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Comparison of Commercial Real-Time PCR Assays for Quantification of Epstein-Barr Virus DNA

Guillermo Ruiz,^* Pilar Peña, Fernando de Ory, and Juan Emilio Echevarría

National Microbiology Center, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo s/n, 28220 Majadahonda, Madrid, Spain

Received 29 September 2004/ Returned for modification 13 December 2004/ Accepted 22 December 2004

Clinical research suggests a role for viral load measurement in predicting and monitoring Epstein-Barr virus (EBV)-associated diseases. The aim of this study was to assess the performance of the recently commercially available quantitative assays for EBV based on real-time PCR: the RealArt EBV LC PCR kit and the LightCycler EBV quantification kit. A total of 87 samples were analyzed: 67 samples were obtained from transplant recipients and patients with EBV-associated diseases, 8 samples were obtained from the Quality Control for Molecular Diagnostics 2002 EBV Proficiency Program, and 12 negative qualitative nested PCR samples were used as negative controls. Inter- and intra-assay variabilities were determined by running replicates of two samples. All samples were run in a LightCycler instrument. The differences between positive and negative results were not considered statistically significant (P = 0.5355). There were no false-positive results using either method for nested PCR negative-control samples. The difference in viral load values using the two different methods was considered statistically significant (P < 0.01). The logarithmic linear correlation for both assays was low (r = 0.449) but significant (P < 0.01). The LightCycler EBV quantification kit showed a wider dispersal in results but produced substantially more-accurate melting temperature profile curves. The bias towards lower measurements was considerable in comparison with higher viral load. The differences in PCR efficiency and the presence of mutations could explain the disparity between the two methods. It was concluded that confidence intervals would be required to report the results rather than plain absolute values of viral load for patient monitoring.

Present address: Department of Clinical Microbiology and Parasitology, University Hospital La Paz, Paseo de la Castellana, 261, 28046 Madrid, Spain.

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