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A high percentage of pleural effusions remain unexplained despite an intensive diagnostic workup. Epstein-Barr virus (EBV) infections occur worldwide and affect the majority of the population. The present study investigated the prevalence and clinical relevance of EBV in pleural effusions. A prospective study was performed in which 60 consecutive patients with pleural effusion were enrolled. Real-time quantitative EBV-PCR was performed on pleural fluid and serum. Pleural fluid was further evaluated using standard biochemical, cytological and microbiological procedures. Demographic data, medical history and medication were recorded. A total of 24 (40%), from 60 pleural fluids tested, were positive in the EBV-PCR. Median EBV-DNA levels for positive samples was 454 genome equivalents (geq).mL-1 (range 36-163,446 geq.mL-1). A total of 20 (59%) out of 34 unexplained pleural effusions were EBV-PCR positive. Serological analysis of all patients with a positive PCR revealed a previous infection. Patients with a positive EBV-PCR on pleural fluid were more likely to have a positive EBV-PCR on serum than patients with a negative PCR on pleural fluid. Epstein-Barr virus reactivation in pleural fluid is a frequent event and the absence of an alternative diagnosis to explain the nature of the effusion in the majority of cases suggests an aetiological role for Epstein-Barr virus in the development of pleural effusion.

Recipients of allogeneic stem cell transplants (SCT) often show active Epstein-Barr virus (EBV) infection, which may progress to EBV-associated lymphoproliferative disorders. It is not known whether these EBV infections are true reactivations of the endogenous EBV strain or re-infections with an exogenous EBV strain. Fifty-three recipients of matched related or matched unrelated donor grafts were studied. EBV monitoring was based on a realtime TaqMan EBV DNA polymerase chain reaction (PCR) assay in plasma. In 17 patients, EBV DNA PCR monitoring was performed in peripheral blood mononuclear cells (PBMCs) as well. Mouth washings (MWs) were collected pre-transplant from all patients and family donors. Both pre-transplant EBV DNA from MWs and post-transplant EBV DNA from plasma or PBMCs were successfully obtained in 6 patients. A nested PCR targeting the EBV latent membrane protein-1 C-terminus gene was used to determine sequence variations enabling EBV strain typing. In 3 of 6 patients, the post-transplant EBV sequence pattern differed from the pre-transplant pattern, indicating a re-infection post-transplant with an exogenous strain instead of a reactivation of the original endogenous EBV strain. In the other 3 patients, the endogenous strain was identified. Active EBV infection resulting from re-infection was more severe compared with active EBV infection because of reactivation. In conclusion, active EBV infections after allogeneic SCT frequently result from re-infection with an exogenous EBV strain instead of a true reactivation of the endogenous strain and are potentially more severe.

Background: Lymphoproliferative disorder (LPD) caused by Epstein-Barr virus (EBV) is a severe complication of bone marrow transplantation. The EBV strain causing LPD is of either donor or recipient origin, however, available data are limited to only a small number of cases. To obtain solid evidence, comparison of the EBV strain that caused the EBV-LPD with pre-stem cell transplantation (SCT) EBV strains of donor and recipient is imperative. Available techniques rely on the production of EBV transformed lymphoblastoid cell lines and lack sensitivity., Objective: The aim of this study was to develop a simple method for EBV sequence analysis on mouth washings (MWs) and peripheral blood mononuclear cells (PBMCs)., Study Design: EBV DNA was extracted from MWs and PBMCs that were collected from 20 healthy individuals. DNA was used for sequence analysis, using a polymerase chain reaction for the C-terminus of the LMP-1 gene., Results: In seropositive individuals EBV DNA could be detected in 11/14 (79%) MWs and in 13/14 (93%) PBMC samples. Sequence analysis showed that in 11 out of 14 (79%) healthy individuals sequence patterns could be established. In these 11 healthy individuals 13 sequence patterns could be detected. Eleven of these 13 patterns (84.6%) were unique. These results encouraged us to explore the feasibility of this method on EBV DNA isolated from plasma from 9 EBV-LPD patients at time of EBV reactivation. In 7 EBV-LPD patients 8 sequence patterns were detected. Six out of 8 sequence patterns (75%) were unique., Conclusion: Our method is suitable for strain identification and we intend to use this technique to evaluate EBV origin in EBV-LPD patients.

Here, the influence of T vs T and B cell depletion on the incidence of EBV-associated lymphoproliferative disorder (EBV-LPD) after bone marrow transplantation (BMT) from a matched unrelated donor (MUD) is analyzed. From 1982 to 1997 the soy bean agglutinin/sheep red blood cell (SBA/SRBC) method was used for T cell depletion. This technique is well established, but the use of SRBC has a risk of transmitting prions or viruses. Therefore, a new T cell depletion method was introduced, using CD2 and CD3 monoclonal antibodies (CD2/3 method) instead of SRBC. Unfortunately, this led to an unexpected high number of EBV-LPDs in patients receiving transplants from MUDs. SBA depletion was reintroduced and combined with the CD2/3 method (SBA/CD2/3) in this patient population, later replaced by B cell-specific (CD19 and CD22) antibodies (CD3/19/22 method). The number of T (x 10(5)/kg) and B (x 10(5)/kg) cells in the graft was 1.5 +/- 0.8 and 2 +/- 1 (T/B ratio 0.75), 2.2 +/- 2.0 and 41 +/- 21 (ratio 0.055), 5.0 +/- 0.0 and 2 +/- 1 (ratio 2.5), 2.5 +/- 1.2 and 10 +/- 6 (ratio 0.25) using the SBA/SRBC, CD2/3, SBA/CD2/3 and CD3/19/22 techniques, respectively. When B cell depletion was performed (SBA/SRBC, SBA/CD2/3, CD3/19/22) four out of 31 patients (13%) receiving a BMT from a MUD developed an EBV-LPD. Without B cell depletion (CD2/3) this occurred in five out of seven patients (71%) (P < 0.05). A T/B cell ratio in the graft of > or = 0.25 seems sufficient to significantly reduce the incidence of EBV-LPD after BMT from MUDs.