Cytomegalovirus (CMV) remains the most common infection that occurs after liver transplantation (LT) in children (1). Human CMV (HCMV) can cause various diseases affecting different organs, and it may contribute to rejection (2), fungal infections (3), and the risk for the Epstein-Barr virus-related posttransplant lymphoproliferative disorders (4). However, recent advances in preventive strategies for CMV have led to a significant decrease in the incidence of HCMV disease (5).
There have been two major approaches to control CMV infection after LT: universal prophylaxis and preemptive therapy. Universal prophylaxis provides antiviral therapy for subjects with a high risk of developing HCMV disease, such as seronegative recipients receiving seropositive grafts, for certain period of time (e.g., 2–14 weeks) (6, 7). In contrast, a preemptive approach provides antiviral therapy for subjects who have positive results for routine monitoring of CMV antigenemia or polymerase chain reaction (PCR). Previous studies have shown the effectiveness of preemptive therapy for solid organ transplant recipients, primarily in adults (7–10), but little data are available for children after LT (11, 12). Furthermore, no study has evaluated a preemptive therapy for children with the highest risk of developing a CMV infection (e.g., donor (D)+/recipient (R)−). Children are at a higher risk of developing a CMV infection because pediatric organ recipients have a higher chance of being seronegative for CMV compared with adult recipients. Thus, more information is necessary to elucidate the importance of preemptive therapy to prevent and treat HCMV infections and disease.
The pediatric LT program at the National Center for Child Health and Development, Tokyo, is the largest pediatric LT program in Japan. The program has performed a universal preemptive therapy monitoring CMV-pp65 antigenemia weekly for CMV prevention for all recipients regardless of their CMV status since November 2005. We herein report the impact of preemptive therapy on the incidence and clinical outcome of HCMV infection and disease in children after live-donor LT.
We performed 113 live-donor LT at our institution between November 2005 and December 2009, and the clinical course and outcome of these patients were monitored for at least 6 months after LT. The baseline information of the 113 children who received live-donor LT is summarized in Table 1. The median age of the recipients was 16 months (range: 1 month–21 years). The most common indication for live-donor LT was biliary atresia (n=49, 43%), followed by metabolic diseases (n=26, 23%), acute liver failure (n=20, 18%), liver cirrhosis (n=10, 9%), vascular abnormalities (n=5, 4%), and hepatic tumors (n=3, 3%). Eleven patients (9.7%) died after live-donor LT during the first year after LT, and the reasons for their deaths were not directly related to HCMV disease.
CMV Serostatus and CMV-pp65 Antigenemia Positivity
The CMV serostatus of the donors and recipients is presented in Table 1. The median time to become positive for CMV-pp65 antigenemia was 33 days postoperatively (interquartile range [IQR]: 17.5 days, range: −8 to 115 days). Of note, two cases were CMV-pp65 antigenemia positive before LT, and both patients were treated with ganciclovir (GCV) pre and postoperatively. The median CMV-positive cells were 3 of 50,000 cells (IQR: 8/50,000 cells, range: 1–201/50,000 cells). The highest CMV-pp65 antigenemia positivity was observed in 63% (22/35) of patients in the D+/R− group, followed by 38% (20/53) in the D+/R+ group. In the D−/R+ and D−/R− groups, only one recipient in each group was positive for CMV-pp65 antigenemia (Table 1). As expected, the proportion of recipients who remained negative for CMV-pp65 antigenemia for the 6 months after LT was significantly affected by the CMV serostatus of the donors and recipients (Figure 1).
Treatment for CMV-pp65 Antigenemia
During the observational period, 44 (39%) recipients had positive tests for CMV-pp65 antigenemia. Among the 44 patients, 38 (86%) patients received intravenous (IV) GCV as an initial therapy. The median duration of GCV therapy was 14 days (range: 6–24 days). IV foscarnet was given to four (9%) recipients when infection by GCV-resistant strains was suspected after GCV induction therapy; however, no GCV resistant strain was identified by the sequence analyses. GCV was switched to valganciclovir for three (8%) recipients after successful GCV induction therapy when valganciclovir became available in 2009. Neutropenia (absolute neutrophile counts <500/μL) was observed in three (7%) recipients, but their neutropenia resolved after discontinuation of therapy. Retreatment with GCV was required for four (9%) recipients. Of note, six (14%) recipients who had positive CMV-pp65 antigenemia after LT did not require antiviral treatment because repeated CMV-pp65 antigenemia in the same week became indeterminate or negative when the patients' immunosuppressive therapy was decreased. Four patients were treated with IV immunoglobulin to control HCMV disease.
