guideline

Management of cytomegalovirus infection in haemopoietic

stem cell transplantation

Vincent Emery,1 Mark Zuckerman,2 Graham Jackson,3 Celia Aitken,4 Husam Osman,5 Anthony Pagliuca,6 Mike Potter,7 Karl
Peggs,8 and Andrew Clark9 on behalf of the British Committee for Standards in Haematology, the British Society of Blood
and Marrow Transplantation and the UK Virology Network
1
Department of Virology, University College London School of Life and Medical Sciences, London, 2Department of Virology, Kings
College Hospital, London, 3Department of Haematology, Freeman Road Hospital, Newcastle, 4West of Scotland specialist virology
centre, Gartnavel General Hospital, Glasgow, 5Birmingham HPA Laboratory, Birmingham Heartlands Hospital, Birmingham,
6
Department of Haematology, Kings College Hospital, London, 7Section of Haemato-oncology, The Royal Marsden NHS Foundation
Trust, London, 8Department of Haematology, University College London Hospitals, London, and 9Blood and Marrow Transplant
Unit, Beatson Oncology Centre, Glasgow, UK

Keywords: stem cell, transplantation, bone marrow, periph-
eral blood stem cells, cytomegalovirus.
List of Recommendations
Cytomegalovirus (CMV) infection and CMV disease
should be diagnosed according to established, interna-
tionally accepted, standardized criteria (Grade 1C).
Risk-adapted patient assessment should inform clinical
management (Grade 1B).
All potential haemopoietic stem cell transplantation
(HSCT) recipients should be tested for the presence of
CMV IgG antibody at diagnosis (Grade 1C).
Once optimum human leucocyte antigen (HLA) match-
ing has been performed, a CMV IgG-negative donor
should be chosen for a CMV IgG-negative recipient and
a CMV IgG-positive donor should be chosen for a CMV
IgG-positive recipient when possible (Grade 1A).
Donors or recipients who are initially found to be CMV
IgG-negative should be retested pre-transplant to
exclude primary CMV infection (Grade 1C).
Apparent CMV seroconversion in potential allograft
recipients who have received unscreened blood products
should be actively investigated to exclude passive acqui-
sition of antibody (Grade 1C)
Any CMV IgG-negative HSCT recipient transplanted
from a CMV IgG-negative donor who develops CMV
infection post-transplant must be reported to the Serious
Hazards of Transfusion (SHOT) scheme (Grade 1C).
Correspondence: Dr Andrew Clark, Bone Marrow Transplant Unit,
Beatson Oncology Centre, West of Scotland Cancer Centre, 1053
Great Western Road, Glasgow G12 0YN, UK.
E-mail: andyclark@doctors.org.uk
Primary prophylaxis with ganciclovir is not generally
recommended as toxicity outweighs efficacy in HSCT
patients (Grade 1B).
Primary prophylaxis with aciclovir or valaciclovir can
be deployed but only in conjunction with appropriate
monitoring of CMV in blood (Grade 1B).
Valaciclovir or valganciclovir are valid treatment
options for secondary prophylaxis with appropriate
monitoring of CMV in blood (Grade 1C).
Intravenous immunoglobulin is not recommended for
prophylaxis of CMV infection (Grade 1A).
Real time quantitative polymerase chain reaction (PCR)
is the preferred choice for monitoring CMV DNA levels
in HSCT patients (Grade 1B).
All diagnostic laboratories should deploy the CMV
international standard to allow viral loads to be
compared between centres (Grade 1C).
Monitoring of CMV load should be undertaken at
least weekly for the first 3 months post-HSCT (Grade
2C).
CMV viral load monitoring should continue for 6-
12 months if the patient has chronic graft-versus-host
disease (GvHD) or prolonged T-cell immunodeficiency
(Grade 1B).
Each transplant centre should have a risk-adapted policy
detailing threshold values for treatment of CMV infec-
tion, taking into account patient factors and PCR meth-
odology (Grade 2C).
Ganciclovir is recommended as first line pre-emptive
therapy for CMV in HSCT patients (Grade 1A).
Oral valganciclovir is a valid alternative when gastroin-
testinal absorption is normal or only minimally
impaired (Grade 1A).
Foscarnet is recommended as an alternative first-line
agent if neutropenia is present or for ganciclovir treat-
ment failure (Grade 1A).

2013 John Wiley & Sons Ltd First published online 6 May 2013
British Journal of Haematology, 2013, 162, 2539 doi:10.1111/bjh.12363
Guideline
Pre-emptive therapy with cidofovir can be considered as
third-line in patients unresponsive to, or intolerant of,
both a ganciclovir preparation and foscarnet (Grade 2B).
In patients in whom CMV DNA loads in blood increase
by 1 log10 over 2 weeks of pre-emptive therapy with a
first line drug, an alternative agent and drug resistance
profiling should be considered (Grade 2C).
Drug resistance should start to be suspected if CMV
loads in the blood fail to respond after 14 d of therapy,
especially in non-lymphopenic or multiply pre-treated
patients (Grade 2C).
Sequence analysis of the UL97 and UL54 genes is the
preferred option for resistance screening for currently
available drugs (grade 1B).
A multidisciplinary approach to management of CMV
disease is required (Grade 1C).
De novo CMV disease should be treated with ganciclovir
or foscarnet monotherapy and intravenous immuno-
globulin (Grade 1B).
