Early Detection of Plasma Cytomegalovirus DNA by Real-Time PCR after Allogeneic Hematopoietic Stem Cell Transplantation

Abstract

Cytomegalovirus (CMV) infection is an important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Therefore, preemptive ganciclovir therapy based on early detection of CMV reactivation is widely used to prevent CMV disease. Real-time polymerase chain reaction (PCR) has been widely used for monitoring CMV reactivation as well as the antigenemia assay that detects CMV structural phosphoprotein with a molecular weight of 65,000 (pp65). We developed a real-time PCR assay system for CMV based on a double-stranded DNA-specific dye, SYBR Green I, and quantified DNA, which was extracted automatically from plasma. This real-time PCR assay and the pp65 antigenemia assay were compared in parallel with 357 blood samples obtained from 64 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). Real-time PCR assay results correlated with those of the pp65 antigenemia assay (p < 0.0001). It is noteworthy that the detection of CMV DNA by PCR preceded the first positive antigenemia by 14 days. In this study, 10 of 64 patients developed CMV disease. The antigenemia assay detected CMV reactivation earlier than the development of CMV disease only in four of 10 patients. In contrast, our real-time PCR detected CMV-DNA before the development of CMV diseases in eight of 10 patients. The real-time PCR with SYBR Green I as a detection signal is simple and readily performed, and may be a useful system for early detection of CMV reactivation after allo-HSCT.