Epstein-Barr virus (EBV) is recognized as a primary pathogen causing Infectious mononucleosis [1]. Monitoring of EBV DNA in peripheral blood is routinely performed in transplant centers because these patients are at higher risk to develop EBV-associated diseases including the potentially life-threatening post-transplant lympho-proliferative disorder (PTLD).　In most cases, PTLD is associated with　EBV infection of B cells, either as a consequence of reactivation of the virus post transplantation [2,3] and intensity and type of immune suppression [4].　The incidence of PTLD reflects the more intensive use of immunosuppressive drugs, possibly in combination with the varied EBV load in the transplanted organ [5]. PTLD is most likely caused by iatrogenic suppression of T-cell activity in transplantation recipients, which leads to inadequate immune surveillance against EBV-induced proliferation of infected B-cells [6]. Currently there is no definitive treatment regimen for PTLD prevention [7].

The study was approved by the institute’s ethics sub-committee. Informed consent was obtained from the patient’s kin. Clinical details were recorded in the proforma made specifically for the study. Samples of peripheral blood (2-3 mL) were collected in plain and EDTA-coated vacutainers. Samples were processed immediately for serological analysis. Samples for molecular detection were stored at minus 80ºC.

In order to confirm the presence of EBV, two PCRs targeting the genes that code for EBV-VCA and EBNA1 were standardized and applied to all samples. All the PCRs were optimized to be carried out in the same thermal profile. 50µL of the PCR mix contained 10 µL of extracted DNA, 100mM of each dNTP, 5 µL of 10× PCR buffer, 1 µM of each forward and reverse primer and 3U/µL Taq DNA polymerase. PCR was carried out denaturing the DNA at 94ºC for 5 minutes followed by amplification for 30 cycles, by secondary denaturation at 94ºC for 1 minute, annealing at 59ºC for 1 minute and extension at 72ºC for 1 minute with final extension for 7 minutes at 72ºC. For the second round of amplification 5µL of the first round product was added to 45µL of the PCR mix containing 10 mM of each dNTP, 10× buffer, 1 µM of each forward and reverse primer and 3 U/µL Taq DNA polymerase. The PCR amplification was carried out for 20 cycles with the same thermal profile as mentioned above. Two controls (a reagent control and a reaction control) were included in each PCR run. The PCR results were considered valid only when the reagent controls were negative and the specific amplified product was obtained with amplified positive controls. To prevent contamination, DNA extraction, PCR cocktail preparation, amplification and analysis of results were carried out in physically separated rooms. Visualization of PCR product was done by subjecting 10 µL of amplified reaction mixture to electrophoresis on a 2% agarose gel incorporating 5µg mL-1 of ethidium bromide in 1×Tris-Borate buffer (pH -8.2-8.6) and documented on gel documentation system (Vilber Lourmat, France). The viral load was estimated in the DNA extracts of all test and control samples using a commercial kit - RoboGene Quantification Kit (Hilden, Germany). The assay was performed on Rotor Gene (Hilden, Germany) real time PCR equipment. The amplification reaction was carried out following the manufacturer’s instructions. PCR was carried out at 50ºC for 30 minutes followed by initial denaturation at 95ºC for 15 minutes followed by 50 cycles of initial denaturation at 95°C for 30 seconds, annealing at 50ºC for 60 seconds and extension at72ºC for 30 seconds. The viral load was expressed as copies/mL. The samples that were found positive for EBV were subjected to genotyping by PCR targeting the EBNA2 and EBNA3C genes. Uniplex PCR for detection of EBNA2, EBNA3C genes was standardized using the EBV-A and EBV-B Standard Strains. Primers targeting genes that codes for EBNA2 and EBNA3C genes were designed using Primer premier Biosoft international, USA, based on consensus sequence obtained with specific sequences of EBV specific genes submitted in GenBank. The nucleotide sequences of the primers and the expected respective product size are given in Table I. All primers and PCR reagents were procured from VBC – Biotech service, Vienna. The PCR positive -amplified products were further subjected to DNA sequencing and compared with the standard strain sequence to determine the homology percentage. Cycle sequencing of the amplified products was performed in a 10µL reaction volume, containing 0.5µL of RR mix, 3.5µL of sequencing buffer, 1µL of forward primer (1:100 diluted), 1µL of reverse primer (1:100 diluted)2 ìL MilliQ water, and 2µL of amplified product. Amplification was carried out in the Perkin- Elmer thermocycler using 25 cycles at 96°C for 10 s, at 50°C for 5s, and at 60ºC for 4 min, with initial denaturation at 96°C for 1 min. The cycle-sequenced products were then purified and sequenced using ABI Prism 3130 AVANT (Applied Biosystems, USA) genetic analyzer, which works based on the principle of Sanger’s dideoxy termination method. The sequences were analyzed by Bio Edit sequence alignment software, (www.softpedia.com/progDownload/BioEdit-Download-174716). BLAST analysis (www.ncbi.nlm.nih.gov/BLAST) was done to compare and confirm the sequenced data with the standard strains and to determine the homology percentage. The nucleotide sequences of the EBNA2 and EBNA3C PCR positive amplified products were analyzed by comparison with EBV standard strain nucleotide sequences using BIOEDIT software. Evolutionary distances were estimated by constructing a phylogram using UPGMA algorithm by performing bootstrap analysis (Replicates 100) in CLC Main Workbench6.71 software. The statistical significance of PCR on diagnosis of EBV in transplant patients was done using Fisher’s exact test. Mean, Standard deviation, median and Box plot for viral load were determined using SPSS14.

