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Seventeen to eighty percent of Epstein-Barr virus (EBV)–naïve pediatric liver recipients become infected,1, 2 usually in the first months following liver transplantation (LT), because of latent virus in lymphocytes within the graft and blood derivatives. The condition of pharmacological immunosuppression in LT patients modifies the normal T-lymphocyte cytotoxic responses during the initial viral lytic phase and subsequent latency. EBV infection can induce uncontrolled lymphocyte B proliferation in transplant recipients. In latently infected cells, the episomal EBV-DNA codes for proteins that inhibit apoptosis and block the ability of interferon to control the outgrowth of EBV-transformed cells.3 The risk of developing EBV-related posttransplant lymphoproliferative disorder (PTLD) is particularly high in children who contract primary EBV infection after transplantation.4, 5 The cumulative incidence of PTLD in the pediatric population is 7% to 10% over long-term follow-up after LT6–8 but increases (17%-22%) for children with EBV infection.1, 2, 9–11 PTLD requires immunosuppression withdrawal to be controlled.12, 13
The high prevalence of EBV infection and its associated morbidity and mortality have resulted in efforts to identify children at risk for these complications. Because of young age and immunosuppression, primary infection after pediatric LT goes unrecognized in 54% to 84%2, 14 unless specific serologic and virologic surveillance is undertaken. Both a sustained persistence of increased viral load and high EBV-DNA levels have been associated with increased risk of PTLD.4, 15, 16 Current care practice in LT settings recommends longitudinal monitoring of the EBV load. Identification of a high viral load prompts tapering of immunosuppression in order to achieve a load decrease, which will reduce the incidence of PTLD.17
Antiviral therapy has been regarded as not useful in post-LT EBV infection because the virus is latent. However, a decreased PTLD incidence has been observed with prophylactic administration of intravenous ganciclovir after LT in children.18 Antivirals have been widely used as part of PTLD treatment12 and might be able to act because latent EBV enters a replicative cycle with B-lymphocyte cellular division.19
Valganciclovir (VGCV) presents better oral bioavailability than ganciclovir.20 In the present study, LT children showing detectable EBV-DNA in their blood, which was sustained over time in most cases, were managed with VGCV. The aim was to decrease the risk of PTLD by blocking EBV replication, which might reduce the number of B-lymphocytes with latent infection.
Pediatric liver transplant patients infected with EBV were treated with VGCV. Forty-seven of 220 surviving pediatric patients who underwent LT at the center between 1990 and 2003 displayed detectable EBV-DNA in their blood. These patients were treated with VGCV, 42 of them having received a previous antiviral treatment. Neither rituximab nor immunoglobulins were used in these patients. They had a follow-up of 12 months from the start of therapy (study period) to assess the efficacy and side effects of VGCV treatment.
The characteristics of the 47 children are summarized in Table 1. The most frequent pre-LT disease was biliary atresia (51%). Fifty-nine percent were <2 years old at the time of transplant. Serologic markers of EBV (immunoglobulin G viral capsid antigen, anti–Epstein-Barr virus nuclear antigen, and immunoglobulin M viral capsid antigen) before LT were negative in 34 (72.3%) patients. The EBV serology of donor and blood derivatives was unknown. All patients received antiviral prophylaxis in the early postoperative period, which consisted of intravenous ganciclovir (5 mg/kg twice daily) during the first 30 days, followed by acyclovir (10 mg/kg three times a day) up to the fourth post-LT month.
Table 1. Patients' Characteristics at the Time of LT
Abbreviations: IS, immunosuppression; LT, liver transplantation; MP, methylprednisolone; Pred, prednisolone; SD, standard deviation.
Alagille syndrome, familial intrahepatic cholestasis type 1, and bile salt export pump defect.
Maple syrup urine disease, alpha-1 antitrypsin deficiency, and cystic fibrosis.
All patients underwent periodic checkups after LT to detect EBV primary infection or reactivation. Serology and genomic amplification in serum were replaced by blood polymerase chain reaction (PCR) tests in 2001. From 2001 onward, surveillance of EBV status at 3- to 6-month intervals was performed with a proprietary qualitative PCR test in blood samples (nested-PCR Herplex, Genomica).
