Gouarin S, Vabret A, Scieux C, Agbalika F, Cherot J, Mengelle C, Deback C, Petitjean J, Dina J, Freymuth F
Laboratory of Virology, University Hospital, Avenue Georges Clemenceau, 14033 Caen Cedex, France.
J Virol Methods. 2007 Dec;146(1-2):147-54. doi: 10.1016/j.jviromet.2007.06.013. Epub 2007 Jul 27.
Automated real-time PCR systems have become the most common method in the quantitation of viral load during cytomegalovirus (CMV) infection in immuno-compromised patients. In order to evaluate a new commercially available CMV real-time PCR assay (CMV R-gene, Argene, France), a pp65 antigenemia assay and four different "in-house" real-time PCR assays were compared to the CMV R-gene for the detection and the quantitation of CMV load in 506 specimens of whole blood from transplant patients in four French hospital laboratories. The CMV R-gene was more sensitive than the pp65 antigenemia: there were 18% antigenemia-negative versus CMV R-gene-positive samples. A significant correlation was found between DNA quantitation by CMV R-gene and the number of positive cells detected by the pp65 antigenemia test (Spearman's rank test, r=0.63, p<0.0001). A CMV DNA load equivalent to 50 pp65-positive cells/200000 polymorphonuclear leukocytes was 5.26log(10)copies/mL of whole blood. When the CMV R-gene kit was compared to the four other "in-house" real-time PCR assays, there were few discordant results (6.7% total for the four laboratories), all detected with a weak positive CMV DNA viral load. Spearman's coefficients showed a good (r=0.82 for laboratory 1, r=0.66 for laboratory 3) to excellent (r=0.99 for laboratory 2, r=0.94 for laboratory 4) correlation between CMV R-gene and the four real-time "in-house" PCR assays. However, the results of CMV DNA viral load generated by CMV R-gene test were constantly higher than those generated by three out of four "in-house" PCR assays. This mean variation in CMV DNA viral load measured by CMV R-gene and "in-house" PCRs was of 0.77log(10), 0.04log(10), 0.77log(10) and 0.97log(10), for laboratories 1, 2, 3 and 4, respectively. We concluded that there was variability between results of different real-time PCR assays for CMV DNA quantitation. This observation emphasized the need of a standardised commercial assay to allow an "inter-laboratory" comparison of results. Our study showed that CMV R-gene is an accurate, efficient, reliable and versatile tool for rapid diagnosis and monitoring of CMV disease in transplantation recipients.
自动化实时荧光定量PCR系统已成为免疫功能低下患者巨细胞病毒（CMV）感染期间病毒载量定量的最常用方法。为了评估一种新的市售CMV实时荧光定量PCR检测方法（CMV R-gene，法国Argene公司），在法国四家医院实验室中，将一种pp65抗原血症检测方法和四种不同的“实验室自建”实时荧光定量PCR检测方法与CMV R-gene检测方法进行比较，以检测和定量506份移植患者全血标本中的CMV载量。CMV R-gene比pp65抗原血症检测更敏感：有18%的样本抗原血症检测为阴性而CMV R-gene检测为阳性。CMV R-gene检测的DNA定量与pp65抗原血症检测所检测到的阳性细胞数之间存在显著相关性（Spearman秩相关检验，r = 0.63，p < 0.0001）。相当于50个pp65阳性细胞/200000个多形核白细胞的CMV DNA载量为全血5.26log(10)拷贝/mL。当将CMV R-gene试剂盒与其他四种“实验室自建”实时荧光定量PCR检测方法进行比较时，不一致的结果很少（四个实验室总计6.7%），所有这些结果均在CMV DNA病毒载量弱阳性时检测到。Spearman系数显示CMV R-gene与四种“实验室自建”实时荧光定量PCR检测方法之间具有良好（实验室1的r = 0.82，实验室3的r = 0.66）至极好（实验室2的r = 0.99，实验室4的r = 0.94）的相关性。然而，CMV R-gene检测所产生的CMV DNA病毒载量结果始终高于四种“实验室自建”PCR检测方法中的三种所产生的结果。对于实验室1、2、3和4，CMV R-gene和“实验室自建”PCR检测所测得的CMV DNA病毒载量的平均差异分别为0.77log(10)、0.04log(10)、0.77log(10)和0.97log(10)。我们得出结论，不同的实时荧光定量PCR检测方法在CMV DNA定量结果上存在差异。这一观察结果强调了需要一种标准化的商业检测方法，以便能够对结果进行“实验室间”比较。我们的研究表明，CMV R-gene是用于快速诊断和监测移植受者CMV疾病的准确、高效、可靠且通用的工具。
