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.

Correlation between systemic lupus erythematosus and cytomegalovirus infection detected by different methods

“…There are 165-252 open reading frames (ORF) on the HCMV genome. HCMV genes, such as pp65 [15], UL54 [16], UL83 [17], UL123 [18], US17 [19], and UL75 [20], which have been used for PCR detection of viral infection in several studies. In this study, UL55 and UL138 were selected as the target genes for the detection of intracellular HCMV infection.…”

Quantitative Real-Time Polymerase Chain Reaction for Evaluating DNAemia due to Cytomegalovirus, Epstein-Barr Virus, and BK Virus in Solid-Organ Transplant Recipients

“…Importantly, both shell vial cell cultures (qualitative) and antigenemia tests (quantitative) have been shown in comparative studies to be less sensitive than PCR. In addition, DNAemia was detected in an earlier stage of nucleic acid amplification [6][7][8][9][10].As an alternative to cell culture and the antigenemia test, implementation of molecular techniques for the rapid and sensitive detection of nucleic acid targets has yielded new levels of capabilities for laboratory diagnosis of many microbial infections [11]. Implementation of these tests has been facilitated by the availability of real-time PCR instrumentation with automation of nucleic acid-target amplification and amplicondetection steps in a "closed system" (i.e., reaction tubes never opened during or after amplification).…”

Optimization of Human Cytomegalovirus LightCycler Real-Time PCR

“…Human Cytomegalovirus (CMV) remains a significant cause of morbidity and mortality in immunocompromised patients, putting them at risk of fatal CMV disease upon viral reactivation [1][2][3][4][5][6]. Realtime PCR has been widely considered as a rapid, efficient, and highly sensitive technique for the evaluation of CMV DNA kinetics [2,[7][8][9][10], and an effective assay for (i) early diagnosis of CMV disease, (ii) antiviral therapy monitoring, (iii) relapse prediction and (iv) antiviral resistance indication [1,5,[11][12][13][14].…”