Epstein–Barr virus DNA quantification and follow-up in Tunisian nasopharyngeal carcinoma patients

The prognostic value of the Epstein–Barr virus (EBV) DNA load in sera of nasopharyngeal carcinoma (NPC) patients measured before any treatment, after treatment and before relapse was assessed. The real-time polymerase chain reaction was used to detect the viral load levels among 74 NPC subjects. Patients were followed up for a period going from 1 to 6 years (median 4 years). Before treatment, the EBV DNA load was correlated with lymph node involvement and advanced stages. After treatment, the viral load level declined significantly and patients presenting a viral load level lower than 1000 copies/ml showed a better overall survival (OS). Moreover, a significant result was found when the 6-year OS rates of patients having fewer or more than 15,000 copies/ml of viral load before relapse were compared. These results suggest that the EBV DNA load quantification after treatment may be a useful predictor of disease progression and survival.     

Quantitative PCR in EBV-infected renal transplant patients

In this study we investigated the levels of Epstein Barr virus (EBV) DNA by quantitative polymerase chain reaction (Q-PCR) in serum, whole blood and peripheral blood mononuclear cells (PBMC) from anti-EA IgG seropositive or anti-EA IgG seronegative EBV infected renal transplant recipients. We compared serological data with the viral load to monitor the risk of developing post-transplant lymphoproliferative disorders (PTLD). All patients were asymptomatic and none of them developed PTLD at the time of the study. EBV DNA quantitation for each patient varied in whole blood and PBMC samples probably due to different numbers of mononuclear cells present in samples from which DNA was extracted (whole blood vs. purified PBMC). In 92% of the serum samples EBV DNA was undetectable probably due to absence of free genomes since the number of DNA copies detected in samples from whole blood and PBMC does not reach very high levels. The correlation between the presence of EA-antibody, considered serological evidence of EBV reactivation, and the viral load showed that 60% of EA-positive patients had quantifiable EBV DNA, whereas in 40% of EA-positive patients EBV DNA was undetectable, showing serological reactivity but no viral replication. Of the remaining EA-negative patients, EBV DNA could be detected in 71% of them, whereas 29% did not show EBV DNA, indicating no EBV replication. In conclusion, our results confirm that the presence of serum IgG anti-EA antibody is not a reliable marker of active EBV infection whereas the evaluation of the viral load in blood samples is a useful diagnostic tool to monitor and to better understand the course of EBV infection in immunocompromised renal transplant patients at risk of developing PTLD.     

Epstein Barr viral load monitoring by quantitative PCR in renal transplant patients

Post-transplant lymphoproliferative disorders (PTLD), ranging from lymphoid hyperplasia to clonal malignancy, are a severe complication arising in solid organ transplant patients. Their reported incidence ranges from 1 to 20%, according to factors such as type of transplanted organ and the age of recipients. A strong correlation between Epstein Barr virus (EBV) infection, the grade and type of immunosuppression and the development of PTLD has been recognized. The detection and quantification of EBV-DNA load in peripheral blood have been utilized as prognostic markers for the development of PTLD, showing a correlation between high levels of EBV-DNA in the blood and the development of PTLD. In this study, we monitored EBV viral load monthly in 15 renal transplant recipients for six months. The number of EBV-DNA copies was measured in peripheral blood mononuclear cells (PBMC) and serum samples by a quantitative PCR protocol developed in our laboratory that employes a previous screening of samples containing a significant number of viral DNA copies (> or =1000 copies/10(5) PBMC or 100 microl serum) by semi-quantitative PCR followed by a precise quantification of the only significant samples by quantitative-competitive (QC)-PCR. Our 15 renal transplant patients neither developed PTLD nor had recurrent acute illnesses or acute graft rejections during the study. The results obtained in the monthly follow up of EB viral load in PBMC samples confirmed its fluctuation in asymptomatic patients reported in literature. In particular, 5/14 (35.7%) of EBV seropositive patients had an EBV-DNA load equal to 1000 EBV copies /10(5) PBMC (roughly corresponding to 10.000 copies/microg PBMC DNA), and 1/14 (7.1%) reached 5000 EBV copies /10(5) PBMC (roughly corresponding to 50.000 copies/microg PBMC DNA), at least once in our study. In the EBV seronegative patient, EBV-DNA in PBMC samples was always undetectable (less than 100 DNA copies/10(5) PBMC). EBV-DNA load in all serum samples was less than threshold value of our quantification protocol (<100 DNA copies/100 microl serum), supporting the literature data. With regard to immunosuppressive treatment, 66.7% of the six patients in whom EBV load reached values equal to or higher than 1000 DNA copies/10(5) PBMC, were on FK506 whereas only 33.3% of them were on CyA. In conclusion, further investigations are needed to better understand the role of EBV infection in the pathogenesis of PTLD in immunosuppressed patients. Given the high positive predictive value of EB viral load in peripheral blood for diagnosis of PTLD reported by several authors, and the described absence of correlation between the serological evidence of EBV reactivation and EB viral load, EBV viral load measurement in PBMC and serum samples using quantitative PCR techniques is a powerful diagnostic tool to monitor transplanted patients at risk to develop PTLD.     

