Journal of Medical Virology 80:12281232 (2008)

Cytomegalovirus Prophylaxis with Valganciclovir in Cytomegalovirus-Seropositive Kidney-Transplant Patients

clawiak,1 Nassim Kamar,1 Catherine Mengelle,2 Joe lle Guitard,1 Laure Esposito,1 Hugo We 1 1 1 ` Laurence Lavayssiere, Olivier Cointault, David Ribes, and Lionel Rostaing1*
1 2

Department of Nephrology, Dialysis and Multiorgan Transplantation, CHU Rangueil, Toulouse, France Department of Virology, CHU Purpan, Toulouse, France

The aims of this prospective, open-label, singlecenter pilot study were to assess the efcacy and safety of human cytomegalovirus (HCMV) prophylaxis using valganciclovir in HCMVseropositive kidney-transplant patients to prevent HCMV infection and disease. Fifty-one HCMV seropositive kidney-transplant patients recipients who received transplants between 1 December 2005 and 30 November 2006 were included in the study. Valganciclovir was given from transplantation up to 114 (37329) days, and was adapted to renal function, i.e., 900 mg/d if calculated creatinine clearance was >60 ml/min, or 450 mg/day if it was <60 ml/min. HCMV DNAemia was assessed every 2 weeks during prophylaxis, and on the same basis for 3 months post-prophylaxis. Immunosuppression was based on calcineurin inhibitors (ciclosporine A 22; tacrolimus 11), with mycophenolate mofetil (n 51), and lowdose steroids. Eighteen patients received no calcineurin-inhibitors, but Belatacept instead. During valganciclovir prophylaxis, asymptomatic HCMV DNAemia was observed in one patient, and no case of HCMV disease occurred. Within 252 days (45425) post-valganciclovir prophylaxis, HCMV DNAemia was detected in 23.5% (n 12) of patients, of whom two had two or more consecutive HCMV DNAemias. Valganciclovir prophylaxis in HCMV-seropositive kidney-transplant patients is effective for preventing cytomegalovirus disease. J. Med. Virol. 80:1228 1232, 2008. 2008 Wiley-Liss, Inc. KEY WORDS: cytomegalovirus; kidney transplantation; organ transplantation; prophylaxis; valganciclovir

1998; Fishman et al., 2007]. Before the era of HCMV prophylaxis, Grattan et al. [1989] showed that HCMV disease in heart-transplant recipients was signicantly associated with the development of coronaryartery vasculopathy. The indirect effects are independent of a high-level of HCMV viremia and result, in part, from the hosts immune response in the setting of long periods of low-level HCMV replication [Fishman et al., 2007]. This translates into organ-allograft injuries, e.g., acute or chronic rejections, and suppression of the systemic immune response, thereby leading to opportunistic infections or facilitating replication of viruses such as Epstein Barr virus (EBV) [Manez et al., 1997] and hepatitis C virus (HCV) [Razonable et al., 2002]. It has been shown that HCMV-related indirect effects were decreased or prevented by HCMV prophylaxis in humans [Opelz et al., 2004; Kalil et al., 2005; Radermacher et al., 2006]. Thus, HCMV prophylaxis can reduce the risk of acute rejection in the following cases: (i), in donor-positive and recipient-negative kidney-transplant patients receiving either valaciclovir [Lowance et al., 1999] or (val)ganciclovir [Paya et al., 2004] HCMV prophylaxis, (ii) in kidneypancreas recipients receiving ganciclovir (GCV) prophylaxis [Ciancio et al., 2004], and (iii) in donor seropositive/ recipient seronegative heart-transplant patients receiving valganciclovir (VGC) plus HCMV hyperimmune globulin prophylaxis [Valantine et al., 1999; Potena et al., 2006]. In heart-transplant recipients, HCMV prophylaxis with VGC can reduce the risk of coronary artery vasculopathy as demonstrated by intravascular ultrasound studies [Potena et al., 2006]. In contrast, in kidney-transplant patients, HCMV pre-emptive

INTRODUCTION After solid-organ transplantation, the human cytomegalovirus (HCMV) can become reactivated to result in direct and indirect effects [Fishman and Rubin,
2008 WILEY-LISS, INC.