CMV disease that develops while on pre-emptive ther-
apy or is clinically progressive requires drug resistance
testing, increased drug doses and/or combination ther-
apy (Grade 1B).
Reduction in immunosuppression, especially reduction
in corticosteroid dose, is strongly recommended when
possible (Grade 1B).
Scope
These evidence-based guidelines expand and adapt previous
guidance (Tomblyn et al, 2009; Andrews et al, 2011). While
specifically focusing on allogeneic haemopoietic stem cell
transplantation (HSCT), they are relevant to other areas of
haematological oncology where there is an increased risk of
cytomegalovirus (CMV) infection, such as haematological
cancers where intense anti-T-cell therapy has been deployed
(OBrien et al, 2006).
Methodology
The production of these guidelines involved the following
steps:
Establishment of a working group comprising experts in
the field of allogeneic transplantation and clinical virology
followed by literature review to 1 May 2012 including
Medline, Pubmed and Cochrane reviews databases.
Development of key recommendations based on ran-
domized, controlled trial evidence. Due to the paucity
of randomized studies, some recommendations are
based on literature review and a consensus of expert
opinion.
The GRADE nomenclature was used to evaluate levels of
evidence and to assess the strength of recommendations
(http://www.bcshguidelines.com/BCSH_PROCESS/EVI-
26
DENCE_LEVELS_AND_GRADES_OF_RECOMMENDA-
TION/43_GRADE.html).
Initial review of manuscript, performed by the UK Clini-
cal virology Network, British Society of Blood and Mar-
row Transplantation (BSBMT) executive committee and
the British Committee for Standards in Haematology
(BCSH) Haem-Onc Task Force.
Final Review by sounding boards of the British Society for
Haematology (BSH) and British Society of Blood and
Marrow Transplantation (BSBMT).
Background
Clinical manifestations of CMV
CMV is a herpes virus. Primary infection is followed by life-
long latency. Clinical manifestations vary widely and should
be diagnosed according to established, internationally
accepted, standardized criteria (Ljungman et al, 2002a).
CMV infection is diagnosed when CMV is detected, most
commonly in the blood, using sensitive screening tools. It is
termed primary CMV infection when infection occurs in a
CMV IgG-negative patient and CMV reactivation when the
patient, or donor, is known to be CMV antibody positive. In
uncomplicated CMV infection, organ-specific signs and
symptoms are absent, although non-specific symptoms, such
as fever and malaise, may occur (Ljungman et al, 2002a,
2011). The diagnosis of CMV disease requires the presence
of symptoms and signs compatible with end organ damage,
together with the detection of CMV by a validated method
in an appropriate clinical specimen (Ljungman et al, 2002a).
If left untreated, asymptomatic CMV infection can progress
to CMV disease, most commonly affecting the lung, gastroin-
testinal tract, eye, liver or central nervous system (CNS).
CMV pneumonia is the most serious complication with
a > 50% mortality (Ljungman, 1995). In addition to specific
organ damage, CMV also has profound, poorly characterized,
indirect immunosuppressive effects, leading to an increased
incidence of fungal and bacterial infection (Nichols et al,
2002; Hakki et al, 2003; Blanquer et al, 2011) as well as
higher rates of both acute and chronic graft-versus-host
disease (GvHD) (Soderberg et al, 1996; Larsson et al, 2004;
Wang et al, 2008). While some recent reports have suggested
reduced relapse rates in selected patients who reactivate
CMV (Behrendt et al, 2009; Elmaagacli et al, 2011), this
finding remains controversial and should not hinder early
aggressive treatment of CMV infection.
Risk factors for CMV infection
Post HSCT, most patients will reactivate latent virus rather
than acquire primary infection. Mechanisms of latency are
not completely understood but prevention of reactivation
appears to be primarily dependent upon a persistent, robust,
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British Journal of Haematology, 2013, 162, 2539

T-cell-mediated, immune response (Sylwester et al, 2005;
Ljungman et al, 2011). Not surprisingly, the intensity of immu-
nosuppression and the degree of T-cell depletion in transplant
protocols both critically affect the risk of reactivation (van Burik
et al, 2007). Recipients of transplants from unrelated or human
leucocyte antigen (HLA)-mismatched donors, where immuno-
suppression is increased and GvHD more likely, are particularly
likely to reactivate CMV and these patients have a survival dis-
advantage. The deployment of T-cell depleting agents, such as
alemtuzumab, or antithymocyte globulin (ATG) or increased/
prolonged courses of immunosuppression to treat GvHD
significantly increases the risk of CMV infection (Schmidt-Hie-
ber et al, 2010). These risk factors can also increase the risk of
persistent, recurrent or late onset disease (Buyck et al, 2010),
usually associated with impaired immune reconstitution (Ha-
kki et al, 2003; Zhou et al, 2009). Conversely, the reconstitution
of specific anti-CMV cytotoxic T-lymphocytes (CTL) has been
shown to be protective (Lamba et al, 2005). CMV disease is as
much of a problem following non-myeloablative transplanta-
tion as it is in the myeloablative setting. Conflicting data have
been reported regarding the relative risk of bone marrow (BM)
compared to peripheral blood stem cells (PBSC) as a stem cell
source (Nakamae et al, 2009; George et al, 2010; Pinana et al,
2010; Guerrero et al, 2012). Published incidence of CMV reacti-
vation and CMV disease after umbilical cord blood transplanta-
tion (UCBT) varies significantly. UCBT recipients have been
reported to be more susceptible to late complications of CMV,
although this is not a universal finding and the relative role of T
cell depletion in UCBT conditioning protocols requires further
investigation (Boeckh et al, 2004; Takami et al, 2005; Walker
et al, 2007; Beck et al, 2010; Brown et al, 2010; Sauter et al,
2011; Chiesa et al, 2012; Mikulska et al, 2012).