TT virus is distributed in various leukocyte subpopulations at distinct levels, with the highest viral load in granulocytes.

When TT virus (TTV) DNA was quantitated in whole blood and plasma aliquots from 27 viremic individuals by real-time detection PCR that can detect essentially all TTV genotypes, the TTV load was 6.9 +/- 3.5 (mean +/- standard deviation)-fold higher in the whole blood than in the plasma samples [P < 0.002 (paired t test)]. To clarify the reason for this difference, peripheral blood cells of various types including red blood cells, granulocytes (CD15+), B cells (CD19+), T cells (CD3+), monocytes (CD14+), and NK cells (CD3-/CD56+) were separated at a purity of 95.4-99.5% from each of three infected individuals with relatively high TTV viremia, and their TTV viral loads were determined. Red blood cells were uniformly negative, but the other cell types were positive for TTV DNA at various titers. In all three patients, the highest TTV load was found in granulocytes (4.2 x 10(4)-3.1 x 10(5) copies/10(6) cells), followed by monocytes (1.4-2.2 x 10(4) copies/10(6) cells) and NK cells (5.4-6.5 x 10(3) copies/10(6) cells); B and T cells were positive, with a low viral load (6.7 x 10(1)-2.7 x 10(3) copies/10(6) cells). These results indicate that TTV is distributed in various peripheral blood cell types at distinct levels, with the highest viral load in granulocytes, and that a significant proportion of the TTV DNA in peripheral blood is not identified by the standard plasma/serum DNA detection methods.     

Anti-Epstein-Barr virus antibody in patients with cancer of various sites and control groups.

Sera collected from patients with nasopharyngeal carcinoma (NPC, 321 cases), cancer of the other sites (297 cases), diseases of the ear, nose and throat (64 cases) and neighborhood controls matched for age and sex (817 cases) were titrated for antibodies to Epstein-Barr virus (EBV) by the indirect immunofluorescence antibody technique. High anti-EBV antibody titers (greater than or equal to 1:640) were noted in 55% in patients with NPC but only less than 31% were noted for the other 3 groups. The differences in the distributions of anti-EBV antibodies are statistically significant. The geometric mean levels of the antibody titers were 1:342 for the patients with NPC but less than 1:178 for the other 3 control groups. The relative risks shows that more than 40 times higher risk for those with equal to or higher than 1:640 antibody titers than those with lower than 1:40. The patients with NPC also show for higher ridit scales than the other 3 groups. The etiology of NPC was discussed with our findings.     

DNA of Epstein-Barr Virus. II. Comparison of the Molecular Weights of Restriction Endonuclease Fragments of the DNA of Epstein-Barr Virus Strains and Identification of End Fragments of the B95-8 Strain

Incubation of the DNA of the B95-8 strain of Epstein-Barr virus [EBV (B95-8) DNA] with EcoRI, Hsu I, Sal I, or Kpn I restriction endonuclease yielded 8 to 15 fragments separable on 0.4% agarose gels and ranging in molecular weight from less than 1 to more than 30 x 10(6). Bam I and Bgl II yielded fragments smaller than 11 x 10(6). Preincubation of EBV (B95-8) DNA with lambda exonuclease resulted in a decrease in the Hsu I A and Sal I A and D fragments, indicating that these fragments are positioned near termini. The electrophoretic profiles of the fragments produced by cleavage of the DNA of the B95-8, HR-1, and Jijoye strains of EBV were each distinctive. The molecular weights of some EcoRI, Hsu I, and Sal I fragments from the DNA of the HR-1 strain of EBV [EBV (HR-1) DNA] and of EcoRI fragments of the DNA of the Jijoye strain of EBV were identical to that of fragments produced by cleavage of EBV (B95-8) DNA with the same enzyme, whereas others were unique to each strain. Some Hsu I, EcoRI, and Sal I fragments of EBV (HR-1) DNA and Kpn I fragments of EBV (B95-8) DNA were present in half-molar abundance relative to the majority of the fragments. In these instances, the sum of the molecular weights of the fragments was in excess of 10(8), the known molecular weight of EBV (HR-1) and (B95-8) DNA. The simplest interpretation of this finding is that each EBV (HR-1), and possibly also (B95-8), DNA preparation contains two populations of DNA molecules that differ in the arrangement of DNA sequences about a single point, such as has been described for herpes simplex virus DNA. Minor fragments could also be observed if there were more than one difference in primary structure of the DNAs. The data do not exclude more extensive heterogeneity in primary structure of the DNA of the HR-1 strain. However, the observation that the relative molar abundance of major and minor fragments of EBV (HR-1) DNA did not vary between preparations from cultures that had been maintained separately for several years favors the former hypothesis over the latter.     