Administration of VGCV
VGCV treatment had the corresponding Health Authority approval on an individual basis and parental signed and informed consent. Dosage was 520 mg/sqm twice daily. Valcyte (Roche) in the form of 450-mg capsules was administered to older patients. For patients needing a dose not fitting capsules, a syrup formulation containing 60 mg/mL was prepared at a hospital pharmacy department, either on site or at the one nearest to the patient's residence.
Initially, treatment was planned for 30 days. However, in view of the results obtained, the administration plan was changed to an indefinite period of time until accomplishment of undetectable EBV-DNA. Thus, 26 children initiated a 1-month VGCV treatment, 6 having a second 1-month treatment and 21 entering a prolonged treatment afterwards, whereas 21 children had the prolonged treatment planned from the start of therapy (Fig. 1).
Immunosuppression During the Study Period
A decrease in immunosuppression was carried out only in those showing PTLD or systemic symptoms. Eighty-three percent of patients kept the same levels of calcineurin inhibitors as had been planned according to the time that elapsed from LT and the individual history of graft rejection. Immunosuppression was increased in 4 cases because of rejection or autoimmune hepatitis and was decreased in 4 cases: 1 case of toxic nephropathy (tacrolimus was lowered), 1 case of monomorphic PTLD (tacrolimus was stopped), 1 case of polymorphic PTLD (cyclosporine was stopped), or 1 suspected case of PTLD (tacrolimus was stopped).
Safety and Efficacy Assessment
During the study period, clinical, biochemical, and virologic tests were performed at 3-month intervals, unless more frequent checks were needed.
The main efficacy endpoints were (1) the rate of new-onset PTLD during the period of study (a high index of suspicion was applied, and biopsy specimens were obtained on clinical grounds), (2) the evolution of symptoms and recording of new symptoms possibly related to EBV infection during the period of study, and (3) virologic evolution in sequential checkups of EBV-DNA in blood during therapy and the relapse rate once the drug was stopped.
Safety was assessed by a comparison of laboratory parameters obtained during treatment to those at baseline and post-therapy. All significant complications and infectious events were recorded.
Statistical analyses of the relationship between the patient baseline characteristics and achievement of undetectable EBV-DNA (2-tail Fisher exact test) and the patient baseline characteristics and responder status (Student t test) were performed. Confidence intervals were set at 95%, and a P value of ≤0.05 was considered significant. A logistic regression multivariate model integrated the candidate variables identified in univariate analysis.
Patients' Baseline Characteristics
First evidence of EBV infection occurred at an average post-LT time of 24 months (Table 2), occurring in 26 (55.3%) children within the first year post-LT. Twenty cases showed symptoms possibly related to EBV, including 3 cases with PTLD (Table 2). Antiviral treatment was administered to 42 children. It consisted of oral acyclovir in 19 (40.4%), oral ganciclovir in 4 (8.5%), oral acyclovir followed by oral ganciclovir in 2, intravenous ganciclovir followed by oral acyclovir or ganciclovir in 15 (32%), and intravenous ganciclovir in 2.
Table 2. Patients' Characteristics at the Onset of EBV Infection
All 47 children showed EBV-DNA in their blood just before VGCV was applied, despite the previous antiviral treatment in 42 of them. The time from first detection of EBV to the start of VGCV therapy was 17 months (median). Patients' characteristics at the moment of treatment are detailed in Table 3. Ages ranged from 6 months to 19 years. The mean time that elapsed from LT was 48 months. Most patients (n = 31, 66%) did not show symptoms related to EBV infection. Three cases had experienced PTLD in the past but were asymptomatic at the time. Symptomatic patients included 1 with monomorphic PTLD (in the initial phase of chemotherapy), 1 with tonsillar polymorphic PTLD who had undergone tonsillectomy, 8 with upper respiratory tract symptoms (tonsillar hypertrophy, otitis media, and regional lymphadenopathy), 2 with systemic involvement (fever, mucositis, and neutropenia) and 1 with graft dysfunction showing hepatitis in liver biopsy.
Table 3. Patients' Characteristics at the Start of Valganciclovir Treatment
Graft function was normal in 27 children (57.4%). Eight children showed a mild disturbance with alanine aminotransferase (ALT) levels less than twice the upper normal value, and 12 (25.5%) had ALT levels higher than 90 U/L. Two patients were jaundiced. None had coagulopathy or signs of decompensated liver disease.
