Epstein-Barr Viral Load Before a Liver Transplant in Children With Chronic Liver Disease

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1 ArtIcle Epstein-Barr Viral Load Before a Liver Transplant in Children With Chronic Liver Disease Nader Shakibazad, 1 Naser Honar, 1 Seyed Mohsen Dehghani, 2 Abdolvahab Alborzi 3 Abstract Objectives: Many children with chronic liver disease require a liver transplant. These patients are prone to various infections, including Epstein-Barr virus infection. This study sought to measure the Epstein- Barr viral load by polymerase chain reaction before a liver transplant. Materials and Methods: This cross-sectional study was done at the Shiraz University of Medical Sciences, Shiraz, Iran, in All patients were aged younger than 18 years with chronic liver disease and were candidates for a liver transplant at the Shiraz Nemazee Hospital Organ Transplant Center. They had been investigated regarding their demographic characteristics, underlying disease, laboratory findings, and Epstein-Barr viral load by real-time TaqMan polymerase chain reaction. Results: Ninety-eight patients were studied and the mean age was 6.5 ± 5.9 years. Cryptogenic cirrhosis was the most-prevalent reason for liver transplant, and the death rate before a transplant was 15%. Among the study subjects, 6 had measurable Epstein-Barr viral load by polymerase chain reaction before the transplant, and 4 of them had considerably higher Epstein-Barr viral loads (more than 1000 copies/ml). From the 1 Department of Pediatrics and the 2 Shiraz Transplant Research Center, Shiraz University of Medical Sciences, School of Medicine, Nemazee Teaching Hospital; and the 3 Professor Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran Acknowledgements: This article was adopted from Nader Shakibazad s thesis for Specialization in Pediatrics. The authors would like to thank the Research Vice-Chancellor of Shiraz University of Medical Sciences, Shiraz, Iran, for financially supporting the study with grant number The Research Improvement Center of Shiraz University of Medical Sciences, and Ms. A. Keivanshekouh are also appreciated for improving the use of English in the manuscript. The authors have no conflicts of interest to declare. Corresponding author: Nader Shakibazad, Department of Pediatrics, Nemazee Hospital, Shiraz University of Medical Sciences, Shiraz, Iran Phone/Fax: Cellular Phone: shakibn@sums.ac.ir Experimental and Clinical Transplantation (2014) 6: Conclusions: With respect to the close prevalence of posttransplant lymphoproliferative disease (6%) and the high Epstein-Barr viral load in the patients before a transplant (4%), high pretransplant Epstein-Barr viral load can be considered a risk factor for posttransplant lymphoproliferative disorder. Key words: Chronic liver disease, Liver transplant, Epstein-Barr virus. Introduction Liver transplant is the standard treatment of choice for end-stage liver diseases. 1 Recently, taking care of children with advanced liver diseases has focused mainly on finding a liver for transplant; however, today, more attention is paid to long-term follow-up of patients, reduction of adverse effects of immunosuppressive drugs, and reaching normal growth. 1 Today, measuring Epstein-Barr Virus (EBV) DNA in the serum is an important means for diagnosing EBV-related diseases because EBV is a latent virus and can be reactivated by immune suppression after transplant. High levels of EBV in the blood (demonstrated by real-time polymerase chain reaction) has a close relation with posttransplant lymphoproliferative disorder (PTLD) and can be considerably reduced by appropriate treatment. 2 Because most children with chronic liver diseases finally require a liver transplant, 3 they are susceptible to various infections, including infection with EBV that can lead to different problems (eg, PTLD), which are accompanied by high death rates. 4,5 Thus, preventing PTLD is critical in reducing mortality rates. Until now, several studies have serially measured EBV viral load after transplant and in case of high EBV viral load, have used treatment strategies to prevent PTLD. 6,7 Copyright Başkent University 2014 Printed in Turkey. All Rights Reserved. DOI: /ect

