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Incidence and characterization of cytomegalovirus resistance mutations among pediatric solid organ transplant patients who received valganciclovir prophylaxis
Journal of Clinical Virology 47 (2010) 321–324
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Incidence and characterization of cytomegalovirus resistance mutations among pediatric solid organ transplant patients who received valganciclovir prophylaxis Mélanie Martin a , Nathalie Goyette a , Jane Ives b , Guy Boivin a,∗ a Research Center in Infectious Diseases, Centre Hospitalier Universitaire de Québec and Laval University, 2705 Blvd Laurier, Room RC-709, Quebec City, Quebec G1V 4G2, Canada b Roche Products, Hexagon Place, 6 Falcon Way, Welwyn Garden City, Hertfordshire AL7 1TW, United Kingdom
a r t i c l e
i n f o
Article history: Received 25 September 2009 Received in revised form 12 January 2010 Accepted 15 January 2010 Keywords: Human cytomegalovirus Ganciclovir Resistance UL97 gene Pediatric Solid organ transplant recipient
a b s t r a c t Background: Drug-resistant cytomegalovirus (CMV) infections can cause significant morbidity among high-risk transplant recipients. Objectives: The aims of this study were to determine the incidence and clinical consequences of CMV mutations conferring ganciclovir resistance in pediatric solid organ transplant (SOT) patients who received valganciclovir oral solution or tablets for prophylaxis of CMV disease. Recombinant CMV mutants were also generated to assess the role of two UL97 mutations of unknown significance. Study design: Genotypic resistance mutations and CMV viral load were sought in blood samples from pediatric SOT recipients who received valganciclovir prophylaxis for 100 days. Recombinant viruses containing novel CMV UL97 mutations were generated using a bacterial artificial chromosome containing the CMV genome to assess ganciclovir susceptibility. Results: Overall, four known resistance UL97 mutations were observed in blood samples from 2 of 46 patients during the study with no development of CMV disease. Two UL97 changes (M615V and V466G) of unknown significance and one UL97 mutation (C603R) associated with ganciclovir resistance, but not yet confirmed by marker transfer, were also detected. Recombinant viruses containing these novel mutations were generated to assess ganciclovir susceptibility. The M615V recombinant virus was susceptible to ganciclovir while the V466G and C603R mutant viruses displayed 3.5-fold and 3.6-fold decreases in susceptibility, respectively. Conclusions: The low incidence of ganciclovir resistance-associated mutations and the absence of clinical consequences associated with drug-resistant viruses observed in this pilot study should encourage the design of larger clinical trials aimed at evaluating the efficacy of valganciclovir prophylaxis and treatment in the pediatric setting. © 2010 Elsevier B.V. All rights reserved.
1. Background Human cytomegalovirus (CMV) causes significant morbidity among high-risk transplant recipients. Ganciclovir, a deoxyguanosine analogue, has been the antiviral of choice for prevention and treatment of CMV disease for two decades. The l-valyl ester prodrug of ganciclovir (valganciclovir) was subsequently developed and shown to have a bioavailability of ∼60%.1 A daily dose of valganciclovir was as clinically effective and well-tolerated as a thrice daily dose of oral ganciclovir for prevention of CMV disease in high-
risk adult solid organ transplant (SOT) recipients.2 Furthermore, valganciclovir was shown to have comparable safety, time of eradication of viremia and clinical efficacy as intravenous ganciclovir for treatment of CMV disease in adult SOT patients.3 However, concerns remain about the emergence of drug-resistant CMV strains among immunocompromised patients receiving antiviral prophylaxis and treatment.4–7 The mechanisms of resistance to ganciclovir imply predominantly mutations in the CMV UL97 protein kinase gene with more occasional alterations in the UL54 DNA polymerase gene.8
2. Objectives Abbreviations: CMV, cytomegalovirus; SOT, solid organ transplant; WT, wildtype; D+/R−, donor CMV seropositive/recipient CMV seronegative; i.v., intravenous; IC50 , inhibitory concentration 50%; Neg, negative; Pos, positive; N/a, not applicable; Blq, below limit of quantification. ∗ Corresponding author. Tel.: +1 418 654 2705; fax: +1 418 654 2715. E-mail address: [email protected] (G. Boivin). 1386-6532/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2010.01.009
The aims of this study were to determine the incidence and clinical consequences of CMV mutations conferring ganciclovir resistance in pediatric SOT patients who received valganciclovir oral solution or tablets for prophylaxis of CMV disease.9 We also
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Table 1 Incidence of CMV mutations at the end of study prophylaxis (Day 100) and during follow-up until 6 months after transplantation (Week 26) among pediatric SOT recipients. At the end of the study prophylaxis (Day 100)a
During follow-up until 6 months after transplantation (Week 26)a
No. of patients with samples available for testing (no. of samples) No. of patients with CMV PCR positive samples (no. of samples) No. of patients with known resistance mutations (% of tested samples)
46 (46) 1 (1) 1 (2.2)
9 (35) 3 (5) 2 (5.7)
Genotype Known UL97 resistance mutations Novel UL97 mutations Natural UL97 polymorphisms Known UL54 resistance mutations Novel UL54 mutations Natural UL54 polymorphisms
M460V, C592G, L595F V466G – – – N685S, A885T, N898D
M460V, C592G, L595F, C603Rb M615V – – – S676G, A885T, S897L, N898D
a b
Compared to sequence of the AD169 strain (GenBank accession number X17403). Another mutation at the same codon (C603W) has been confirmed to confer ganciclovir resistance.
