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

Abstract

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.Epstein-Barr virus (EBV) is a human herpesvirus included in the Gammaherpesvirinae subfamily and is the only human species belonging to the genus Lymphocryptovirus (11). EBV infects more than 90% of the world's population, leaving the majority of individuals with a lifelong silent infection (14). Although most primary EBV infections are asymptomatic, the virus is the causative agent of infectious mononucleosis, a mild and self-limited lymphoproliferative disease (13). EBV is associated with the development of two epithelial tumors, nasopharyngeal carcinoma (5) and oral hairy leukoplakia, seen mainly in human immunodeficiency virus infection (3), as well as with various lymphoid carcinomas, lymphoproliferative disease in immunosuppressed patients, Burkitt's lymphoma, Hodgkin's disease, and T-cell non-Hodgkin's lymphoma (25).EBV DNA is present in a small fraction of lymphoid cells, and healthy virus carriers harbor 1 to 50 EBV genomes per 10 6 mononuclear cells, with B lymphocytes representing the major cellular reservoir (9). Qualitative PCR assays are unable to distinguish between active and latent infection. Consequently, clinical interpretation of positive results is difficult. However, clinical research suggests a role for viral load measurement in predicting and monitoring EBV-associate...

Evaluation of the Epstein-Barr Virus R-Gene Quantification Kit in Whole Blood with Different Extraction Methods and PCR Platforms

“…This problem of accurately quantifying low EBV DNA load (ie, between 2 and 3 log copies/ml) has already been reported for other commercial or inhouse EBV DNA quantification assays tested with the previous EBV QCMD Proficiency panel 2002. 16,20,21 Together, these results emphasize that evaluating new commercial EBV DNA quantification assays relies both on quality controls and clinical studies. They also confirm that it is more important to monitor EBV DNA load in follow-up samples and to correlate its dynamic decrease or increase with clinical events rather than to base the interpretation of viral load on absolute EBV levels only.…”

Comparison of Commercial Extraction Systems and PCR Assays for Quantification of Epstein-Barr Virus DNA Load in Whole Blood

“…In one case (patient H), with VL above 3 log 10 copies/ ml, the quantification differed repeatedly by more than 2 log 10 copies/ml between the Artus (5.69 log 10 copies/ml) and Argene (3.45 log 10 copies/ml) PCRs, despite the use of the same extraction process and the same PCR platform. Mutations involving the primers or the probe annealing site could account for this difference, since the Argene PCR and the laboratory-developed PCR target the EBV thymidine kinase gene, and the Artus PCR targets the EBNA1 gene, but the thymidine kinase gene was sequenced, and no mutations were observed (results not shown) (17). Targeting of genes present in multiple repeated copies may also cause this type of discrepancy, but to our knowledge, the thymidine kinase and EBNA1 genes are single-copy genes (15).…”

“…This heterogeneity is a barrier to the optimal use of quantitative PCR assays. The automation of nucleic acid extraction procedures and the use of commercialized PCR assays could decrease this heterogeneity and improve the agreement and the clinical utility of EBV load measurements in routine settings (5,15,17).…”

Delay in B-lymphocyte recovery and function following rituximab for EBV-associated lymphoproliferative disease early post-allogeneic hematopoietic SCT

“…14 In case of a positive signal or for follow-up of established EBV replication, EBV load was measured and monitored at similar intervals in PBMCs and plasma samples by means of a real-time quantitative PCR (artus EBV RG PCR, Qiagen, Hilden, Germany) as described in detail elsewhere. 15,16 The results were expressed as the number of copies per microgram of DNA for PBMCs and as the number of copies per milliliter of plasma, respectively.…”

“…), administered on days þ 1, þ 3, þ 6, þ 11. Primary engraftment was achieved in all patients and occurred after a median of 22.5 days (range, [16][17][18][19][20][21][22][23][24][25][26][27][28]; two patients had secondary graft failure at days 98 and 225 (patient nos. 2 and 4).…”