2 Nader Shakibazad et al/experimental and Clinical Transplantation (2014) 6: Although various studies have checked the serology of EBV before transplant, 5-7 no studies have yet measured EBV viral load using polymerase chain reaction before transplant. Therefore, the present study sought to investigate EBV viral loads using polymerase chain reaction before transplant; so that in cases where there is a high viral load and a determined relation with PTLD preventive measures should be started earlier. Materials and Methods This cross-sectional study was conducted at the Shiraz University of Medical Sciences, Shiraz, Iran, in The study subjects included all inpatients and outpatients who were younger than 18 years, who had consecutively been referred to the Organ Transplant Center of Nemazee Hospital in Shiraz, Iran. Subjects were assessed for their demographic information, underlying diseases, laboratory findings, Pediatric End-Stage Liver Disease/Model for End-Stage Liver Disease (PELD/MELD), Child-Turcotte-Pugh (CTP) scores, and EBV viral loads in serum. Of note, patients who required a liver transplant because of acute liver failure or causes other than chronic liver diseases, including Crigler Najjar syndrome, familial hypercholesterolemia, and primary hyperoxaluria were excluded from the study. Patients PELD scores (< 12 years old) and MELD scores ( 12 years old) also were computed before the transplant. According to PELD/MELD scores, patients were divided into 5 groups of PELD/MELD scores < 10, 10 to 19, 20 to 29, 30 to 39, and more than 40. epstein-barr viral load measurement Written, informed consent was obtained from the patients or their parents before the study. The study was approved by the Local Ethics Committee of Shiraz University of Medical Sciences. All protocols conform to the ethical guidelines of the 1975 Helsinki Declaration. A 5-mL sample of venous blood withdrawn from the participants was sent to Professor Alborzi Clinical Microbiology Research Center, Nemazee Hospital, Shiraz, Iran, where EBV viral load was measured. It must be mentioned that measuring the EBV viral load was done with the patients sera via the TaqMan real-time polymerase chain reaction assay. In various studies, according to the serology of EBV before transplant, various cutoff values of EBV viral load (range, copies/ml) have been stated for diagnosing and treating PTLD. 8,9 In this study, 1000 copies/ml and higher was considered an important regarding progression toward PTLD. Statistical analyses Statistical analyses were performed with SPSS software (SPSS: An IBM Company, version 17.0, IBM Corporation, Armonk, NY, USA). The quantitative data were compared using the t and Mann-Whitney U tests, while the chi-square test was used for qualitative data. A value for P <.05 was considered statistically significant. Results Ninety-eight patients with chronic liver disease were enrolled in the study. Subjects ages ranged from 36 days to 18 years (mean, 6.5 ± 5.9 y). In addition, 19 participants were younger than 1 year (19%), 29 were 1 to 4 years old (29%), 26 were 5 to 11 years old (26%), and 24 were older than 12 years (24%). Most of study subjects were between 1 and 4 years old. The mean age of subjects with biliary atresia who were on the liver transplant wait list was 1.4 ± 1.3 years, which was significantly different from other chronic liver diseases except biliary atresia (5.9 ± 5.6 y; 95% CI: ; P =.003). Regarding sex distribution, 53 participants were male (54%), while 45 were female (46%). The mean age of male and female subjects was 6.3 ± 5.7 and 6.8 ± 6.1 years; however, no statistically significant difference was observed between the sexes regarding mean age (P =.6). Chronic liver diseases included 28 with cryptogenic cirrhosis (28%); 17 with biliary atresia (17%); 9 with autoimmune hepatitis (9%); 18 with metabolic liver diseases including glycogen storage disease types I and IV, and tyrosinemia type I (18%); 15 with Wilson disease (15%); 6 with secondary biliary cirrhosis (6%); 1 with Alagille syndrome (1%); 3 with progressive familial intrahepatic cholestasis (3%); and 1 with hepatitis B (1%). Moreover, 34 patients (34%) had malnutrition and growth failure (below the third percentile in the weight/age growth curve). In fact, 39% of patients with cryptogenic cirrhosis, 28% with metabolic liver disease, 11% with autoimmune hepatitis, 27% with Wilson disease, 53% with biliary atresia, and 36% with other chronic liver diseases had malnutrition

