Real-time PCR assays based on distinct genomic regions for cytomegalovirus reactivation following hematopoietic stem cell transplantation

Abstract

Summary:Real-time PCR has many advantages compared with antigenemia and qualitative PCR assays for detecting cytomegalovirus (CMV) infection in patients following SCT. However, the procedure used in each report was not standardized. This study compares the CMV load detected by real-time PCR assays amplifying distinct genomic regions. Real-time PCR assays based on US17, UL65, immediate early protein (IE) and glycoprotein B(gB) were selected and comparisons were made between each genomic region, and with antigenemia and nested PCR (IE region) in 18 SCT patients. The CMV load detected by real-time PCR using all combinations of primers targeting distinct genomic regions and by antigenemia assays correlated well. However, US17 and UL65-PCR could detect CMV earlier than gB-PCR, antigenemia and nested PCR assays. In longitudinal analysis, gB-PCR demonstrated a trend for showing a lower viral load in some patients than US17-, UL65-and IE-PCR. Moreover, the results suggest that a cutoff level of 500 copies/ml might be used to decide when to initiate treatment. We propose that monitoring should be carried out using real-time PCR assays targeting the US17 region and that a CMV load of 500 copies/ml could be used as a cutoff value for initiating treatment in patients following SCT, receiving immunoglobulin prophylaxis.

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

“…In our study, the CMV DNA copy number determined by real-time CMV PCR using SYBR Green I correlated with the number of pp65-positive cells, in agreement with the results of other studies using a TaqMan-based assay (Gault et al 2001;Sanchez et al 2001;Leruez-Ville et al 2003;Li et al 2003;Ikewaki et al 2005). ROC analysis showed that this real-time CMV PCR exhibited adequate sensitivity and specificity, using the pp65 antigenemia assay as a reference standard.…”

“…Real-time PCR, one modality of quantitative PCR, is a simple, reliable, cost-effective, and time-saving alternative strategy (Holland et al 1991). Many institutes have developed real-time PCR assays for monitoring CMV, and have reported encouraging results (Gault et al 2001;Cortez et al 2003;Li et al 2003;Nitsche et al 2003;Ikewaki et al 2005), where a dual-labeled fluorogenic hybridization probe or two singlelabeled probes have been used to monitor PCR product formation.…”

Detection of CMV Infection in Allogeneic SCT Recipients: The Multiple Assays

“…The differences among assays are based on the method for nucleic acid extraction, the specimen type, the target genomic region, primers and probes used for amplification and www.intechopen.com detection Ikewaki et al, 2005). Although, the variability in the results obtained with commercial assays (kits or ASRs) are proven to be lower than when using inhouse developed methods ).…”

“…In the absence of standardization the current clinical guidelines recommend to each individual laboratory to establish their own viral thresholds for CMV management, (Kotton et al, 2010;Razonable & Emery, 2004), threshold that cover a wide range of viral loads varying from 200-500 copies/mL in some laboratories (Ikewaki et al, 2005;Mori et al, 2002) to 2000-5000 copies/mL in others (Humar et al, 1999).…”

A Commutable Cytomegalovirus Calibrator Is Required to Improve the Agreement of Viral Load Values between Laboratories

“…For this reason, thresholds for predicting CMV disease in preemptive programs are institution or laboratory specific and cannot be generalized. These thresholds vary from 200 -500 copies/mL in some laboratories (6,7 ) to 2000 -5000 copies/mL in others (1 ). There are several factors responsible for differences in CMV values between measurement methods.…”