The impact of targeting repetitive BamHI-W sequences on the sensitivity and precision of EBV DNA quantification

Armen Sanosyan (1) , Alexis Fayd’herbe de Maudave (1) , Karine Bolloré (1) , Valérie Zimmermann (2) , Vincent V. Foulongne (3, 1) , Philippe van de Perre (1) , Edouard Tuaillon (1)

1 Pathogénèse et contrôle des infections chroniques (PCCI)
2 IGMM - Institut de Génétique Moléculaire de Montpellier
3 Laboratoire de Virologie

Background: 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. 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. Methods: Repetitive 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. Results BamHI-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. Conclusions: Targeting 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.

Mots clés: Epstein-Barr virus; Repeated sequences; Viral load; Polymerase chain reaction; Raji cells; Blood; Blood plasma; DNA extraction

Domaines: Virologie; Ingénierie biomédicale

Armen Sanosyan, Alexis Fayd’herbe de Maudave, Karine Bolloré, Valérie Zimmermann, Vincent V. Foulongne, et al.. The impact of targeting repetitive BamHI-W sequences on the sensitivity and precision of EBV DNA quantification. PLoS ONE, 2017, 12 (8), pp.e0183856. ⟨10.1371/journal.pone.0183856⟩. ⟨hal-01890162⟩