Comparison of commercial extraction systems and PCR assays for quantification of Epstein-Barr virus DNA load in whole blood

Abstract

The automation of DNA extraction and the use of commercial quantitative real-time PCR assays could help obtain more reliable results for the quantification of Epstein-Barr virus DNA loads (EBV VL). This study compared two automated extraction platforms and two commercial PCRs for measurement of EBV VL in 10 EBV specimens from Quality Control for Molecular Diagnostics (QCMD) and in 200 whole-blood (WB) specimens from transplant (n = 137) and nontransplant (n = 63) patients. The WB specimens were extracted using the QIAcube or MagNA Pure instrument; VL were quantified with the EBV R-gene quantification kit (Argene) or the artus EBV RG PCR kit (Qiagen) on the Rotor-Gene 6000 real-time analyzer; and the results were compared with those of a laboratory-developed PCR. DNA was extracted from the QCMD specimens by use of the QIAamp DNA minikit and was quantified by the three PCR assays. The extraction platforms and the PCR assays showed good correlation (R, >0.9; P, <0.0001), but as many as 10% discordant results were observed, mostly for low viral loads (<3 log(10) copies/ml), and standard deviations reached as high as 0.49 log(10) copy/ml. In WB but not in QCMD samples, Argene PCR tended to give higher VL values than artus PCR or the laboratory-developed PCR (mean difference for the 200 WB VL, -0.42 or -0.36, respectively). In conclusion, the two automated extraction platforms and the two PCRs provided reliable and comparable VL results, but differences greater than 0.5 log(10) copy/ml remained between the two commercial PCRs after common DNA extraction.

Fig 1

Bland-Altman analysis of EBV DNA loads positive by both technologies. (A) Comparison of extraction robots. MagNA the plus the laboratory-developed PCR on a LightCycler instrument (MP-Lab) was compared with QIAcube plus the laboratory-developed PCR on a LightCycler instrument (Q-Lab). A total of 159 samples were used. (B) Comparison of Q-Lab with Q-A (QIAcube plus the artus EBV RG PCR kit on a Rotor-Gene instrument). A total of 154 samples were used. (C) Comparison of Q-Lab with Q-R (QIAcube plus the Argene EBV R-gene quantification kit on a Rotor-Gene instrument). A total of 156 samples were used. (D) Comparison of Q-A and Q-R. A total of 151 samples were used. The bold line represents the mean differences; the thin lines represent the mean ± 1.96 standard deviation.

Fig 2

Follow-up, using four different EBV DNA load technologies, for eight patients with various pathologies. Patient 1 was a hematopoietic stem cell transplantation recipient with non-Hodgkin lymphoma; patient 2, a hematopoietic stem cell transplantation recipient (donor EBV serological status, positive; recipient serological status, negative [D⁺ R⁻]); patients 3, 4, and 5, kidney transplant recipients (D⁺ R⁻). Patient 6 had HIV; patient 7, hypogammaglobulinemia with primary EBV infection; patient 8, EBV encephalitis. MP, MagNA Pure extraction; Q, QIAcube extraction; Lab, laboratory-developed real-time PCR (on a LightCycler amplification platform); A, Qiagen artus EBV RG PCR kit (on a Rotor-Gene amplification platform); R, Argene EBV R-gene kit (on a Rotor-Gene amplification platform).