Comparison of quantitative HCMV DNAemia in whole blood and leukocytes, and Real-Time PCR in a BMT patient

Abstract

Background. HCMV is the most opportunistic viral agent in the transplantation of bone marrow and solid organ. Early therapy based on the detection of HCMV pp65 antigen in peripheral blood leukocytes, has led to a significant reduction in the incidence of related HCMV diseases.The pp65 antigenemia is difficult to standardize, while the quantification of HCMV DNA by Real-Time PCR is an alternative diagnostic approach with greater sensitivity and reproducibility, providing important information in the management of infected patients. Objectives. The aim of this study was the comparative analysis of quantitative HCMV DNAemia in leukocytes and in whole blood, with Real-time PCR, in a BMT patient with HCMV post-transplant reactivation, in order to analyze the relationship between levels of viral DNA at different time-points obtained in the two biological matrices. Study Design. The presence of HCMV DNA was detected in whole blood and peripheral blood leukocytes in a patient who underwent allogeneic marrow transplant for Ph1 + acute lymphoblastic leukemia in molecular remission. After 5-6 months, the patient has increased molecular Bcr-Abl (Philadelphia chromosome). It was activated immune reaction by means of tapering (lowering the dose of cyclosporine) that uses the GvL effect to turn negative the Philadelphia chromosome positivity (Bcr/Abl negativity). Later, the patient develops GvHD and cortisone is administered. The persistence of grafts treated with immunosuppressants periodically reactivates HCMV infection. The DNA extraction from whole-blood was performed by automatic extractor QIACUBE (Qiagen), while extraction from leucocytes was performed on a standard number of leukocytes (EXTRAcell- Nanogen).The extracted DNA was amplified by Real-Time Alert Q-PCR (Nanogen).The samples were analyzed weekly for about 5 months from 1 year after transplantation. Results. The patient at 1 year after transplantation, has three HCMV reactivation at 56th, 62th and 67th week. In all reactivations a good overlap values of viraemia in both whole blood leukocytes is shown. Conclusions. Our data confirm that the whole blood is a biological matrix with diagnostic similar performance not only to pp65 antigenaemia, as supported by extensive scientific literature, but also to the DNAemia in leukocytes that has been for a long time considered the elective matrix for evaluating the post-transplant HCMV infection, for pre-emptive therapy particularly in BMT patients.

Keywords

• HCMV, DNAemia, Real-Time PCR, BMT