generated recombinant CMV mutants to assess the role of two UL97 mutations of unknown significance. 3. Study design 3.1. Patients Clinical trial WV16726 was a single arm, non-comparative multicenter study (18 centers in 7 countries) of the safety and pharmacokinetics of valganciclovir oral solution in the prevention of CMV disease in pediatric SOT recipients. Patients (38% donor CMV seropositive/recipient CMV seronegative or D+/R−) with a heart (n = 12), kidney (n = 33), liver (n = 17) or kidney and liver (n = 1) allograft received valganciclovir oral solution or tablets once daily at a dose determined by an algorithm.9 3.2. Genotypic analysis of resistance Blood samples were routinely obtained at the end of prophylaxis (Day 100) and during suspicion of CMV disease up to 6 months after transplantation. In some centers, blood samples were also collected for resistance testing at the time of a routine post-transplant biopsy. DNA was extracted from whole blood samples using the MagNA Pure LC system (Roche Diagnostics). For UL97 genotyping, the region including codons 363–698 was amplified by a semi-nested PCR protocol.6 In addition, a region of the UL54 gene encompassing codons 184–1017 was amplified by a nested PCR protocol.10 An automated method with a chromatogram readout (ABI 3720xl) was used as a sequencing method for three independent PCR products of the UL97 and UL54 genes. 3.3. CMV viral load Five microliters of extracted nucleic acid from whole blood samples were added to 15 L of a reaction mixture containing 1 mmol/L of MgCl2 , 0.4 mol/L of each primer, 0.3 mol/L of fluorescein hybridization probe (Tib Molbiol), 0.6 mol/L of LC Red 640 probe (Tib Molbiol), 5% dimethyl sulfoxide and 2 L of LightCyclerFaststart DNA Master hybridization probes kit (Roche Diagnostics). Amplification primers for the CMV glycoprotein B gene were as previously reported.11 The hybridization probe sequences were as follows: TTA AAG GTG TGC GCC ACG ATG TTG CG-fluorescein and LC Red 640-TTG TAG ACC ACC ATG ATG CCC T-phosphate. Viral loads were normalized for human beta globin gene using the LightCycler Control Kit DNA (Roche Diagnostics). 3.4. Generation of recombinant CMV mutants Mutagenesis of the CMV/BAC plasmid (pHB5, a gift from Dr M. Messerle, Germany) in Escherichia coli was performed by using a
two-step recombination protocol.12 For each recombinant virus generated, the UL97 and UL54 genes and the homologous regions where recombination events occurred were sequenced to confirm the appropriate genotype. Susceptibility testing of the recombinant wild-type (WT) and mutant viruses was performed using a standardized plaque reduction assay.13 Recombinant viruses were considered to be resistant to ganciclovir if their IC50 value was at least 3 times greater than that of the WT recombinant virus.12 4. Results 4.1. Genotypic analysis of resistance At the end of prophylaxis (Day 100), samples from 46 of 63 SOT patients (aged 4 months to 16 years) were available for ganciclovir resistance testing. 17 out of 46 patients (37%) were high-risk (D+/R−) recipients. Only 1 tested sample was CMV positive by PCR. Seven amino acid changes relative to the comparator sequence were observed within the CMV UL97 and UL54 genes in this sample (Table 1). Of these, three were known UL97 mutations conferring ganciclovir resistance (M460V, C592G and L595F),8 one was a UL97 mutation of unknown significance (V466G) and three were known UL54 natural polymorphisms (N685S, A885T and N898D).14 Overall, the incidence of ganciclovir resistance mutations at the end of study prophylaxis was 2.2% (1/46). Thirty-five samples from 9 patients were collected after prophylaxis up to 6 months post-transplant. Only 7 of these samples were from patients with suspected CMV disease whereas the other 28 were collected during routine post-transplant biopsy or at the end of follow-up (Week 26). Of these, 5 samples from 3 patients were CMV positive by PCR (including the patient described above). A total of nine amino acid changes were observed in these samples (Table 1). The same three UL97 mutations conferring ganciclovir resistance were detected at the end of the study follow-up (Week 26) from the previous patient with UL97 mutations at Day 100. An additional resistance-associated UL97 mutation, C603R, was observed in 1 sample from a second patient at Week 20. One novel UL97 amino acid change of unknown significance, M615V, was observed in 1 sample from a third patient at Week 26. Four known UL54 natural polymorphisms (S676G, A885T, S897L and N898D)14 were detected in 1 patient at Week 26. 4.2. Clinical impact of CMV UL97 mutations Kinetics of emergence of CMV UL97 mutations and corresponding CMV viral loads are presented in Table 2. Although Patient A (a 3-year-old liver D+/R− transplant recipient) was asymptomatic for CMV throughout the study, samples for genotyping were nevertheless collected at the end of prophylaxis (Day 100) and at Weeks 2, 6,
M. Martin et al. / Journal of Clinical Virology 47 (2010) 321–324
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Table 2 Kinetics of emergence of known ganciclovir resistance and novel CMV UL97 mutations in three high-risk D+/R− patients. Patients (age and transplanted organ)
Time after transplantation
UL97 PCR result
UL97 resistance mutations
UL97 novel mutations
CMV, copies/104 blood cells
A (3-year-old liver transplant)
Week 2 Week 6 Week 10 Day 100 Week 16 Week 20 Week 26
Neg Neg Neg Neg Pos Pos Pos
n/a n/a n/a n/a No mutation C603R a No mutation
n/a n/a n/a n/a No mutation No mutation No mutation
n/a n/a n/a n/a 2.68 × 102 1.11 × 102 7.34 × 101
B (6-month-old heart transplant)
Day 100 Week 26
Pos Pos
M460V, C592G and L595Fa M460V, C592G and L595Fa
V466Ga No mutation
2.54 × 104 Blq
C (16-year-old heart transplant)
Day 100 Week 16 Week 20 Week 26
Neg Neg Neg Pos
n/a n/a n/a No mutation
n/a n/a n/a M615V
n/a n/a n/a 7.75 × 103
Note: Day 100, end of prophylaxis. Week 26, end of study follow-up. Neg, negative; Pos, positive; N/a, not applicable due to negative PCR result; Blq, below limit of quantification. a Mixed (mutant and wild-type) viral populations.
