Kulkarni A, Westmoreland D, Fox J D
Department of Virology, Cardiff Public Health Laboratory, University Hospital of Wales, Cardiff, CF14 4XN, UK.
Clin Microbiol Infect. 2001 Apr;7(4):179-86. doi: 10.1046/j.1198-743x.2001.00227.x.
Molecular assays are now considered to be the "gold standard" for assessment of human cytomegalovirus (CMV) infection and disease in those at risk from severe associated clinical manifestations. There is, however little consistency in the methods used in different centres. This study was undertaken to compare different qualitative molecular-based approaches for assessment of CMV activation from latency in samples from immunosuppressed transplant recipients.
Nucleic acid amplification techniques based on the polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA) were undertaken for the assessment of CMV replication and associated disease in immunosuppressed transplant recipients. Samples from 32 transplant recipients were tested during this study using three molecular-based strategies: (1) detection of CMV DNA in whole blood extracts (positive after a single round of PCR considered "high-level" positive, N = 55); (2) detection of cell-free CMV DNA in plasma (two methods, N = 55 for each); and (3) detection of late pp67 CMV mRNA after NASBA (N = 51). Results using a commercial pp65 antigenemia assay were available for comparison from 40 samples.
Seven samples were positive for CMV by all methods and 36 were negative by all methods undertaken. The other 12 samples gave discordant results using different molecular methods. The correlation between whole blood "high-level" PCR, NASBA for pp67 mRNA and antigenemia results was generally good. Results presented show that plasma PCR results do not always correlate with methods utilizing whole blood as the substrate and that inhibitors in these samples could be problematic. Whole blood PCR gave more positive results than the other assays but use of a nested assay on whole blood or plasma led to detection of CMV in individuals who had no other indicators of virus replication and who did not develop associated disease (low specificity). Although the number of confirmed CMV disease episodes was low in this study, the problems of low positive predictive value for sensitive, qualitative PCR assays was clearly demonstrated.
Assays based on qualitative detection of viral nucleic acid may provide information useful for management of CMV but caution is necessary when making comparisons between results using different molecular strategies. It remains to be proven in large, comparative clinical studies in which the approach and method give the best balance between sensitivity, specificity and clinical relevance for different patient groups.
对于存在严重相关临床表现风险的人群，分子检测目前被视为评估人巨细胞病毒（CMV）感染及疾病的“金标准”。然而，不同中心所采用的方法缺乏一致性。本研究旨在比较不同的基于分子的定性方法，以评估免疫抑制移植受者样本中潜伏状态的CMV激活情况。
采用基于聚合酶链反应（PCR）和基于核酸序列的扩增（NASBA）的核酸扩增技术，对免疫抑制移植受者的CMV复制及相关疾病进行评估。在本研究中，使用三种基于分子的策略对32名移植受者的样本进行检测：（1）检测全血提取物中的CMV DNA（一轮PCR后呈阳性被视为“高水平”阳性，N = 55）；（2）检测血浆中游离的CMV DNA（两种方法，每种方法N = 55）；（3）NASBA后检测晚期pp67 CMV mRNA（N = 51）。40份样本可获得使用商业pp65抗原血症检测的结果用于比较。
所有方法检测均为阳性的样本有7份，所有方法检测均为阴性的样本有36份。其他12份样本使用不同分子方法得出了不一致的结果。全血“高水平”PCR、NASBA检测pp67 mRNA与抗原血症检测结果之间的相关性总体良好。结果表明，血浆PCR结果并不总是与以全血为底物的方法相关，且这些样本中的抑制剂可能存在问题。全血PCR比其他检测得出更多阳性结果，但对全血或血浆使用巢式检测会导致在没有其他病毒复制指标且未发生相关疾病的个体中检测到CMV（特异性低）。尽管本研究中确诊的CMV疾病发作数量较少，但灵敏的定性PCR检测阳性预测值低的问题得到了明确证明。
基于病毒核酸定性检测的方法可能为CMV管理提供有用信息，但在比较不同分子策略的结果时需要谨慎。在大型比较临床研究中，哪种方法和策略能在不同患者群体中实现敏感性、特异性和临床相关性的最佳平衡，仍有待证实。