Renal function, assessed by the creatinine level and the Schwartz formula for the glomerular filtration rate, was normal in all but 6 patients. Four suffered from calcineurin inhibitor toxic nephropathy, and 2 suffered from renal hypoplasia (Alagille syndrome). Only 1 case showed a glomerular filtration rate below 50 mL/minute.
Before treatment, the leukocyte count was low (<3000 × 109 cells/L) in 1 case, and 2 patients showed neutropenia (<1250 × 109 cells/L).
Twenty-six patients received VGCV for 30 days. Ten (38.4%) had undetectable EBV-DNA in the following analysis (1-3 months later). However, 8 of them relapsed during the third to tenth month post-treatment. A second course of 30 days of therapy was applied to 6 patients. Two were previous responders and did not show a response. Negative EBV-DNA was achieved in 1 of 4 previous nonresponders, but a relapse was observed in later follow-up. Overall, 32 courses of short treatment were applied to 26 patients. Undetectable EBV-DNA was observed in 11/32 (34.3%), but 82% relapsed.
Twenty-one patients (80.7%) went into a longer period of VGCV treatment. As for the other 5 cases, who were followed to the 12th month, 3 maintained positive EBV-DNA and 2 had sustained negative EBV-DNA since therapy.
Forty-two patients, 21 of whom had been on previous short therapy, received VGCV therapy until undetectable EBV-DNA was achieved on 1 or more visits. The mean duration of therapy was 7.1 ± 3.2 months (median: 8 months; range: 2-12).
Twenty patients (47.6%) achieved undetectable EBV-DNA (Fig. 2A) with treatment for 7.1 ± 3.4 months. Two of them experienced an on-treatment breakthrough. Five were still treated at the last follow-up visit, and 15 had VGCV stopped. Nine of these 15 responders (60%) had sustained negative EBV-DNA in the follow-up off therapy ranging from 1 to 9 months (median: 5.5) within the study period. An extended follow-up period showed that 65% of 19 responders maintained negative EBV-DNA off therapy (Fig. 2B). Nonresponders were still on treatment at the last follow-up visit (therapy duration: 7 ± 3.1 months).
Thirty-four asymptomatic patients at baseline (including 3 with a previous history of PTLD) remained asymptomatic during the 12-month observation period. Among the 13 symptomatic patients at baseline, 9 became asymptomatic, including the Burkitt lymphoma case, who exhibited cytopenia related to chemotherapy.
Three cases remained symptomatic. One child with liver dysfunction at baseline maintained ALT disturbances, and although liver biopsy showed hepatitis, EBV-RNA (EBV encoded RNA [EBER] sequence) was negative. Upper respiratory symptoms persisted in 2 patients, who showed tonsillar hypertrophy and lymphadenopathy, although at a very mild degree. One case evolved with fever, worsening liver dysfunction, and hypergammaglobulinemia. Liver biopsy showed severe fibrosis and portal lymphocytic infiltrate (T-cell predominance, negative EBER). Immunosuppression was increased by means of a higher steroid dose (baseline 0.3 mg/kg to 2 mg/kg tapered to 0.5 mg/g in 3 months) plus mycophenolate; tacrolimus was maintained at previous levels (10-15 ng/mL). No improvement in liver function was observed. Fever, diarrhea, and malnutrition prompted a thorough study to exclude PTLD, which included CT scan, liver, and jejunum histology with negative findings. Lactate dehydrogenase levels were high (633 U/L), and liver biopsy showed no changes in lesions. Because of overall conditions, even in the absence of a definite diagnosis, the treatment strategy was changed to calcineurin inhibitor withdrawal at the end of the study period. The child's condition improved in further follow-up and normalized completely; the child has now been off calcineurin inhibitors for 3 years. Thus, only 1 (2.1%) new suspected PTLD case was observed in the 12-month period of study.
There were no deaths or severe events attributable to VGCV, and no patient stopped treatment because of the drug's toxic effects.
Hemoglobin, leukocyte, neutrophil, and platelet mean values did not change during therapy in comparison with the baseline (Table 4). Nine cases displayed hematological disturbances. One had pancytopenia attributed to chemotherapy, 6 had neutropenia requiring filgastrim (granulocyte colony-stimulating factor), 1 had mild thrombocytopenia, and 1 had an increase in the hemoglobin level.
