Download Human cytomegalovirus resistance to antiviral drugs: diagnosis

Journal of Antimicrobial Chemotherapy (2003) 52, 324–330
DOI: 10.1093/jac/dkg354
Advance Access publication 29 July 2003
Human cytomegalovirus resistance to antiviral drugs: diagnosis,
monitoring and clinical impact
Fausto Baldanti1,2 and Giuseppe Gerna1*
1Servizio
di Virologia, 2Laboratori Sperimentali di Ricerca, IRCCS Policlinico San Matteo, 27100 Pavia, Italy
The incidence of human cytomegalovirus (HCMV) disease in AIDS patients decreased dramatically after the
introduction, a few years ago, of highly active antiretroviral combination therapy. As a consequence, the
emergence of drug-resistant HCMV strains is no longer a major problem in HIV-infected individuals. However, HCMV resistance to antiviral drugs is presently recognized as an emerging problem in transplantation
settings. The mechanisms of HCMV drug resistance will be analysed along with the clinical features relevant
to the emergence of drug-resistant HCMV strains during antiviral treatment of patients receiving either solid
organ or haematopoietic stem cell transplantation.
Keywords: HCMV, antivirals, drug resistance
Introduction
Until very recently, the large majority of immunocompromised
patients receiving antiviral treatment for human cytomegalovirus
(HCMV) infection or disease was represented by patients with
AIDS. Thus, it is not surprising that emergence of drug-resistant
HCMV strains was mostly reported in this patient population.1–18 The
striking clinical benefit of the recently introduced highly active
antiretroviral combination therapies (HAART) in HIV-infected
individuals is demonstrated by the sharp decrease in the incidence of
HCMV infections as well as other opportunistic infections.19–23 Consequently, from an epidemiological standpoint, HCMV drug resistance has become a less dramatic problem. However, a substantial
number of drug-resistant HCMV strains has now been recovered
from either transplant recipients24–37 or other immunosuppressed
patient populations.1,38,39 Thus, physicians and clinical virologists are
still facing the need for a timely identification of drug-resistant
HCMV strains when in the presence of virological failure of antiHCMV treatment. In addition, the development of new antivirals
with different mechanisms of action is still recognized as a major
priority for the effective treatment of immunocompromised patients.
This report will describe the mechanism of action of anti-HCMV
drugs and the molecular basis of HCMV drug resistance. In addition,
techniques for monitoring HCMV infection and response to antiviral
treatment and for detecting the emergence of drug-resistant HCMV
strains will be reviewed. Special attention will be paid to discuss the
advantages and drawbacks of the techniques for phenotypic and
genotypic drug resistance determination and to highlight the peculiar
clinical features relevant to the emergence of drug-resistant HCMV
strains in transplantation settings.
Assays for diagnosis and monitoring of HCMV
infection
Viral parameters currently utilized for monitoring of HCMV infection and evaluation of the response to antiviral treatment are: (i)
viraemia, (ii) antigenaemia, (iii) circulating cytomegalic endothelial
cells (CCEC), (iv) DNAaemia and (v) RNAaemia.
Viraemia consists of a rapid virus isolation technique based on
16–24 h co-cultivation of 2 × 105 peripheral blood leucocytes (PBL)
with human fibroblast monolayers grown in shell vials, followed by
counting of infected fibroblasts after nuclear staining with a monoclonal antibody (MAb) directed to the major HCMV immediateearly (IE) antigen p72.40
Antigenaemia consists of quantitative determination of HCMV
pp65-positive cells in 2 × 105 PBL examined in a cytospin preparation.41
The number of HCMV-infected CCEC is determined in the cytospin preparations used for antigenaemia quantification after staining
with pp65-specific MAbs.42
DNAaemia can be determined by quantitative PCR either in
PBLs,43 plasma43,44 or whole blood45 and is expressed as HCMV DNA
genome equivalents (or copy number) per 2 × 105 PBL, or a determined volume of whole blood or plasma, respectively. Other methods, such as the branched DNA (bDNA) technique, have been used
for HCMV DNA quantification in immunocompromised patients.46
RNAaemia consists of qualitative or quantitative determination of
IE- or late-mRNAs in blood by RT–PCR47 or NASBA (nucleic acid
sequence based amplification).48,49
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*Corresponding author. Tel: +39-0382-502644; Fax: +39-0382-502599; E-mail [email protected]
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© The British Society for Antimicrobial Chemotherapy 2003; all rights reserved.
