Epstein‐Barr viral load monitoring for diagnosing post‐transplant lymphoproliferative disorder in pediatric liver transplant recipients

This study aimed to investigate the incidence of PTLD in pediatric liver transplant recipients and the risk factors for the development of PTLD. We also determined clinically useful quantitative EBV PCR parameters for aiding in the diagnosis of EBV‐associated PTLD in the pediatric liver transplant recipients at our institute. We reviewed children < 18 years old who had undergone liver transplantations and quantitative analysis of whole blood EBV load at our institute from January 2006 to March 2015. A total of 142 liver transplant recipients were included, and their median age was 1.5 years. Clinically significant high‐level EBV DNAemia ≥ 10 000 copies/mL at least twice was observed in 53.5% and PTLD occurred in 9.9%. Among PTLD group, graft failure and mortality rate were as high as 21.4% and 14.3%, respectively. Deceased donor, presence of high‐level EBV DNAemia, and primary CMV infection following transplant were associated with an increased risk for PTLD in the multivariate analysis. The peak titer at 10 875 copies/mL could be used as a cutoff value with a sensitivity of 92.9% and a specificity of 37.9%; the rate of increase in EBV load suggested a sensitivity of 64.3% and a specificity of 70.9% at the cutoff value of 44 000 copies/mL/week. In conclusion, the incidence of PTLD following liver transplant in children was as high as 10%. PTLD is associated with significant morbidity and mortality. Close monitoring of EBV DNAemia is crucial for the early diagnosis and proper treatment of PTLD in pediatric liver transplant recipients.     

Epidemiology and outcome of chronic high Epstein‐Barr viral load carriage in pediatric kidney transplant recipients

The development of EBV infection and PTLD is normally associated with a high EBV viral load in peripheral blood. Observations have previously identified existence of a CHL carrier state that demonstrated variable outcomes based upon the organ which was transplanted. Data defining the incidence and outcome of CHL in pediatric KTx are not well described. The charts of children undergoing isolated KTx at Children's Hospital of Pittsburgh between January 2000 and December 2014 were retrospectively reviewed. EBV loads in the peripheral blood were routinely measured as part of surveillance protocols at our center. CHL was defined as the presence of high load for >50% of samples for ≥6 months. PTLD was defined histologically using WHO definitions. Of 188 isolated KTx recipients, we identified a total of 16 (8%) children who developed CHL carrier state. No patient developed EBV‐driven late‐onset PTLD. Age at the time of KTx was significantly lower in the CHL group (median 3.9 years, interquartile range: IQR 2.9‐6.6, P = .0004). Children in the CHL group were more likely to be EBV‐seronegative prior to KTx (94%, 15/16), compared to the UVL and LVL groups (55% and 50%, respectively, P < .002). The median duration of CHL carrier state was 20 months (IQR 10.7‐35.8). Fifteen of the 16 CHL carriers experienced spontaneous resolution of CHL carrier state. Children in the CHL group were younger at the time of primary EBV infection (P = .023). Finally, antiviral medication was not effective in either preventing or decreasing the EBV viral load in blood (P = .84). Overall incidence of late‐onset PTLD is very low compared to heart and intestinal transplant, even though KTx recipients can develop CHL carrier state. The CHL carriers in KTx recipients were EBV‐seronegative prior to transplant and were younger both at the time of KTx and at the time of primary EBV infection compared to those in the UVL and HVL groups. Antivirals did not prevent EBV infection or decrease EBV viral loads.     

Long‐term monitoring of Epstein–Barr virus DNA load and humoral parameter abnormalities in pediatric liver transplant recipients before development of malignancy

Gregorek H, Jankowska I, Dzier?anowska‐Fangrat K, Teisseyre J, Sawicka A, Kasztelewicz B, Paw?owska J. Long‐term monitoring of Epstein–Barr virus DNA load and humoral parameter abnormalities in pediatric liver transplant recipients before development of malignancy.
Pediatr Transplantation 2010: 14:629–635. © 2010 John Wiley & Sons A/S. Abstract: EBV loads and abnormalities of humoral responses were monitored in 51 pediatric liver transplant recipients as a proposed non‐invasive laboratory tool for early detection of changes preceding severe clinical complications. EBV DNA load, concentrations of IgM, IgG, IgA, and monoclonal proteins were determined in each blood sample. EBV DNA was detected in 70.6% of the children, dysgammaglobulinemia of one or more Ig isotype was present in 41.2% of them. MG detected in 43.1% of patients correlated with the presence of EBV DNA (p = 0.003) and was usually preceded by hypergammaglobulinemia. The median maximum EBV load was significantly higher in EBV DNA+/MG+ patients than in EBV DNA+/MG‐ patients (p = 0.04), although there was no correlation between current viral load and appearance of MG. Four of 15 EBV DNA‐negative patients developed MG, preceded by hypergammaglobulinemia in two. Minimization or cessation of immunosuppression in 42 patients, in whom abnormal biomarkers and/or clinical symptoms raised suspicion of disease progression, permitted complete resolution of abnormalities in all but one patient who developed B‐NHL and died. Simultaneous monitoring of protein profiles and EBV DNA load together with thorough physical evaluation of children after LTx is important for early implementation of suitable preemptive therapy.     

