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A prospective and diagnostic marker for the development of posttransplant lymphoproliferative disorders (PTLD), especially in Epstein-Barr virus (EBV)-seronegative organ transplant recipients who receive anti-lymphocyte globulin for induction immunosuppression and OKT-3 treatment for early rejection
Primary infection with Epstein-Barr virus (EBV), a DNA-containing virus classified among the family Herpesviridae, may cause infectious mononucleosis resulting in a benign lymphoproliferative condition characterized by fever, fatigue, sore throat, and lymphadenopathy. Infection occurs early in life, and by 10 years of age, 70% to 90% of children have been infected with this virus. Usually, infection in children is asymptomatic or mild and may be associated with minor illnesses such as upper respiratory tract infection, pharyngitis, tonsillitis, bronchitis, and otitis media. The target cell for EBV infection is the B lymphocyte. Immunocompromised patients, lacking antibody to EBV, are at risk for acute EBV infection that may cause lymphoproliferative disorders in organ transplant recipients (posttransplant lymphoproliferative disorders [PTLD]) and AIDS-related lymphoma.(1) The incidence of PTLD ranges from 1% for renal transplant recipients, to as high as 9% for heart/lung transplants, and 12% for pancreas transplant patients.
EBV DNA can be detected in the blood of patients with this viral infection; however, quantitative evaluation of EBV DNA has been shown to correlate highly with the subsequent (3-4 months) development of PTLD in susceptible patients.(2) Organ transplant recipients seronegative (risk for primary EBV infection) for EBV (frequently children) who receive anti-lymphocyte globulin for induction immunosuppression and OKT-3 treatment for early rejection are at highest risk for developing PTLD compared to immunologically normal individuals with prior infection with this virus.(4,5)
Increasing copy levels of Epstein-Barr virus (EBV) DNA in serial specimens may indicate possible posttransplant lymphoproliferative disorders (PTLD).
Positive results are quantitated in copies/mL.
Reportable range is 2,000 to 200,000,000 copies/mL. Specimens with results <5,000 copies EBV DNA/mL include a disclaimer that states: "Results may not be reproducible due to low copy number." Blood specimens of normal blood donors for EBV infection usually have low or undetectable levels of viral DNA.
Serial determination of blood specimens from patients may be necessary to monitor increasing (risk of development of posttransplant lymphoproliferative disorders: PTLD) or decreasing (treatment efficacy) levels of Epstein-Barr virus (EBV) DNA.
The reference range is typically "negative" for this assay, and 20 whole blood samples from "normal" asymptomatic donors were negative for the presence of EBV DNA by this assay. However, viremia or viral shedding may occasionally be detected in asymptomatic individuals. Therefore, this assay is only to be used for patients with a clinical history and symptoms consistent with EBV infection, and must be interpreted in the context of the clinical picture. This test should not be used to screen asymptomatic patients.
Twenty-five specimens from patients with prior infection with Epstein-Barr virus (EBV) were assayed for EBV-DNA by conventional PCR (gel and Southern blot). Quantitative levels of EBV-DNA were determined using a competitive target (plasmid containing target EBV-DNA). The results were recorded as EBV copies/mL in the following range: 0-50; 51-500; 501-5000; 5001-50,000. This preliminary conventional PCR assay has been formatted to a real-time LightCycler PCR which allows the determination of specific numeric copy levels of EBV-DNA.
All 25 samples, indicated above, were tested by LightCycler PCR for EBV-DNA. Specific copy levels obtained for all 25 samples correlated with the ranges obtained by conventional PCR.
To supplement the above data, 50 whole blood samples submitted to the Mayo Clinic Virology Lab were tested by a Mayo laboratory-developed EBV PCR assay and the Roche EBV ASR PCR reagent. After resolution of 1 discrepant sample (noted to be very low copy number), the sensitivity and specificity were 100% and 86%, respectively.
Analytical Sensitivity/Limit of Detection (LoD):
The lower limit of detection (LoD) for this assay is 1 target/ microliter in whole blood.
No PCR signal was obtained from extracts of 40 bacterial and viral isolates that could cause similar symptoms including herpes simplex virus (HSV) 1 and 2, cytomegalovirus (CMV), varicella-zoster virus (VZV) and human herpesvirus (HHV) HHV6, HHV 7 and HHV 8. Additionally, 20 whole blood samples from "normal" asymptomatic donors were negative for the presence of EBV DNA.
Inter-assay precision was 100% and intra-assay precision was 100%. The Standard Deviation did not exceed 0.1.
Reportable (Linear) Range/Range of Quantification:
This LC PCR assay is linear from 2,000 copies/mL to 200,000,000 copies/mL. The lowest and highest calibrators (expected LLoQ and ULoQ) fall within the linear range. Results will be reported as <2,000 copies/mL, a numeric value between 2,000 copies/mL and 200,000,000 copies/mL or >200,000,000 copies/mL.
1. Paya CV, Fung JJ, Nalesnik MA, et al: Epstein-Barr virus-induced posttransplant lymphoproliferative disorders. ASTS/ASTP EBV-PTLD Task Force and the Mayo Clinic Organized International Consensus Development Meeting. Transplantation 1999;68(10):1517-1525
2. Kenagy DN, Schlesinger K, Weck JH, et al: Epstein-Barr virus DNA in peripheral blood leukocytes of patients with posttransplant lymphoproliferative disease. Transplantation 1995;60(6):547-554
3. Green MJ, Bueno D, Rowe G, et al: Predictive negative value of persistent low Epstein-Barr virus viral load after intestinal transplantation in children. Transplantation 2000;70(4):593-596
4. Kogan DL, Burroughs M, Emre S, et al: Prospective longitudinal analysis of quantitative Epstein-Barr virus polymerase chain reaction in pediatric liver transplant recipients. Transplantation 1999;67(7):1068-1070
5. Green M, Cacciarelli TV, Mazariegos GV, et al: Serial measurement of Epstein-Barr viral load in peripheral blood in lymphoproliferative disease. Transplantation 1998;66(12):1641-1644
6. Lau AH, Soltys K, Sindhi RK, et al: Chronic high Epstein-Barr viral load carriage in pediatric small bowel transplant recipients. Pediatr Transplant 2010 Jun;14(4):549-553