10, 16, 20 and 26. Notably, this patient was withdrawn from study treatment on Day 35 due to intestinal obstruction. The patient was found to be CMV positive on Day 86 which prompted the administration of intravenous (i.v.) ganciclovir for 10 days. The virus load was undetectable at Day 100, but raised to 2.68 × 102 , 1.11 × 102 and 7.34 × 101 CMV copies/104 blood cells at Weeks 16, 20 and 26, respectively. The UL97 resistance mutation C603R was observed at Week 20, but was not observed at Day 100 (immediately post-i.v. ganciclovir treatment) and had disappeared by Week 26. For Patient B (a 6-month-old heart D+/R− transplant recipient), CMV was initially detected on Day 44 post-transplant in a blood sample collected during a hospitalization for prolonged vomiting. The CMV positive result was not considered significant by the investigator and, therefore, no adjustment was made to the patient antiviral prophylaxis. The patient was asymptomatic for CMV disease at the time the three known GCV resistance mutations (M460V, C592G and L595F) and the novel V466G UL97 mutation were initially observed on Day 100. The patient remained asymptomatic until the end of follow-up (Week 26) despite persistence of CMV mutations. The Day 100 sample (viral load: 2.54 × 104 CMV copies/104 blood cells) also contained three known UL54 polymorphisms (N685S, A885T and N898D). The viral load was below the limit of quantification at Week 26. Although Patient C (a 16-year-old heart D+/R− transplant recipient) had been asymptomatic throughout the study, samples for genotyping were collected during routine visits on Day 100 and at Weeks 16, 20 and 26. The patient was found to be CMV positive during a routine heart biopsy on Day 173 (Week 25). He was then treated with valganciclovir, but the viremia remained unresolved (viral load: 7.75 × 103 CMV copies/104 blood cells) a week later at the end of study follow-up (Week 26). The novel CMV UL97 change M615V was observed at Week 26. In addition, four UL54 natural polymorphisms (S676G, A885T, S897L and N898D) were also detected in this sample. Clinical consequences of the novel UL97 change M615V are unknown because of absence of follow-up beyond Week 26.
4.3. Ganciclovir susceptibilities of recombinant mutant viruses Ganciclovir susceptibilities of the two novel UL97 changes and one suspected resistance-associated mutation not yet confirmed by marker transfer experiments were determined. Two distinct recombinant viruses per mutation were generated and each of these was tested in triplicate. The recombinant viruses containing the UL97 mutation M615V had a mean ganciclovir IC50 value comparable to that of the WT virus (i.e. 1.15-fold increase compared
to WT). Consequently, M615V can be considered a natural polymorphic variant. On the other hand, recombinant mutant viruses containing the UL97 mutation V466G exhibited a mean IC50 value of 6.32 mol/L while mutant C603R had a mean IC50 value of 6.48 mol/L, which were 3.5 and 3.6 times greater than that of the susceptible WT virus, respectively. The UL97 V466G and C603R mutants were thus considered to be resistant to ganciclovir.
