Standard and clinical samples
The study was carried out in accordance with the Declaration of Helsinki. This study was a non-interventional study with no addition sampling to usual procedures. Biological material and clinical data were obtained only for standard viral diagnostic following physicians’ prescriptions (no specific sampling, no modification of the sampling protocol). Data analyses were carried out using an anonymized database. According to the French Health Public Law (CSP Art L 1121–1.1), such protocol was exempted from informed consent application.
Leftover EDTA K2 tube (BD Vacutainer®) samples sent to the Virology unit of Saint Louis Hospital for EBV monitoring were used in this study. A total of 282 whole blood (WB) specimens received in the laboratory for EBV load quantification were collected from 196 patients including 95 hematopoietic stem cell transplant recipients, 22 kidney transplant recipients, 29 patients with immunological or heamatological diseases, 27 patients from general medicine, 10 HIV infected patients, 9 patients from intensive care unit and 4 patients hospitalized in infectious disease department. The clinical samples were selected prospectively and retrospectively from aliquots frozen at − 80 °C. All clinical consecutive whole blood samples received for EBV quantification within 7 days (124) were tested with the two assays. In order to obtain sufficient positive samples to enable a correlation analysis of EBV loads between the two assays, additional positive whole blood samples were selected retrospectively within 8 months. The samples were tested in separate runs both with the Abbott RealTime EBV assay (RT assay) and EBV PCR Kit V1 assay (V1 assay). Furthermore, 75 additional frozen samples at − 80 °C of 11 HSCT recipients were tested in order to analyze EBV DNA kinetics after rituximab injection in the two quantitative real time PCR assays.
AcroMetrix™ EBV controls high and low
Thermo Scientific™ AcroMetrix™ EBV low positive-control (catalog number 961230, lot 513,101) and high-positive-control (catalog number 961231, lot 417,503) were used for analytical evaluation of RT assay. The manufacturer’s instructions mentioned that expected results when using the AcroMetrix™ EBV Low and High controls must be established by the end user for their particular EBV DNA assay.
The Quality Control for Molecular Diagnosis (QCMD) 2016 EBV WB challenge 1 and 2 and 2017 EBV WB challenge 1 and 2 proficiency panels were composed of 5 frozen WB samples. Expected values correspond to mean consensus (log10 IU/mL) calculated from data returned by participants from different laboratories after removing outliers.
WHO international standard
The first WHO international standard for EBV for nucleic acid amplification techniques (National Institute for Biological Standards and Control NIBSC code 09/260; Potters Bar, Hertfordshire, Great Britain) is a lyophilized whole virus preparation of the EBV B95–8 strain (type 1). The material has been assigned a concentration of 5 × 106 IU/mL when reconstituted in 1 mL of nuclease-free water.
Quantitative real time PCR assays
The quantification of EBV in WB was carried out on the Abbott m2000 platform for the two assays. This platform includes the m2000 sp. instrument for automated extraction of DNA and the m2000 rt. instrument for real-time PCR of series of 48 samples.
Abbott RealTime EBV assay (RT assay)
The amplification target is a highly conserved region of the BLLF1 gene which encodes the gp350/220 envelope glycoprotein. The Abbott RealTime EBV assay (Abbott Molecular Inc., Des Plaines, IL, USA) uses three reagent kits, the amplification reagent kit, the calibrator kit for the standard curve and the control kit for external control. An internal control is also supplied to check the overall internal process, including DNA extraction and possible PCR inhibition. Extraction of DNA was done on the m2000sp system. DNA extraction was performed from 300 μL of WB and eluted in 250 μL. Extraction was followed by automated addition of 25 μL of master mix and 35 μL of purified DNA into the PCR plate. In each run, one negative control and two positive controls (Low and High) were included. Two calibrators (A and B) were used to determine the standard curve. The results are expressed in IU/mL. Manufacturer lower limit of quantification (LLQ) is reported as 150 IU/mL and limit of detection (LOD) as 115 IU/mL.
Artus EBV PCR kit V1 assay (V1 assay)
EBV DNA quantification with the Artus EBV PCR Kit V1 assay (Qiagen, MD, USA, previously commercialized by Abbott Molecular) was also performed on the m2000 platform in batches of 48 tests. The PCR amplification reagent targets a conserved region within the gene coding for Epstein Barr virus Nuclear Antigen (EBNA1). The EBV PCR Kit V1 includes an internal control to check the overall process including DNA extraction and possible PCR inhibition. Automated DNA extraction and PCR reaction set up were performed on the Abbott m2000sp instrument. Briefly, DNA was purified from 300 μL of WB and eluted in 250 μL. The EBV quantification was performed with 20 μL of purified DNA. Sealed PCR plates were loaded on the Abbott m2000rt instrument for real-time PCR. Four calibrators (QS1, QS2, QS3 and QS4) were used to establish a calibration curve. Every run included one low calibrator (QS3). The results were expressed in copies/mL. For comparison with the RT assay, conversion factor previously calculated  was used to obtain IU/mL. The LLQ of the assay was 1000 copies/mL corresponding to 310 IU/mL.
Quantitative real time PCR assays interpretation
For both assays, the results were classified as follows: target not detected, target detected but not quantifiable (< LLQ) and target detected and quantifiable (>LLQ and in the range of linearity).
Analytical performances of the Abbott RealTime EBV assay
All dilutions were performed in EBV negative whole blood.
Limit of detection
The LOD was estimated by using serial dilutions of the WHO international standard at expected value of 500, 100 and 20 IU/mL. Each dilution was tested 10 times. The LOD is defined as the EBV DNA concentration detected with a probability of 95% or more.
The assay linearity was verified with dilutions of a highly EBV DNA positive sample in EBV negative WB at expected value of 1,000,000, 100,000, 10,000, 1000 and 100 IU/mL. Each dilution was quantified with the RT assay 3 times and the mean EBV concentration of each sample was calculated.
The repeatability was determined with the AcroMetrix™ EBV Plasma Control High (4.76 log10 IU/mL) and two EBV DNA positive WB clinical samples (7.20 log10 IU/mL - “Blood High”- and 4.09 log10 IU/mL - “Blood Low” quantified with Abbott RealTime EBV assay). Ten replicates of each sample were tested in the same run. For each sample, intra assay coefficient of variation (CV) was estimated.
The reproducibility was determined with the AcroMetrix™ EBV Plasma Control High and Low. Sixteen replicates of AcroMetrix High and Low were tested on a period of 16 days by four different operators. For each sample, inter assay CV was estimated.
A panel of 30 samples consisting of alternate phosphate buffered saline as negative samples and High-load EBV DNA WB (mean = 4.29 log10 IU/mL) were assayed on the three m2000 platforms.
Concordance on qualitative results between the RT assay and the V1 assay was established by Cohen’s kappa statistic. The evaluation of quantitative correlation between the two assays included results positive in both and was estimated by using linear regression analysis and Bland-Altman plots. Statistical analysis was performed using GraphPad Prism6 software . Differences were considered statistically significant at p-values below 0.05.