Treatment of Recipients at High Risks for CMV
Among the highest risk group for CMV (D+/R−), 20 of 35 (57%) recipients required GCV due to positivity for CMV-pp65 antigenemia, with a median duration of 14 days (IQR: 3.8 days). Two (6%) patients did not receive GCV because the repeated CMV-pp65 antigenemia evaluation in the same week dropped to within an indeterminate range. In contrast, 15 of 35 (43%) recipients were able to avoid the use of GCV. Similarly, among the patients in the D+/R+ group, 16 of 53 (30%) recipients required GCV therapy for positive CMV-pp65 antigenemia, with a median duration of 14 days (IQR: 3.0 days). Four patients (8%) did not receive GCV because the repeated CMV-pp65 antigenemia evaluation in the same week was negative or within an indeterminate range. In contrast, 37 of 53 (70%) recipients were able to avoid the use of GCV that they would have received if they were on universal prophylaxis.
Comparison Between Recipients Positive for CMV-pp65 Antigenemia and Those Negative for CMV-pp65 Antigenemia
We compared the baseline characteristics of recipients who were positive for CMV-pp65 antigenemia and those who were negative for CMV-pp65 antigenemia (Table 2). No differences were found between the two groups in age or sex. Recipients who received LT for acute liver failure tended to have a higher rate of positive CMV-pp65 antigenemia (70%, 14/20), in contrast, recipients with cholestatic liver diseases tended to have a lower rate of positive CMV-pp65 antigenemia (20%, 10/49). Among patients with positive CMV-pp65 antigenemia, 95% of recipients (42/44) were in either D+/R− or D+/R+ groups.
All patients were successfully treated with GCV for 2 to 4 weeks, and their symptoms and signs resolved and CMV-pp65 antigenemia became negative when the treatment was completed. Overall, only 6 of 113 (5%) recipients developed HCMV disease including CMV syndrome (n=3), hepatitis (n=1), enterocolitis (n=1), and pneumonitis (n=1). No complications or sequelae were noted in these four recipients with HCMV disease. Seven (6%) recipients were retreated with GCV when CMV-pp65 antigenemia became positive after the completion of the initial GCV therapy.
Acute rejection was observed in 39 (35%) recipients after LT. The median duration from LT to the time when the diagnosis of acute rejection was made was 12 days (range: 6–50 days) postoperatively. No significant difference was found between the time for acute rejection and the CMV serostatus; 11.5 days (range: 7–50 days) in the D+/R− group, 12 days (range: 6–37 days) in the D+/R+ group, 22.5 days (range: 13–32 days) in the D−/R+ group, and 16 days (range: 8–30 days) in the D−/R− group (P=0.39). In addition, no association was found between the incidence of acute rejection and CMV-pp65 antigenemia; among 39 patients positive for CMV-pp65 antigenemia, 17 patients (44%) developed acute rejection, whereas 27 of 74 (36%) of the patients negative for CMV-pp65 antigenemia after LT developed acute rejection (P=0.46). Only two patients (both were D+/R+) developed acute rejection subsequent to positive CMV-pp65 antigenemia.
Overall, 11 of 113 (9.7%) patients died after the live-donor LT due to sepsis (n=5, 45%), graft failure (n=5, 45%), and tumor recurrence (n=1, 10%). Among children with graft failure, no recipients had positive CMV-pp65 antigenemia after LT. Therefore, no recipients died due to HCMV infection or disease.
This is the first study to demonstrate that a universal preemptive therapy was effective to prevent the development of HCMV disease in children after LT, notably in children with the highest risk for HCMV disease (e.g., D+/R−). This universal approach was successful to target therapy to the recipients who developed early evidence of CMV reactivation and thereby decrease drug costs and toxicity for patients who do not need the treatments.
Reviewing the current guidelines proposed by the Transplantation Society International CMV Consensus Group (13), the administration of GCV for 12 weeks is the recommended regimen for any D+/R− transplants and R+ liver transplants, and no preemptive approach is recommended. The only recommendation by some experts for a preemptive approach is 2 to 4 weeks of prophylaxis, followed by preemptive therapy as an alternative therapy (14). No current recommendation is available with respect to a universal preemptive approach for pediatric LT recipients (15).