5. Discussion We evaluated the incidence and clinical consequences of CMV resistance-associated mutations observed in pediatric SOT patients who received valganciclovir prophylaxis for 100 days. A challenge in evaluating the incidence of CMV resistance-associated mutations in prophylactic studies is the lack of a baseline comparator sequence that makes it impossible to differentiate novel resistance-associated mutations versus uncharacterized polymorphisms. Consequently, novel UL97 changes detected in this study were introduced into recombinant viruses and phenotypically characterized. The incidence of patients harboring CMV UL97 mutations conferring ganciclovir resistance was 2.2% (1/46) at the end of prophylaxis with an additional case detected during follow-up. In one case (Patient A), it remains unclear whether the resistanceassociated mutation developed during the shorter 35 days of prophylaxis or whether it developed during 10 days of ganciclovir treatment because the mutation was only observed transiently post-treatment. In the other case (Patient B), prolonged vomiting may have resulted in suboptimal ganciclovir exposure facilitating the development of resistance. The 2 patients who harbored confirmed ganciclovir resistance mutations did not develop CMV disease and had uneventful clinical outcomes. Overall, no patient developed CMV disease during this study. We previously reported a prospective study of high-risk adult SOT patients who received either oral ganciclovir or valganciclovir for CMV prophylaxis during 100 days in which no ganciclovir resistance mutations were observed in the valganciclovir arm.15 Similar results were observed in the present pediatric study with minimal breakthrough viremia and development of resistance during prophylaxis and subsequent follow-up. In adult SOT patients, mismatch (D+/R−) lung transplant recipients appear to have the highest risk of developing ganciclovirresistant CMV infections and to present the most important clinical complications.4,7,10 It is noteworthy that none of the 46 patients from our study received a lung transplant and that only 38% were high-risk (D+/R−) recipients which may explain the low level of resistance and low incidence of CMV disease observed. More-
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over, the immunosuppression regimens used in different types of pediatric SOT recipients varied among centers, which may have influenced clinical outcome of CMV infection. The absence of clinical CMV disease despite the presence of ganciclovir resistance mutations has been reported elsewhere.10 It is probable that the development of CMV disease is influenced by viral replicative capacities as well as by host and iatrogenic factors. The uneventful clinical courses associated with CMV resistance mutations could also potentially be explained by the fact that routine screening for mutations was performed in all patients at the end of prophylaxis. In clinical practice, resistance testing would not be indicated for patients showing no clinical signs suggestive of CMV disease or without sustained viremia. In this study, two novel CMV UL97 changes of unknown significance (V466G and M615V) were observed. The UL97 mutations V466M (instead of V466G)6 and M615V16 have been previously observed in patients with AIDS. These changes lie outside the documented hot-spot regions for ganciclovir resistance (codons 460, 520 and 590–607).8 While the mean IC50 value obtained for recombinant virus M615V was comparable to that of the WT virus, the V466G mutant had a 3.5-fold increase in IC50 value. Of note, a V466M mutant was susceptible to ganciclovir (unpublished). The C603R mutant, previously associated with ganciclovir resistance but not yet confirmed by marker transfer,17 had a 3.6-fold increase in IC50 value compared to the susceptible recombinant virus. Such decrease in susceptibility for the C603R mutant is smaller than what was observed for mutant C603W (8-fold increase in IC50 value),18 but comparable to that obtained for mutant M460I (3.9fold increase in IC50 value) that we generated using the same CMV/BAC system.12 Since CMV genotyping has essentially replaced CMV phenotype testing in the clinical setting, it is a concern that two out of six mutations found in the CMV UL97 gene were novel mutations. This supports the use of recombinant viruses to assess mutations of unknown significance12 as well as the development of a virtual database containing all published UL97 and UL54 mutations.19 In conclusion, the low incidence of ganciclovir resistanceassociated mutations and the absence of clinical consequences associated with drug-resistant viruses observed in this pilot study should encourage the design of larger clinical trials aimed at evaluating the efficacy of valganciclovir prophylaxis and treatment in the pediatric setting. Conflicts of interest J.I. is an employee of Roche. The other authors report no potential conflicts of interest. Acknowledgments This study was presented in part at the 48th Interscience Conference of Antimicrobial Agents and Chemotherapy on October 27th 2008 in Washington, DC, USA (abstract V-3561). This study was supported by research grants from Roche Products Ltd. and the Canadian Institutes of Health Research (CIHR; MOP-86583) to G.B.