Recommendations
CMV infection and CMV disease should be diagnosed
according to established, internationally accepted, stan-
dardized criteria (Grade 1C).
Risk-adapted patient assessment should inform clinical
management (Grade 1B).
Impact of host and donor CMV serostatus
CMV screening
CMV screening should be performed using commercially
available CMV IgG assays. There is no gold standard assay
and it is important that laboratories running these tests par-
ticipate in external and internal quality assurance schemes.
CMV IgG-negative donor recipient pairs (R /D )
CMV-negative recipients of grafts from CMV-negative
donors (R /D ) very rarely develop major CMV-related
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British Journal of Haematology, 2013, 162, 2539
Guideline
complications and a CMV IgG-negative donor should be
chosen in these circumstances when possible. There is
debate about the relative importance of CMV serostatus
and HLA compatibility in donor selection. Specifically,
while HLA matching at HLA-A,B,C and DR remains the
most important factor in donor selection, the value of
choosing a CMV IgG-negative donor over mismatches at
other loci, such as HLA DQ or DP, or in protocols with
aggressive T cell depletion remains unresolved (Boeckh &
Ljungman, 2009). It is also recognized that donor selection
can be a complex process and optimum CMV matching
may not always be possible (Kollman et al, 2001; Spellman
et al, 2012). Provision of CMV-safe blood products is stan-
dard practice when both host and donor are negative, either
derived from CMV IgG-negative recipients or achieved by
leucodepletion. Both techniques are effective, although rare
incidences of transfusion-transmitted infection have
occurred using both approaches (Bowden et al, 1995; Ljung-
man et al, 2002b; Nichols et al, 2003). In the UK, universal
leucodepletion has largely replaced blood products from
CMV-negative donors as of May 2012. It is important to be
aware of the need to check the baseline CMV IgG status of
potential transplant candidates at the time of initial diagno-
sis, to avoid confusion caused by passive transmission of
antibodies with transfusion of CMV IgG-positive blood
products to negative recipients. Any transplant recipient
who converts from CMV IgG-negative to CMV IgG-positive
status pre-transplant will require careful assessment to sepa-
rate passive antibody from true seroconversion, as this has
major implications for donor selection. In this setting, a
falling titre with time is suggestive of passively acquired
antibody. Pre-allograft, it is recommended that samples for
IgM antibody and CMV polymerase chain reaction (PCR)
are sent as soon as a change in CMV status is suspected. If
both are negative then this suggests the presence of pas-
sively acquired antibody. Blood products must be leucode-
pleted in the blood bank facility (Ratko et al, 2001) and
evidence of quality control for leucoreduction should be
available to all transplant centres for JACIE [Joint Accredi-
tation Committee International Society for Cellular Ther-
apy (Europe) & European Group for Blood and Marrow
Transplantation (EBMT)] accreditation purposes. R /D
transplants still require to be monitored for CMV infection.
Any cases of CMV infection occurring in this group of
patients should be reported to the Serious Hazards of
Transfusion (SHOT) scheme.
CMV IgG-positive donor or recipient
CMV seropositivity in either host or donor pre-transplant
continues to be associated with a poorer overall survival
post-allogeneic transplantation as a result of increased
non-relapse mortality (NRM) (Broers et al, 2000; Kroger
et al, 2001; Albano et al, 2006; Tomonari et al, 2008). The
degree of this risk is determined primarily by the CMV IgG
27
Guideline
status of the recipient. If a CMV IgG-negative recipient
receives cells from a CMV IgG-positive donor (R /D+) then
CMV infection occurs in 2030% of cases. CMV disease is
unusual but NRM is increased, probably through the indirect
effects of CMV on immune status post-transplant (Nichols
et al, 2002; Ljungman et al, 2011; Pergam et al, 2012). There
is a greater risk of CMV reactivation and progression to
CMV disease in CMV seropositive recipients, where 80% are
likely to reactivate CMV, irrespective of CMV status of
donor (Ljungman et al, 2011). However, in most studies,
major complications were further increased when the donor
wass CMV IgG-negative (Ozdemir et al, 2007; Zhou et al,
2009; Ugarte-Torres et al, 2011).
Recommendations
All Potential HSCT recipients should be tested for the
presence of CMV IgG antibody at diagnosis (Grade 1C).
Once optimum HLA matching has been performed, a
CMV IgG-negative donor should be chosen for a CMV
IgG-negative recipient and a CMV IgG-positive donor
should be chosen for CMV IgG-positive recipient when
possible (Grade 1A).
Donors or recipients who are initially found to be CMV
IgG-negative should be retested pre-transplant to
exclude primary CMV infection (Grade 1C).
Apparent CMV seroconversion in potential allograft
recipients who have received unscreened blood products
should be actively investigated to exclude passively
acquired antibody (Grade 1C)
Any CMV IgG-negative HSCT recipient transplanted
from a CMV IgG-negative donor who develops CMV
infection post-transplant must be reported to SHOT
(Grade 1C).