The major advantage of preemptive therapy is to limit the periods of antiviral treatment in children in whom antivirals are necessary for preventing HCMV disease. Gerna et al. (12) demonstrated equal efficacy of universal prophylaxis and preemptive therapy in 21 pediatric liver transplant recipients for preventing HCMV diseases, although the duration of antiviral therapy was significantly shorter in the preemptive group compared with the universal prophylaxis group (18 vs. 30 days). Madan et al. (11) demonstrated the usefulness of a combination of a short course of antiviral prophylaxis (≥14 days) for high-risk patients (D+/R−) and preemptive monthly CMV PCR monitoring in children after LT. With this approach, 12 subjects (9.8%) developed HCMV disease, and there were no mortalities secondary to CMV. This approach spared a total of 39% of the patients from the use of antiviral medications beyond their initial postoperative prophylactic period. In this study, we were able to avoid the use of GCV in 13 of 35 (37%) recipients in the D+/R− group and 37 of 53 (70%) recipients in the D+/R+ group. In addition, the long-term use of GCV or valganciclovir for the treatment or prevention of CMV in children is a concern. The major toxicities in patients receiving GCV are bone marrow suppression and renal toxicity (16). Additionally, animal studies have suggested that GCV may cause gonadal toxicity (17) or carcinogenicity (18). Finally, the long-term use of antivirals is costly and may increase the chance of developing GCV-resistant virus strains after prolonged exposure (19). The major advantages of universal prophylaxis are that there is no need for virologic monitoring for CMV infection after LT. Regular monitoring of CMV-pp65 antigenemia or CMV-DNA PCR is costly and labor intensive; however, a few studies suggested that the cost of the preemptive approach is actually lower than the cost of universal prophylaxis in adults (20, 21). In preemptive approach, a delayed response to the test results may delay the treatment for patients, thus resulting in increased diseases and higher costs of treatment.
Given the challenge of characterizing the recipient serostatus in those less than 18 months of age, any CMV seropositive recipient who is less than 18 months of age should be considered to be seronegative, as maternal antibodies may account for this finding. Therefore, CMV urine culture or nucleic acid amplification testing for all seropositive recipients is recommended (13). However, negative tests do not exclude the exposure to CMV, because negative tests may result from the intermittent shedding of the virus. In this study, not all recipients less than 18 months of age with positive CMV antibodies were tested for CMV-pp65 antigenemia at baseline; therefore, we analyzed the data solely using their CMV serostatus. In this study, 28 (53%) patients among the 53 D+/R+ patients were less than 18 months of age, and we assumed that the majority of patients were seronegative (D+/R−). This assumption favors the use of a preemptive approach because it allows for the inclusion of more recipients with the highest risk (D+/R−) of developing CMV reactivation.
Oral valganciclovir was used for three patients after successful GCV induction therapy. Valganciclovir has excellent oral bioavailability, resulting in systemic drug levels that are comparable with IV GCV in patients after LT (22). The oral bioavailability of valganciclovir in neonates and infants has also been confirmed by other studies (23). However, the current guideline does not recommend preemptive therapy using oral valganciclovir for LT recipients (15) due to the absence of adequate pharmacokinetics and efficacy data. In addition, one study indicated that the incidence of HCMV disease was higher in the patients receiving valganciclovir compared with GCV in the subgroup analysis of CMV D+/R− solid organ transplant recipients (24). Because CMV infection usually becomes apparent approximately 3 weeks after LT, the majority of recipients can tolerate oral valganciclovir by that time, and the option of using oral medication is practical and reasonable. Further studies to compare these two medications are needed to demonstrate the effectiveness of valganciclovir in children after LT.
There are a few limitations to this study. First, because of the study design, we had a limited number of recipients after LT and were unable to directly compare universal prophylaxis and a universal preemptive therapy for HCMV disease in children. Second, we have used the cutoff for a positive CMV-pp65 antigenemia as ≥5/50,000 cells, but this is not the standardized value. Further studies are necessary to determine the optimal cutoff value of CMV-pp65 antigenemia and to compare it with the results of CMV-DNA PCR. Third, this study included only subjects who received live-donor LT, which requires less immunosuppression compared with cadaveric LT; therefore, caution is required when this approach is used for the patients who receive a cadaver liver for the LT. Finally, judicious use of IV sodium was not performed in four recipients. Sodium was initiated when CMV-pp65 antigenemia persistently increased after more than 2-week course of GCV induction therapy; however, the event of delayed antigenemia decrease after appropriate treatment with GCV without GCV resistant virus has been clearly documented (25, 26).
In conclusion, a universal preemptive therapy in children after live-donor LT was safe and successful at the largest pediatric LT center in Japan. A further prospective study is necessary to identify the best approach to preventing and treating HCMV disease in children after LT.
MATERIALS AND METHODS
The study subjects comprised 113 children who received live-donor LT at the National Center for Child Health and Development in Tokyo, the largest pediatric LT center in Japan, between November 2005 and December 2009. This study was a retrospective evaluation of the standard protocol, which has been performed at our institution. The following information was extracted from the medical record database; age, gender, and HCMV serostatus of the donors and recipients, baseline diseases, reasons for LT, the first postoperative date positive for HCMV-pp65 antigenemia, antiviral treatment and its duration, HCMV disease, clinical outcome, and mortality.