G.B. is a national research scholar of “Le Fonds de Recherche en Santé du Québec” (FRSQ) and holds the Canada Research Chair on Emerging Viruses and Antiviral Resistance. M.M. has a Ph.D. scholarship from the FRSQ. References 1. Pescovitz MD, Rabkin J, Merion RM, Paya CV, Pirsch J, Freeman RB, et al. Valganciclovir results in improved oral absorption of ganciclovir in liver transplant recipients. Antimicrob Agents Chemother 2000;44:2811–5. 2. Paya C, Humar A, Dominguez E, Washburn K, Blumberg E, Alexander B, et al. Efficacy and safety of valganciclovir vs. oral ganciclovir for prevention of cytomegalovirus disease in solid organ transplant recipients. Am J Transplant 2004;4:611–20. 3. Asberg A, Humar A, Rollag H, Jardine AG, Mouas H, Pescovitz MD, et al. Oral valganciclovir is noninferior to intravenous ganciclovir for the treatment of cytomegalovirus disease in solid organ transplant recipients. Am J Transplant 2007;7:2106–13. 4. Limaye AP, Corey L, Koelle DM, Davis CL, Boeckh M. Emergence of ganciclovirresistant cytomegalovirus disease among recipients of solid-organ transplants. Lancet 2000;356:645–9. 5. Jabs DA, Enger C, Dunn JP, Forman M. Cytomegalovirus retinitis and viral resistance: ganciclovir resistance. J Infect Dis 1998;177:770–3. 6. 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;184:1598–602. 7. 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;185:20–7. 8. Gilbert C, Boivin G. Human cytomegalovirus resistance to antiviral drugs. Antimicrob Agents Chemother 2005;49:873–83. 9. Vaudry W, Ettenger R, Jara P, Varela-Fascinetto G, Bouw MR, Ives J, et al. Valganciclovir dosing according to body surface area and renal function in pediatric solid organ transplant recipients. Am J Transplant 2009;9:636–43. 10. Boivin G, Goyette N, Gilbert C, Humar A, Covington E. Clinical impact of ganciclovir-resistant cytomegalovirus infections in solid organ transplant patients. Transplant Infect Dis 2005;6:1–5. 11. Bai X, Hosler G, Rogers BB, Dawson DB, Scheuermann RH. Quantitative polymerase chain reaction for human herpesvirus diagnosis and measurement of Epstein-Barr virus burden in posttransplant lymphoproliferative disorder. Clin Chem 1997;43:1843–9. 12. 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;194:579–83. 13. Landry ML, Stanat S, Biron K, Brambilla D, Britt W, Jokela J, et al. A standardized plaque reduction assay for determination of drug susceptibilities of cytomegalovirus clinical isolates. Antimicrob Agents Chemother 2000;44:688–92. 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;43:1500–2. 15. Boivin G, Goyette N, Gilbert C, Roberts N, Macey K, Paya C, et al. Absence of cytomegalovirus-resistance mutations after valganciclovir prophylaxis, in a prospective multicenter study of solid-organ transplant recipients. J Infect Dis 2004;189:1615–8. 16. Foulongne V, Turriere C, Diafouka F, Abraham B, Lastere S, Segondy M. Ganciclovir resistance mutations in UL97 and UL54 genes of human cytomegalovirus isolates resistant to ganciclovir. Acta Virol 2004;48:51–5. 17. Lurain NS, Bhorade SM, Pursell KJ, Avery RK, Yeldandi VV, Isada CM, et al. Analysis and characterization of antiviral drug-resistant cytomegalovirus isolates from solid organ transplant recipients. J Infect Dis 2002;186:760–8. 18. Chou S, Marousek G, Guentzel S, Follansbee SE, Poscher ME, Lalezari JP, et al. Evolution of mutations conferring multidrug resistance during prophylaxis and therapy for cytomegalovirus disease. J Infect Dis 1997;176:786–9. 19. Chevillotte M, von Einem J, Meier BM, Lin FM, Kestler HA, Mertens T. A new tool linking human cytomegalovirus drug resistance mutations to resistance phenotypes. Antiviral Res 2009;(October) [Epub ahead of print].
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Incidence and characterization of cytomegalovirus resistance mutations among pediatric solid organ transplant patients who received valganciclovir prophylaxis Mélanie Martin a , Nathalie Goyette a , Jane Ives b , Guy Boivin a,∗ a Research Center in Infectious Diseases, Centre Hospitalier Universitaire de Québec and Laval University, 2705 Blvd Laurier, Room RC-709, Quebec City, Quebec G1V 4G2, Canada b Roche Products, Hexagon Place, 6 Falcon Way, Welwyn Garden City, Hertfordshire AL7 1TW, United Kingdom
a r t i c l e
i n f o
Article history: Received 25 September 2009 Received in revised form 12 January 2010 Accepted 15 January 2010 Keywords: Human cytomegalovirus Ganciclovir Resistance UL97 gene Pediatric Solid organ transplant recipient
a b s t r a c t Background: Drug-resistant cytomegalovirus (CMV) infections can cause significant morbidity among high-risk transplant recipients. Objectives: The aims of this study were to determine the incidence and clinical consequences of CMV mutations conferring ganciclovir resistance in pediatric solid organ transplant (SOT) patients who received valganciclovir oral solution or tablets for prophylaxis of CMV disease. Recombinant CMV mutants were also generated to assess the role of two UL97 mutations of unknown significance. Study design: Genotypic resistance mutations and CMV viral load were sought in blood samples from pediatric SOT recipients who received valganciclovir prophylaxis for 100 days. Recombinant viruses containing novel CMV UL97 mutations were generated using a bacterial artificial chromosome containing the CMV genome to assess ganciclovir susceptibility. Results: Overall, four known resistance UL97 mutations were observed in blood samples from 2 of 46 patients during the study with no development of CMV disease. Two UL97 changes (M615V and V466G) of unknown significance and one UL97 mutation (C603R) associated with ganciclovir resistance, but not yet confirmed by marker transfer, were also detected. Recombinant viruses containing these novel mutations were generated to assess ganciclovir susceptibility. The M615V recombinant virus was susceptible to ganciclovir while the V466G and C603R mutant viruses displayed 3.5-fold and 3.6-fold decreases in susceptibility, respectively. Conclusions: The low incidence of ganciclovir resistance-associated mutations and the absence of clinical consequences associated with drug-resistant viruses observed in this pilot study should encourage the design of larger clinical trials aimed at evaluating the efficacy of valganciclovir prophylaxis and treatment in the pediatric setting. © 2010 Elsevier B.V. All rights reserved.