Prevention of CMV disease occurrence
Prophylactic and pre-emptive strategies have both been used
to reduce the incidence of CMV disease. Universal monitor-
ing of CMV levels in the blood is essential irrespective of
whether prophylaxis is administered.
Primary prophylaxis for CMV
Intravenous immunoglobulin
There is no evidence that intravenous immunoglobulin
(IVIG), CMV-specific hyperimmune immunoglobulin or
anti-CMV monoclonal antibodies are useful alone or in com-
bination with antiviral agents in primary prophylaxis against
CMV infection (Bowden et al, 1991; Ruutu et al, 1997;
Boeckh et al, 2001). A recent Cochrane review in solid organ
transplant did not recommend prophylactic immunoglobulin
(Hodson et al, 2007).
28
Adoptive immunotherapy
There have been several small studies published using CD4
or CD8 T cells (Walter et al, 1995; Einsele et al, 2002; Peggs
et al, 2003, 2009; Hanley et al, 2011; Sili et al, 2012), or
CMV peptide-loaded dendritic cell vaccination (Grigoleit
et al, 2007) for treatment or prophylaxis of CMV infection,
but too little evidence currently exists to make any recom-
mendation at present although ongoing prospective studies
are addressing this issue.
Antiviral agents
Aciclovir prophylaxis has been extensively studied post-
HSCT. A large randomized trial of 310 patients initially
appeared to suggest a reduced incidence and delayed onset
of CMV infection as well as a significant improvement in
survival. More mature follow up has shown no significant
difference in CMV reactivation between groups, although
reactivation did occur later in the prophylactic group. A
modest survival benefit was still seen in the most aggressively
treated patients, though it is difficult to attribute this to
anti-CMV activity alone (Prentice et al, 1994, 1997).
Improved survival in allograft patients who received aciclovir
prophylaxis post-engraftment was also shown in a meta-
analysis in which the vast majority of donor/recipient pairs
were CMV IgG-positive (Yahav et al, 2009). However, the
impact of aciclovir on CMV reactivation/disease rates in the
studies included was again minimal and survival advantage
was potentially mediated through anti-herpes simplex effects.
Subsequently, valaciclovir, 2 g four times a day, was
compared with oral aciclovir at 800 mg four times a day, in
727 patients following high dose intravenous (iv) aciclovir in
the immediate post-transplant period (Ljungman et al,
2002c). In this study, valaciclovir significantly reduced CMV
infection and disease rates (P < 0
0001). There was a 50%
reduction in the use of pre-emptive therapy although there was
no difference in overall survival (Ljungman et al, 2002c). Similar
results were shown in a smaller case controlled study using valac-
iclovir at a dose of 1 g three times a day (Vusirikala et al, 2001).
The studies described above predominantly used myeloablative
conditioning and T-cell-replete BM as the stem cell source. Stud-
ies in PBSC recipients, though smaller, suggested that the benefi-
cial effects of high dose aciclovir prophylaxis appeared to be
maintained (Verma et al, 2003; Hazar et al, 2004). However, aci-
clovir was shown to be significantly less effective in T-cell-
depleted BM transplant recipients, where 83% of T-cell-depleted
recipients still had a CMV reactivation compared to 41% of
unmanipulated stem cell sources (P < 0
0001) (Nakamura
et al, 2002). As most of these studies predated widespread
use of pre-emptive therapy based on quantitative PCR, their
significance is questionable in terms of current management
of CMV, though a compelling argument can be made for its
use for suppression of herpes simplex virus infection.
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Ganciclovir prophylaxis significantly reduced the incidence
of CMV infection and disease during the period of prophy-
laxis (Goodrich et al, 1993; Boeckh et al, 1996). However
neutropenia occurred in up to 30% of cases treated (Salzber-
ger et al, 1997) and infective complications were increased
(Boeckh et al, 1996). Ganciclovir was less effective in T-cell-
depleted transplants and heavily immunosuppressed recipi-
ents (Maltezou et al, 1999). In a prospective randomized trial
of ganciclovir versus aciclovir, cumulative rates of CMV
disease were equivalent, although more patients in the aciclo-
vir group required pre-emptive therapy (P = 0
2) (Burns
et al, 2002). Post-prophylaxis, late onset CMV disease
remained a problem (Boeckh et al, 2003) and prolonged
exposure of CMV to ganciclovir, especially in the setting of
T-cell depletion may encourage resistance, as occurs in solid
organ transplantation (Eid et al, 2008). Valganciclovir pro-
phylaxis has been reported to reduce risk of CMV disease in
cord blood transplants (Montesinos et al, 2009).
More intensive prophylactic regimens involving pre-trans-
plant ganciclovir combined with high dose aciclovir prophy-
laxis or a combination of ganciclovir and foscarnet have
been employed in high-risk paediatric and cord blood trans-
plants where both have been reported to be successful in
reducing CMV infection and disease (Shereck et al, 2007;
Milano et al, 2011).
Maribavir, when given from engraftment, initially showed
favourable results in phase II studies; but, at the dose chosen,
failed to show any effect on CMV disease or initiation of
CMV pre-emptive therapy compared to placebo in a larger
phase III study. There was a small impact on CMV DNA
loads in plasma (Winston et al, 2008; Marty et al, 2011).
Letermovir (AIC246), a maturation inhibitor of CMV, has
been studied in phase 2 trials as anti-CMV prophylaxis in
133 HSCT patients with potentially encouraging results
(Goldner et al, 2011). Neither of these drugs can be recom-
mended for prophylaxis at present.
