Amplicon size and non‐encapsidated DNA fragments define plasma cytomegalovirus DNA loads by automated nucleic acid testing platforms: A marker of viral cytopathology?

Abstract

AbstractManagement of cytomegalovirus (CMV) in transplant patients relies on measuring plasma CMV‐loads using quantitative nucleic acid testing (QNAT). We prospectively compared the automated Roche‐cobas®6800‐CMV and Roche‐CAP/CTM‐CMV with laboratory‐developed Basel‐CMV‐UL54‐95bp, and Basel‐CMV‐UL111a‐77bp. Roche‐cobas®6800‐CMV and Roche‐CAP/CTM‐CMV were qualitatively concordant in 142/150 cases (95%). In‐depth comparison revealed higher CMV‐loads of the laboratory‐developed assay and correlated with smaller amplicon size. After calibration to the 1.WHO‐approved CMV international standard, differences were reduced but remained significant. DNase‐I pretreatment significantly reduced CMV‐loads for both automated Roche‐CAP/CTM‐CMV and Roche‐cobas®6800‐CMV assays, whereby 90% and 95% of samples became undetectable. DNase‐I pretreatment also reduced CMV‐loads quantified by Basel‐CMV‐UL54‐95bp and Basel‐CMV‐UL111a‐77bp, but remaining detectable in 20% and 35%, respectively. Differences were largest for 110 samples with low‐level CMV‐DNAemia being detectable but not‐quantifiable by Roche‐cobas®6800‐CMV, whereby the smaller amplicon sizes yielded higher viral loads for concordant positives. We conclude that non‐encapsidated fragmented CMV‐DNA is the major form of plasma CMV‐loads also measured by fully‐automated platforms. Amplicons of <150 bp and calibrators are needed for reliable and commutable QNAT‐results. We hypothesize that non‐encapsidated fragmented CMV‐DNA results from lysis of CMV‐replicating cells and represent a direct marker of viral cell damage, which contribute to delayed viral load responses despite effective antivirals.