3 536 Nader Shakibazad et al/experimental and Clinical Transplantation (2014) 6: Exp Clin Transplant table 1. Subjects Mean Age, Weight, CTP and PELD/MELD Scores Before Transplant Chronic Liver Cryptogenic Biliary Atresia AIH Wilson Disease Metabolic Liver Other Chronic Total Disease Cirrhosis Disease Except Liver Disease Parameters Wilson Disease Numbers Mean Age (y) ± SD 5.9 ± ± ± ± ± ± ± 5.9 Mean CTP ± SD 9.5 ± ± ± ± ± ± ± 2.3 Mean WT (kg) ± SD 21.9 ± ± ± ± ± ± ± 16.7 Mean PELD/MELD ± SD 19.8 ± ± ± ± ± ± ± 14.3 Abbreviations: AIH, autoimmune hepatitis; CTP, Child-Turcotte-Pugh; PELD/MELD, pediatric end-stage liver disease/model for end-stage liver disease; SD, standard deviation; WT, weight *Metabolic liver diseases except Wilson disease, including glycogen storage disease I and IV and tyrosinemia type I. Other chronic liver diseases including secondary biliary cirrhosis,alagille syndrome, progressive familial intrahepatic cholestasis, and hepatitis B. and growth failure. The highest rate of malnutrition and growth failure was seen in the patients with biliary atresia. Subjects means of age, weight, and CTP and PELD/MELD scores before transplant are presented in Table 1. According to the results, patients CTP and PELD/MELD mean scores were 9.1 ± 2.3 (range, 5-14) and 19.9 ± 14.3 (range, 1-66). Before the transplant, 15 subjects died of complications of chronic liver diseases including gastrointestinal bleeding, hepatic encephalopathy, sepsis, and multiorgan failure. In fact, patients mortality rate was 15% before the transplant. The prevalence of clinical symptoms and the complications of chronic liver diseases among study subjects and their comparison with deaths before transplant are shown in Table 2. Anemia was the most-common complication of chronic liver diseases among study subjects (70%). According to these results, mortality rates of patients with and without encephalopathy were 41% and 4% (P =.001). Besides, mortality rates of subjects with and without spontaneous bacterial peritonitis were 35% and 11% (P =.01; Table 2). table 2. Prevalence of Clinical Symptoms and Complications of Chronic Liver Diseases Among Study Subjects Compared With Mortality Before Transplant Mortality Before Transplant Parameters n (%) Yes (%) No (%) P Value Hepatic Yes 29 (29) 12 (41) 17 (58).001 encephalopathy No 69 (70) 3 (4) 66 (95) Gastrointestinal Yes 36 (36) 8 (22) 28 (77).14 bleeding No 62 (63) 7 (11) 55 (88) Ascites Yes 68 (69) 10 (14) 58 (85).4 No 30 (30) 15 (16) 25 (83) SBP Yes 17 (17) 6 (35) 11 (64).01 No 81 (82) 9 (11) 72 (88) Growth failure Yes 34 (34) 6 (17) 28 (82).63 No 64 (65) 9 (14) 55 (85) Anemia Yes 69 (70) 11 (15) 58 (84).78 No 29 (29) 4 (13) 25 (86) Thrombocytopenia Yes 54 (55) 9 (16) 45 (83).67 No 44 (44) 6 (13) 38 (86) Abbreviations: SBP, spontaneous bacterial peritonitis Considering the CTP scores, among the 98 patients under study, 16 patients (16%), 32 patients (32%), and 50 patients (51%) were classified as Child A, Child B, and Child C. Additionally, among the 15 patients who died before the transplant, 2 were Child B (13%) and 13 were Child C (86%). Mean CTP score for patients who died before transplant was 11.1 ± 1.6, while it was 8.8 ± 2.2 for those who did not die (P =.001). Furthermore, the mean PELD/MELD score of patients who died before transplant was 39.6 ± 12.6, while it was 16.3 ± 11.4 for those who did not die (P =.001). The PELD/MELD score of 30 and above had the sensitivity of 80% and specificity of 93.4% for predicting mortality before the transplant. In fact, 80% of patients who died before transplant had PELD/MELD score above 30. The frequency of the groups on the PELD/MELD tests, and the mortality rates in different ranges of the PELD/MELD score are given in Table 3. table 3. Mortality Rates in Different Ranges of PELD/MELD Scores PELD/MELD scores Number (%) Mortality n (%) (12) 9 (60) (9) 3 (20) (23) 3 (20) (25) 0 <10 29 (29) 0 Abbreviations: PELD/MELD, pediatric end-stage liver disease/model for end-stage liver disease Among the study subjects, 6 subjects (6%) who had measurable EBV levels before the transplant, and in 4 of them (4%) the number of EBV was considerably high (> 1000 copies/ml). All patients with considerably high EBV viral loads before the transplant had cryptogenic cirrhosis. Overall, the mean EBV viral load in patients with measurable viral load through the polymerase chain reaction method in serum was ± ranging from 100 to copies/ml. The mean age of the patients with high EBV viral load