HCMV drug resistance
Figure 1. Chemical structures of ganciclovir, foscarnet and cidofovir.
Anti-HCMV drugs available clinical practice
Ganciclovir is a nucleoside analogue inhibitor of HCMV DNA polymerase (encoded by UL54). In order to be active, the drug requires a
triple phosphorylation, the first being carried out by a viral phosphotransferase (encoded by UL97), whereas the second and third
phosphorylations are carried out by cellular enzymes.3,50
Ganciclovir is usually administered at the dosage of 5 mg/kg body
weight twice a day for induction treatment, whereas primary or
secondary prophylaxis treatments (see below) are usually carried out
with half this dosage. Ganciclovir is currently administered intravenously (iv), although an oral formulation of the drug is available.
Valganciclovir is a recently developed oral prodrug of ganciclovir
with improved pharmacokinetic characteristics.51 In a recent study, it
was shown that using valganciclovir at a dosage of 900 mg twice a
day for induction treatment and 900 mg once a day for maintenance
treatment, systemic exposure to ganciclovir was similar to that
obtained following iv administration of ganciclovir, eventually leading to a comparable efficacy of the two drugs.51
Foscarnet is a DNA polymerase inhibitor acting as a competitor of
pyrophosphate. Foscarnet does not require metabolic activation and
is administered at a dosage of 90 mg/kg body weight twice a day for
induction treatment and at half that dosage for primary or secondary
prophylaxis. The administration is iv.
Cidofovir is a nucleotide analogue inhibiting the viral DNA polymerase. Being already phosphorylated, it does not require activation
by UL97. A peculiar characteristic of cidofovir is its extended halflife in vivo allowing weekly or bi-weekly administration. The drug
is administered iv at a dosage of 5 mg/kg once a week during
induction and 5 mg/kg once every other week during maintenance
treatment.
A schematic of the chemical structures of ganciclovir, foscarnet
and cidofovir is shown in Figure 1.
ment was usually administered at a reduced dosage lifelong. However, in the absence of effective control of HIV replication with
concomitant decay of the immune system, HCMV recurrences were
not prevented, eventually leading to the emergence of HCMV drugresistant strains.1–18 Moreover, in these highly immunocompromised
patients, sequential treatments with different anti-HCMV drugs led
in many cases to the accumulation of multiple genetic alterations with
the final emergence of multidrug-resistant strains.5,9,52,53 Following
the introduction of HAART, the incidence of HCMV disease in HIVinfected individuals dropped sharply.19–23 In parallel, the emergence
of drug-resistant HCMV strains in this clinical setting appears to be a
memory of the past. However, the number of patients failing HAART
for multiple reasons (drug-related toxicity, intolerance, poor adhernce to treatment, emergence of HIV drug-resistant strains) is
increasing. Thus, in the future, detection of HCMV drug-resistant
strains in HAART-treated individuals might not remain just an
anecdote.54
In solid organ (SOTR) or haematopoietic stem cell (HSCTR) transplant recipients, pre-symptomatic treatment approaches are usually
adopted. In particular, both primary prophylaxis and pre-emptive
therapy of HCMV infections have been utilized. Primary prophylaxis
is based on the administration of ganciclovir to SOTR for several
weeks after transplantation in order to suppress viral replication
during a period of time when the patient is considered to be at high
risk for development of HCMV infection. In contrast, pre-emptive
therapy is based on virological monitoring of patients in the posttransplant period in order to identify and treat only those actually at
risk for HCMV disease since reaching threshold values of viral load.
