[Epstein Barr viral load monitoring in mononuclear lymphocytes and serum of renal transplant recipients using a quantitative PCR protocol].
BACKGROUND
Post-transplant lymphoproliferative disorders (PTLD), ranging from lymphoid hyperplasia to clonal malignancy, are severe complications arising in solid organ transplant patients; their reported incidence ranges from 1 to 20%, according to factors such as type of transplanted organ and age of recipients. A strong correlation between Epstein-Barrvirus (EBV) infection, the grade and type of immunosuppression and the development of PTLD has been recognized. The detection and quantification of EBV-DNA load in peripheral blood have been utilized as prognostic markers for the development of PTLD, showing a correlation between high levels of EBV-DNA in the blood and the development of PTLD. In this study, we monthly monitored EBV viral load in 15 renal transplant recipients for six months. The number of EBV-DNA copies was measured in peripheral blood mononuclear cells (PBMC) and serum samples by a quantitative PCR protocol developed in our laboratory.


METHODS
Our EBV-DNA quantification protocol employs a previous screening of samples containing a significant number of viral DNA copies (>=1000 copies/105 PBMC or 100 mL serum) by semi-quantitative PCR followed by a precise quantification of the only significant samples by quantitative-competitive (QC)-PCR.


RESULTS
Our 15 renal transplant patients neither developed PTLD nor had recurrent acute illnesses or acute graft rejections during the study. The results obtained in the monthly follow up of EB viral load in PBMC samples confirmed its fluctuation in asymptomatic patients reported in the literature. In particular, 5/14 (35.7%) of EBV seropositive patients had an EBV-DNA load equal to 1000 EBV copies /105 PBMC, and 1/14 (7.1%) reached 5000 EBV copies /105 PBMC at least once in our study. In the EBV seronegative patient, EBV-DNA in PBMC samples was always undetectable (less than 100 DNA copies/105 PBMC). EBV-DNA load in all serum samples was less than threshold value of our quantification protocol (<100 DNA copies/100 mL serum). With regard to the immunosuppressive treatment, it should be noted that 66.7% of the six patients in whom EBV load reached values equal to or higher than 1000 DNA copies/105 PBMC, were on FK506 whereas only 33.3% of them were on CyA.


CONCLUSIONS
Since the high positive predictive value of EB viral load in peripheral blood for diagnosis of PTLD reported by several Authors, and the described absence of correlation between the serological evidence of EBV reactivation and EB viral load, EBV viral load measurement in PBMC and serum samples using quantitative PCR techniques is a powerful diagnostic tool to monitor transplanted patients at risk of developing PTLD.
Dip. Sanita' Pubblica e Microbiologia, S.C. Virologia, Universita' di Torino, Torino, Italy. chiara.merlino@unito.it
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D016572:Cyclosporine / Q000009:adverse effects / Q000627:therapeutic use; D004279:DNA, Viral / Q000097:blood*; D020031:Epstein-Barr Virus Infections / Q000097:blood* / Q000821:virology; D005260:Female; D005500:Follow-Up Studies; D004854:Herpesvirus 4, Human / Q000302:isolation & purification*; D006801:Humans; D007166:Immunosuppressive Agents / Q000009:adverse effects / Q000627:therapeutic use; D016030:Kidney Transplantation; D007963:Leukocytes, Mononuclear / Q000821:virology*; D008232:Lymphoproliferative Disorders / Q000175:diagnosis* / Q000821:virology; D008297:Male; D008775:Methylprednisolone / Q000009:adverse effects / Q000627:therapeutic use; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D011183:Postoperative Complications / Q000175:diagnosis* / Q000821:virology; D011237:Predictive Value of Tests; D020123:Sirolimus / Q000009:adverse effects / Q000627:therapeutic use; D016559:Tacrolimus / Q000009:adverse effects / Q000627:therapeutic use; D019562:Viral Load; D014766:Viremia / Q000821:virology*
D004279:DNA, Viral; D007166:Immunosuppressive Agents; D016572:Cyclosporine; D020123:Sirolimus; D016559:Tacrolimus; D008775:Methylprednisolone