Development of Polymerase Chain Reaction for Cytomegalovirus, Herpes Simplex Vir

由于缺乏敏感的方法来检测最可能的病原体，因此在免疫功能低下的宿主（如艾滋病或癌症患者）中诊断脑膜炎是困难的。这些环境中的生物体，如巨细胞病毒（CMV）、单纯疱疹病毒（HSV）和水痘-带状疱疹病毒（VZV），很难从集落刺激因子（CSF）中生长，并且没有有效的抗原检测技术可用。在过去的几年里，人们越来越清楚地认识到，通过聚合酶链反应（PCR）方法检测这些病毒因子可能提供最敏感和最特异的诊断测试。因此，我们已着手开发一系列针对CMV、HSV和VZV的内部PCR检测试剂盒，以便将其用作NIH患者CSF的常规服务检测。1997年，脊髓液中CMV PCR检测的开发和临床验证完成，该检测开始作为常规分子诊断检测提供。1998年，完成了脊髓液中HSV PCR检测的最终开发和验证，该检测现在也作为常规分子诊断检测提供。脊髓液上的HSV测定将特异性地确定HSV 1、HSV 2或两种病毒DNA的存在。将继续开发与现有CMV和HSV检测试剂盒具有相同性能特征的VZV检测试剂盒。所有三种检测试剂盒均设计为使用内部模拟物来评价单个PCR管中的扩增效率，并检测PCR抑制，以消除假阴性结果的来源。用铕标记的荧光杂交探针进行终点检测，检测下限为3 - 5个病毒基因组当量。CMV和HSV检测试剂的评价显示出极佳的特异性。分子诊断技术的灵敏度和可靠性不仅取决于扩增和检测方法的效率，还取决于样品制备方法的性能。在过去的一年里，开展了一个大规模项目，调查从人类脊髓液、呼吸道样本或血液中制备核酸的六种不同方法的相对效率和可靠性。我们已经确定了一种样品制备方法，似乎是高度可靠的，并提供了提高效率的DNA回收。该方法现已被纳入所提供的临床分子诊断测定。开发并验证了用于检测患者CSF样品中VZV的PCR测定法。该检测试剂盒目前与先前开发的脊髓液CMV、HSV-1和HSV-2检测试剂盒一起沿着常规用于患者检测。

Diagnosis of meningitis in immunocompromised hosts, such as those with AIDS or cancer, is difficult because of the unavailability of sensitive methods for detecting the most likely pathogens. Organisms in these settings, such as cytomegalovirus (CMV), Herpes simplex virus (HSV), and varicella-zoster virus (VZV), are difficult to grow from colony-stimulating factor (CSF), and no effective antigen detection techniques are available. Over the past few years, it has become increasingly clear that detecting these viral agents by the polymerase chain reaction (PCR) method may provide the most sensitive and specific diagnostic tests. Therefore, we have undertaken development of a battery of in- house PCR assays for CMV, HSV, and VZV so that they will be available as routine service tests for CSF from NIH patients. In 1997, development and clinical validation of the PCR assay for CMV in spinal fluid was completed and the test began to be offered as a routine molecular diagnostic assay. During 1998, final development and validation of the PCR assay for HSV in spinal fluid was completed and the test is now also offered as a routine molecular diagnostic assay. The HSV assay on spinal fluid will specifically determine the presence of HSV1, HSV2, or both viral DNAs. Development of a VZV assay with the same performance characteristics as the existing CMV and HSV assays will continue. All three assays are designed to use internal mimics to evaluate the efficiency of amplification within individual PCR tubes and to detect PCR inhibition to eliminate this source of false-negative results. End-point detection by europium-labeled fluorescence hybridization probes allows a lower limit of detection of three to five viral genome equivalents. Evaluations of the CMV and HSV assays show excellent specificity. The sensitivity and reliability of molecular diagnostic techniques depend not only on the efficiencies of the amplification and detection methods, but also on the performance of the sample preparation methods. In the past year, a large-scale project was undertaken to investigate the relative efficiencies and reliabilities of six different methods for preparing nucleic acids from human spinal fluid, respiratory samples, or blood. We have identified a sample preparation method that appears to be highly reliable and offers improved efficiency for DNA recovery. This method has now been incorporated into the clinical molecular diagnostic assays offered. A PCR assay for the detection of VZV in patient CSF samples was developed and validated. This assay is now routinely offered for patient testing along with the previously developed CMV, HSV-1, and HSV-2 assays for spinal fluid.