Original article Cytomegalovirus resistance in solid organ transplant recipients treated with intravenous ganciclovir or oral valganciclovir

Antiviral Therapy 14:697 704 Original article Cytomegalovirus resistance in solid organ transplant recipients treated with intravenous ganciclovir or oral valganciclovir Guy Boivin 1,2 *, Nathalie Goyette 1, Halvor Rollag 3, Alan G Jardine 4, Mark D Pescovitz 5, Anders Asberg 6, Jane Ives 7, Anders Hartmann 8 and Atul Humar 9 1 Infectious Disease Research Center of the Centre hospitalier universitaire de Québec, Québec City, QC, Canada 2 Laval University, Québec City, QC, Canada 3 Institute of Microbiology, University of Oslo, Oslo, Norway 4 Department of Medicine, University of Glasgow, Glasgow, UK 5 Department of Surgery and Microbiology, Indiana University, Indianapolis, IN, USA 6 Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway 7 Roche Products Ltd, Welwyn Garden City, UK 8 Department of Medicine, Rikshospitalet-Radiumhospitalet Medical Centre, University of Oslo, Olso, Norway 9 Department of Medicine, University of Alberta, Edmonton, AB, Canada *Corresponding author: e-mail: Guy.Boivin@crchul.ulaval.ca Background: The rate of cytomegalovirus (CMV) mutations conferring ganciclovir resistance was assessed in a trial comparing intravenous ganciclovir and oral valganciclovir for treatment of CMV disease in solid organ transplant (SOT) recipients. Methods: Viral genes (UL97 and UL54) conferring ganciclovir resistance were amplified and sequenced from blood samples collected at days 0 (before therapy), 21 (end of induction) and 49 (end of maintenance). Results: The overall risk of developing a confirmed or probable ganciclovir resistance mutation during treatment was similar for patients treated with ganciclovir (2.3%) and valganciclovir (3.6%; P=0.51). A persistent viral load at day 21 was associated with a significant risk of ganciclovir resistance by day 49 (odds ratio 11.83; P=0.022). In multivariate analyses, presence of a confirmed ganciclovir resistance mutation was independently associated with virological failure (viral load 600 copies/ml) at days 21 and 49. One-third (3/9) of patients with confirmed CMV resistance mutations had recurrent CMV disease. The plasma half-life of confirmed ganciclovir-resistant UL97 mutants was significantly longer than that of wild-type strains, polymorphic variants and strains with mutations of unknown significance (P=0.045). Multiple UL54 mutations of unknown significance were found in clinical strains. Viral kinetic analysis of these latter strains revealed no effect (negative or positive) on in vivo viral fitness. Conclusions: Treatment with oral valganciclovir or intravenous ganciclovir results in similar and low rates of resistance mutations in SOT recipients. Patients with drug-resistant CMV strains often have virological failure and might have unfavourable clinical outcomes. Introduction Cytomegalovirus (CMV) is a significant cause of morbidity in solid organ transplant (SOT) recipients [1]. Despite various prevention strategies, CMV disease still develops in patients who have not received or who have completed antiviral prophylaxis [2]. Intravenous ganciclovir has been the mainstay of therapy for CMV disease over the past two decades [3]. Valganciclovir is an oral prodrug of ganciclovir with a systemic exposure from 900 mg once daily, similar to that of 5 mg/kg/day intravenous ganciclovir [4,5]. A recent study has indicated that oral valganciclovir is as effective as intravenous ganciclovir for treatment of CMV disease in SOT recipients [6]. Prolonged use of antiviral agents might lead to the development of CMV resistance [2,7]. Ganciclovir resistance is primarily conferred by mutations in the viral UL97 phosphotransferase gene responsible for ganciclovir monophosphorylation and, more rarely, by 2009 International Medical Press 1359-6535 (print) 2040-2058 (online) 697

G Boivin et al. mutations in the viral UL54 DNA polymerase gene that is the target of ganciclovir triphosphate [8]. Although our previous study indicated no CMV mutations associated with ganciclovir resistance in SOT patients who received valganciclovir prophylaxis for 100 days after transplantation [9], the problem of CMV resistance could be more important when the drug is administered in a therapeutic setting (that is, in presence of high viral load). In addition, numerous mutations have been reported in both UL97 and UL54 genes that do not necessarily confer ganciclovir resistance based on in vitro marker transfer experiments [8]. For UL54, such mutations might occur in conserved and nonconserved regions, and are often referred to as natural