No effect of treatment with intravenous ganciclovir on Epstein‐Barr virus viremia demonstrated after pediatric liver transplantation

EBV after pediatric LT is a risk factor for PTLD. We wanted to evaluate the effect of intravenous ganciclovir on EBV viremia and to identify risk factors for chronic EBV viremia. All pediatric patients who underwent LT in Norway from 2002 until 2015 were reviewed. Twenty‐two of 38 patients with viremia were treated with intravenous ganciclovir for a median of 22 (21‐38) days. Treated and untreated patients were not different with respect to EBV seroconversion prior to transplantation or age at transplantation, but treated patients had significantly earlier viremia after transplantation (P=.005). There was no difference in the proportion of patients with reduction in virus load in patients treated with ganciclovir compared to untreated patients at 8 weeks. After 1 year, five of 19 patients treated with ganciclovir and six of 14 untreated patients had reduced virus load compared to start of viremia (P=.27). In conclusion, treatment with intravenous ganciclovir did not change the proportion of patients with reduction in EBV load at 8 weeks and 1 year after viremia. Younger age at transplantation, short time from transplantation to viremia, and lack of EBV seroconversion prior to transplantation were significant predictors of chronic EBV viremia.     

Chronic high Epstein–Barr viral load carriage in pediatric small bowel transplant recipients

Lau AH, Soltys K, Sindhi RK, Bond G, Mazariegos GV, Green M. Chronic high Epstein–Barr viral load carriage in pediatric small bowel transplant recipients.
Pediatr Transplantation 2010: 14:549–553. © 2010 John Wiley & Sons A/S. Abstract: The development of EBV infection and PTLD is normally associated with a high EBV load in peripheral blood. Often, children undergoing primary or reactivation of EBV infection subsequent to ITx will have chronically elevated EBV loads. To better understand this phenomenon and its consequences, we retrospectively reviewed the records of children who underwent ITx (either isolated or part of multivisceral transplantation) at our center from 1992 to 2007, to identify chronic high EBV load carriers in this population. CHL state was defined as the presence of high load for >50% of samples for greater than or equal to six months following either asymptomatic infection or complete clinical resolution of EBV disease/PTLD. Thirty‐five CHL carriers were identified from our patient population. Pretransplant serologies were available on 34 of these patients: 17 were EBV negative and 17 seropositive; one had unknown EBV serostatus prior to transplant. Seven of the 17 seronegative patients developed their CHL carrier state at the time of their primary EBV infection. Thirteen of the 35 (37%) HLC patients developed EBV disease after meeting the definition of high‐load carrier states. EBV‐related diseases developing in CHL carriers included EBV adenitis (n = 1), EBV enteritis (n = 7), PTLD (n = 4), and EBV+ spindle cell tumor (n = 1). Disease was seen in 7/17 of the seronegative (one PTLD) and 6/17 of the seropositive patients (three PTLD). Thirteen of 35 patients (37%) resolved their CHL state without apparent sequelae while nine remain asymptomatic CHL carriers. Three children have had more than one episode of CHL. These data provide important information about the outcome of chronic EBV high‐load carriage in pediatric intestinal transplant recipients.     

Post‐transplant lymphoproliferative disorder in pediatric intestinal transplant recipients: A literature review

Intestinal transplantation is a successful treatment for children with intestinal failure, but has many potential complications. PTLD, a clinically and histologically diverse malignancy, occurs frequently after intestinal transplantation and can be fatal. The management of this disease is particularly challenging. The rejection‐prone intestinal allograft requires high levels of immunosuppression, a precondition for PTLD. While EBV infection clearly plays a role in disease pathogenesis, the relatively naïve immune system of children is another likely contributor. As a result, pediatric intestine recipients have a higher risk of developing PTLD than other solid organ recipients. Other risk factors for disease development such as molecular and genomic changes that precipitate malignant transformation are not fully understood, especially among children. Studies on adults have started to describe the molecular pathogenesis of PTLD, but the genomic landscape of the malignancy remains largely undefined in pediatric intestinal transplant patients. In this review, we describe what is known about PTLD in pediatric patients after intestinal transplant and highlight current knowledge gaps to better direct future investigations in the pediatric population.     

