Cytomegalovirus DNA Measurement in Blood and Plasma Using Roche LightCycler CMV Quantification Reagents

Abstract

Cytomegalovirus (CMV) is a common cause of morbidity and mortality in immunosuppressed patients. Newly available commercial systems facilitate the measurement of CMV DNA in whole blood or plasma, as a means of detecting and monitoring active disease. We evaluated the performance characteristics of a quantitative polymerase chain reaction that relies on analyte-specific reagents and instruments from Roche Diagnostics. DNA was extracted using a MagNaPure instrument from blood and matched plasma specimens of patients with active CMV disease (defined by another molecular assay) and from controls. Viral load was measured against Roche's standards on a LightCycler instrument, followed by melt curve analysis to confirm product specificity. Dual hybridization probes targeted the CMV UL54 gene and a control sequence that was spiked before extraction. Accuracy and linearity were established using spiked DNA from the Towne-strain CMV. The assay was linear across 6 logs, and it detected CMV DNA in 67/70 blood samples (96%) from patients who were considered to have an active CMV infection, including all 67 viral loads above 208 CMV copies/mL, suggesting that it was sensitive enough to detect clinically significant infections in immunosuppressed patients. Virus levels in plasma correlated reasonably well with the levels in whole blood (r=0.5756), suggesting that levels in either plasma or blood level were indicative of active infection. It was important to verify the calculated values by visualizing the amplification plot and using the melt curve analysis to resolve discrepancies. The LightCycler CMV assay is rapid, sensitive, and linear for quantifying CMV viral load, and it seems to be useful in the diagnosis and monitoring of affected patients.