4 DISCUSSION
The results of the present study show that human cytomegalovirus UL21.5 mRNA is detectable in plasma during clinically significant episodes of HCMV DNAemia in immunocompromised patients. The mRNA detected in plasma is mostly virion-encapsulated, and its levels correlate with those of HCMV DNAemia. UL21.5 mRNA is absent in plasma of patients showing transient self-resolving DNAemia blips during LTV prophylaxis (i.e. abortive infection), whereas it is detectable in patients with HCMV DNAemia because of HCMV infection resistant to LTV or MBV (i.e. productive virus replication).
HCMV DNAemia monitoring have been used for more than 20 years to monitor HCMV infection in immunocompromised patients. Detection of HCMV DNA is generally considered a direct marker of active viral replication [13,14] and is widely used to guide pre-emptive therapy of HCMV infection in transplant recipients [15,16] and to monitor the efficacy of antiviral treatment [14,17].
However, the novel antiviral drugs recently introduced, such as LTV and MBV inhibit the production of infectious viruses without interfering with HCMV DNA synthesis, which accumulates in infected cells as non-infectious DNA or immature viral particles [18-20]. Consequently, viral DNA not associated with infectious virions can be released in the bloodstream by infected cells undergoing lysis. Therefore, the presence of HCMV DNA in blood during antiviral treatment with these antiviral agents should be considered with caution [4], and alternative markers of productive HCMV infection should be investigated.
The detection of infectious viruses in blood through its isolation from virus-carrying leukocytes (i.e. viremia) would be the ideal parameter for the identification of productive viral infection. However, the assay for viremia quantification is not sensitive enough for the early diagnosis of active infection episodes.
In order to discriminate between the presence of free-floating DNA or virion-associated DNA, pre-treatment of plasma with DNAse has been proposed [3,4]. Free-floating DNA is disrupted by DNAse treatment, while virion-associated DNA remains detectable. Thus, detection of HCMV DNA in plasma after DNAse treatment may represent a surrogate marker of the presence of infectious HCMV in blood.
In addition to genomic DNA, mRNA molecules are also packaged into virions, specifically into the tegument compart. Originally, five transcripts were detected in the virions, UL21.5, UL 106–109, TRL/IRL 2–5, TRL/IRL 7 and TRL/IRL 13 [21]. It was later found that other viral and cellular transcripts are also non-specifically incorporated into virions as well as non-infectious enveloped particles and dense bodies [8,22]. Packaging of transcripts into virions has been observed in different HCMV strains [23] and in Herpes Simplex-1 virus [24].
Initially, the detection of HCMV late transcripts UL21.5 and UL65 (pp67) or immediate early transcript UL123 (p72) in leukocytes or whole blood was proposed for monitoring HCMV infection [5,6,25]. HCMV transcripts can be detected in leukocytes or plasma. The HCMV late transcripts found in leukocytes are uptaken from infected endothelial cells, while the immediate early transcripts are both uptaken and synthesized in the leukocytes [26]. Transcripts found in plasma may be released after the lysis of infected cells or can be packaged in virions. Recently, Piccirilli and colleagues reported the presence of UL21.5 mRNA in plasma of transplant recipients with active HCMV infection [27]. Our aim was to extend this observation and define the form in which UL21.5 mRNA is present in plasma, either as RNA packaged in virions or as free-floating RNA. Using the RNAse protection assay, we observed that most of the UL21.5 mRNA in plasma is resistant to RNAse digestion, proving that it is mainly packaged in virions. Indeed, the median reduction in the amount of UL21.5 mRNA after RNAse treatment was within 0.5 Log10 (i.e. less than 30%), with a median reduction of 0.1 Log10. Therefore, although mRNA can also be released in plasma by disrupted cells, it is likely that naked mRNA potentially released from infected cells is degraded by endogenous plasma ribonuclease activity [28]. If it were not the case, the furtherex vivo treatment of plasma with RNAse would have dramatically reduced the amount of UL21.5 mRNA with respect to that detected before RNAse treatment. Thus, UL21.5 mRNA in plasma is mostly virion-associated and represents a marker for productive HCMV infection leading to the release of virions in plasma. A discrepancy between HCMV DNAmia and UL21.5 mRNA in plasma was observed in the five patients here examined with DNAemia blips associated with an abortive replications during LTV prophylaxis. In these cases, DNAemia breakthrough was not associated with resistance to antiviral drugs and resolved spontaneously. Determination of UL21.5 mRNA gave negative results in these patients. Conversely, it was always positive in case of HCMV infection non-responsive to LTV or MBV because of drug resistance. This further indicates that the presence of UL21.5 mRNA in plasma is associated with productive infection.
Although UL21.5 mRNA in plasma appears virion-associated, we found a weak correlation between mRNA levels and viremia. However, it should be considered that the viremia assay measures the number of leukocytes carrying infectious virus (it is not possible to isolate the virus from plasma), whereas the UL21.5 mRNA in plasma is determined as a marker for virions released in the extracellular compartment. In addition, the viremia assay is poorly standardized and is affected by blood transport and storage conditions.
Plasma UL21.5mRNA levels correlate with WB and PL DNAemia. In addition, it appears in plasma 1-2 weeks prior to preemptive therapy and is usually detectable when WB DNAemia is ≥10,000 copies/ml or PL DNAemia is pre-emptive therapy in hematopoietic stem cell transplant recipients [29]. Therefore, although it appears less sensitive than DNAemia in detecting HCMV in blood, UL21.5 mRNA in plasma may be considered a useful parameter for identifying patients in need of pre-emptive antiviral treatment, since all patients requiring antiviral treatment here analysed were positive for mRNA in plasma some days before the initiation of treatment. We can hypothesize that a potential strategy involving this parameter for initiation of pre-emptive therapy would not lead to a delayed start of treatment.
The limitation of this study is its retrospective nature and relatively small sample size. However, UL21.5 mRNA was detected in all patients with clinically significant DNAemia, and RNAse-resistant mRNA was detected in almost all samples analyzed.
In conclusion, our results suggest that UL21.5 mRNA in plasma is virion-associated and represents a marker for productive HCMV infection. Prospective studies are warranted to verify whether UL21.5 mRNA alone or in combination with HCMV DNAemia can improve current strategies for the treatment of HCMV infection in immunocompromised patients.