*Correspondence to: Lionel Rostaing, Department of Nephrology, Dialysis and Multiorgan Transplantation, CHU Rangueil, ` s, TSA 50032, 31059 Toulouse Cedex 9, France. 1 av. Jean Poulhe E-mail: rostaing.l@chu-toulouse.fr Accepted 21 February 2008 DOI 10.1002/jmv.21183 Published online in Wiley InterScience (www.interscience.wiley.com)

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TABLE I. Patients characteristics Kidney-transplant patients (n 51) Age, median (years) Gender, male/female Weight, mean (kg) Donor serostatus Donor seropositive Donor seronegative Immunosuppressive regimen Tacrolimus Cyclosporine A Belatacept Steroids Mycophenolate mofetil 53 (2276) 33/18 66 31 20 11 22 18 51 51

therapy with VGC is not able to reduce the indirect effects of HCMV as compared to ganciclovir prophylaxis [Radermacher et al., 2006]. HCMV prophylaxis may also modify the natural posttransplant history of some viruses such as human Herpes viruses 6, 7, and 8, Varicelle zoster virus, EBV, polyomavirus, and adenovirus [Humar, 2006]. The use of valganciclovir as HCMV prophylaxis has been approved by regulatory authorities in donor seropositive/recipient seronegative organ-transplant patients: this is based on the results of the PV16000 study where donor seropositive/recipient seronegative organ-transplant patients were randomized to receive either valganciclovir or oral ganciclovir within the rst 3 post-transplant months. These treatments reduced dramatically the rate of HCMV replication during prophylaxis, even though some degree of HCMV replication was observed with ganciclovir therapy and after prophylaxis was stopped [Paya et al., 2004]. To date, no data are available regarding the potential benet of HCMV prophylaxis with valganciclovir in seropositive solid-organ transplant recipients. This prompted a pilot study where HCMV-seropositive kidney-transplant patients were offered valganciclovir prophylaxis within the rst 4 months post-transplant, and the preliminary results from the rst 51 patients are described. The aim of this prospective study was to address the efcacy and safety of valganciclovir to prevent both HCMV infection (assessed by HCMV DNAemia using ultrasensitive polymerase chain reaction) and disease in HCMV-seropositive kidney-transplants.

PATIENTS AND METHODS Study Design Between 1 December 2005 and 30 November 2006, 106 kidney transplantations were performed in Toulouse University Hospital. Of these, 51 patients were included in this present study, i.e., patients who died or lost their allografts within the rst 3 months post-transplant were excluded. However, none of the latter experienced positive HCMV DNAemia between the time of receiving their transplant and death or graft loss. Fifty-one patients were kidneytransplant recipients (Table I). The patients received valganciclovir prophylaxis for the rst 34 months posttransplantation. This was initiated within 10 days post-transplantation, as soon as creatinine clearance (according to the Cockcroft and Gault formula) was >20 ml/min. With regards to valganciclovir dosage, the daily dose was 900 mg a day if creatinine clearance was >60 ml/min; if it was below 60 ml/min then dosage was 450 mg once a day. In cases of neutropenia, i.e., below 1000/mm3, valganciclovir was withheld for 1 week. Assessments During follow-up, HCMV infection was monitored by regular sampling of whole blood. HCMV DNAemia

was assessed every 2 weeks until day 120, and thereafter at 23-weeks intervals until day 180. At the same time, the following parameters were assessed: alanine (ALT), aspartate (AST) aminotransferase, gamma glutamyl transpeptidase (gGT), alkaline phosphatase (AP), serum creatinine levels, creatinine clearance and hemoglobin levels, as well as platelet, whiteblood-cell (WBC) and polymorphonuclear-cell (PMNC) counts. Patients were also monitored for symptoms of HCMV disease when HCMV DNAemia was found to be positive. Symptomatic CMV disease was dened according to agreed criteria [Ljungman, 1995] and included either a fever greater than 388C for 48 h in the absence of bacterial or fungal infection or rejection, and/or a progressive falling neutrophil count for 3 days, and/or thrombocytopenia at <100 103/mm3 or the involvement of an organ. CMV reactivation was dened on the basis of at least one viraemia without any of the previous symptoms/signs. For every patient, the treatment of CMV DNAemia episodes (if any), daily dosage of valganciclovir at the time of viremia, acute organ rejection, and adverse events attributable to valganciclovir were recorded. Virology HCMV DNAemia: Serial samples were collected into potassium EDTA tubes for quantitative real-time PCR, on DNA extracted from whole blood as previously described [Mengelle et al., 2003]. The detection limit of this method was 500 copies/mL (2.69 log10/mL). Serological markers: HCMV serology was assessed in the donor as well as in the recipient on the day of transplantation and at 6 months after. IgG and IgM were detected with ETI-CYTOK-G Plus and ETI-CYTO-M reverse (DIA Sorin, Antony, France) assays, which were performed according to the manufacturers instructions. Immunosuppression Regimen Immunosuppression was based on 33 cases with calcineurin-inhibitor agents, i.e., ciclosporine A (n 22) with blood C2 levels of between 800 and 1500 ng/ml for the rst 3 months, or tacrolimus
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clawiak et al. We