Table 4. Analytical Evolution During Long Treatment with VGCV (Levels: Median Value)
Three patients entered the study with graft disease (1 with autoimmune hepatitis and 2 with rejection with jaundice) that required high immunosuppression, but their liver condition resolved during the period of study.
Three cases developed rejection; 2 resolved with increased steroids1 or increased tacrolimus level,1 and 1 progressed to chronic rejection despite further treatment (methylprednisolone bolus, mycophenolate, and basiliximab). Overall ALT levels did not change throughout therapy with VGCV (Table 4).
Creatinine levels did not change in the study period (Table 4). The only patient with severe disturbance had her VGCV dosage adjusted to 520 mg/m2 once daily, and her renal function improved with a decrease in the tacrolimus level.
Infections During the Observation Period
Most children (33/47, 70%) showed at least 1 episode of infection in the 12-month period. Upper respiratory tract infections were the most frequent. There were 16 patients showing 1 or more upper respiratory infections, which, by their clinical characteristics, were considered unrelated to EBV. Other infections included gastrointestinal infections (9 cases), mucositis (1), pneumonia (1), urinary tract infections (1), cholangitis (3), and febrile infections of unidentified origin (4). Tests aimed at detecting EBV-DNA allowed also the detection of 1 cytomegalovirus case and 7 human herpesvirus 6 cases. Varicella symptoms appeared in 2 cases, and herpes zoster appeared in 1; none of these cases were on VGCV treatment at the time. Two patients developed a transient salivary gland enlargement mimicking mumps and had a fine needle aspiration biopsy performed to exclude PTLD.
A child complained of muscular cramps while on VGCV therapy.
Factors Related to the Virologic Response to VGCV
Response to Short Treatment
Responders (achieving negative EBV-DNA) to the first course of VGCV treatment had a trend of EBV infection onset at a later time post-LT than nonresponders and had significantly more symptoms with the infection and a longer time since LT at therapy initiation (P < 0.01). No differences in the distribution of calcineurin inhibitors were observed. Those on tacrolimus (n = 18) had similar trough levels at therapy initiation (5 responders: 8 ± 3.7 ng/mL, 13 nonresponders: 8.4 ± 4.5 ng/mL). Immunosuppression was reduced during VGCV in 1 responder and 1 nonresponder and was increased in 1 nonresponder (not significant). Liver function was significantly less disturbed at baseline in responders; only 1/10 had abnormal ALT versus 8/16 nonresponders (P = 0.03).
Response to Long Treatment
The statistical significance in the univariate analysis of several factors of virologic response is detailed in Table 5. Those on tacrolimus showed a trend of lower trough levels at baseline in the group of responders but no differences thereafter. No differences could be observed regarding pharmacological immunosuppression change between responders and nonresponders.
Table 5. Factors Analyzed for Their Possible Influence on the Viral Response to Long VGCV Administration
Nonresponders (Remaining EBV-DNA–Positive)
Abbreviations: ACV, acyclovir; EBV, Epstein-Barr virus; GCV, ganciclovir; IgG VCA, immunoglobulin G viral capsid antigen; IS, immunosuppression; LT, liver transplantation; NS, not significant; VGCV, valganciclovir.
Boxes: significant factors in the multivariate analysis.
Median age at LT (years)
IgG VCA pre-LT, negative (n)
Initial IS (n)
Maintenance IS (n)
IS at onset of EBV infection (n)
Median time post-LT when EBV started (months)
Symptoms when EBV started (n)
Antiviral treatment before VGCV (n)
Short VGCV course prior to long treatment (n)
Median time from start of EBV to start of VGCV (months)
Symptoms of EBV infection at the start of valgancyclovir were more frequent in the group of responders, almost reaching significance in the multivariate analysis (P = 0.08). Together with a longer time after transplantation (P = 0.03), the absence of infections during the observation period was a main characteristic of responders (P = 0.038) in the multivariate analysis. Tacrolimus levels were lower in children who had some episode of infection in comparison with children without infections. Liver function was not different between responders and nonresponders at the start of long treatment. Liver dysfunction was not considered to be caused by EBV at baseline except in 1 case. However, the evolution of ALT levels in responders showed a significant decrease during treatment that was not seen in nonresponders (Table 6).