polymorphisms. However, the in vivo significance of such mutants has not been previously assessed on a large scale. We sought to evaluate the rate and clinical significance of CMV mutations associated with ganciclovir resistance in a large trial that compared the use of oral valganciclovir and intravenous ganciclovir for treatment of CMV disease after SOT. Our secondary objective was to assess the in vivo fitness effect of viral mutations in UL97 and UL54 genes. Methods Study design and virological endpoint The resistance study was part of a randomized openlabel multicentre trial in adult SOT recipients with tissue invasive CMV disease or syndrome (the Victor study) [6]. In the clinical trial, the median time from transplant to onset of CMV disease was 73 days (range 13 9,257, interquartile range 49 143). Patients were randomized to treatment with 900 mg twice-daily oral valganciclovir or 5 mg/kg twice-daily intravenous ganciclovir (both drugs adjusted for renal function) for an induction period of 21 days, followed by 900 mg oncedaily oral valganciclovir until day 49 in both arms. The primary outcome for this study was the eradication of CMV viraemia at day 21, assessed using the COBAS Amplicor CMV Monitor test (Roche Molecular Diagnostics, Branchburg, NJ, USA) with a cutoff value set at the lower level of reliable quantification (that is, 600 copies/ml of plasma). Additional viral load measurements were performed at days 0, 3, 7, 10, 14, 17, 28, 35, 42, 49, 90 and 180, and at time of relapsing CMV disease. For the resistance study, samples with detectable CMV viraemia (that is, viral load threshold >200 copies/ml) were retrospectively tested for viral mutations at specific times. Human experimentation guidelines of the US Department of Health and Human Services and/or those of the participating institutions were followed in the conduct of the trial (ClinicalTrials. gov NCT00431353). Genotypic resistance studies PCR amplification and DNA sequencing studies of CMV UL97 [10] and UL54 [9] genes were performed at a central laboratory on PCR-positive whole blood samples collected at days 0 (prior to treatment), 21 (end of induction therapy) and 49 (end of maintenance therapy). Additional samples were analysed in patients with relapsing CMV disease during the year after initial treatment and in those with confirmed CMV resistance mutations. All sequences were compared with those of reference strain AD169 (X17403) and clinical CMV isolates or recombinant viruses with confirmed ganciclovir resistance phenotypes [11 15]. On the basis of comparative sequence analyses, CMV mutations were classified into one of the following five categories: confirmed ganciclovir resistance mutation (previouslyreported mutations with phenotypes confirmed by marker transfer experiments), probable ganciclovir resistance mutation (a new mutation at a codon previously implicated in drug resistance), unknown mutation located in a catalytic domain or a conserved gene region, unknown mutations not located in a catalytic or conserved gene region, and known naturally occurring polymorphic mutations [14 16]. Viral load kinetics Early phase viral load kinetics were calculated by modelling a best fit curve for the early part of viral decay using the day 0, 3, and 7 viral loads from plasma measurements as previously described [17]. Based on an exponential decay curve with the equation y=y 0 e -ax, the viral load half-life can be calculated by the equation T 1/2 =(ln2)/a [17]. Statistical analyses Statistical analyses were performed using either the χ 2 test with odds ratio (OR) and 95% confidence interval (CI) estimates or the multivariate binomial logistic regression analysis. Median viral load half-lives were compared using the Mann Whitney U test. Results Description of ganciclovir resistance mutations The resistance substudy included 275 (86%) of the original 321 patients enrolled in the clinical trial. Patients were excluded if they had invalid viral load at time of CMV disease. There is no evidence that the clinical outcome was different in the excluded patients compared with evaluated patients when CMV disease activity was monitored by investigators at days 21 (P=0.38) and 49 (P=0.72). Among the 275 SOT recipients monitored for resistance (140 valganciclovir and 135 ganciclovir recipients), a total of 13 (4.7%) patients had confirmed or probable ganciclovir 698 2009 International Medical Press