In summary, post-HSCT, in contrast to solid organ trans-
plantation, ganciclovir-induced myelosuppression limits its
use for prophylaxis. Routine use of aciclovir or valaciclovir is
relatively non-toxic but will result in some patients being
overtreated and the effect in T-cell-depleted transplants is
small. However, the potential benefits of prophylaxis using
these drugs in selected patients include reducing the need for
hospital admission, for iv pre-emptive therapy, reducing
indirect effects of CMV reactivation on immune status post-
transplant and delaying CMV reactivation until the patient
has recovered from the toxicity associated with the transplant
and is no longer on immunosuppression.
Secondary prophylaxis for CMV
In patients who have had previous CMV disease prior to
transplant or with recurrent episodes of CMV infection,
especially in the context of T-cell depletion or GvHD, sec-
ondary prophylaxis should be considered, in conjunction
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British Journal of Haematology, 2013, 162, 2539
Guideline
with prolonged CMV viral screening. If prophylaxis is given,
then oral valaciclovir 2 g three times a day or valganciclovir
900 mg daily is an option (Boeckh & Ljungman, 2009).
Recommendations
Primary prophylaxis with ganciclovir is not generally
recommended as toxicity outweighs efficacy in HSCT
patients (Grade 1B).
Primary prophylaxis with aciclovir or valaciclovir can
be deployed but only in conjunction with appropriate
monitoring of CMV in the blood (Grade 1B).
Valaciclovir or valganciclovir are valid treatment
options for secondary prophylaxis with appropriate
monitoring of CMV in the blood (Grade 1C).
Intravenous immunoglobulins are not recommended for
prophylaxis of CMV infection (Grade 1A).
Pre-emptive therapy
The current mainstay for managing CMV infection after
HSCT is the rapid introduction of therapy, based on
evidence of CMV replication in blood (Goodrich et al,
1991). Success of pre-emptive therapy is dependent on the
availability of a rapid, sensitive assay to allow early treatment
at low levels of viral infection.
Diagnostic tests for early detection of CMV infection
Historically, the CMV antigenaemia assay provided a rapid
way to detect CMV infection and is still used as a cost-
effective screening tool in many centres worldwide. However,
it suffers from low sensitivity, is not accurately quantitative
and, in HSCT patients, is limited by the leucopenia evident in
the early stages post-transplant. Here, CMV antigenaemia
testing may be negative despite active viral replication
(Gondo et al, 1994; Koehler et al, 1995). The availability of
real time quantitative PCR (RQ-PCR) approaches has revolu-
tionized the area of CMV DNA monitoring. RQ-PCR tech-
niques provide rapid, high throughput platforms that are
significantly less affected by white cell counts (Einsele et al,
1995; Emery et al, 2000; Fishman et al, 2007; Gimeno et al,
2008). The choice of sample type for routine monitoring lies
between plasma and whole blood and remains controversial.
The choice is often based on practical issues surrounding
local sample handling and preparation. There are few pub-
lished reports supporting the superiority of one sample type
over the other. In the past, plasma was regarded as an easier
sample to extract, but this is now less of an issue as there are
a number of automated extraction platforms that can handle
whole blood. Several studies have shown that more CMV
DNA can be extracted from whole blood samples than from
plasma (Garrigue et al, 2006; Koidl et al, 2008; Bravo et al,
2011a). Responses to therapy may also be more predictable in
29
Guideline
whole blood compared to plasma given the higher CMV
DNA loads in the former (Atkinson & Emery, 2011). Other
body fluids, where white cells may be scanty, such as urine,
bronchoalveolar lavage, endotracheal aspirates, amniotic fluid
and cerebrospinal fluid can also be rapidly analysed using
RQ- PCR. However, to date, RQ-PCR has not been validated
for diagnosis of CMV end-organ disease and no recommen-
dations can be made for the use of this technology in this
setting.
Methodological issues
A diverse range of in-house and commercially available
RT-PCR assays are used in many diagnostic laboratories
throughout the UK. The primers, probes and assay condi-
tions used in these tests vary. Laboratories running in-
house assays will regularly make up their own new
reagents as part of the assay master mix, as well as a set
of log dilutions of a separately amplified quantification
standard, also known as a calibrator. This is used in order
to produce a standard curve from which the number of
copies of CMV DNA per ml of sample can be reported. A
number of controls must be used when running a diag-
nostic PCR assay including:
A positive control that is detected at the lower limit of
the assay.
At least one negative control to monitor for assay con-
tamination.
An internal amplification control that is an unrelated target
to monitor for any substances inhibitory to the assay.
A run control to monitor inter- and intra-assay variation.
Run controls are available commercially although some
laboratories will produce their own controls. Standardiza-
tion of nucleic acid extraction involves running an extrac-
tion control in each assay as well as an inhibition control
for each sample. Recently, a World Health Organization
standard for genome amplification of CMV from clinical
samples was produced by the National Institute for Biolog-
ical Standards and Control (Freyer et al, 2010; Bravo et al,
2011a). There are still significant differences in CMV DNA
load values reported by different laboratories. These differ-
ences have previously been attributed to the variety of in-
house methods used. However, it is likely that differences
will still be seen using commercial assays as well. A recent
comparison showed substantial differences in the nucleic
acid extraction efficiency of a number of automated sys-
tems and differences in the commercial RQ PCR assays
included in the study. This was most marked at the lower
CMV DNA loads (Bravo et al, 2011a). It is likely that the
international standard will be increasingly used to calibrate
assays on a regular basis. This will allow CMV DNA levels
to be compared between different centres and aid multi-
centre trials (Hirsch et al, 2013). Involvement with national
external quality assurance schemes is strongly advised.