The latter approach was proven to be able to prevent development of
HCMV-related symptoms with the advantage of avoiding unnecessary treatment and related drug toxicity. Although pre-emptive therapy is now widely utilized, no consensus on standardized treatment
protocols has been reached. The major reason for this is the fact that
different viral parameters have been used in different laboratories to
monitor HCMV infection. The most utilized viral parameters have
been antigenaemia and DNAaemia. However, these techniques still
lack standardization and patients at risk for HCMV disease are thus
far defined mostly on the basis of locally established threshold values. In addition, different patient populations are treated on the basis
of different threshold values of viral load. In this respect, it is
common to treat HSCTR at the first confirmed appearance of HCMV
in blood,55 whereas in SOTR higher viral load levels have been set
as a threshold for treatment initiation. An exception is represented
by treatment of donor-positive, recipient-negative (D+/R–) SOTR
which is treated upon the first appearance of HCMV in blood.55
Monitoring of HCMV infection and response to
antiviral treatment
Approaches to treatment of HCMV infection in
immunocompromised patients
Different treatment approaches have been applied to different populations of immunocompromised patients. In the pre-HAART era,
given the very high prevalence of HIV-infected individuals with
low CD4 cell counts showing positivity for HCMV in blood and the
unfeasibility of weekly monitoring of HCMV infections in asymptomatic individuals, specific treatment was administered in the presence of overt HCMV disease (symptomatic treatment). Response to
treatment was monitored both clinically and virologically. However,
the high risk of HCMV disease relapse prompted the introduction of
secondary prophylaxis or maintenance treatment. Maintenance treat-
In immunocompromised patients, correct monitoring of the response
to antiviral treatment of HCMV infections may be the most powerful
tool for detecting the emergence of drug-resistant HCMV strains. In
fact, following treatment initiation, a sharp decrease in level of viral
parameters is consistently observed in patients infected by drugsusceptible HCMV strains. In contrast, the emergence of drug-resistant strains is associated with an increase in viral load or the lack of
decrease in level of different viral parameters.4,5,9,11,12,26–28,33–35,54,56–61
The most reliable parameter for the identification of the emergence
of drug-resistant strains appears to be viraemia. In fact, positive
viraemia levels during treatment document active viral replication
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F. Baldanti and G. Gerna
and may anticipate the increase in viral load as determined by quantitative determination of viral components (pp65, DNA).3,4,8,12,28 However, increase in levels of viral parameters can also be documented
following short (few days) interruptions of treatment or when therapy
is administered at a reduced dosage because of drug toxicity or intolerance. Thus, results of the virological response to treatment should
always be interpreted for each patient with great caution and within
the clinical context. In addition, the emergence of drug-resistant
HCMV strains should always be proven before shifting to an
alternative treatment. Thus, several assays (either phenotypic or
genotypic) have been developed in recent years to rapidly detect
the presence of drug-resistant HCMV strains directly in biological
specimens (see below). A clinical situation stressing this warning is
represented by the peculiar behaviour of antigenaemia during treatment of primary HCMV infection in SOTR. In this subset of patients,
a dissociation between increasing antigenaemia and decreasing
DNAaemia and viraemia levels is often observed.55 This phenomenon appears to be restricted to patients treated with ganciclovir, and