Table 6. Status at the End of the Study Period in Children Receiving Long Valganciclovir Treatment (Levels: Mean and Standard Deviation)
The results of this study of VGCV treatment in liver-transplanted children showing detectable EBV-DNA in their blood must be interpreted with caution as no control group was considered and the relatively short follow-up precludes us from making definitive conclusions regarding EBV outcomes. However, the data indicate a low rate of new PTLD in a high-risk population observed for 12 months that was not managed with a decrease in the immunosuppressor regime, with few exceptions. Tolerability to the drug was excellent, and no severe adverse effect attributable to VGCV was observed, even in long periods of treatment.
The major role of the host immune response in avoiding the development of PTLD during EBV infection is well known. Therefore, a decrease in calcineurin inhibitors is considered the main point in managing EBV infection after LT. However, the risk of graft rejection has to be balanced, and only high EBV viremia should prompt an immunosuppression decrease. The availability of commercial assays to quantify EBV viremia helps to make this decision. Appropriate function of T-lymphocytes for controlling EBV-driven B-lymphocyte proliferation is not related to a certain level of cyclosporine or tacrolimus. Thus, to optimize the care of these children, complementary assessment of T-lymphocyte function is important,4, 21 and other clinical data of infections or rejection must be taken into consideration.
Longitudinal measurement of the EBV load in peripheral blood detects a substantial proportion of patients displaying a high viral load. In a series of 77 Italian children, 16% of children with reactivation and 41% of children with a primary infection displayed more than 500 genomes/105 peripheral blood mononuclear cells14; other authors found such high levels in 66% of primary infections in a long follow-up after LT. Equivalent high levels (>4000 copies/μg of DNA) were detected in 28% of specimens taken from 18 prospectively studied LT children.21 Some authors have reported high EBV viremia persisting during prolonged periods of time despite lowering of immunosuppression; however, others have observed a rapid response.
Monitoring high viremia and acting in response do not prevent PTLD in all cases.17 A high viral load is usually found in PTLD but lacks specificity, and some children will develop rejection in the period of decreased immunosuppression. In addition, surveillance of EBV infection has shown a high proportion (77%) of children with sustained detection of EBV in their blood.16 This feature poses a risk for developing PTLD and currently has no therapeutic recommendations.
In the present study, most patients were affected by a chronically increased viral load because positive EBV-DNA persisted 17 months (median) after EBV was detected for the first time. As no quantification was available then, the decision was to treat with VGCV without modification of the immunosuppression regime.
The results suggest that VGCV may have had a role in decreasing EBV viremia because negative DNA was achieved in a substantial proportion of patients. However, the main factors associated with achievement of undetectable DNA indicate that the host immune response plays an important role. These factors were a long time elapsing from the moment of transplantation and a lower incidence of intervening infections. Response was not associated with a lower level of immunosuppression, which was estimated by the doses and levels of drugs. The absence of intervening infections during the study period was unrelated to calcineurin inhibitor blood levels; indeed, such levels were lower in children who had infections in comparison with those who did not. The significantly higher rate of negative EBV-DNA during VGCV therapy in children less affected by infections probably reflects the importance of the host immune response.
The prevalence of PTLD in the risk group of children with EBV primary infection or reactivation after LT has been reported to be 17% to 22%, the first year after transplantation being the time of highest risk. The incidence of PTLD in the long term, to parallel the context of our study, was 21.4% (3/14) in 47 months (median) after LT among patients with sustained viral detection after primary infection.16 However, the difference in observation times does not allow a comparison with the 0% to 2.1% incidence of suspected PTLD in VGCV-treated patients.
Prevention of PTLD has alternative approaches. A German retrospective study with renal transplant recipients showed absence of lymphoma during the first posttransplantation year in patients who received a short course of anti-cytomegalovirus immunoglobulin early after transplantation. These results suggest a protective effect of immunoglobulin preparations containing antibodies against EBV proteins.22 Once high EBV-DNA is detected, treatment with anti-CD20 monoclonal antibody (rituximab) has been shown to clear the viral load in recipients of hematopoietic stem cell transplantation. However, recurrence of a high EBV load and PTLD development occur in half of those patients.23
In conclusion, EBV infection creates anxiety in families and physicians because of the risk of PTLD. The current approach applies immunosuppression reduction to those with a high viral load, which is associated with risk but lacks specificity in predicting PTLD. VGCV has been shown to be safe in the current study, and the population of treated children did not develop PTLD; this warrants future trials to confirm a positive effect in the management of EBV infection.