4 Nader Shakibazad et al/experimental and Clinical Transplantation (2014) 6: (> 1000 copies/ml) was 8.8 ± 5.8 years, while it was 6.4 ± 5.8 years for those with low viral load (< 1000 copies/ml) (P =.42). Moreover, the mean CTP score of subjects with high EBV viral load was 8.7 ± 2.2, while it was 9.2 ± 2.3 in those with a low viral load (P =.7). Furthermore, the mean PELD/MELD score of patients with high viral loads was 13.2 ± 9.8, while it was 20.2 ± 14.4 for those with low viral loads (P =.36). Discussion Epstein-Barr virus infection and PTLD are among the main causes of mortality and morbidity in solidorgan transplant patients. 4 Therefore, in addition to investigating patients before liver transplants, the status of EBV viral loads also were evaluated in chronic liver patients before the liver transplant. According to the results, most children on a liver transplant wait list at our center were in the range of 1 to 5 years old (mean age, 6.5 years), which agrees with Mesquita and associates study in 2008 of 84 patients (mean age, 6.6 y before liver transplant). 10 However, in the study performed by Haberal and associates in 2008 in Turkey, most participants were younger than 1 year. 11 In the current study, the most-prevalent indication for liver transplant was cryptogenic cirrhosis (28%), followed by metabolic liver diseases (18%), with tyrosinemia being the most prevalent among metabolic live diseases and biliary atresia was the third most prevalent (17%). Most studies done on the issue, have shown biliary atresia in children as the most prevalent indication for a liver transplant. 12,13 Nevertheless, in the study performed by Haberal and associates, in Turkey, in 2008, the most-common indication for liver transplant was metabolic liver disorders. 11 The low prevalence of biliary atresia in this study compared with others may be the fact that the Kasai operation for biliary atresia patients is performed in higher age groups at this center (it usually leads to cirrhosis and death when done before transplant at an early stage of life). It also may be that we select children who are older for a liver transplant. Considering the fact that the highest rate of malnutrition and growth failure was observed in patients with biliary atresia (53%), early diagnosis of such patients, and appropriate nutritional support can improve their growth, increase the success rate of the Kasai operation, and decrease the rates of mortality and morbidity in patients on a liver transplant wait list. In this study, the observable complications of chronic liver patients include gastrointestinal bleeding, abdominal ascites, coagulation disorder, hepatic encephalopathy, anemia (most prevalent, 70%), thrombocytopenia, and spontaneous bacterial peritonitis. This is consistent with the study by McHutchison and associates in 2006 that reported a prevalence rate of anemia as 75% in chronic liver patients before transplant. 14 In general, anemia in chronic liver patients may result from acute or chronic gastrointestinal bleeding, and hypersplenism due to portal hypertension. 15 In this study, the mortality rate of the patients with liver cirrhosis was 15.3% before the transplant; its major causes were sepsis and multiorgan failure. In the study performed by Dehghani and associates in 2007, at the same center, the mortality rate before transplant was reported as 31.3%. 16 At our center, developing new surgical techniques, such as split liver transplant, availability of organs for transplant, improving interventions before transplant, and better selection of patients for liver transplant have reduced the death rate before transplant. In the study by Mesquita and associates in 2010, in Brazil, the death rate before liver transplant was approximately 50% and was due mainly to sepsis (38.1%). 10 In this study, patients with chronic liver disease complications (eg, hepatic encephalopathy and spontaneous bacterial peritonitis), had statistically significantly higher death rates (P =.001 and P =.01). Moreover, the mortality rate of patients with hepatic encephalopathy before the transplant was 15.5-fold greater than it was for those without encephalopathy (95% CI: ; OR 15.5). Spontaneous bacterial peritonitis in chronic liver patients also was an important risk factor for increasing the mortality rate before transplant (95% CI: ; OR 4.3). So, these 2 factors should be considered prioritizing liver transplant. Before the transplant, the mean PELD/MELD score was ± (range, 1-66) which was lower than the study performed by Aydogdu, 17 which might be due to the selection of patients with better conditions for transplant. Nevertheless, the mean CTP score was consistent with Aydogdu and associates study. 17 In fact, PELD/MELD and CTP scores are 2 prognostic factors of mortality before