CMV resistance in SOT recipients resistance mutations either at baseline or during the 49 days of treatment (Table 1). Ten patients had only UL97 mutations, two had only UL54 mutations, and one had both a UL97 and a UL54 mutation. Of note, 5/13 (38%) patients had confirmed or probable ganciclovir resistance mutations present at day 0; they had received previous antiviral prophylaxis (n=4; 76 243 days of oral ganciclovir or valganciclovir) or pre-emptive therapy (n=1; 106 days of valganciclovir or intravenous ganciclovir). In many cases, the specific ganciclovir resistance mutations persisted for several weeks after treatment (Table 1). Overall, no significant difference in the incidence of CMV resistance mutations (either confirmed or probable) that appeared between days 0 and 49 was observed between the ganciclovir (2.3%) and valganciclovir (3.6%) arms of the study (Table 2). Risk factors for development of ganciclovir resistance mutations As shown in Table 3, there was no statistical evidence that the type of transplanted organ affected the probability of finding ganciclovir resistance mutations. However, there was a trend towards a higher incidence of confirmed or probable resistance mutations (17.6%) in the small number of lung transplant recipients. Ganciclovir resistance mutations were found more frequently in patients with the CMV serostatus combination of donor-positive/recipient-negative (D + /R - ) at the time of last transplant than in other serogroups (P=0.015; Table 3). The probability of finding ganciclovir resistance mutations increased substantially with time from last transplant as shown in Figure 1. Association between viral load and ganciclovir resistance mutations The effect of baseline viral load (using dichotomized values of >10,000 or 10,000 copies/ml) on the subsequent risk of exhibiting ganciclovir resistance mutations was analysed after excluding patients with day 0 mutations. There was a trend between high baseline viral load (>10,000 copies/ml) and subsequent emergence of confirmed or probable resistance mutations (P=0.06) by day 49. Persistent viraemia at day 21 (OR 11.83, 95% CI 1.41 97.75; P=0.022) but not at day 14 (OR 3.43, 95% CI 0.42 28.40; P=0.252) was associated with Table 1. Confirmed or probable cytomegalovirus mutations conferring ganciclovir resistance in solid organ transplant recipients First Last Day 0 viral load, Day 21 viral load, Patient Mutation Gene detection, days a detection, days a Antiviral copies/ml copies/ml 57 Lys599Glu b UL97 21 NA VGCV 110,500 5,450 82 Del596(mix) UL97 49 NA VGCV 260,500 3,390 83 Leu595Ser UL97 49 90 VGCV 86,000 16,000 358 Leu595Ser UL97 35 >90 GCV 308,000 30,800 370 Lys599Glu b UL97 21 21 VGCV 246,000 3,480 371 Leu595Ser UL97 0 >90 GCV 1,145 910 390 Cys603Trp(mix) UL97 0 NA VGCV 750,000 NA 523 Met460Val UL97 0 >42 GCV 35,900 10,450 526 Cys603Trp UL97 0 >180 GCV 204,000 4,400 601 Cys603Trp UL97 21 49 VGCV 54,000 1,880 524 Cys603Trp UL97 0 >49 VGCV 5,300 1,775 Ala987Gly UL54 0 >49 27 Gly841Ser(mix) b UL54 21 NA GCV 29,700 1,755 605 Pro522Ala(mix) UL54 21 21 GCV 3,125 360 a Days after treatment onset. b Probable mutation, that is, a different amino acid change at the same codon is associated with ganciclovir (GCV) resistance. mix, mixture of wild-type and mutated amino acid; NA, not available because of missing sample or information, or study exclusion; VGCV, valganciclovir. Table 2. Incidence of confirmed or probable ganciclovir resistance mutations according to the antiviral induction regimen Populations or patients evaluated Valganciclovir, n (%) Ganciclovir, n (%) P-value Monitored population 164 157 No data on resistance 24 22 Resistance population 140 135 Baseline resistance a 2 (1.4) 3 (2.2) 0.62 Induced resistance b 5 (3.6) 3 (2.3) 0.51 a Resistance mutations at day 0 (before induction treatment). b Resistance mutations that appeared between day 0 and day 49 (end of maintenance therapy). Antiviral Therapy 14.5 699

G Boivin et al. Table 3. Type of transplanted organ or serological status and incidence of ganciclovir resistance mutations Incidence of resistance mutations No evidence of confirmed or Evidence of confirmed or Variable probable mutations, n (%) probable mutations, n (%) P-value Transplanted organ 0.077 Heart 19 (95.0) 1 (5.0) Kidney 208 (96.3) 8 (3.7) Liver 22 (95.7) 1 (4.3) Lung 14 (82.4) 3 (17.6) Donor/recipient CMV serological status 0.015 a D + /R - 45 (86.5) 7 (13.5) All others 142 (96.6) 5 (3.4) There were 76 patients with incomplete serological data. a Odds ratio for incidence of resistance mutations was 4.42, and the 95% confidence interval for incidence of resistance mutations was 1.34 14.60. CMV, cytomegalovirus; D + /R -, donor-positive/recipient-negative. ganciclovir resistance. Also, the presence of a confirmed or probable resistance mutation significantly increased the risk of virological failure at day 21 (OR 11.04 95% CI 1.38 88.40; P=0.024) and at day 49 (OR 6.82, 