There are two types of Epstein Barr virus (EBV): EBV-1 and EBV-2, distinguished by genomic polymorphism in the genes encoding the nuclear antigens (EBNA-2, -3A, -3B, -3C). Latent membrane protein 1 (LMP-1) is an EBV protein with known oncogenic properties. Different variants had been described; among them, a 30 base pair (bp) deletion (del-LMP-1) had been reported in benign and malignant pathologies, but there is little information about its frequency in healthy populations. The aim of this study was to determine the distribution of the EBV genotypes and the 30 bp deletion frequency, in EBV healthy carriers from Argentina. Analysis of EBNA-3C and LMP-1 genes were done by polymerase chain reaction (PCR) followed by Southern blot hybridization on DNA of peripheral blood mononuclear cells (PBMCs) from blood bank donors. EBV-1 was present in 75.9% of samples, EBV-2 in 14.6%, and co-infections with both types in 6.5%. The deleted LMP-1 variant was found in 7.4% of analyzed samples, corresponding 3.2% to deleted variant alone and 4.2% to co-infections with non-deleted form. The non-deleted variant was found in 64.6% whereas in the remaining 28%, no PCR product was detected. These results showed that EBV-1 was the more prevalent type in healthy carriers of Argentina, similar to reports from others countries. A predominance of the non-deleted LMP-1 variant was observed. The presence of co-infections with both types and variants demonstrated that healthy individuals may also harbor multiple EBV infections.

High levels of circulating EBV load are used as a marker of post-transplant lymphoproliferative disorders (PTLD). There is no consensus regarding the threshold level indicative of an increase in peripheral EBV DNA. The aim of the study was to clinically validate a developed EBV quantification assay for early PTLD detection. Transversal study: paired peripheral blood mononuclear cells (PBMC), plasma and oropharyngeal lymphoid tissue (OLT) from children undergoing a solid organ transplant with (n=58) and without (n=47) PTLD. Retrospective follow-up: 71 paired PBMC and plasma from recipients with (n=6) and without (n=6) PTLD history. EBV load was determined by real-time PCR. The diagnostic ability to detect all PTLD (categories 1-4), advanced PTLD (categories 2-4) or neoplastic PTLD (categories 3 and 4) was estimated by analyzing the test performance at different cut-off values or with a load variation greater than 0.5log units. The higher diagnostic performance for identifying all, advanced or neoplastic PTLD, was achieved with cut-off values of 1.08; 1.60 and 2.47log EBVgEq/10(5) PBMC or 2.30; 2.60; 4.47loggEq/10(5) OLT cells, respectively. EBV DNA detection in plasma showed high specificity but low (all categories) or high (advanced/neoplastic categories) sensitivity for PTLD identification. Diagnostic performance was greater when: (1) a load variation in PBMC or plasma was identified; (2) combining the measure of EBV load in PBMC and plasma. The best diagnostic ability to identify early PTLD stages was achieved by monitoring EBV load in PBMC and plasma simultaneously; an algorithm was proposed.

During HIV infection PBMC-EBV load rises in comparison to healthy carriers, but decreases when immunosuppression progresses and CD4+ T cell count becomes <50/mm(3). Circulating EBV is mainly cell-associated in the HIV-infected population. Neither PBMC-EBV nor plasma-EBV loads would be useful to diagnose brain lymphoma in AIDS patients.

114 HIV-negative individuals. EBV genotypes and variants in LMP-1 gene were detected by polymerase chain reaction (PCR)-Southern blot on DNA extracted from peripheral blood mononuclear cells and brain biopsies. In PCNSL, the presence of EBV was confirmed by EBER RNA in situ hybridization, and DNA sequencing of 3' end LMP-l gene of PCR products was performed. In HIV-infected patients, EBV-1 was detected in 48.6%, EBV-2 in 18.8%, and co-infection with both genotypes in 32.6%. In control group, EBV-1 was present in 74.3%, EBV-2 in 12.4%, and co-infection in 13.3%. Del-LMP-1 was found in 44.4% of HIV-infected patients samples (20.7% alone and 23.7% co-infection with non-deleted form) while it was found in 25.3% (6.3% alone and 19% with co-infection) in HIV-negative individuals. In HIV-infected patients EBV-2, co-infection and 30-bp deletion are more prevalent than in control group. In all, PCNSL brain biopsies samples, del-LMP-1 always was detected with EBV-2, but more cases would have to be included to draw definitive conclusions.

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The performance of the assay was suitable for the required clinical application. The assay may be useful to monitor EBV infection in transplant patients, in particular in laboratories from low-income regions that cannot afford to use commercial assays.

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