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Detection of hepatotropic viruses has no impact on the prognosis after Kasai procedure
Journal of Pediatric Surgery (2012) 47, 1828–1832
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Detection of hepatotropic viruses has no impact on the prognosis after Kasai procedure Nagoud Schukfeh a,⁎, Ali Al-Gamrah b , Claus Petersen a , Joachim Friedrich Kuebler a a
Department of Pediatric Surgery of Hannover Medical School, Hannover, Germany Department of Pediatric Surgery, University of Sana'a, Sana'a, Yemen
b
Received 18 December 2011; revised 2 April 2012; accepted 21 April 2012
Key words: Extrahepatic biliary atresia; Cholestasis; Risk factor; Liver; Portoenterostomy; Cytomegalovirus; Rotavirus; Epstein-Barr virus
Abstract Background/Purpose: A viral origin of biliary atresia (BA) is discussed, and several studies have demonstrated different viral strains in liver biopsies of patients undergoing Kasai portoenterostomy. We hypothesized that the presence of hepatotropic viruses in patients undergoing portoenterostomy contributes to the progression of the disease and negatively affect the outcome. Methods: Liver biopsies were prospectively taken from 70 patients undergoing portoenterostomy in our department from April 1996 to April 2004. Samples were screened by polymerase chain reaction for all common hepatic viruses. Primary outcome parameter was survival with the native liver. Secondary parameters were postoperative serum activity of liver enzymes and serum bilirubin levels at different time points. Patients underwent regular follow-up until October 2008. Results: Twenty-eight patients (40%) were positive for 1 or more hepatotropic viruses. Four patients were lost to follow-up. In the remaining 66 patients, there was no significant difference in survival with their native liver between virus-positive and virus-negative patients. After a mean follow-up of 7.7 years (range, 4.6-16.1 years), 15 (23%) of 66 patients still lived with their native liver. There was no difference in liver enzymes, C-reactive protein, or bilirubin at any time point between both groups. Conclusion: A significant number of our patients tested positive for hepatotropic viruses in liver biopsies at the time of the Kasai procedure, but the presence of virus had no influence on the course of BA. This suggests that the ongoing inflammatory process of BA leading to liver cirrhosis in most Kasaitreated patients is not affected by hepatotropic viruses. Our data question the necessity to aggressively screen for and treat viral infections in patients with BA. © 2012 Elsevier Inc. All rights reserved.
Biliary atresia (BA) is the most common neonatal cholestatic disorder, occurring in approximately 1 of 8000 (Asian countries) to 1 of 18,000 (European countries) live ⁎ Corresponding author. Department of Pediatric Surgery, Universitaetsklinikum Essen, Hufelandstr. 55, D-45147 Essen, Germany. Tel.: + 49 201 723 1110; fax: +49 201 723 1131. E-mail address: [email protected] (N. Schukfeh). 0022-3468/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jpedsurg.2012.04.024
births [1], with a female preponderance, characterized by complete fibrotic obliteration of the lumen of all or part of the extrahepatic biliary tree within 3 months of life. The etiology of BA remains unclear. Several factors have been suggested in the pathogenesis of extrahepatic BA. Since the findings of Landing [2] that indicated that BA could represent the result of a virally induced process of the liver and the hepatobiliary tree, several observations have pointed
Hepatotropic virus' impact on prognosis after Kasai procedure toward a potential association between viral infection and the development of BA. Riepenhoff-Talty et al [3] detected group C rotavirus in infants with extrahepatic BA, and a viral infection was considered as a potential initiator for the obliteration of the bile ducts caused by progressive fibrosing inflammation [4-6]. This hypothesis has been supported by findings of individual viral strains in patients with BA. In liver samples of patients undergoing the Kasai procedure, cytomegalovirus (CMV) [7-11], human papilloma virus [8], reovirus type 3 [12-14], and rotavirus [3,15] were observed. In a large retrospective study of 74 patients, we detected hepatotropic viruses in only 50% of the patients at the time of the Kasai procedure, and those patients with detectable viral RNA/DNA in their liver biopsies were significantly older than virus-free patients [16], suggesting that viral infection could be a secondary phenomenon. However, it remains disputed whether the presence of virus in livers of patients with BA at the time of the Kasai procedure could negatively affect the outcome [17,23]. If the presence of viruses at the time of the Kasai operation would influence the outcome, this would be a rational for screening for hepatotropic viruses and eventually using antiviral medication. This clinically relevant aspect was not sufficiently answered by our previous study looking at the viral incidence in Kasai patients. Therefore, in this study, we followed up these patients to investigate whether viral presence at the time of the Kasai procedure affects the outcome of patients with BA.