5 538 Nader Shakibazad et al/experimental and Clinical Transplantation (2014) 6: Exp Clin Transplant transplant. In this study, the mean PELD/MELD and CTP scores were significantly higher among the patients who died before the transplant (P <.05). The PELD/MELD score of above 30 was an important risk factor for mortality in the present study (95% CI: ; OR 30). Compared with other chronic liver diseases, patients with cryptogenic cirrhosis were more likely to have a high EBV viral load (95% CI: 2-163; OR 18). Thus, EBV can be considered a causative factor in patients who have liver cirrhosis with no specific cause. In line with the current study, Tanaka and associates 18 also introduced chronic, persistent EBV infection with probable autoimmune mechanism as a scarce cause of liver fibrosis and chronic liver failure. The findings of the present study revealed no significant relation between EBV viral load and the patients age, sex, and mortality before transplant. However, considering the fact that since 1993, 19 four hundred liver transplants have been performed on children and adolescents in the Iranian liver transplant center, and 24 individuals (6%) had PTLD, the prevalence of PTLD has been reported from 2% to 20% in different centers, 20 and most PTLD patients have more than 1000 copies/ml EBV. 8 Considering the close prevalence of PTLD (6%) to that of high EBV viral load before the transplant (4%) in our center, a high EBV viral load before transplant can be mentioned as a risk factor for PTLD. The serologic test of EBV infection in the EBV-seronegative recipient is not reliable in patients receiving immunosuppressive therapy. Also, it is not great worth in the EBV-seropositive recipient. But the elevated EBV viral load measured by polymerase chain reaction of EBV-DNA in the peripheral blood of transplant patients is an approved risk factor for EBV-associated PTLD. The shortages of EBV viral load measurement are included in low specificity in recurrent EBV-associated PTLD and lack of a global reference standard. 21 Therefore, the prevalence of PTLD can be reduced by measuring EBV viral load before transplant, identifying the high-risk individuals for PTLD, and carrying out preventive treatments using acyclovir or gancyclovir and low doses of immunosuppressive drugs. References 1. Spada M, Riva S, Maggiore G, Cintorino D, Gridelli B. Pediatric liver transplantation. World J Gastroenterol. 2009;15(6): Green M, Cacciarelli TV, Mazariegos GV, et al. Serial measurement of Epstein-Barr viral load in peripheral blood in pediatric liver transplant recipients during treatment for posttransplant lymphoproliferative disease. Transplantation. 1998;66(12): O Leary JG, Lepe R, Davis GL. Indications for liver transplantation. Gastroenterology. 2008;134(6): Lee TC, Savoldo B, Rooney CM, et al. Quantitative EBV viral loads and immunosuppression alterations can decrease PTLD incidence in pediatric liver transplant recipients. Am J Transplant. 2005;5(9): Green M, Soltys K, Rowe DT, Webber SA, Mazareigos G. Chronic high Epstein-Barr viral load carriage in pediatric liver transplant recipients. Pediatr Transplant. 2009;13(3): D Antiga L, Del Rizzo M, Mengoli C, Cillo U, Guariso G, Zancan L. Sustained Epstein-Barr virus detection in paediatric liver transplantation. Insights into the occurrence of late PTLD. Liver Transpl. 2007;13(3): Jang JY, Kim KM, Lee YJ, Lee SG, Chi HS. Quantitative Epstein-Barr virus viral load monitoring in pediatric liver transplantation. Transplant Proc. 2008;40(8): Holmes RD, Sokol RJ. Epstein-Barr virus and post-transplant lymphoproliferative disease. Pediatr Transplant. 2002;6(6): Tanaka E, Sato T, Ishihara M, et al. Asymptomatic high Epstein-Barr viral load carriage in pediatric renal transplant recipients. 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Epstein-Barr Virus-related posttransplant lymphoproliferative disorders: pathogenetic insights for targeted therapy. Am J Transplant. 2011;11(5):