1. Background Human cytomegalovirus (CMV) causes significant morbidity among high-risk transplant recipients. Ganciclovir, a deoxyguanosine analogue, has been the antiviral of choice for prevention and treatment of CMV disease for two decades. The l-valyl ester prodrug of ganciclovir (valganciclovir) was subsequently developed and shown to have a bioavailability of ∼60%.1 A daily dose of valganciclovir was as clinically effective and well-tolerated as a thrice daily dose of oral ganciclovir for prevention of CMV disease in high-
risk adult solid organ transplant (SOT) recipients.2 Furthermore, valganciclovir was shown to have comparable safety, time of eradication of viremia and clinical efficacy as intravenous ganciclovir for treatment of CMV disease in adult SOT patients.3 However, concerns remain about the emergence of drug-resistant CMV strains among immunocompromised patients receiving antiviral prophylaxis and treatment.4–7 The mechanisms of resistance to ganciclovir imply predominantly mutations in the CMV UL97 protein kinase gene with more occasional alterations in the UL54 DNA polymerase gene.8
2. Objectives Abbreviations: CMV, cytomegalovirus; SOT, solid organ transplant; WT, wildtype; D+/R−, donor CMV seropositive/recipient CMV seronegative; i.v., intravenous; IC50 , inhibitory concentration 50%; Neg, negative; Pos, positive; N/a, not applicable; Blq, below limit of quantification. ∗ Corresponding author. Tel.: +1 418 654 2705; fax: +1 418 654 2715. E-mail address: [email protected] (G. Boivin). 1386-6532/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2010.01.009
The aims of this study were to determine the incidence and clinical consequences of CMV mutations conferring ganciclovir resistance in pediatric SOT patients who received valganciclovir oral solution or tablets for prophylaxis of CMV disease.9 We also
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Table 1 Incidence of CMV mutations at the end of study prophylaxis (Day 100) and during follow-up until 6 months after transplantation (Week 26) among pediatric SOT recipients. At the end of the study prophylaxis (Day 100)a
During follow-up until 6 months after transplantation (Week 26)a
No. of patients with samples available for testing (no. of samples) No. of patients with CMV PCR positive samples (no. of samples) No. of patients with known resistance mutations (% of tested samples)
46 (46) 1 (1) 1 (2.2)
9 (35) 3 (5) 2 (5.7)
Genotype Known UL97 resistance mutations Novel UL97 mutations Natural UL97 polymorphisms Known UL54 resistance mutations Novel UL54 mutations Natural UL54 polymorphisms
M460V, C592G, L595F V466G – – – N685S, A885T, N898D
M460V, C592G, L595F, C603Rb M615V – – – S676G, A885T, S897L, N898D
a b
Compared to sequence of the AD169 strain (GenBank accession number X17403). Another mutation at the same codon (C603W) has been confirmed to confer ganciclovir resistance.
generated recombinant CMV mutants to assess the role of two UL97 mutations of unknown significance. 3. Study design 3.1. Patients Clinical trial WV16726 was a single arm, non-comparative multicenter study (18 centers in 7 countries) of the safety and pharmacokinetics of valganciclovir oral solution in the prevention of CMV disease in pediatric SOT recipients. Patients (38% donor CMV seropositive/recipient CMV seronegative or D+/R−) with a heart (n = 12), kidney (n = 33), liver (n = 17) or kidney and liver (n = 1) allograft received valganciclovir oral solution or tablets once daily at a dose determined by an algorithm.9 3.2. Genotypic analysis of resistance Blood samples were routinely obtained at the end of prophylaxis (Day 100) and during suspicion of CMV disease up to 6 months after transplantation. In some centers, blood samples were also collected for resistance testing at the time of a routine post-transplant biopsy. DNA was extracted from whole blood samples using the MagNA Pure LC system (Roche Diagnostics). For UL97 genotyping, the region including codons 363–698 was amplified by a semi-nested PCR protocol.6 In addition, a region of the UL54 gene encompassing codons 184–1017 was amplified by a nested PCR protocol.10 An automated method with a chromatogram readout (ABI 3720xl) was used as a sequencing method for three independent PCR products of the UL97 and UL54 genes. 3.3. CMV viral load Five microliters of extracted nucleic acid from whole blood samples were added to 15 L of a reaction mixture containing 1 mmol/L of MgCl2 , 0.4 mol/L of each primer, 0.3 mol/L of fluorescein hybridization probe (Tib Molbiol), 0.6 mol/L of LC Red 640 probe (Tib Molbiol), 5% dimethyl sulfoxide and 2 L of LightCyclerFaststart DNA Master hybridization probes kit (Roche Diagnostics). Amplification primers for the CMV glycoprotein B gene were as previously reported.11 The hybridization probe sequences were as follows: TTA AAG GTG TGC GCC ACG ATG TTG CG-fluorescein and LC Red 640-TTG TAG ACC ACC ATG ATG CCC T-phosphate. Viral loads were normalized for human beta globin gene using the LightCycler Control Kit DNA (Roche Diagnostics). 3.4. Generation of recombinant CMV mutants Mutagenesis of the CMV/BAC plasmid (pHB5, a gift from Dr M. Messerle, Germany) in Escherichia coli was performed by using a
