Epstein-Barr virus infection in paediatric liver transplant recipients: detection of the virus in post-transplant tonsillectomy specimens

Abstract

Aims: Post-transplant lymphoproliferative disease (PTLD) is an important and serious complication in transplant patients. Recent studies have suggested that quantitative assessment of Epstein-Barr virus (EBV) infection in transplant patients might help to identify those at risk of developing PTLD. Therefore, tonsils from paediatric liver transplant recipients were studied for evidence of EBV infection.

Methods: Tonsils were studied by in situ hybridisation for the detection of the small EBV encoded nuclear RNAs (EBERs). The phenotype of EBV infected cells was determined by double labelling in situ hybridisation and immunohistochemistry. The expression of viral latent and lytic antigens was determined by immunohistochemistry. Tonsils from patients without known immune defects were studied as controls.

Results: Tonsils from transplant patients showed pronounced follicular hyperplasia and minor paracortical hyperplasia. In situ hybridisation revealed variable numbers of EBV infected B cells in the tonsils from transplant patients (range, 2-1000/0.5 cm(2); mean, 434/0.5 cm(2); median, 105/0.5 cm(2)). Lower numbers were detected in the control tonsils (range, 1-200/0.5 cm(2); mean, 47/0.5 cm(2); median, 9/0.5 cm(2)). The latent membrane protein 1 (LMP1) of EBV was not detected and there were only rare cells in two cases showing expression of the EBV encoded nuclear antigen 2 (EBNA2). There was no evidence of lytic infection. None of the patients developed PTLD within a follow up period of up to five years.

Conclusions: These data indicate that tonsillar enlargement in paediatric liver transplant patients does not necessarily imply a diagnosis of PTLD. Furthermore, the presence of increased numbers of EBV infected cells in tonsils from liver transplant recipients by itself does not indicate an increased risk of developing PTLD.

Figure 1

(A,B) In situ hybridisation with ³⁵S labelled small Epstein-Barr virus (EBV) encoded nuclear RNA (EBER) specific probes identifies numerous (A; case 9) and isolated (B; case 3, arrow) EBV positive cells in palatine tonsils from two paediatric liver transplant patients. (C) Using digoxigenin labelled EBER probes (red nuclear signal) the presence of EBV in small lymphocytes (long arrows) and in larger blast cells (short arrow; case 9) is demonstrated. (D) Double labelling immunohistochemistry (red signal) and in situ hybridisation (black grains) shows the presence of EBV in CD79a positive B cells (arrows). (E) Using immunohistochemistry, numerous EBN2A positive cells are detected in a tonsil from a patient with acute infectious mononucleosis, whereas (F) only isolated EBNA2 positive cells are seen in the tonsils from transplant patients (inset). (G) Numerous latent membrane protein 1 (LMP1) positive cells are present in a tonsil from a patient with infectious mononucleosis, whereas (H) LMP1 expression is not detected in the tonsils from transplant patients.

Figure 2

PCR analysis of IgH gene rearrangement in tonsils from liver transplant recipients. M, 10 bp ladder (bar, 100 bp); lanes 1 to 11, patients 13, 8, 6, 3, 7, 10, 5, 9, 1, 2, 12, respectively (table 1 ▶); lane 12, adult liver transplant patient; lane 13, positive control (BJAB cell line); lane 14, negative control (H₂O).