Abstract
BackgroundViral load monitoring and early Epstein-Barr virus (EBV) DNA detection are essential in routine laboratory testing, especially in preemptive management of Post-transplant Lymphoproliferative Disorder. Targeting the repetitive BamHI-W sequence was shown to increase the sensitivity of EBV DNA quantification, but the variability of BamHI-W reiterations was suggested to be a source of quantification bias. We aimed to assess the extent of variability associated with BamHI-W PCR and its impact on the sensitivity of EBV DNA quantification using the 1st WHO international standard, EBV strains and clinical samples.MethodsRepetitive BamHI-W- and LMP2 single- sequences were amplified by in-house qPCRs and BXLF-1 sequence by a commercial assay (EBV R-gene™, BioMerieux). Linearity and limits of detection of in-house methods were assessed. The impact of repeated versus single target sequences on EBV DNA quantification precision was tested on B95.8 and Raji cell lines, possessing 11 and 7 copies of the BamHI-W sequence, respectively, and on clinical samples.ResultsBamHI-W qPCR demonstrated a lower limit of detection compared to LMP2 qPCR (2.33 log10 versus 3.08 log10 IU/mL; P = 0.0002). BamHI-W qPCR underestimated the EBV DNA load on Raji strain which contained fewer BamHI-W copies than the WHO standard derived from the B95.8 EBV strain (mean bias: - 0.21 log10; 95% CI, -0.54 to 0.12). Comparison of BamHI-W qPCR versus LMP2 and BXLF-1 qPCR showed an acceptable variability between EBV DNA levels in clinical samples with the mean bias being within 0.5 log10 IU/mL EBV DNA, whereas a better quantitative concordance was observed between LMP2 and BXLF-1 assays.ConclusionsTargeting BamHI-W resulted to a higher sensitivity compared to LMP2 but the variable reiterations of BamHI-W segment are associated with higher quantification variability. BamHI-W can be considered for clinical and therapeutic monitoring to detect an early EBV DNA and a dynamic change in viral load.
Highlights
Epstein-Barr virus (EBV) is a member of gamma herpesviruses subfamily which infects more than 95% of adults [1]
BamHI-W qPCR underestimated the EBV DNA load on Raji strain which contained fewer BamHI-W copies than the World Health Organization (WHO) standard derived from the B95.8 EBV strain
Comparison of BamHI-W qPCR versus LMP2 and BXLF-1 qPCR showed an acceptable variability between EBV DNA levels in clinical samples with the mean bias being within 0.5 log10 international units (IU)/mL EBV DNA, whereas a better quantitative concordance was observed between LMP2 and BXLF-1 assays
Summary
Epstein-Barr virus (EBV) is a member of gamma herpesviruses subfamily which infects more than 95% of adults [1]. EBV DNA measurement in peripheral blood compartments became routine practice to help diagnose, monitor, prevent and treat the post-transplant lymphoproliferative disorder (PTLD) in immunocompromised recipients [4,5]. Sensitive and accurate detection of EBV DNA at the early stages of the PTLD remains crucial in the monitoring of transplanted patients [3,5,7]. Viral load monitoring and early Epstein-Barr virus (EBV) DNA detection are essential in routine laboratory testing, especially in preemptive management of Post-transplant Lymphoproliferative Disorder. We aimed to assess the extent of variability associated with BamHI-W PCR and its impact on the sensitivity of EBV DNA quantification using the 1st WHO international standard, EBV strains and clinical samples
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