two-step recombination protocol.12 For each recombinant virus generated, the UL97 and UL54 genes and the homologous regions where recombination events occurred were sequenced to confirm the appropriate genotype. Susceptibility testing of the recombinant wild-type (WT) and mutant viruses was performed using a standardized plaque reduction assay.13 Recombinant viruses were considered to be resistant to ganciclovir if their IC50 value was at least 3 times greater than that of the WT recombinant virus.12 4. Results 4.1. Genotypic analysis of resistance At the end of prophylaxis (Day 100), samples from 46 of 63 SOT patients (aged 4 months to 16 years) were available for ganciclovir resistance testing. 17 out of 46 patients (37%) were high-risk (D+/R−) recipients. Only 1 tested sample was CMV positive by PCR. Seven amino acid changes relative to the comparator sequence were observed within the CMV UL97 and UL54 genes in this sample (Table 1). Of these, three were known UL97 mutations conferring ganciclovir resistance (M460V, C592G and L595F),8 one was a UL97 mutation of unknown significance (V466G) and three were known UL54 natural polymorphisms (N685S, A885T and N898D).14 Overall, the incidence of ganciclovir resistance mutations at the end of study prophylaxis was 2.2% (1/46). Thirty-five samples from 9 patients were collected after prophylaxis up to 6 months post-transplant. Only 7 of these samples were from patients with suspected CMV disease whereas the other 28 were collected during routine post-transplant biopsy or at the end of follow-up (Week 26). Of these, 5 samples from 3 patients were CMV positive by PCR (including the patient described above). A total of nine amino acid changes were observed in these samples (Table 1). The same three UL97 mutations conferring ganciclovir resistance were detected at the end of the study follow-up (Week 26) from the previous patient with UL97 mutations at Day 100. An additional resistance-associated UL97 mutation, C603R, was observed in 1 sample from a second patient at Week 20. One novel UL97 amino acid change of unknown significance, M615V, was observed in 1 sample from a third patient at Week 26. Four known UL54 natural polymorphisms (S676G, A885T, S897L and N898D)14 were detected in 1 patient at Week 26. 4.2. Clinical impact of CMV UL97 mutations Kinetics of emergence of CMV UL97 mutations and corresponding CMV viral loads are presented in Table 2. Although Patient A (a 3-year-old liver D+/R− transplant recipient) was asymptomatic for CMV throughout the study, samples for genotyping were nevertheless collected at the end of prophylaxis (Day 100) and at Weeks 2, 6,
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Table 2 Kinetics of emergence of known ganciclovir resistance and novel CMV UL97 mutations in three high-risk D+/R− patients. Patients (age and transplanted organ)
Time after transplantation
UL97 PCR result
UL97 resistance mutations
UL97 novel mutations
CMV, copies/104 blood cells
A (3-year-old liver transplant)
Week 2 Week 6 Week 10 Day 100 Week 16 Week 20 Week 26
Neg Neg Neg Neg Pos Pos Pos
n/a n/a n/a n/a No mutation C603R a No mutation
n/a n/a n/a n/a No mutation No mutation No mutation
n/a n/a n/a n/a 2.68 × 102 1.11 × 102 7.34 × 101
B (6-month-old heart transplant)
Day 100 Week 26
Pos Pos
M460V, C592G and L595Fa M460V, C592G and L595Fa
V466Ga No mutation
2.54 × 104 Blq
C (16-year-old heart transplant)
Day 100 Week 16 Week 20 Week 26
Neg Neg Neg Pos
n/a n/a n/a No mutation
n/a n/a n/a M615V
n/a n/a n/a 7.75 × 103
Note: Day 100, end of prophylaxis. Week 26, end of study follow-up. Neg, negative; Pos, positive; N/a, not applicable due to negative PCR result; Blq, below limit of quantification. a Mixed (mutant and wild-type) viral populations.
10, 16, 20 and 26. Notably, this patient was withdrawn from study treatment on Day 35 due to intestinal obstruction. The patient was found to be CMV positive on Day 86 which prompted the administration of intravenous (i.v.) ganciclovir for 10 days. The virus load was undetectable at Day 100, but raised to 2.68 × 102 , 1.11 × 102 and 7.34 × 101 CMV copies/104 blood cells at Weeks 16, 20 and 26, respectively. The UL97 resistance mutation C603R was observed at Week 20, but was not observed at Day 100 (immediately post-i.v. ganciclovir treatment) and had disappeared by Week 26. For Patient B (a 6-month-old heart D+/R− transplant recipient), CMV was initially detected on Day 44 post-transplant in a blood sample collected during a hospitalization for prolonged vomiting. The CMV positive result was not considered significant by the investigator and, therefore, no adjustment was made to the patient antiviral prophylaxis. The patient was asymptomatic for CMV disease at the time the three known GCV resistance mutations (M460V, C592G and L595F) and the novel V466G UL97 mutation were initially observed on Day 100. The patient remained asymptomatic until the end of follow-up (Week 26) despite persistence of CMV mutations. The Day 100 sample (viral load: 2.54 × 104 CMV copies/104 blood cells) also contained three known UL54 polymorphisms (N685S, A885T and N898D). The viral load was below the limit of quantification at Week 26. Although Patient C (a 16-year-old heart D+/R− transplant recipient) had been asymptomatic throughout the study, samples for genotyping were collected during routine visits on Day 100 and at Weeks 16, 20 and 26. The patient was found to be CMV positive during a routine heart biopsy on Day 173 (Week 25). He was then treated with valganciclovir, but the viremia remained unresolved (viral load: 7.75 × 103 CMV copies/104 blood cells) a week later at the end of study follow-up (Week 26). The novel CMV UL97 change M615V was observed at Week 26. In addition, four UL54 natural polymorphisms (S676G, A885T, S897L and N898D) were also detected in this sample. Clinical consequences of the novel UL97 change M615V are unknown because of absence of follow-up beyond Week 26.