Monitoring of CMV-pp65 Antigenemia
CMV-pp65 antigenemia was measured by a direct immunoperoxidase technique using a horseradish peroxydase-conjugated F(ab′)2 fragment of human monoclonal (humab C7), designated horseradish peroxydase-C7 (27). The measurements were performed weekly for the first 3 months postoperatively, while the recipients were hospitalized, then monthly in an outpatient setting until 6 months after LT. We determined that the presence of more than 5 CMV antigen-positive cells/50,000 cells indicated that the patient was positive, and 1 to 5 positive cells/50,000 cells were considered to be an indeterminate result. Once the result was positive, a repeated CMV-pp65 antigenemia was performed within the same week in some of the patients, particularly when the value was close to the cutoff. The test results were returned to the primary care physicians less than 48 hr after the sample was taken and then were used to select the patients' treatment strategies.
Immunosuppressive and Antiviral Therapies
After LT, standard immunosuppression consisted of corticosteroids and tacrolimus. The corticosteroid was started intraoperatively (10 mg/kg/dose) and continued with tapering for the first 3 months after LT (1.0 mg/kg/day IV [days 1–3], 0.5 mg/kg/day IV [days 4–6], 0.3 mg/kg/day IV [day 7], 0.3 mg/kg/day orally (PO) [days 8–28], 0.1 mg/kg/day PO [days 29–90]). Tacrolimus was also started 1 day before surgery, and the dose was adjusted to maintain a trough level of 10 to 15 mg/L for the first 2 weeks, followed by 8 to 10 mg/L (days 15–28 after LT), 6 to 8 mg/L (days 29 to 90), and 4 to 6 mg/L (after day 91). If CMV-pp65 antigenemia was noted to be positive, then a dosage of tacrolimus was reduced down to 75% of the regular dosage, and IV GCV (5 mg/kg/dose, every 12 hr) was initiated for the first 2 weeks, followed by a maintenance dose of IV GCV (5 mg/kg/dose, every 24 hr), and the treatment continued until CMV-pp65 antigenemia became negative. If CMV-pp65 antigenemia was indeterminate, immunosuppressive therapy was reduced as far as possible, and CMV-pp65 antigenemia was reevaluated twice a week. We also switched from GCV to valganciclovir (16 mg/kg/dose PO every 12 hr, available from August 2008), which is an oral tablet that was crushed and dissolved in syrup and used as a maintenance therapy when recipients (1) were able to tolerate to oral medication and (2) demonstrated a trend of decrease in CMV-pp65 antigenemia after GCV induction therapy. The dosing of valganciclovir in maintenance was based on the study performed for controlling HCMV disease in infants (23, 28). Sodium (100 mg/kg/dose, every 24 hr) was empirically used when CMV-pp65 antigenemia did not improve after decreasing the dose of immunosuppressants and administering a 2- to 3-week course of GCV induction therapy. When sodium was empirically started for possible GCV-resistant CMV, the UL97 genetic sequencing or by an analysis of other known genetic sequences related to resistance was examined (29). IV immunoglobulin was used when HCMV disease was not able to be controlled by GCV or sodium.
CMV Serostatus of the Donor and Recipient
The CMV serostatus of donors and recipients was determined by the CMV IgG determined by preoperative enzyme immunoassay. Patients were defined as seropositive if the antibody titer was ≥1:4 and negative if the antibody titer was less than 1:4.
Diagnosis of Acute Rejection and Treatment
Acute rejection was clinically diagnosed on the bases of an increase in liver enzymes (more than three times the upper limit of normal range or an increase of more than 50% over the previous record) with or without histological evidence. Histological diagnosis and grading of acute rejection were performed according to the standardized criteria (30). Graft biopsies were considered when other etiologies other than rejection were suspected in clinical course. All the rejection episodes were treated with a corticosteroid bolus injection. Steroid-resistant rejection was treated with additional immunosuppressants, such as mycophenolate mofetil.
The primary endpoint of this study was the proportion of recipients with event-free survival 6 months after LT. Events were defined as the occurrence of CMV infection based on the CMV-pp65 antigenemia, active HCMV disease, or death from any cause. HCMV disease was defined when recipients developed the following diseases with positive CMV-pp65 antigenemia; CMV syndrome without any evidence of acute rejection, enterocolitis, pneumonitis, and meningitis.
We used the SPSS 15.0 software package (SPSS, Inc., Chicago, IL) for all analyses. The χ2 test and Fisher's exact test were used to compare categorical variables between the groups. The Mann-Whitney U test and the Kruskal-Wallis test were used to compare continuous variables between two groups and more than three groups, respectively. P values were calculated as two trailed and were considered to be significant if P less than 0.05.
The authors acknowledge Dr. Naoki Inoue at the National Institute of Infectious Diseases for sequencing CMV genes and Yayoi Nakasato, the coordinating nurse at the National Center for Child Health and Development, for dedication to the families and ensuring that the children received the best treatment possible.
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