The efficacy of valganciclovir for prevention of infections with cytomegalovirus and Epstein‐Barr virus after kidney transplant in children

This study evaluated the efficacy of prophylactic ValGCV in preventing CMV and EBV infections in a single‐center pediatric kidney transplant population (2008‐2014). Therapy duration was determined according to donor/recipient serostatus. EBV monitoring was performed using monthly plasma PCR for 18 months post‐transplant and for CMV, monthly for 6 months after prophylaxis cessation. Data were collected on 35 children, median age 10.6 years. There were 15 (42.9%) and 11 (31.4%) recipients seronegative for CMV or EBV, respectively, who received a kidney from a seropositive donor. Prophylaxis was ceased by 6 months in 24 (69%), between seven and 13 months in 10 (29%) children. Fourteen (40%) and eight (23%) children experienced CMV and EBV DNAemia, respectively. Ten of the 14 (71%) episodes of CMV DNAemia occurred in the first 6 months following cessation of prophylaxis. Shorter prophylaxis was associated with increased CMV DNAemia (P = 0.044). There was an inverse correlation between adjusted ValGCV dose and EBV incidence/timing. Neutropenia was more common if ValGCV dosage was ≥10% of the dose predicted (by BSA and creatinine clearance). ValGCV prevents CMV and may modify EBV infection risk. Frequent dosing adjustment for BSA and creatinine clearance is required to optimize safety and efficacy.     

Epstein–Barr virus‐associated smooth muscle tumors in children following solid organ transplantation: A review

EBV‐SMT are a rare entity following organ transplantation. Given the rarity of the tumor, there is no standard approach to diagnosis and treatment. A literature search identified 28 reported cases of EBV‐SMT in addition to our own experience with one case. The aim of this review is to summarize the existing data regarding pathogenesis, diagnosis, and treatment.     

Post‐transplant lymphoproliferative disorders and other malignancies after pediatric intestinal transplantation: Incidence,clinical features and outcome

PTLDs are a well‐recognized and potentially fatal complication after intestinal transplantation. We analyzed the incidence, clinical features, and outcome in a 63 intestinal transplantation series performed in our unit between October 1999 and July 2011. Types of graft included ISB (n = 23), LSB (n = 20), and MV (n = 20). Patients were categorized into three groups of immunosuppression: I (n = 43) received basiliximab, tacrolimus, and steroids; II (n = 11) thymoglobulin and tacrolimus, and III (n = 9) alemtuzumab and tacrolimus. EBV status was serially assessed. All PTLD cases were biopsied to establish histopathological diagnosis. The incidence of PTLD was 14.2% (9/63). Median onset of PTLD after transplant was four months (range: 0.5–28), within first postoperative year in 6 (66.6%) patients. Fever was the most common symptom. Graft removal was needed in four patients (44%). The patient survival rate was 66.6% (6/9). We have not found any association between PTLD and immunosuppression regimen or transplant type. However, there was a statistical association with EBV active infection.     

Impact of melphalan dose during reduced‐intensity conditioning on engraftment of cord blood transplantation for chronic Epstein‐Barr virus‐associated T or NK cell lymphoproliferative diseases

The rejection rate in cord blood transplants for chronic Epstein‐Bar virus‐associated T or natural killer cell lymphoproliferative diseases using our standard reduced‐intensity conditioning “LPAM140 regimen,” which includes fludarabine, melphalan (LPAM), etoposide, and antithymocyte globulin, has been high. To ensure better engraftment, we increased the LPAM dose to 210 mg/m² (“LPAM210 regimen”). Patient data (n = 22; LPAM140, n = 7; LPAM210, n = 15) were analyzed retrospectively. The engraftment rate after the LPAM210 regimen (100.0%) was significantly higher than that after the LPAM140 regimen (57.1%; P = .002). Fludarabine combined with melphalan (210 mg/m²) had a favorable impact on engraftment.     

Hypogammaglobulinemia in pediatric liver transplant recipients

Hypogammaglobulinemia has been reported after solid organ transplantation in adults, however immunoglobulin replacement [intravenous immunoglobulins (IVIG)] is only necessary in a minority of affected patients. We here present three pediatric patients with severe post-transplant hypogammaglobulinemia following liver transplantation (LTx) receiving a cyclosporine-based standard immunosuppression. Patient 1 was transplanted at the age of 10 months for biliary atresia. Eight weeks post-Ltx the serum IgG was 1.7 g/L. Patient 2 was transplanted at the age of 12 yr for acute liver failure. Four weeks post-Ltx the IgG dropped to 2.6 g/L. Patient 3 was transplanted at the age of 4 months for biliary atresia. Ten weeks post-Ltx severe hypogammaglobulinemia (IgG < 1.48 g/L) was diagnosed during a severe infectious complication. Patients 1 and 3 received a steroid bolus therapy for acute graft rejection. All patients had normal IgG concentrations prior to Ltx and lymphocyte subsets were post-operatively in the normal range. There was no extensive loss of protein by ascites. IGIV were replaced in the three patients monthly without further complications. In two of the patients (1 and 3) IVIG therapy was discontinued 8 and 10 months after Ltx when the immunosuppression has been reduced and serum IgG concentrations were found in the normal range without further immunoglobulin replacement. Severe hypogammaglobulinemia is a rare phenomenon following pediatric LTx and seems to be mainly caused by immunosuppressive drugs, however, the exact underlying mechanisms are unclear. A screening for hypogammaglobulinemia is useful after pediatric LTx, especially in patients with an intensified immunosuppression. Moreover, further immunologic research in affected patients is necessary.     