the reasons for this event occurring are still unclear. Considering that
D+/R– is also the subset of SOTR in whom almost all reported drugresistant HCMV strains have been detected,25–28,33,36,37 it appears
evident that separating an abnormal rise of antigenaemia alone from a
true increase in viral load during therapy is of critical importance for
correct diagnosis of drug resistance. In this respect, the concomitant
determination of DNAaemia and viraemia is of crucial help: emergence of drug-resistant strains in these patients is associated with a
concomitant increase in all viral parameters, which, in contrast, are
shown to decrease in parallel when an effective treatment is administered.28
Molecular basis for drug resistance
Ganciclovir
Acquisition of mutations in UL97 phosphotransferase (Figure 2)
appears to be a crucial step in the selection of ganciclovir-resistant
HCMV strains. HCMV strains with mutation in key regions (domains
VIII, VI and IX) of the viral enzyme have been proven to be highly
resistant to ganciclovir.6–8,14,17,18,25–28,31,56,62–68 In addition to UL97,
mutations in UL54 impact ganciclovir susceptibility.13,15,53,56,69,70
However, mutations in UL54 are less common and have been
reported only in patients harbouring ganciclovir-resistant UL97mutant strains maintained on ganciclovir treatment.53 The isolates
with mutations in both UL97 and UL54 showed very high levels of
resistance to ganciclovir and simultaneous cross-resistance to cidofovir,53 but retained foscarnet susceptibility. Finally, it is worth mentioning that, according to the limited data so far available,
valganciclovir does not appear to select for an increased number of
ganciclovir-resistant strains.71
Foscarnet
Mutations in domains II, III and VI of HCMV DNA polymerase
(Figure 2) are responsible for foscarnet resistance.5,57,66,69,70 Interestingly, no cross resistance to either ganciclovir or cidofovir is
associated with foscarnet-induced UL54 mutations.66,69,70 Thus, the
reported emergence of double ganciclovir- and foscarnet-resistant
HCMV strains was due to the accumulation of specific mutations in
the two viral genes induced by sequential treatment with the two
drugs.9,5,66 However, mutations in domain III have been proven to
induce simultaneous resistance to foscarnet, ganciclovir and cidofovir.56
Cidofovir
Mutations in the HCMV DNA polymerase are responsible for simultaneous resistance to cidofovir and ganciclovir.66,70 Because of its
high renal toxicity, cidofovir is not widely utilized for treatment of
HCMV infection. Thus, selection of cidofovir-resistant strains is an
extremely rare event and it appears mostly driven by sustained ganciclovir administration.53
Methods for phenotypic determination of antiviral
drug resistance
Reduction of HCMV replication in the presence of increasing drug
concentrations can be determined by several methods.
Plaque reduction assay (PRA)
This assay measures the reduction in number of cytopathic effect foci
following infection of fibroblast monolayers with titrated virus isolates (CPE-PRA).72 Alternatively, the number of IE antigen or late
antigen plaques can be determined (IEA- or LA-PRA).4,73 This assay
is regarded as the reference method for HCMV drug susceptibility
determination and, following titration of the virus isolate, requires
4–10 days to be completed. In detail, IEA-PRA provides results in 4
days, LA-PRA is completed in 6–7 days and CPE-PRA takes longer.
Although PRA has been known for several years and widely utilized,
it is not yet standardized and comparison of results from different
laboratories appears sometimes difficult. The lack of standardization
is primarily related to:(i) the use of different fibroblast strains, (ii) the
use of cell-free virus as inoculum versus cell-associated virus, (iii)
the virus plaque staining technique; (iv) the use of laboratory adapted
HCMV strains (AD169, Towne) versus clinical isolates as controls,
(v) the subjectivity in reading test results.