95% CI 2.14 21.72; P=0.001; Table 4). A multivariate logistic regression analysis was performed to identify the significant predictors of virological failure (viral load 600 copies/ml) at days 21 and 49. The model included variables shown in Table 5. The only significant predictors of virological failure at day 21 were: presence of baseline viral load >10,000 copies/ml (OR 23.95), presence of confirmed or probable resistance mutations (OR 32.47) and absence of anti-cmv immunoglobulin G antibodies at baseline (OR 3.35). Only the presence of confirmed or probable resistance mutations (OR 8.50) and baseline viral load >10,000 copies/ml (OR 5.89), were associated with virological failure at day 49. Clinical outcome of patients with ganciclovir resistance mutations The 13 patients with confirmed or probable ganciclovir resistance CMV mutations included 8 kidneys, 3 lungs, 1 heart and 1 liver transplant recipients. Two-thirds of these patients had a D + /R CMV serostatus at the time of transplant. Their mean baseline CMV load was 161,090 copies/ml (range 1,145 750,000) and 69.2% (9/13) had received previous anti-cmv therapy prior to the study including antiviral prophylaxis (n=2), preemptive therapy (n=2) and prophylaxis plus a prior course of treatment (n=5). Day 0 ganciclovir-resistant UL97 strains had a significantly longer plasma half-life than all other categories of UL97 mutants or wild-type virus (P=0.045; Table 6). The Met460Val and Leu595Ser mutants had the longest half-lives (>49 days), whereas Lys599Glu had the lowest (<5 days) and Cys603Trp had intermediate values (7 30 days). Three (23.1%) patients with resistance mutations discontinued treatment prematurely before day 49: one for a gastrointestinal problem (day 28), one for Figure 1. Rates of ganciclovir resistance mutations by time of cytomegalovirus disease after transplantation Patients, % 15 10 Monitored patients, n Confirmed or probable mutations, n 5 0 2.6% 60 116 3 2.6% 7.3% 61 120 121 180 Days from last transplant 12.5% 181 Rates of patients with confirmed or probable cytomegalovirus mutations are shown according to time (days) from last transplant. refractory anaemia (day 3) and one for leukopaenia (day 33). On day 21, 11/12 (91.7%) patients with available data were deemed to have clinical success defined as resolution of CMV disease by local investigators (versus 16.7% of virological success) whereas 10/10 (100%) patients with available data on day 49 achieved clinical success (versus 55.6% of virological success). Three (33.3%) of the nine patients with extended follow-up (including two CMV-related deaths) required additional anti-cmv treatment for recurrent CMV disease: one received intravenous ganciclovir for 16 days, one received valganciclovir for 172 days and foscarnet for 63 days, and data were 78 2 41 3 40 5 700 2009 International Medical Press

CMV resistance in SOT recipients Table 4. Rates of cytomegalovirus viral load eradication according to the presence or absence of ganciclovir resistance mutations Rates of CMV eradication No evidence of confirmed or Evidence of confirmed or Eradication outcome probable mutations, n (%) probable mutations, n (%) OR (95% CI) P-value Day 21 eradication 11.04 (1.38 88.40) 0.024 Success a 146 (55.1) 1 (10) Failure b 119 (44.9) 9 (90) Day 49 eradication 6.82 (2.14 21.72) 0.001 Success a 213 (81.0) 5 (38.5) Failure b 50 (19.0) 8 (61.5) a Patients who had an available sample with viral load at day 21 or day 49 below the limit of quantification (<600 copies/ml) and who had a valid viral load ( 600 copies/ml) at baseline were classified as success. b Missing samples, patients without confirmed baseline viral load 600 copies/ml and those with viral load at day 21 or day 49 >600 copies/ml were classified as failure. CI, confidence interval; CMV, cytomegalovirus; OR, odds ratio. Table 5. Probability of virological failure at days 21 and 49 from multivariate logistic regression analysis Day 21 Day 49 Explanatory factor P-value OR (95% CI) P-value OR (95% CI) Baseline viral load (>10,000 copies/ml) <0.001 23.95 (9.98 57.49) 0.001 5.89 (2.04 16.95) Confirmed or probable resistance 0.05 32.47 (2.94 358.87) 0.001 8.50 (2.28 31.65) Negative CMV IgG at time of CMV disease 0.021 3.35 (1.20 9.36) 0.815 1.15 (0.35 3.76) Transplanted organ 0.147 0.087 Sex 0.159 1.65 (0.82 3.31) 0.134 1.86 (0.83 4.21) Age group a 0.441 0.965 Treatment b 0.451 0.78 (0.40 1.50) 0.266 0.64 (0.29 1.41) Diagnosis c 0.893 0.96 (0.49 1.88) 0.398 1.40 (0.64 3.08) For multilevel predictors, no odds ratio (OR) is reported unless statistically significant. a Age groups were 40, 41 55 and >55 years. b Valganciclovir versus ganciclovir. c Syndrome