Simultaneous or sequential gastrectomy in pediatric liver transplant recipients

The association between LT and gastrectomy is not common. Only two studies reported the gastrectomy/LT association in children. Here, we report three children who underwent LT who required a concomitant or sequential gastrectomy for different reasons. Patient 1, a 16‐yr‐old boy, during the LT, underwent a partial gastrectomy due to extensive injury to the duodenum. He had a previous and unusual portoenterostomy performed in the duodenum. Bowel reconstruction was performed using an intestinal loop that was first used for the bilio‐enteric anastomosis and then connected to the gastric stump. Patient 2, a 22‐month‐old female child, underwent a partial gastrectomy with a Roux‐en‐Y reconstruction during a retransplantation. She had a large perforated gastric ulcer blocked by the allograft liver. Patient 3, a 26‐month‐old male child, five yr after living donor LT, was submitted to a partial gastrectomy because of gastric outlet obstruction. The histopathology was compatible with eosinophilic gastritis. The association between LT and gastrectomy in the pediatric population is extremely rare. Appropriate knowledge of the previous transplantation technique is very important. Further studies are required to assess the outcomes of the different types of gastric reconstruction in pediatric recipients.     

The use of everolimus in pediatric liver transplant recipients: first experience in a single center

The role of mTOR inhibitors, such as EVL, has not been established for pediatric liver transplant recipients up to now, although data from adult solid organ graft transplantation are very promising. Major complications following pediatric liver transplantation in the long-term course include chronic graft rejection and CNI-derived nephrotoxicity. The purpose of our study was to report first results using EVL as a rescue therapy in pediatric liver transplant recipients for the following indications: chronic graft dysfunction n=12, suspected CNI toxicity n=3, hepatoblastoma n=2, and recurrence of primary sclerosing cholangitis post-Ltx n=1. Four patients with chronic graft dysfunction developed completely normal liver function tests using EVL, six patients showed partial improvement, and two patients did not respond at all. One patient with CNI-induced nephropathy showed a slightly improved GFR. Both patients with hepatoblastoma did not develop any metastasis post-Ltx. First experience with EVL in pediatric liver transplant recipients shows promising results in patients with chronic graft failure when standard immunosuppression has failed. The future role of EVL in immunosuppressive protocols for children post-Ltx has to be proven by controlled clinical trials.     

Monitoring of Epstein–Barr virus load and antibody in pediatric renal transplant patients

Background: Epstein–Barr virus (EBV) infection can lead to life-threatening post-transplant lymphoproliferative disorder (PTLD). The aim of the present study was to establish EBV monitoring methods to prevent PTLD.
Methods: EBV-DNA load was investigated, using real-time polymerase chain reaction (PCR) and anti-EBV antibody titers, in peripheral blood mononuclear cells of 21 renal transplant patients (seven recipients who were EBV-seronegative, R[−]; 14 who were EBV-seropositive, R[+]) before grafting. The mean age at entry and the mean follow-up period was 7.8 years of age (range, 3.3–12.0 years) and 1.8 years (range, 0.4–4.0 years), respectively, in the R(−) group, and 12.5 years of age (range, 3.9–17.7 years) and 3.8 years (range, 0.8–8.2 years) in the R(+) group, respectively.
Results: The mean maximum load of the EBV genome was 1071 copies/μg DNA (range, 106–20700 copies/μg DNA) in the R(−) group, and 61 copies/μg DNA (range, <50–552 copies/μg DNA) in the R(+) group. During follow up no patient in the R(+) group had any noticeable symptoms that could be related to EBV, but three recipients in the R(−) group developed EBV-related symptoms including adenoid hypertrophy, cervical lymphadenopathy, and PTLD (B cell lymphoma), in one patient each. In the R(−) group the first leukocyte-associated viremia was detected at 30–180 days, and seroconversion at 43–266 days after transplantation.
Conclusions: Viral DNA detection using PCR is a useful tool for EBV surveillance, but the maximum EBV load was not markedly elevated (2474 copies/μg DNA) in a patient with PTLD. Therefore, EBV surveillance using only monitoring of EBV load in peripheral leukocyte may be insufficient. Histology may therefore be necessary to accurately diagnose PTLD.