosis of toxoplasmic encephalitis in a resource-poor setting.