Cytomegalovirus quantification in plasma by an automated real-time PCR assay

Abstract

Background Sensitive quantitation of cytomegalovirus (CMV) DNA in blood is helpful for the diagnosis of CMV infection or reactivation and the monitoring of transplanted patients. Objectives We compared a new PCR assay coupled with an automated extraction system (CMV real-time PCR, Abbott Molecular, Des Plaines, IL, USA) to a previously validated method (ultrasensitive Cobas Amplicor CMV DNA Monitor, Roche Molecular, Indianapolis, IN, USA). Results Using limiting dilutions of CMV DNA positive plasma, the two assays had a similar detection threshold ranging between 20 and 45 copies/ml. Coefficients of variation of CMV real-time PCR assay varied from 1 to 12% for CMV DNA levels between 10,000 and 20 copies/ml. Viral loads assessed by the two methods on 179 clinical samples showed an overall concordance of 89% and an excellent correlation ( R = 0.94). Discrepancies were only observed for samples with low CMV DNA levels (<300 copies/ml); 18 samples were positive by CMV real-time PCR only, and 2 samples by ultrasensitive Cobas CMV only. Values obtained by CMV real-time PCR were on average 0.4 log higher than those of ultrasensitive Cobas CMV. Successive samples of transplanted patients with evidence of CMV infection/reactivation revealed that CMV real-time PCR assay was positive earlier and for a longer period of time after treatment initiation. Conclusions Although both assays had similar analytical performances, the CMV real-time PCR assay has the advantages of automated extraction and higher dynamic range, and shows a trend for an improved sensitivity that might impact on clinical decisions.