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.