Consensus is lacking in the definition of drug resistance: some
authors indicate 6 µM ganciclovir and 400 µM foscarnet as ID50
cut-off values for ganciclovir- and foscarnet-sensitive HCMV strains,
respectively. In our laboratory we define as drug-resistant the HCMV
isolates showing ID50 values ≥5-fold the mean ID50 values determined on a series of HCMV isolates from untreated patients.4,9,12,28,74
Other authors consider as resistant HCMV isolates with ID50 values
increased by ≥2 S.D. of the mean ID50 values determined on a series of
HCMV control field isolates.6
Recently, a rapid drug susceptibility screening assay has been
developed based on the concept of PRA.58 This assay measures the
reduction in IEA-plaques following inoculation onto fibroblast
monolayers of a predetermined number of PBL and incubation of cell
cultures for 4–6 days in the presence of a single drug concentration
(ganciclovir, 20 µM; foscarnet, 400 µM). A decrease of ≤50% in
the number of IEA-plaques with respect to the no-drug control is
considered as associated with the presence of drug resistance.74 A
similar approach has been described by Pepin et al.75
In situ ELISA
This technique measures the reduction in LA production in fibroblast
cultures infected with cell-free HCMV, which reflects the number
of cells undergoing a complete virus replication cycle. The assay is
carried out in 96-well microtitre plates and viral titration can be
carried out in the context of the assay by simultaneously challenging
increasing virus dilutions with decreasing drug concentrations in the
same plate. After 4–6 days of incubation, the amount of viral antigen
in each well is determined by immunoenzymic staining followed by
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HCMV drug resistance
Figure 2. Schematic of HCMV DNA polymerase (encoded by UL54) and phosphotransferase (encoded by UL97). DNA polymerase codons mutated in ganciclovirand cidofovir-cross-resistant, foscarnet-resistant and multidrug-resistant HCMV strains are indicated by solid, dashed, and dotted vertical bars, respectively. UL97
codons mutated in ganciclovir-resistant HCMV strains are shown by vertical solid bars.
spectrophotometric determination of absorbance. The assay is completed in 4–6 days.76,77
Virus yield reduction assay
The effect of antiviral drugs on viral replication can also be determined by quantifying the reduction in infectious viral titres, measured either as cell-free or cell-associated virus.78 This technique is
cumbersome and extremely time-consuming and is currently utilized
for research in drug development.
Flow cytometry-based assays
The reduction in viral antigen production can also be determined
using flow cytometry by counting the number of trypsinized fibroblasts expressing late viral proteins.79,80
DNA reduction assay
This assay measures the reduction in viral DNA synthesis in fibroblast cultures in the presence of increasing drug concentrations.81
Quantification of HCMV DNA is obtained by hybridization of the
viral genome with a 125I-labelled probe followed by counting the
γ-emission by the bound probe. Following preparation of a titrated
virus stock, results are available in 1 week. However, this assay is not
widespread, primarily because of the need for dedicated facilities and
expensive equipment (γ-counter).
Methods for genotypic determination of antiviral
drug resistance
The presence of drug-resistant HCMV strains can be determined by
identifying specific mutations in the viral phosphotransferase (UL97)
or DNA polymerase (UL54) coding sequences.
Sequencing
Analysis of UL97 and UL54 genes is usually carried out following
amplification of gene fragments from viral isolates or directly from
clinical samples by PCR. When using an automated sequencer, results
are available in a couple of days. This assay is the reference technique
for the identification of mutations emerging during treatment failure.
However, despite its powerful diagnostic potential, sequence analysis is currently restricted to specialized laboratories, requiring
expensive equipment and personnel with specific expertise.
Restriction fragment length polymorphism (RFLP)
This assay allows detection of specific mutations associated with
drug resistance by evaluating the restriction pattern of PCR products
digested with endonucleases, in which restriction sites have been
modified (suppressed or generated) by single mutations.28,63 In
addition, deletions modifying the size of digested fragments can be
readily detected.64 Thus far, this technique has been widely applied
for identification of UL97 mutations conferring ganciclovir resistance.