versus disease. CI, confidence interval; CMV, cytomegalovirus; IgG, immunoglobulin G. incomplete for the third patient. Two lung transplant recipients with CMV resistance mutations died during the follow-up period. The first patient with dual UL97 (Cys603Trp) and UL54 (Ala987Gly) mutations detected on days 1, 21 and 49 died on day 293 of respiratory failure and graft loss. The second patient with a single UL97 mutation (Cys603Trp) detected from day 0 to 180 died of recurrent CMV disease on day 328. Notably, the latter two patients had persistent viral load on last evaluation (day 180), that is, 9,250 and 1,120 copies/ml, respectively. Of note, the two patients with UL54 mutations alone had negative viral load on day 49 with no subsequent relapse of CMV disease. Mutations outside known resistance loci At day 0, any CMV UL97 mutations were found in 48/255 (18.8%) strains when compared with the wildtype strain (AD169; Table 6). The median number of mutations was 0 (range 0 2) for UL97. For UL97, 12/255 (4.7%) patients had mutations of unknown significance (non-catalytic site) and 31/255 (12.2%) had mutations that were known natural polymorphisms. Median baseline viral load in patients with unknown mutations was 13,600 copies/ml versus 23,450 copies/ml in patients with wild-type virus (P=0.11). Analysis of clearance kinetics demonstrated similar viral load half-lives in each group compared with wild-type virus (Table 6). For UL54, mutations were extremely common, compared with wildtype, being present in 244/255 (95.7%) of patients and including unknown mutations in catalytic (n=9; 3.5%) and non-catalytic (n=56; 21.9%) regions and known natural polymorphisms (n=178; 69.8%). The median number of mutations per patient was 4 (range 0 9). Again, baseline viral loads were similar across all categories (data not shown). Analysis of plasma half-lives demonstrated no significant effect of these mutations compared with wild-type virus (Table 6). We also performed a revised analysis by considering variants present in >33% of patients as being wild-type strains. Again, the virtually identical half-lives shown in Table 6 indicate that natural polymorphisms as well as mutations of unknown significance had neither a negative or positive effect on viral fitness parameters. Also, increasing the number of UL54 polymorphic mutations had no effect on viral fitness (for example, plasma half-life in patients with 7 polymorphic mutations was 6.7 days compared with 7.8 days for wild-type virus; P-value non-significant). Antiviral Therapy 14.5 701

G Boivin et al. Table 6. Median half-lives of cytomegalovirus strains at baseline Gene Category of mutations Number of strains Median plasma half-life, days UL97 Confirmed resistance 5 30.14 a Unknown significance (catalytic) 0 NA Unknown significance (non-catalytic) 12 4.20 Known polymorphism 31 6.65 Wild-type (AD169) sequence 207 5.27 UL54 Confirmed resistance 1 20.27 Unknown significance (catalytic) 9 7.76 Unknown significance (non-catalytic) 56 5.24 Known polymorphism 178 5.38 Wild-type (AD169) sequence 11 7.75 Revised polymorphism b 96 5.93 Revised wild type sequence b 93 5.14 a P=0.045 for confirmed UL97 resistance versus other UL97 mutations. b Revised classification, that is, mutations found in 33% of viral strains were considered wild-type sequences. NA, not applicable. Discussion In this large trial, we show that ganciclovir resistance rates are similar in SOT recipients with CMV disease initially treated with either oral valganciclovir or intravenous ganciclovir. Drug resistance mutations have a major effect on viral load eradication as well as on early phase kinetic parameters (resulting in very long calculated half-lives), but they are not always associated with unfavourable clinical outcomes. Finally, we report for the first time that a high degree of polymorphism is present mainly in the CMV DNA polymerase (UL54) gene but this has neither a negative or positive effect on in vivo viral fitness. The rate of ganciclovir resistance among SOT recipients has not been precisely defined and appears to vary according to the specific population studied, the immunosuppressive agents, the antiviral regimen (that is, prophylaxis, pre-emptive therapy or treatment) and the method to define resistance (that is, phenotypic versus genotypic assays) [8]. Limaye [7] identified the following risk factors for ganciclovir-resistant CMV strains after SOT: D + /R - status, prolonged drug exposure, potent immunosuppression, suboptimal ganciclovir levels, high viral load and specific populations, such as kidney-pancreas or lung transplant recipients. Rates of ganciclovir-resistant CMV mutations have varied