(n 11) with trough target levels of 812 ng/ml, in association with mycophenolate mofetil (n 51) and steroids (n 51). Eighteen patients received belatacept instead of calcineurin inhibitors within the framework of a phase III protocol. All patients received an induction therapy of either antithymocyte globulins (n 8) or anti-CD25 monoclonal antibodies (n 43). In case of suspicion of acute rejection the patients underwent kidney allograft biopsy. If acute rejection was conrmed the patient was treated by methylprednisolone pulses (10mg/kg each for 3 consecutive days). If the acute rejection was steroid-resistant the second line of therapy was antithymocyte globulins. All patients received Pneumocystis jiroveci prophylaxis within the rst 6 months post-transplantation with sulfamethoxazole/trimetoprim (400 mg/80 mg) every other day. RESULTS There were 51 (33 men and 18 females) kidneytransplant patients with a median age 53 years (2276) (see Table I). Concerning the donors, HCMV serostatus was positive in 31 patients. The median time of prophylaxis with VGC was 113 (37329) days (Table II). Overall, three patients are still being administered VGC prophylaxis after 8 months because of profound CD4 lymphopenia. VGC was denitively stopped in two cases (3.9%) before 100 days because of hematological toxicity, i.e., sustained neutropenia <1000 mm3. During prophylaxis, two patients (3.1%) temporarily withdrew VGC due to transient neutropenia <1000 mm3. Transplant recipients were followed for a median time of 252 (45425) days after HCMV prophylaxis was stopped. Under prophylaxis HCMV DNAemia occurred in only one patient, i.e., 2% of transplant patients. The onset of HCMV viremia occurred 3 months after the initiation of VGC, but was not associated with any symptoms of HCMV disease. The level of HCMV DNAemia was 3.72 log10/mL. However, there was an error in VGC dosage: it was given at 450 mg once daily, instead of 900 mg/d, whereas creatinine clearance was >60 ml/min. In addition, onset of HCMV DNAemia was analyzed after cessation of prophylaxis. Overall, it occurred in 12 patients, i.e., 23.5%, of whom two had two or more consecutive CMV DNAemias. The mean time until detection of HCMV DNAemia was 87 (40212) days. None of these patients developed symptoms of

HCMV disease. Their median viral load was 3.15 log10 (2.444.49) copies/ml. Each episode of viral reactivation was treated with either ganciclovir IV for 2 weeks (n 6) or a double dose of VGC (n 6), both adapted to renal function. Of these 12 patients, ten had received induction therapy with anti-CD25 and only two had received rabbit antithymocyte globulins. With respect to HCMV reactivation and immunosuppression of those on tacrolimus-based immunosuppression only one (9.1%) presented with HCMV reactivation as compared to six (27%) and ve (27.1%) for those on cyclosporine-based or belatacept-based immunosuppression (P ns). Median tacrolimus trough levels were at 8.9 (6.114.8) ng/mL, whereas median cyclosporine C2 levels were at 889 (3521208) ng/mL. Regarding the cyclosporine patients, there was no difference in those with or without HCMV reactivation. With respect to acute rejection this occurred in two patients who developed HCMV reactivation (16.6%) as compared to 10 patients (25.6%) in those who did not present HCMV reactivation (P ns). Overall, after a median follow-up of VGC prophylaxis of 252 days (45425) no CMV disease was observed. Finally, the predictive factors for developing at least one HCMV DNAemia either during or after valganciclovir prophylaxis were searched for. In a univariate analysis, donor variables (age, HCMV status, sex), as well as recipient variables (cold ischemia time, HLA matching, type of immunosuppression, type of induction therapy, and various biological parameters at different post-transplant time points as listed in the Patients and Methods section), were taken into account. The only two signicant factors predictive of at least one HCMV DNAemia were the recipients older age (59 11 years vs. 50 13.4 years; P 0.01) and a lower calculated creatinine clearance at month 2 post-transplant (52.5 13.4 ml/min vs. 64.3 27.3 ml/min; P 0.03). Because of the low number of patients with at least one HCMV DNAemia, a multivariate analysis was not performed. DISCUSSION To the best of the authors knowledge this is the rst study to show that HCMV valganciclovir prophylaxis in HCMV seropositive kidney-transplant patients is safe and associated with no HCMV disease. So far, HCMV prophylaxis has been successful for prevention of HCMV disease in solid-organ transplant recipients at highest

TABLE II. Valganciclovir prophylaxis and its effects upon HCMV infection/disease HCMV DNAemia Time on VGC prophylaxis (days) Kidney-transplant patients (n 51) 113 (37329) Overall HCMV disease (%) 0 (0) During VGC (%) 1 (1.9) After VGC (%) 12 (23.5) Follow-up since VGC withdrawal (days) 254 (45420)

KT, kidney-transplant patients; VGC, valganciclovir; HCMV, human cytomegalovirus.