30
Timing of CMV screening samples
There is no clinical trial evidence demonstrating how often
CMV DNA load in blood samples should be determined
(Freyer et al, 2010). However, weekly assessment following
allogeneic transplantation is advisable for the first 36 months
(Hebart et al, 1997). The duration of monitoring will depend
on the level of immunosuppression, the degree of immune
reconstitution, the presence of GvHD and response to antiviral
therapy. In these clinical settings, prolonged monitoring for
612 months may be required.
Recommendations
Real-time quantitative PCR is the preferred option for
monitoring CMV DNA levels in HSCT patients (Grade
1B).
All diagnostic laboratories should deploy the CMV
international standard to allow CMV DNA loads to be
compared between centres (Grade 1C).
Monitoring of CMV DNA load should be undertaken at
least weekly for the first 3 months post-HSCT (Grade 2C).
CMV viral load monitoring should continue to 6
12 months if the patient has chronic GvHD or pro-
longed T-cell immunodeficiency (Grade 1B).
Treatment thresholds
There is no consensus concerning the CMV load at which
pre-emptive treatment should be initiated (Boeckh & Ljung-
man, 2009; Ljungman et al, 2011). Some centres will start
antiviral therapy once CMV DNA has been detected at any
level in two consecutive samples, while others have set
much higher thresholds on the basis of local assays and
clinical outcome data. Unfortunately, these thresholds will
remain difficult to compare between centres until PCR test-
ing is fully standardized. A randomized trial comparing
antigenaemia with RQ-PCR has indicated that a CMV load
>10 000 copies/ml may be an acceptable threshold for initi-
ation of therapy. (Gerna et al, 2008; Boeckh & Ljungman,
2009; Mullier et al, 2009). This allows some patients to gen-
erate effective immune responses and avoid toxic drug ther-
apy while still treating susceptible patients pre-emptively,
before the onset of CMV disease. Demonstration of active
CMV-specific T-cell responses may help select patients who
do not require treatment (Cwynarski et al, 2001; Hebart
et al, 2002; Ozdemir et al, 2002). In addition to the peak
viral load there is evidence that the initial viral load and
the rate of viral load increase are important predictors of
development of CMV disease (Emery et al, 2000). Risk-
adapted strategies are likely to be needed and early treat-
ment is advised in R+/D- patients, especially in those with
severe lymphopenia, high initial viral loads (e.g. >34 log10
copies/ml), rapid viral load doubling times (e.g. >1 log10
2013 John Wiley & Sons Ltd
British Journal of Haematology, 2013, 162, 2539
 Guideline

copies/ml in 3 d) or those who are heavily immunocompro- bioavailability of active drug compared to iv ganciclovir
mised. 5 mg/kg bd and showed equivalent efficacy (Lim et al, 2009).
Good absorption was also shown in the context of intestinal
GvHD grade III (Einsele et al, 2006). Large prospective ran-
Recommendations
domized trials comparing valganciclovir and ganciclovir are
Each transplant centre should have a risk-adapted pol- lacking in HSCT patients. However, there are now several
icy detailing threshold values for treatment of CMV single centre randomized studies published, including over
infection, taking into account patient factors and local 150 patients, comparing the use of oral valganciclovir
PCR methodology (Grade 2C). 900 mg bd with 5 mg/kg bd iv ganciclovir. All these studies
showed no difference in any measure of efficacy although the
power of each individual study to exclude non-inferiority
Antiviral agents used in pre-emptive therapy was limited (Einsele et al, 2006; van der Heiden et al, 2006;
Lim et al, 2009; Chawla et al, 2011; Ruiz-Camps et al, 2011).
Ganciclovir. Ganciclovir requires phosphorylation by the UL
Further evidence of valganciclovir effectiveness was derived
97 viral kinase. It is virustatic. It inhibits viral replication by
from several single arm case series. These studies reported
competing with deoxyguanosine triphosphate as a substrate
response rates comparable to historic controls using ganciclo-
for viral DNA polymerase (UL 54) (Sia & Patel, 2000). Resis-
vir (Ayala et al, 2006; Rzepecki et al, 2008; Liu et al, 2010).
tance is mediated by mutations in genes encoding either of
CMV resistance rates appeared low following pre-emptive
these enzymes (Chou, 2008). Ganciclovir has been extensively
therapy with valganciclovir for CMV in HSCT (Allice et al,
used to treat CMV infection and disease (Goodrich et al,
2009). In solid organ transplants, where valganciclovir was
1991; Manteiga et al, 1998). It is still regarded as first choice
used extensively for prophylaxis it was shown to be non-infe-
pre-emptive therapy in most HSCT centres (Pollack et al,
rior to ganciclovir in preventing both CMV infection and
2011). The standard dose is 5 mg/kg intravenously twice
disease, with low rates of CMV resistance (Andrews et al,
daily for 14 d with maintenance of 5 mg/kg daily for a fur-
2011). After pre-emptive therapy in solid organ transplants
ther 714 d. Side effects and supportive therapies are listed
the kinetics of decrease in viral load were the same as for
in Table I.
intravenous ganciclovir (Mattes et al, 2005).