from 0% in CMV-seropositive kidney transplant patients to 27% in D + /R - lung transplant recipients [2,7,8,18 20]. However, previous observational reports might have overestimated the risk of ganciclovir resistance, as clinicians usually request resistance analyses in case of clinical and/or virological failures. Our large systematic study describes a more precise risk estimate of developing drug resistance using a molecular approach over a single treatment course; such risk was lowest in kidneys (3.7%), intermediate in livers and hearts (4.3 5.0%) and highest in lungs (17.6%). We also confirm an important bidirectional relationship between drug resistance mutations and viral load [21,22]. First, a high viral load before treatment and at day 21 was associated with ganciclovir resistance mutations. Second, the presence of resistance mutations was an important factor (along with the baseline viral load) associated with virological failure at both days 21 and 49. Similar to another study [2], we report an increased rate of ganciclovir resistance in patients who developed late onset CMV disease. There was a stepwise increase in the rate of confirmed resistance mutations from 2.6% (CMV disease within the first 2 months after transplantation) to 12.5% (disease occurring after 6 months). A novel aspect of this study was the extensive analysis of the in vivo effect of mutations outside known resistance loci. We demonstrate that numerous unknown mutations and polymorphisms commonly occur especially in the UL54 gene. In confirmation of some in vitro marker transfer experiments, such mutations (as a group) do not impart in vivo ganciclovir resistance. In addition, on the basis of the analysis of viral kinetics in these patients, such mutations or polymorphisms do not confer any in vivo evidence of negative fitness. This is true even in patients with high numbers of mutations ( 7) in the UL54 gene. A full understanding of the clinical effect of mutations and naturally occurring polymorphisms is important to understand the evolution of ganciclovir resistance in patients with CMV infections. Valganciclovir has been shown to be non-inferior to intravenous ganciclovir for treatment of CMV disease in both AIDS patients [23] and SOT recipients [6]. Here, we report that a 21-day induction course with either drug was associated with similar rates of confirmed or probable resistance mutations over our study period (49 days; that is, 3.6% and 2.3% for valganciclovir 702 2009 International Medical Press

CMV resistance in SOT recipients and intravenous ganciclovir, respectively) if we exclude mutations already present at baseline, which probably reflect previous antiviral prophylaxis. It must be noted, however, that the clinical study was not primarily designed to explore resistance, although this was a predefined secondary endpoint. Comparable rates of resistance for these two drugs have been reported by our group in AIDS patients with CMV retinitis [10]. Most ganciclovir-resistant CMV strains detected in our study contained a mutation in the viral UL97 gene, although occasional viruses (15.3%) harboured a mutation in the UL54 gene only. Notably, only one patient had a CMV strain containing both types of CMV mutations. The presence of dual UL97 and UL54 mutations has been associated with higher levels of phenotypic ganciclovir resistance [24,25]. In addition, some UL54 mutations can confer resistance to other antiviral agents. In particular, the Pro522Ala mutation, found in one of our patients, has been associated with additional resistance to cidofovir whereas another UL54 mutation, Gly841Arg (Gly841Ser in our patient), confers additional resistance to both foscarnet and cidofovir [8,26]. There is considerable evidence that ganciclovirresistant strains can be fully pathogenic [2,7,27 29]. In a review of 13 SOT recipients with ganciclovirresistant CMV infections, Isada et al. [28] reported a high rate of tissue-invasive CMV disease (85%) and mortality (69%) caused by CMV or its complications. However, not all clinical failures with ganciclovir can be attributed to drug resistance and, conversely, clinical improvement can occur despite ganciclovir resistance [9,18,30,31]. Our group previously reported an adverse clinical outcome in lung but not in other transplant patients carrying viruses with known ganciclovir resistance mutations [31]. The clinical effect of ganciclovir-resistant CMV strains was not systematically investigated in our trial because of limited follow-up after the end of therapy (day 49). However, it is noteworthy that only 3 of 9 (33%) evaluable patients with established ganciclovir resistance mutations received