Reliable quantification of Cytomegalovirus DNAemia in Letermovir treated patients

Polymerase chain reaction (PCR) based methods are a fast and sensitive approach to detect and monitor viral load in Cytomegalovirus (CMV) patients. Letermovir (LMV) acts at a late stage during the CMV replication cycle and does not inhibit CMV DNA replication per se. Therefore, quantitative nucleic acid amplification testing might lead to the overestimation of viral load in patients treated with LMV and underestimate treatment success. To study this discrepancy, we treated infected cells with LMV or Ganciclovir (GCV) and compared viral progeny DNA levels. Prior to nucleic acid extraction and qPCR measurements we pretreated cell lysates and cell culture supernatants from infected cells with DNase I. This step assumes the degradation of DNA which is not protected from a viral capsid. LMV treatment did not reduce genomic copies (GC) in samples from whole cell lysates compared to samples treated with GCV. DNase treatment prior to DNA extraction, decreased GC in the LMV treated group to comparable levels as seen in the GCV group. In cell culture supernatants, LMV or GCV treatment led to an equivalent reduction of CMV GC. In this case, DNase treatment exerted a negligible effect on both groups. We conclude that the accumulation of concatemeric DNA within cells seems to be a confounding variable when monitoring LMV efficacy via qPCR. However, qPCR shows to be a reliable method to evaluate antiviral efficacy of LMV in cell free specimens. These results have strong clinical implications for the monitoring of CMV therapy during LMV treatment.