4.3. Ganciclovir susceptibilities of recombinant mutant viruses Ganciclovir susceptibilities of the two novel UL97 changes and one suspected resistance-associated mutation not yet confirmed by marker transfer experiments were determined. Two distinct recombinant viruses per mutation were generated and each of these was tested in triplicate. The recombinant viruses containing the UL97 mutation M615V had a mean ganciclovir IC50 value comparable to that of the WT virus (i.e. 1.15-fold increase compared
to WT). Consequently, M615V can be considered a natural polymorphic variant. On the other hand, recombinant mutant viruses containing the UL97 mutation V466G exhibited a mean IC50 value of 6.32 mol/L while mutant C603R had a mean IC50 value of 6.48 mol/L, which were 3.5 and 3.6 times greater than that of the susceptible WT virus, respectively. The UL97 V466G and C603R mutants were thus considered to be resistant to ganciclovir.
5. Discussion We evaluated the incidence and clinical consequences of CMV resistance-associated mutations observed in pediatric SOT patients who received valganciclovir prophylaxis for 100 days. A challenge in evaluating the incidence of CMV resistance-associated mutations in prophylactic studies is the lack of a baseline comparator sequence that makes it impossible to differentiate novel resistance-associated mutations versus uncharacterized polymorphisms. Consequently, novel UL97 changes detected in this study were introduced into recombinant viruses and phenotypically characterized. The incidence of patients harboring CMV UL97 mutations conferring ganciclovir resistance was 2.2% (1/46) at the end of prophylaxis with an additional case detected during follow-up. In one case (Patient A), it remains unclear whether the resistanceassociated mutation developed during the shorter 35 days of prophylaxis or whether it developed during 10 days of ganciclovir treatment because the mutation was only observed transiently post-treatment. In the other case (Patient B), prolonged vomiting may have resulted in suboptimal ganciclovir exposure facilitating the development of resistance. The 2 patients who harbored confirmed ganciclovir resistance mutations did not develop CMV disease and had uneventful clinical outcomes. Overall, no patient developed CMV disease during this study. We previously reported a prospective study of high-risk adult SOT patients who received either oral ganciclovir or valganciclovir for CMV prophylaxis during 100 days in which no ganciclovir resistance mutations were observed in the valganciclovir arm.15 Similar results were observed in the present pediatric study with minimal breakthrough viremia and development of resistance during prophylaxis and subsequent follow-up. In adult SOT patients, mismatch (D+/R−) lung transplant recipients appear to have the highest risk of developing ganciclovirresistant CMV infections and to present the most important clinical complications.4,7,10 It is noteworthy that none of the 46 patients from our study received a lung transplant and that only 38% were high-risk (D+/R−) recipients which may explain the low level of resistance and low incidence of CMV disease observed. More-
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over, the immunosuppression regimens used in different types of pediatric SOT recipients varied among centers, which may have influenced clinical outcome of CMV infection. The absence of clinical CMV disease despite the presence of ganciclovir resistance mutations has been reported elsewhere.10 It is probable that the development of CMV disease is influenced by viral replicative capacities as well as by host and iatrogenic factors. The uneventful clinical courses associated with CMV resistance mutations could also potentially be explained by the fact that routine screening for mutations was performed in all patients at the end of prophylaxis. In clinical practice, resistance testing would not be indicated for patients showing no clinical signs suggestive of CMV disease or without sustained viremia. In this study, two novel CMV UL97 changes of unknown significance (V466G and M615V) were observed. The UL97 mutations V466M (instead of V466G)6 and M615V16 have been previously observed in patients with AIDS. These changes lie outside the documented hot-spot regions for ganciclovir resistance (codons 460, 520 and 590–607).8 While the mean IC50 value obtained for recombinant virus M615V was comparable to that of the WT virus, the V466G mutant had a 3.5-fold increase in IC50 value. Of note, a V466M mutant was susceptible to ganciclovir (unpublished). The C603R mutant, previously associated with ganciclovir resistance but not yet confirmed by marker transfer,17 had a 3.6-fold increase in IC50 value compared to the susceptible recombinant virus. Such decrease in susceptibility for the C603R mutant is smaller than what was observed for mutant C603W (8-fold increase in IC50 value),18 but comparable to that obtained for mutant M460I (3.9fold increase in IC50 value) that we generated using the same CMV/BAC system.12 Since CMV genotyping has essentially replaced CMV phenotype testing in the clinical setting, it is a concern that two out of six mutations found in the CMV UL97 gene were novel mutations. This supports the use of recombinant viruses to assess mutations of unknown significance12 as well as the development of a virtual database containing all published UL97 and UL54 mutations.19 In conclusion, the low incidence of ganciclovir resistanceassociated mutations and the absence of clinical consequences associated with drug-resistant viruses observed in this pilot study should encourage the design of larger clinical trials aimed at evaluating the efficacy of valganciclovir prophylaxis and treatment in the pediatric setting. Conflicts of interest J.I. is an employee of Roche. The other authors report no potential conflicts of interest. Acknowledgments This study was presented in part at the 48th Interscience Conference of Antimicrobial Agents and Chemotherapy on October 27th 2008 in Washington, DC, USA (abstract V-3561). This study was supported by research grants from Roche Products Ltd. and the Canadian Institutes of Health Research (CIHR; MOP-86583) to G.B.
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