additional anti-cmv therapy beyond day 49 for relapsing CMV disease. Two of the latter patients died of CMV disease or graft failure; both cases were lung transplants with persistent viral load over several months [17] and one of the two had dual UL97/UL54 mutations. Thus, pathogenicity of ganciclovir-resistant CMV strains can vary according to numerous factors, such as the fitness of the viral strain [22], the immunosuppressive state of the recipient and host factors such as Toll-like receptor polymorphisms [32]. Our resistance study has some limitations. First, we did not generate recombinant CMV mutants to assess the role of unknown mutations and were unable to correlate the levels of ganciclovir resistance with virological and clinical endpoints. Also, the absence of clinical endpoints in our trial and the relatively short follow-up period limit our understanding of the long-term consequences of ganciclovir-resistant CMV infections. However, the strengths of our study are its prospective design where resistance was a predefined secondary endpoint, the analysis of a large number of patients with diverse transplanted organs and the complementary in vivo study of viral fitness. In conclusion, although the incidence of ganciclovirresistant CMV strains is low in SOT patients who receive either valganciclovir or intravenous ganciclovir for treatment of CMV disease, the effect of drug-resistant viruses appears important when using virological endpoints for assessing treatment success. By contrast, the relationship between drug resistance and clinical outcome is less straightforward; that is, not all patients with drug-resistant viruses experience recurrent or lifethreatening CMV diseases. In clinical practice, a high viral load at baseline could require a more prolonged induction treatment to achieve complete viral eradication before maintenance whereas a persistently high viral load beyond day 21 of treatment might necessitate genotypic susceptibility studies and/or alternate therapy. Numerous mutations might be present outside of known resistance determinants, but they do not generally influence in vivo viral fitness. Acknowledgements This study has been supported financially by F Hoffmann La Roche. Results of this study have been presented in part at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, 17 20 September 2007, Chicago, IL, USA. The authors would like to thank Angelo Bignamini from Hyperphar Research (Milan, Italy) for statistical analyses as well as the other clinical investigators of the Victor study group. Disclosure statement JI is an employee of F Hoffmann La Roche. GB, AH, AGJ, MDP, AA, HR and AH have performed consultancy work for F Hoffmann La Roche. All other authors have no financial disclosure. References 1. Fishman JA, Rubin RH. Infection in organ-transplant recipients. N Engl J Med 1998; 338:1741 1751. 2. Limaye AP, Corey L, Koelle DM, Davis CL, Boeckh M. Emergence of ganciclovir-resistant cytomegalovirus disease among recipients of solid-organ transplants. Lancet 2000; 356:645 649. 3. Preiksaitis JK, Brennan DC, Fishman J, Allen U. Canadian society of transplantation consensus workshop on cytomegalovirus management in solid organ transplantation final report. Am J Transplant 2005; 5:218 227. Antiviral Therapy 14.5 703

G Boivin et al. 4. Pescovitz MD, Rabkin J, Merion RM, et al. Valganciclovir results in improved oral absorption of ganciclovir in liver transplant recipients. Antimicrob Agents Chemother 2000; 44:2811 2815. 5. Wiltshire H, Hirankarn S, Farrell C, et al. Pharmacokinetic profile of ganciclovir after its oral administration from its prodrug, valganciclovir, in solid organ transplant recipients. Clin Pharmacokinet 2005; 44:495 507. 6. Asberg A, Humar A, Rollag H, et al. Oral valganciclovir is noninferior to intravenous ganciclovir for the treatment of cytomegalovirus disease in solid organ transplant recipients. Am J Transplant 2007; 7:2106 2113. 7. Limaye AP. Ganciclovir-resistant cytomegalovirus in organ transplant recipients. Clin Infect Dis 2002; 35:866 872. 8. Gilbert C, Boivin G. Human cytomegalovirus resistance to antiviral drugs. Antimicrob Agents Chemother 2005; 49:873 883. 9. Boivin G, Goyette N, Gilbert C, et al. Absence of cytomegalovirus-resistance mutations after valganciclovir prophylaxis, in a prospective multicentre study of solid-organ transplant recipients. J Infect Dis 2004; 189:1615 1618. 10. Boivin G, Gilbert C, Gaudreau A, Greenfield I, Sudlow R, Roberts NA. Rate of emergence of cytomegalovirus (CMV) mutations in leukocytes of patients with acquired immunodeficiency syndrome who are receiving valganciclovir as induction and maintenance therapy for CMV retinitis. J Infect Dis 2001; 184:1598 1602. 