Patients with systemic lupus erythematosus have abnormally elevated Epstein-Barr virus load in blood

Various genetic and environmental factors appear to be involved in systemic lupus erythematosus (SLE). Epstein-Barr virus (EBV) is among the environmental factors that are suspected of predisposing to SLE, based on the characteristics of EBV itself and on sequence homologies between autoantigens and EBV antigens. In addition, higher titers of anti-EBV antibodies and increased EBV seroconversion rates have been observed in SLE patients as compared with healthy control individuals. Serologic responses do not directly reflect EBV status within the body. Clarification of the precise status of EBV infection in SLE patients would help to improve our understanding of the role played by EBV in this disease. In the present study we determined EBV types in SLE patients (n = 66) and normal control individual (n = 63) by direct PCR analysis of mouthwash samples. We also compared EBV load in blood between SLE patients (n = 24) and healthy control individuals (n = 29) using semiquantitative PCR assay. The number of infections and EBV type distribution were similar between adult SLE patients and healthy control individuals (98.5% versus 94%). Interestingly, the EBV burden in peripheral blood mononuclear cells (PBMCs) was over 15-fold greater in SLE patients than in healthy control individuals (mean +/- standard deviation: 463 +/- 570 EBV genome copies/3 microg PBMC DNA versus 30 +/- 29 EBV genome copies/3 microg PBMC DNA; P = 0.001), suggesting that EBV infection is abnormally regulated in SLE. The abnormally increased proportion of EBV-infected B cells in the SLE patients may contribute to enhanced autoantibody production in this disease.     

Replication of Viral Deoxyribonucleic Acid and Breakdown of Cellular Deoxyribonucleic Acid in Epstein-Barr Virus Infection

Infection of Raji cells with Epstein-Barr virus (EBV) causes suppression of cellular deoxyribonucleic acid (DNA) synthesis and fragmentation of the cellular DNA. About 1,000 copies of EBV DNA of normal size (about 5 x 10(7) daltons in a single strand, as shown in an alkaline gradient) are synthesized per cell.     

Nasopharyngeal carcinoma patients from Norway show elevated Epstein-Barr virus IgA and IgG antibodies prior to diagnosis

BackgroundIgA antibodies against few Epstein-Barr virus (EBV) proteins are established serological markers for nasopharyngeal carcinoma (NPC). We recently validated a novel, comprehensive EBV marker panel and showed that IgA, but also IgG antibodies against multiple EBV proteins are highly sensitive and specific for EBV-positive NPC at diagnosis. However, data about these novel biomarkers as prospective markers for NPC are sparse.MethodsThis study included 30 incident NPC cases and 60 matched controls from the Norwegian Janus Serum Bank. For 21 NPCs, molecular EBV and human papillomavirus (HPV) status were assessed by EBER-ISH and HPV DNA/RNA testing by PCR, respectively. IgA and IgG serum antibodies against 17 EBV antigens were analyzed in prediagnostic sera of cases (median lead time 14 years) and controls using multiplex serology. Sensitivities were calculated using receiver operating characteristic analysis pre-specified to yield 90% specificity in the control group. From 10 cases, serial samples were available.ResultsQuantitative EBV antibody levels were significantly elevated among all cases (p < 0.05) for three IgA and six IgG antibodies. The highest sensitivities for defining 12 EBER-ISH-positive NPCs were observed for BGLF2 IgA (67%) and BGLF2 IgG (83%). Increased IgA and IgG antibody levels between the first and last draw before diagnosis were observed for EBER-ISH positive, but not for EBER-ISH negative NPCs. Among 21 molecularly analyzed NPCs, 4 EBER-ISH negative NPCs showed concomitant positivity to HPV type-specific DNA and RNA; 3 NPCs were HPV16 and 1 NPC was HPV18 positive.ConclusionBoth, EBV IgA and IgG antibody levels are significantly elevated many years before diagnosis of EBV-positive NPCs in Norway, an NPC low-incidence region. This study provides insights into one of the largest available prospective sample collections of NPCs in a non-endemic country.     

Use of plasma Epstein-Barr virus DNA monitoring as a tumor marker in follow-up of patients with nasopharyngeal carcinoma: preliminary results and report of two cases

Recent studies suggest that plasma Epstein-Barr virus (EBV) DNA may reflect tumor burden in patients with nasopharyngeal cancer. A prospective study was initiated to investigate this correlation in 125 patients (34 pretreatment [Group A], 78 in remission [Group B] and 13 relapsed [Group C]) and 19 healthy controls. In group A, EBV DNA was detected in plasma samples of 24 (70%) patients. In Group B, EBV DNA was detected in 7 patients (range 77-13,731 copies/mL) and further imaging in all but one of these patients revealed active disease confirmed by ultrasound-guided fine-needle biopsy. There was only one false-positive case; this patient is currently under follow-up. Here we describe 2 of the 7 patients with detectable plasma EBV DNA in whom recurrence was documented by PET scan during follow-up. Our results showed that in group B the positive predictive value of quantitative analysis of plasma EBV DNA was 85%. Quantitative analysis of EBV DNA in plasma seems to become an integral part of screening, staging, monitoring, and prediction of relapse in patients with nasopharyngeal carcinoma. However, previous studies cannot be considered definitive and more reports on the use of this technique are urgently needed from both endemic and non-endemic regions.