Valganciclovir. Valganciclovir is the valine ester of ganciclo-
Foscarnet. Foscarnet does not require phosphorylation and
vir, it is hydrolysed to ganciclovir after oral absorption. A
is virustatic through inhibition of viral DNA polymerase
small randomized trial in HSCT patients showed that oral
(Sia & Patel, 2000). Two randomized studies have compared
valganciclovir 900 mg twice daily led to higher effective
the outcomes of foscarnet versus ganciclovir pre-emptive
therapy of CMV infection post-allograft (Moretti et al,
Table I. Side effects of drugs used to treat CMV infection. 1998; Reusser et al, 2002). Foscarnet 90 mg/kg was com-
pared with ganciclovir 5 mg/kg, both administered
Drug Side effect Treatment/support
12 hourly iv, using CMV antigenaemia as a guide to com-
Ganciclovir Myelosuppression Granulocyte mencement of therapy (Moretti et al, 1998). Of 40 patients
colony-stimulating were randomly allocated to either treatment. There were no
factor statistically significant differences in rates of resolution of
Valganciclovir Haemolysis Toxicity-specific dose antigenaemia, treatment failure or progression to CMV
Nausea/vomiting reductions disease (510% in both groups). Both drugs required dose
Fever Regular twice daily
reductions of at least 20% in approximately half of all
Rash 5HT3 inhibitors
patients. A larger EBMT study compared 60 mg/kg foscar-
Mental state changes
net (n = 110) with ganciclovir 5 mg/kg (n = 103) both 12
Urinary symptoms
Foscarnet Electrolyte abnormalities Aggressive electrolyte hourly using either RQ-PCR or antigenaemia as a trigger to
Renal impairment replacement commence therapy (Reusser et al, 2002). No differences in
Nausea/vomiting Fluid support CMV disease occurrence, treatment-related mortality (TRM)
Genitourinary Regular twice daily or event-free survival were seen. Renal insufficiency was
Urinary symptoms 5HT3 inhibitors more common with foscarnet and myelosuppression with
Toxicity-specific dose ganciclovir. Further evidence of foscarnet efficacy comes
reductions from case series. In one, 313 patients were treated for CMV
Cidofovir Renal impairment Probenecid disease (n = 65), or were given pre-emptive therapy of
Nausea/vomiting Aggressive prehydration
CMV reactivation in related- donor transplants (n = 248)
Ocular Regular twice daily
(Asakura et al, 2010). Of 194 patients had failed previous
5HT3 inhibitors
ganciclovir due to lack of efficacy (n = 99) or myelosup-
Ophthalmology review
pression in (n = 95). Fifty two percent of patients with

2013 John Wiley & Sons Ltd 31

British Journal of Haematology, 2013, 162, 2539
Guideline
CMV disease and 90% of pre-emptively treated cases
responded. More limited data were reported supporting
foscarnet use in cord blood transplantation (Narimatsu
et al, 2007; Takami et al, 2007).
Cidofovir. Cidofovir is a nucleotide analogue that does not
require phosphorylation by viral UL97 kinase for activation.
The largest case series using cidofovir has been published by
the infectious disease working party of EBMT (Ljungman
et al, 2001). 82 patients were treated as primary (n = 24) or
secondary (n = 38) pre-emptive therapy for CMV infection
or for CMV disease (n = 20). Most patients were treated
with 5 mg/kg per week (range 15) for a median duration of
3 weeks. Response rates were 50% for CMV disease, 66% for
primary and 62% for secondary CMV infection. Other smal-
ler series demonstrated that cidofovir was effective but
response rates varied from 50 to 90% with most series
reporting significant failure rates (Chakrabarti et al, 2001;
Platzbecker et al, 2001; Cesaro et al, 2005). Side effects are
shown in Table I. A more tolerable alternative dosing regi-
men of 1 mg/kg three times a week has been used to treat
patients with adenovirus infection (Lindemans et al, 2010)
but there is no information using this strategy for the treat-
ment of CMV. Modified hexadecyloxypropyl-cidofovir
(HDP-CDV/CMX001), or other liposomal preparations of
cidofovir have been developed but their use has only been
studied in small numbers of patients (Hostetler, 2010; Bravo
et al, 2011b; Gokulgandhi et al, 2012) and no recommenda-
tions are possible.
Combination anti-viral drug therapy
The theoretical advantages of in vivo combination therapy
are reduced toxicity, and increased efficacy, both demon-
strated in vitro (Manischewitz et al, 1990; Manion et al,
1996): the primary disadvantage is generation of resistance,
particularly in the T-cell-deficient, immunocompromised
host, post-allograft. The combination of foscarnet and ganci-
clovir (both at half dose) versus full dose ganciclovir was
evaluated in a small randomized study (n = 48) in transplant
patients, including HSCT recipients (Mattes et al, 2004).
There was less myelosuppression but overall increased toxic-
ity, primarily renal, in the combination arm. Importantly
though underpowered to show a clinical effect 71% of the
ganciclovir group reached the primary clinical endpoint of
viral clearance at 14 d compared to 50% in the combination
group. At present, not enough evidence exists to support
treatment using these two drugs at sub-conventional doses.
