A semiquantitative PCR method (SQ-PCR) to measure Epstein-Barr virus (EBV) load: its application in transplant patients

Abstract

Background: High Epstein-Barr virus load has been related to an increased risk of Posttransplant Lymphoproliferative Disorders (PTLD) in transplant recipients.

Objectives: Development of a method to quantitate EBV DNA levels in peripheral blood mononuclear cells (PBMC) and evaluate its usefulness in transplant patients.

Study design: We designed a semiquantitative nested PCR based on a limiting dilution analysis to detect high viral loads in PBMC. This method was applied to 25 healthy carriers, and 85 solid organ transplant recipients as follows: (A) 53 asymptomatic patients; (B) 24 symptomatic patients; (C) eight patients with PTLD.

Results: In healthy carriers the reciprocal of the limiting dilution (RLD) ranged between non-detected (ND) and 1, the median RLD was ND, which is equivalent to a viral load of <1 copy per 10(5) PBMC. In the transplant population the medians RLD (range) were: (A) asymptomatic group: ND (ND-64), median equivalent to a viral load of <1 copy per 10(5) PBMC; (B) symptomatic group: 4 (ND-256), median equivalent to a range of viral load of 4-64 copies per 10(5) PBMC. (C) PTLD group: 256 (16-16384), median equivalent to a range of viral load of 256-4096 copies per 10(5) PBMC. Statistically significant differences were found between all groups: A+B vs. C (P<0.0001); A vs. B (P<0.0001); A vs. C (P<0.0001), B vs. C (P<0.0001). We also observed a good correlation between viral loads and clinical findings in four follow-up patients. Considering the RLD=256 as a cutoff point to detect transplant patients with PTLD, resulted in sensitivity 75%, specificity 96.7%, positive predictive value 60%, negative predictive value 98.3%.

Conclusion: This SQ-PCR method enables us to differentiate between transplant patients with and without PTLD; therefore, it could be applied as a marker for early detection of this pathology.