11. Martin M, Gilbert C, Covington E, Boivin G. Characterization of human cytomegalovirus (HCMV) UL97 mutations found in a valganciclovir/oral ganciclovir prophylactic trial by use of a bacterial artificial chromosome containing the HCMV genome. J Infect Dis 2006; 194:579 583. 12. Gilbert C, Bestman-Smith J, Boivin G. Resistance of herpesviruses to antiviral drugs: clinical impacts and molecular mechanisms. Drug Resist Updat 2002; 5:88 114. 13. Chou S, Lurain NS, Thompson KD, Miner RC, Drew WL. Viral DNA polymerase mutations associated with drug resistance in human cytomegalovirus. J Infect Dis 2003; 188:32 39. 14. Chou S, Lurain NS, Weinberg A, Cai GY, Sharma PL, Crumpacker CS. Interstrain variation in the human cytomegalovirus DNA polymerase sequence and its effect on genotypic diagnosis of antiviral drug resistance. Antimicrob Agents Chemother 1999; 43:1500 1502. 15. Chou S, Waldemer RH, Senters AE, et al. Cytomegalovirus UL97 phosphotransferase mutations that affect susceptibility to ganciclovir. J Infect Dis 2002; 185:162 169. 16. Fillet AM, Auray L, Alain S, et al. Natural polymorphism of cytomegalovirus DNA polymerase lies in two nonconserved regions located between domains delta-c and II and between domains III and I. Antimicrob Agents Chemother 2004; 48:1865 1868. 17. Humar A, Kumar D, Boivin G, Caliendo AM. Cytomegalovirus (CMV) virus load kinetics to predict recurrent disease in solid-organ transplant patients with CMV disease. J Infect Dis 2002; 186:829 833. 18. Limaye AP, Raghu G, Koelle DM, Ferrenberg J, Huang ML, Boeckh M. High incidence of ganciclovir-resistant cytomegalovirus infection among lung transplant recipients receiving preemptive therapy. J Infect Dis 2002; 185:20 27. 19. Lurain NS, Bhorade SM, Pursell KJ, et al. Analysis and characterization of antiviral drug-resistant cytomegalovirus isolates from solid organ transplant recipients. J Infect Dis 2002; 186:760 768. 20. Bhorade SM, Lurain NS, Jordan A, et al. Emergence of ganciclovir-resistant cytomegalovirus in lung transplant recipients. J Heart Lung Transplant 2002; 21:1274 1282. 21. Humar A, Kumar D, Preiksaitis J, et al. A trial of valganciclovir prophylaxis for cytomegalovirus prevention in lung transplant recipients. Am J Transplant 2005; 5:1462 1468. 22. Emery VC, Griffiths PD. Prediction of cytomegalovirus load and resistance patterns after antiviral chemotherapy. Proc Natl Acad Sci U S A 2000; 97:8039 8044. 23. Martin DF, Sierra-Madero J, Walmsley S, et al. A controlled trial of valganciclovir as induction therapy for cytomegalovirus retinitis. N Engl J Med 2002; 346:1119 1126. 24. Erice A, Gil-Roda C, Perez JL, et al. Antiviral susceptibilities and analysis of UL97 and DNA polymerase sequences of clinical cytomegalovirus isolates from immunocompromised patients. J Infect Dis 1997; 175:1087 1092. 25. Smith IL, Cherrington JM, Jiles RE, Fuller MD, Freeman WR, Spector SA. High-level resistance of cytomegalovirus to ganciclovir is associated with alterations in both the UL97 and DNA polymerase genes. J Infect Dis 1997; 176:69 77. 26. Chou S, Marousek GI, Van Wechel LC, Li S, Weinberg A. Growth and drug resistance phenotypes resulting from cytomegalovirus DNA polymerase region III mutations observed in clinical specimens. Antimicrob Agents Chemother 2007; 51:4160 4162. 27. Boivin G, Chou S, Quirk MR, Erice A, Jordan MC. Detection of ganciclovir resistance mutations and quantitation of cytomegalovirus (CMV) DNA in leukocytes of patients with fatal disseminated CMV disease. J Infect Dis 1996; 173:523 528. 28. Isada CM, Yen-Lieberman B, Lurain NS, et al. Clinical characteristics of 13 solid organ transplant recipients with ganciclovir-resistant cytomegalovirus infection. Transpl Infect Dis 2002; 4:189 194. 29. Eckle T, Prix L, Jahn G, et al. Drug-resistant human cytomegalovirus infection in children after allogeneic stem cell transplantation may have different clinical outcomes. Blood 2000; 96:3286 3289. 30. Chmiel C, Speich R, Hofer M, et al. Ganciclovir/ valganciclovir prophylaxis decreases cytomegalovirusrelated events and bronchiolitis obliterans syndrome after lung transplantation. Clin Infect Dis 2008; 46:831 839. 31. Boivin G, Goyette N, Gilbert C, Humar A, Covington E. Clinical impact of ganciclovir-resistant cytomegalovirus infections in solid organ transplant patients. Transpl Infect Dis 2005; 7:166 170. 32. Kijpittayarit S, Eid AJ, Brown RA, Paya CV, Razonable RR. Relationship between Toll-like receptor 2 polymorphism and cytomegalovirus disease after liver transplantation. Clin Infect Dis 2007; 44:1315 1320. Accepted for publication 7 April 2009 704 2009 International Medical Press