Halonen P, Herholzer J, Ziegler T
Department of Virology and MediCity, University of Turku, FIN-20520 Turku, Finland.
Clin Diagn Virol. 1996 May;5(2-3):91-100. doi: 10.1016/0928-0197(96)00210-3.
Advances have been made in selecting sensitive cell lines for isolation, in early detection of respiratory virus growth in cells by rapid culture assays, in production of monoclonal antibodies to improve many tests such as immunofluorescence detection of virus antigens in nasopharyngeal aspirates, in highly sensitive antigen detections by time-resolved fluoroimmunoassays (TR-FIAs) and biotin-enzyme immunoassays (BIOTH-E), and, finally, in the polymerase chain reaction (PCR) detection of respiratory virus DNA or RNA in clinical specimens. All of these advances have contributed to new or improved possibilities for the diagnosis of respiratory virus infections.
This review summarizes our experiences during the last 15 years in the development of diagnostic tests for respiratory virus infections, and in use of these tests in daily diagnostic work and in epidemiological studies.
Immunofluorescence tests based on monoclonal antibodies, all-monoclonal TR-FIAs, and biotin-enzyme immunoassays (EIAs) have about the same sensitivities and specificities. They compare well with the sensitivity of virus culture. PCR followed by liquid-phase hybridization is a sensitive method for detecting adenovirus DNA and enterovirus and rhinovirus RNA in clinical specimens. IgG EIA on paired acute and convalescent phase sera is the most sensitive serological test for respiratory virus infections and is a valuable reference method when evaluating the sensitivity of new diagnostic tests. The IgG avidity test can distinguish primary infections from re-infections at least in respiratory syncytial virus (RSV) infections. IgM antibody assays, on the other hand, had low sensitivities in our studies.
The choice of diagnostic methods for respiratory virus infections depends on the type and location of the laboratory, the number of specimens tested, and the previous experience of the laboratory. Virus culture, whenever possible, should be the basic diagnostic method; the results, including identification of the virus, should be available no more than 24 h later than the results of rapid diagnostic tests. In small laboratories, especially in hospitals where specimen transportation is well organized, immunofluorescence may be the best choice for antigen detection with the provision that an experienced microscopist and a good UV microscope are available. If the laboratory receives a large number of specimens and has previous experience with EIAs, then biotin-EIAs or TR-FIAs may be the most practical techniques. Their advantages include the stability of the antigens in clinical samples since intact, exfoliated epithelial cells are not required, treatment of specimens is practical, testing of large numbers of specimens is possible, and reading the printed test result is less subjective than reading fluorescence microscopy. The larger role of PCR in the diagnosis of respiratory virus infections depends on future developments such as practical methods to extract DNA or RNA and to purify the extracts from nonspecific inhibitors, plus further improvements to minimize cross-contamination. Group-specific detection of enteroviruses and rhinoviruses is an example of the potential for PCR technology. In experienced laboratories. EIA IgG antibody tests should be available. Recombinant antigens may be a useful part of such assays.
在选择用于分离的敏感细胞系、通过快速培养试验早期检测细胞中呼吸道病毒的生长、生产单克隆抗体以改进许多检测方法（如免疫荧光检测鼻咽抽吸物中的病毒抗原）、通过时间分辨荧光免疫测定法（TR-FIA）和生物素-酶免疫测定法（BIOTH-E）进行高灵敏度抗原检测以及最后通过聚合酶链反应（PCR）检测临床标本中的呼吸道病毒DNA或RNA等方面均取得了进展。所有这些进展都为呼吸道病毒感染的诊断带来了新的或改进的可能性。
本综述总结了我们在过去15年中开发呼吸道病毒感染诊断检测方法以及在日常诊断工作和流行病学研究中使用这些检测方法的经验。
基于单克隆抗体的免疫荧光检测、全单克隆TR-FIA和生物素-酶免疫测定法（EIA）具有大致相同的灵敏度和特异性。它们与病毒培养的灵敏度相当。PCR结合液相杂交是检测临床标本中腺病毒DNA以及肠道病毒和鼻病毒RNA的灵敏方法。急性期和恢复期配对血清的IgG EIA是呼吸道病毒感染最灵敏的血清学检测方法，在评估新诊断检测的灵敏度时是一种有价值的参考方法。IgG亲和力检测至少在呼吸道合胞病毒（RSV）感染中可区分初次感染和再次感染。另一方面，在我们的研究中IgM抗体检测灵敏度较低。
呼吸道病毒感染诊断方法的选择取决于实验室的类型和地点、检测的标本数量以及实验室的既往经验。只要有可能，病毒培养应作为基本诊断方法；其结果，包括病毒鉴定，应在不迟于快速诊断检测结果24小时后获得。在小型实验室，尤其是在标本运输组织良好的医院，免疫荧光可能是抗原检测的最佳选择，前提是有经验丰富的显微镜检查人员和良好的紫外线显微镜。如果实验室接收大量标本且有EIA的既往经验，那么生物素-EIA或TR-FIA可能是最实用的技术。它们的优点包括临床样本中抗原的稳定性，因为不需要完整的、脱落的上皮细胞，标本处理简便，可检测大量标本，并且读取打印的检测结果比读取荧光显微镜结果主观性更小。PCR在呼吸道病毒感染诊断中发挥更大作用取决于未来的发展，如提取DNA或RNA以及从非特异性抑制剂中纯化提取物的实用方法，以及进一步改进以尽量减少交叉污染。肠道病毒和鼻病毒的组特异性检测就是PCR技术潜力的一个例子。在有经验的实验室，应提供EIA IgG抗体检测。重组抗原可能是此类检测的有用组成部分。