Probe-specific hybridization or primer-specific amplification
Probes discriminating single nucleotide substitutions can be used
for hybridization of PCR products. Alternatively, primers can be
designed to anneal only with the wild-type or the mutant sequence,
thus allowing specific amplification by PCR or ligase chain reaction
(LCR).82,83
Advantages and disadvantages of phenotypic testing
for drug resistance
Methods determining the level of inhibition of viral replication in
vitro in the presence of a drug provide direct evidence of susceptibility or resistance to antiviral compounds. However, ‘susceptibility’ or ‘resistance’ are not absolute concepts, but indicate levels
of inhibition of viral replication which are comparable or significantly lower than those observed using reference strains, respectively. Indeed, the selection of appropriate controls represents one of
the most debated issues, and lack of consensus on this topic represents the major obstacle for the standardization of phenotypic assays
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F. Baldanti and G. Gerna
for HCMV drug resistance determination. AD169 or Towne strains,
which most laboratories use as drug-sensitive reference strains,
appear to be different from clinical HCMV isolates in different
aspects. In fact, the replication and spread in cell culture of AD169
and Towne strains are different from those of clinical isolates, as
shown by the faster viral kinetics and the higher release of cell-free
virus in cell culture supernatant, which contrasts with the slower
growth rate and the preferential cell-to-cell transmission of recent
clinical isolates. In addition, AD169 and Towne strains present important genetic alterations,84 which likely occurred during the fibroblast
adaptation process and which might be accompanied by more subtle
mutations in other genes. The characteristics of reference strains
might also affect their susceptibility to antiviral drugs. Interestingly,
AD169 recombinants carrying mutations from clinical isolates often
show ID50 values substantially different from those of the parental
drug-resistant strain.56,57,64,66,67,69,70 Besides other factors already
mentioned above, other factors should be taken into account in an
attempt to standardize the phenotypic drug resistance testing, i.e. the
presence of multiple viral variants in the viral isolate, and the possible
growth deficits of drug-resistant viral strains. The last two points
have direct implications in the interpretation of results of drug
susceptibility testing. In fact, in the presence of mixtures of wild-type
and mutated virus strains, the isolation procedure may favour the
recovery of a single viral variant, sometimes delaying the isolation of
the resistant strain.12,28,64 In addition, when in the presence of a mixed
viral population in vivo including drug-resistant strains with impaired
growth in fibroblast cultures,4,8,9,60,61,66,69 the sensitive strains may
overgrow the resistant ones in cell culture, thus hampering the
reliability of the drug susceptibility testing.
Advantages and disadvantages of genotypic testing
for drug resistance
The use of molecular biology techniques for detection of drug resistanceassociated mutations in UL97 and UL54 can greatly shorten the time
required for the identification of HCMV drug-resistant strains. In
addition, molecular techniques provide the opportunity to quantify
the mutant (resistant) viral variants in the context of a mixed viral
population. Finally, genotypic testing of drug susceptibility appears
in need of better standardization. However, identification of HCMV
strains with specific mutations in target gene products is only indirect
evidence of drug resistance. In this respect, problems in result
interpretation might arise in the face of new mutations or constellations
of mutations. In these cases, great caution must be applied before
labelling as resistant a strain with a new mutation in UL97 or UL54.
In fact, although UL97 regions involved in ganciclovir recognition
and processing appear to be well defined, confirmatory marker transfer experiments still appear to be mandatory in order to elucidate
the role of new mutations in these regions in conferring ganciclovir
resistance. As for UL54, although distinct patterns of mutations
conferring resistance to foscarnet and cross-resistance between
ganciclovir and cidofovir have been described, some amino acid
changes appear to confer multiple drug resistance. Thus, the impact
of new UL54 mutations in decreasing the susceptibility of clinically
available anti-HCMV compounds is not a priori predictable, and
marker transfer confirmation is needed.
Conclusions
In conclusion, as emerging from the above-reported considerations,
no wonder tool is available at present for rapid detection of the emergence of drug-resistant HCMV strains in immunocompromised patients
failing anti-HCMV treatment. On the contrary, an integrated programme including: (i) careful virological monitoring of HCMV
infection and response to treatment, (ii) the use of rapid phenotypic
screening assays for drug susceptibility testing and molecular techniques for detection of specific genetic mutations, and (iii) the application of confirmatory drug susceptibility assays on viral isolates
obtained during virological follow-up, still appears to be the best
approach for the timely detection of drug-resistant HCMV strains in
the clinical setting.
Acknowledgements
This work was partially supported by Ministero della Salute, Istituto
Superiore di Sanità, Programma Nazionale di Ricerca sull’AIDS
(grant 50D.12), Ministero della Salute, Ricerca Finalizzata 2001
(convenzione 126).
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