Capaul S E, Gorgievski-Hrisoho M
Institute for Infectious Diseases, University of Bern, Friedbühlstrasse 51, CH-3010 Bern, Switzerland.
J Clin Virol. 2005 Mar;32(3):236-40. doi: 10.1016/j.jcv.2004.08.006.
Rapid detection of enterovirus (EV) infections is essential in the management of aseptic meningitis. Molecular approaches have opened the way to such rapid, but also specific and sensitive, diagnostic tests. The aim of this study was to compare the performance of the CE marked NucliSens EasyQ Enterovirus assay with an in-house two-step RT-PCR assay using cerebrospinal fluid (CSF) and throat swab samples. In addition, specificity was tested with clinical isolates positive for viruses with clinical importance in CSF samples. For nucleic acid extraction, the NucliSens miniMAG and NucliSens magnetic extraction reagents were used. Subsequently real-time nucleic acid sequence-based amplification (NASBA) RNA amplification was performed using NucliSens EasyQ basic kit reagents and NucliSens EasyQ Enterovirus reagents. An EV-specific internal homologous control (IC) RNA was used to monitor the entire NucliSens EasyQ procedure at the individual sample level. No IC but an external inhibition control was available for the RT-PCR method. For the NucliSens EasyQ procedure, amplification and real-time detection reactions were carried out in the NucliSens EasyQ analyzer. The real-time NASBA enterovirus detection was based on NASBA amplification and real-time molecular beacon technology. Data were analyzed using the manufacturer's software on the NucliSens EasyQ analyzer. For the in-house assay, RT-PCR amplicons were detected using agarose gel analysis. The analysis of clinical samples positive for HSV-1, HSV-2, adenovirus, CMV, VZV, mumps and rhinovirus were all negative by NucliSens EasyQ Enterovirus assay. Three rhinovirus samples were, however, strongly positive in RT-PCR. A total of 141 clinical samples were retrospectively tested, including 126 cerebrospinal fluid (CSF) samples and 15 throat swabs. The 91 CSF samples were negative by both methods, 31 CSF samples and 14 throat swab samples were positive by both methods. The four CSF samples were positive by RT-PCR only. One throat swab sample was negative in NucliSens EasyQ but positive in RT-PCR. The sensitivity and specificity of both methods seem to be more or less comparable. However, the in-house RT-PCR assay appears to amplify some rhinovirus strains and should therefore not be used for throat swab samples. NucliSens EasyQ Enterovirus assay gave more invalid results than the in-house RT-PCR, which is obvious taken into account the difference in quality control between the CE marked NucliSens EasyQ Enterovirus assay and the in-house enterovirus assay. The NucliSens EasyQ procedure can be completed within 5h versus 9.5h for the RT-PCR. NucliSens EasyQ Enterovirus assay showed to be a standardized, rapid, specific, sensitive and reliable procedure for the detection of enterovirus RNA.
快速检测肠道病毒（EV）感染对于无菌性脑膜炎的管理至关重要。分子方法为这种快速、特异且灵敏的诊断测试开辟了道路。本研究的目的是比较CE标记的NucliSens EasyQ肠道病毒检测法与使用脑脊液（CSF）和咽拭子样本的内部两步RT-PCR检测法的性能。此外，还使用CSF样本中对具有临床重要性的病毒呈阳性的临床分离株测试了特异性。对于核酸提取，使用了NucliSens miniMAG和NucliSens磁珠提取试剂。随后，使用NucliSens EasyQ基础试剂盒试剂和NucliSens EasyQ肠道病毒试剂进行实时基于核酸序列的扩增（NASBA）RNA扩增。使用EV特异性内部同源对照（IC）RNA在个体样本水平监测整个NucliSens EasyQ程序。RT-PCR方法没有IC，但有外部抑制对照。对于NucliSens EasyQ程序，扩增和实时检测反应在NucliSens EasyQ分析仪中进行。实时NASBA肠道病毒检测基于NASBA扩增和实时分子信标技术。使用NucliSens EasyQ分析仪上制造商的软件分析数据。对于内部检测法，使用琼脂糖凝胶分析检测RT-PCR扩增产物。通过NucliSens EasyQ肠道病毒检测法对HSV-1、HSV-2、腺病毒、CMV、VZV、腮腺炎病毒和鼻病毒呈阳性的临床样本分析均为阴性。然而，三个鼻病毒样本在RT-PCR中呈强阳性。总共对141份临床样本进行了回顾性检测，包括126份脑脊液（CSF）样本和15份咽拭子。91份CSF样本两种方法均为阴性，31份CSF样本和14份咽拭子样本两种方法均为阳性。四份CSF样本仅RT-PCR呈阳性。一份咽拭子样本在NucliSens EasyQ中为阴性，但在RT-PCR中为阳性。两种方法的灵敏度和特异性似乎大致相当。然而，内部RT-PCR检测法似乎能扩增一些鼻病毒株，因此不应用于咽拭子样本。与内部RT-PCR相比，NucliSens EasyQ肠道病毒检测法产生的无效结果更多，考虑到CE标记的NucliSens EasyQ肠道病毒检测法和内部肠道病毒检测法在质量控制上的差异，这是显而易见的。NucliSens EasyQ程序可在5小时内完成，而RT-PCR需要9.5小时。NucliSens EasyQ肠道病毒检测法显示是一种用于检测肠道病毒RNA的标准化、快速、特异、灵敏且可靠的程序。
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