TABLE I  List of Primers Used for Amplification of Genes That Code for VCA, EBNA1, EBNA2 and EBNA3C of EBV

Peripheral blood samples were obtained from 70 pediatric solid organ transplant recipients; 24 were renal and 46 were liver transplant recipients. The most common clinical conditions presented were acute renal failure, Hepatitis, encephalitis, interstitial nephritis and Glomerulo-nephritis. Eight of the 70 patients were positive for IgG VCA. Two of the 70 patients were positive for IgM VCA and seven of the 70 patients were positive for IgG EBNA VCA. None of the 70 patients were positive for IgM EBNA. Serological tests for detection of other viruses showed IgM CMV in five, and IgM and IgG to CMV in six patients. Seven patients were positive for IgG HSV1 and one of the patients was positive for IgM HSV1. None of the controls tested positive to IgM VCA, whereas nine samples tested positive to IgG VCA.

Thirty-five samples (50%) tested positive for both VCA and EBNA1. Eight (13.3%) control samples tested positive for EBNA1 PCR. None of the controls tested positive f22or EBV VCA and no detectable copy numbers were found by Real time PCR. All the test samples that tested positive by nPCR were also tested positive by real-time PCR. The mean viral load for EBV PCR positive patients who did not develop PTLD was 50,424 copies/mL (Lowest Viral Load: 14 copies/mL and Highest Viral Load: 8,20,955 copies/mL). One of the 70 post-transplant patients developed PTLD four months post-transplant. The patient had CMV infection acquired 3-6 weeks post liver transplant which was successfully treated with gancyclovir. The PTLD coincided with strongly increased levels of EBV DNA load in the peripheral blood.　 The highest titer value of 11,63,900 copies/mL was detected in the blood collected from this PTLD patient. Real-time PCR EBV titre from lymphoid biopsy of this patient was 89,14,188 copies/mL. The viral load for this patient prior diagnosis of PTLD was 11,63,900 copies//mL, which was significantly higher compared to the load in the liver transplant recipients who did not develop PTLD. The clinical presentations of EBV positive pediatric renal and liver transplant recipients are given in Web Table I.

Genotyping of type A and type B was done by targeting EBNA2, EBNA3C genes. Type A was detected in thirty two (45.7%) and type B in blood of two (2.9%) samples (Table II). The blood and lymphoid tissue of the patient who developed PTLD revealed mixed subtypes, a co-infection with both A and B EBV genotypes. Both samples that tested positive for EBV Type-B genotype were found to have higher titre values than all of the EBV type-A positive samples (1,23,714 copies /mL and 8,20,955 copies/mL). Eight control sample tested EBNA1 PCR positive revealed prevalence of EBV Type A genotype by both genotyping PCRs (Table II). All PCR positive samples were subjected to sequencing. Sequencing of the samples re-confirmed the PCR results. The full length sequences were submitted to Genbank database and the assigned accession numbers are KC884748 – KC884757 and KF429681 – KF429706. Comparison of EBNA2 and EBNA3C sequences with EBV standard strain sequence, showed type A to form a unique clade with B95_8 strain and type B to form a separate clade with EBV Type B standard strain Ag876.

TABLE II  Viral Load in EBV-Positive Patients

In this study of 70 solid organ transplant recipients, we found EBV type A to be more prevalent in pediatric transplant patients as compared to EBV type B. Samples positive for EBV type B had significantly higher titre values than Type A samples. The sample with co-infection had the highest titer values and this patient also developed PTLD. All the patients except the patient who developed PTLD responded to the drugs and recovered from EBV illness (EBV titer reduced).

The limitations in our current study were the follow-up samples were not collected or diagnosed for all the patients. The immunological response during active EBV infection was not detected. Knowledge about the pathogenic factors of PTLD may help in the development prognostic markers and therapeutic strategies for treating EBV induced PTLD in immunocompromised post-transplant patients.

Every center should have a high-risk group which would include patients based on previous studies and the centers own experience. Factors for high-risk patients could include EBV sero-negativity at the time of transplant, active primary EBV infection at the time of transplant, underlying disease leading to transplantation, prior splenectomy, second transplant, patient age (children and older adults), co-infection by cytomegalovirus and other viruses, acute or chronic graft-versus-host disease, immunosuppressive drug regimen and intensity, cytokine polymorphisms, HLA type and extent of HLA mismatch, and the presence of multiple risk factors on this list [12]. Levels often rise before clinical diagnosis of PTLD, allowing pre-emptive intervention in high-risk patients who are routinely monitored for EBV levels [12]. In our study, the viral load was higher in the patient who developed PTLD before the diagnosis was made compared to the samples received from the patient after diagnosis was made and treatment had started. Overall EBV DNA load in this patient decreased from 1, 40,392 copies /mL before diagnosis of PTLD to 1032 copies/mL blood, after diagnosis and treatment and finally EBV negative.

Despite having only one patient who developed PTLD we suggest that EBV viral load could act as a good diagnostic tool to improve prediction of PTLD in transplant patients. Developing better PTLD prediction tools using high and low risk patient groups will surely improve patient treatment. Patients who fall in the High-risk group can be focused on for regular follow-up and treatment.