J. Med. Virol. DOI 10.1002/jmv

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risk, i.e., seronegative recipients of an organ from seropositive donors [Lowance et al., 1999; Paya et al., 2004] and for those who have received antithymocyte globulins as their induction regimen [Said et al., 2004]. Originally, intravenous ganciclovir was the gold standard for both prevention and treatment of HCMV; this was relayed when possible by oral ganciclovir, though this has poor bioavailability. Recently, valganciclovir, the L-valyl ester prodrug of ganciclovir, has been developed: compared to ganciclovir, its bioavailability is markedly improved [Einsele et al., 2006]. Recent studies have reported its clinical efciency for the prevention (900 mg once daily) as well as for the treatment of HCMV disease (900 mg twice daily) in solid-organ transplant recipients in high-risk groups (donor seropositive/recipient seronegative) [Paya et al., 2004; Asberg et al., 2007] as compared to oral ganciclovir. In contrast, HCMV prophylaxis for all HCMV() organ-transplant patients is not a consensual approach. In this setting, most of the centers utilize pre-emptive therapy in which frequent monitoring for viremia is carried out, utilizing either the antigenemia or the polymerase chain reaction assay [Lopau et al., 2007]. As soon as it becomes positive, antiviral therapy is initiated until antigenemia/DNAemia becomes negative. However, this allows HCMV replication to occur, which may lead to HCMV-mediated indirect effects and can impair long-term allograft outcome [Fishman and Rubin, 1998; Fishman et al., 2007]. On the other hand, HCMV prophylaxis in this setting might unnecessarily expose a huge number of patients to the potential sideeffects of prophylactic drugs. Nonetheless, in situations such as lung and heartlung transplantations, HCMV prophylaxis in HCMV() recipients has been shown to be efcient in reducing the risk of acute and chronic rejection [Halme et al., 1998; Wreghitt et al., 1999]. Conversely, in HCMV() kidney-transplant recipients, almost no data regarding HCMV prophylaxis therapy are available. Thus, the only study published so far describing HCMV() kidney-transplant patients compared oral ganciclovir given either prophylactically or in a pre-emptive fashion, i.e., as soon as HCMV viremia became positive [Radermacher et al., 2006]. It showed that HCMV prophylaxis was associated with better patient and graft survival at 4 years post-transplant compared to pre-emptive therapy. In this prospective study, it has been demonstrated that HCMV prophylaxis in HCMV seropositive recipients is efcient, i.e., it is associated with an absence of HCMV disease, and with a very low level of HCMV replication both on and after therapy. Within a median follow-up of 9 months after withdrawal of prophylaxis, HCMV DNAemia was detected in 2% of patients during prophylaxis and in 23.5% of patients after cessation of prophylaxis. The major concern when using oral HCMV prophylaxis with (val)ganciclovir is the emergence of HCMV mutant strains that might be resistant to (val)ganciclovir. In lung transplantations, where HCMV prophylaxis is given for long periods, i.e., up to 1 year,

the long-term use of ganciclovir has been associated with a high rate of mutations, such as UL97 one [Bhorade et al., 2002]. In the PV16000 study, comparison of HCMV prophylaxis in high-risk solid-organ transplant patients (donor seropositive/recipient seronegative) who received either ganciclovir or valganciclovir, it was found that HCMV-resistant strains were only observed in the ganciclovir arm. Moreover, in this study, valganciclovir prophylaxis resulted in virtually no HCMV DNAemia whereas it occurred in $5% of those receiving ganciclovir [Boivin et al., 2004]. The safety prole of valganciclovir in this study was excellent, with only three cases of mild neutropenia occurring during prophylaxis, despite the fact that patients were receiving mycophenolate mofetil and sulfamethoxazoletrimetoprim prophylaxis. This good hematological tolerance was possibly related to the fact that all patients studies were on steroid-based therapy; thus, this may have helped maintain normal neutrophil counts in these transplant patients. In conclusion, this study has shown that HCMV prophylaxis with valganciclovir in HCMV seropositive kidney-transplant patients is well tolerated, is associated with no HCMV disease, and has a very low level of HCMV replication after its cessation.

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J. Med. Virol. DOI 10.1002/jmv