Bacigalupo et al (1996a) have also reported combining
foscarnet and ganciclovir at full dose, adjusted for organ
function, aiming for 180 mg/kg/d foscarnet and 10 mg/kg/d
ganciclovir and delivering 5060% of these doses. Drugs were
delivered together at full dose for 2 weeks followed by main-
tenance, where drugs were given at full dose but on alternate
days. This approach was effective and the authors suggested
32
a possible reduction in TRM compared with historical con-
trols (Bacigalupo et al, 1996b). However, not enough
evidence exists to make a recommendation regarding combi-
nation therapy in this context.
Recommendations
Ganciclovir is recommended as first line pre-emptive
therapy for CMV in HSCT patients (Grade 1A).
Oral valganciclovir is a useful alternative when gastroin-
testinal absorption is normal or minimally impaired
(Grade 1B).
Foscarnet is recommended as an alternative first line
agent if neutropenia is present or for ganciclovir treat-
ment failures (Grade 1A).
Pre-emptive therapy with cidofovir can be considered as
third line in patients unresponsive or intolerant of a
ganciclovir preparation or foscarnet (Grade 2B).
Switching pre-emptive therapy
The doubling time of CMV DNA load in untreated patients
is 12 d on average but may be even more rapid (Emery
et al, 1999; Buyck et al, 2010) At the start of pre-emptive
therapy, before drugs can have a major impact, increases in
CMV DNA load occur in approximately one-third of
patients due to underlying immunosuppression and rapidity
of replication (Buyck et al, 2010; Park et al, 2011). This
should be appreciated and no changes in therapy instituted
in the first 14 d, in the absence of overt CMV disease or
dramatic rises in viral PCR log values. It is difficult to be
dogmatic about triggers for changing pre-emptive antiviral
drugs, as responses to treatment can be very slow in pro-
foundly lymphopenic patients. An increase in viral load by
one log after 2 weeks of therapy is one option. Switching
therapy is seldom recommended earlier, and can often be
delayed further, even in high-risk patients, unless disease
has progressed clinically, or viral copy number is increasing
rapidly.
Recommendations
In patients where CMV DNA loads in blood increase by
1 log10 over 2 weeks of pre-emptive therapy with a first
line drug, an alternative agent and drug resistance profil-
ing should be considered (Grade 2C).
Antiviral drug resistance
Drug resistance is relatively uncommon in the stem cell trans-
plant setting. Resistance to ganciclovir is estimated at 28% of
treated patients and generally occurs after 23 months of
prolonged therapy (Marfori et al, 2007) but can occur after
initial therapy (van der Beek et al, 2012; Kim et al, 2012).
2013 John Wiley & Sons Ltd
British Journal of Haematology, 2013, 162, 2539

Close liaison between transplant physicians and clinical virolo-
gists is essential. Resistance is less common in drug-naive
patients than in patients experiencing repeated episodes of late
onset CMV infection. Resistance should be suspected in
patients on antiviral drugs where the CMV DNA load has been
static or has increased for more than 2 weeks, although clini-
cal resistance is still more likely in lymphopenic drug-nave
patients. If drug resistance is suspected, samples should be sent
for CMV UL97 and UL54 sequencing.
Recommendations
Drug resistance should be considered if the CMV DNA
load in blood fails to respond after 14 d of therapy,
especially in non-lymphopenic or multiply pre-treated
patients (Grade 2C).
Sequence analysis of the UL97 and UL54 genes is the
preferred option for monitoring resistance to currently
available drugs (Grade 1B).
Management of CMV disease
A multidisciplinary approach to the management of CMV
disease is important. Specific investigations and treatment
depends on which organs are affected and whether disease
has developed de novo or from asymptomatic CMV infection
while on first-line therapy. De novo CMV disease can be trea-
ted with ganciclovir 5 mg/kg, or foscarnet 90 mg/kg twice
daily for 2 weeks, followed by maintenance, although more
prolonged treatment may be necessary. Careful ophthalmo-
logical review is mandatory as intraocular therapy may be
required (Song et al, 2008). Higher baseline CMV DNA
loads, faster kinetics of replication and a slower viral decay
rate after starting therapy are associated with increased rates
of treatment failure (Mattes et al, 2004) and mandate closer
monitoring of these patients with lower thresholds for
diagnostic interventions. If CMV disease occurs during first
line pre-emptive therapy or if de novo CMV pneumonia is
progressive at any time, then alternative strategies should be
considered. Failure to respond is more likely to be related to
the hosts inability to control the virus as a result of
impaired T cell immunity than to drug resistance, unless
multiple courses of therapy have been given previously
(van der Beek et al, 2012). However, in critically unwell
patients, where available, drug resistance testing should be
performed early to optimize pharmacological management.
Alternative treatment approaches in this setting include
increasing ganciclovir doses to 7
5 mg/kg twice a day (Bo-
eckh & Ljungman, 2009) or switching ganciclovir to foscar-
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2013 John Wiley & Sons Ltd
British Journal of Haematology, 2013, 162, 2539
Guideline
net (or vice versa). Combination therapy is theoretically
attractive but may prove difficult to deliver (Manion et al,
1996). Foscarnet and ganciclovir can be combined, at full
dose (Bacigalupo et al, 1996a). Cidofovir is an alternative to
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testing, increased drug doses and/or a change of drug
(Grade 1B).
Reduction in immunosuppression, especially corticoste-
roid dosage, is strongly recommended (Grade 1B).
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The authors report no conflicts of interest.
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