1829 Table 1 Summary of the different hepatotropic virus strains in patients with BA at the time of the Kasai procedure Detection of virus strains Reovirus positive CMV positive Adenovirus positive Enterovirus positive Virus positive
20/70 8/70 2/70 1/70 26/70
29% 11% 3% 1% 37%
atively. During the Kasai procedure, liver biopsies were taken in all patients. Specimens were fixed in 4% buffered formalin for immunostaining or immediately snap frozen in liquid N2 and stored at − 80°C for polymerase chain reaction (PCR) analysis. The samples were screened by PCR for common hepatic viruses: herpes simplex virus, Epstein-Barr virus (EBV), varicella zoster virus (VZV), CMV, adenovirus, parvovirus B19, enteroviruses, papillomavirus, rotavirus, and reovirus 3, as previously described [16], and detection of DNA viruses (herpes simplex virus, EBV, VZV, CMV, adenovirus, parvovirus B19, and human papillomavirus). The DNA of 50 mg of frozen liver tissue was extracted using a commercial kit (DNeasy Tissue Kit; Qiagen, Duesseldorf, Germany), and the resuspended DNA was stored at −20°C. A LightCycler (Roche Molecular Systems, Pleasanton, USA) was used for real-time quantitative PCR. Detection of RNA viruses (enterovirus, rotavirus, and reovirus 3); RNA was extracted using TRIzol reagent (Gibco BRL, San Francisco, CA, USA) using 25 to 50 mg of frozen liver tissue.
Patients and methods 1,0
The study design was reviewed and approved by the local ethics committee.
Virus positive patients Virus negative patients
0,8
Biopsies from 70 patients with BA (30 male and 40 female) treated at the Hannover Medical School, Germany, between April 1996 and April 2004 were prospectively included in the study. Biliary atresia was diagnosed after the exclusion of other cholestatic diseases of the newborn by preoperative endoscopic retrograde cholangiopancreatography, as described previously [18]. The diagnosis was confirmed by intraoperative cholangiography in doubtful cases. All patients underwent our in-house workup for neonatal cholestasis including blood samples for direct and indirect serum bilirubin, liver enzymes, blood count, coagulation parameters, α-1–antitrypsin, hepatitis, and TORCH (Toxoplasmosis, Others, Rubella, Cytomegalovirus, Herpes Simplex) serology. Abdominal and cardiac sonography was performed to exclude associated malformations. Biliary atresia was then confirmed by performing endoscopic retrograde cholangiopancreatography in all patients preoper-
Survival rate (%)
Patient samples 0,6
0,4
0,2
0,0 0
2,5
5
7,5
10
Time (years)
Fig. 1 Kaplan-Meier survival curve. Displayed is the time of survival of patients with BA with their native liver in years after the Kasai procedure. The points indicate the end of follow-up. Log-rank test, P = .907; there is no statistically significant difference.
1830
N. Schukfeh et al.
Outcome assessment
Results
Survival with the native liver was determined as a primary end point. The course of serum bilirubin, aspartate aminotransferase, alanine aminotransferase, γglutamyltransferase, and C-reactive protein (CRP) as secondary end points was monitored at the time of the Kasai procedure and at 3 days, 7 days, 1 month, and 3 months after the procedure and at last follow-up. The mean follow-up after Kasai procedure was 7.7 years (range, 4.6-16.1 years).
Patient characteristics In the 70 patients who underwent a Kasai procedure, 28 (40%) were positive (11 male and 17 female) and 42 (60%) were negative (19 male and 23 female) for different hepatotropic viruses. At the time of the Kasai procedure, the mean age of the patients was 55 days (range, 19-122 days) for all patients, 64 days (range, 19-122 days) for viruspositive and 51 days (19-91 days) for virus-negative patients, respectively. The difference between both groups with an older age of virus-positive patients was statistically significant (Student's t test, P = .037). Five patients underwent treatment with intravenous antibiotics owing to the postoperative development of cholangitis. Eleven patients (15.7%; 3, virus positive; 8, virus negative) had syndromatic BA with associated nonhepatic malformations. Associated malformations included atrial septal defect in 3 patients and ventricular
Statistical analysis Significance was assessed by twin-tailed unpaired Student's t test, with significance assumed at P b .05. Survival rates were compared by log-rank analysis based on KaplanMeier survival curves. Descriptive analyses were performed using the χ2 test or Fisher's Exact test, respectively.
Total n = 66
Virus positive n = 26 (39%)
Bilirubin 3m <20µmol/l n = 13 (50%)
Own liver Mean: 8 years n=4 (31%)
LTX/Death n=9 (69%)
Virus negative n = 40 (61%)
Bilirubin 3m >20µmol/l n = 13 (50%)
Own liver Mean: 6 years n=2 (15%)
LTX/Death n = 11 (85%)
Bilirubin 3m <20µmol/l n = 10 (25%)
Own liver Mean: 7 years n=3 (30%)
LTX/Death n=7 (70%)
Bilirubin 3m >20µmol/l n = 30 (75%)
Own liver Mean: 7 years n=6 (20%)
LTX/Death n = 24 (80%)
Fig. 2 Flowchart. Outcome of all patients. Bilirubin 3 m: serum bilirubin level at 3 months after the Kasai procedure. Own liver: number and mean survival in years of patients still living with their native liver. LTX/Death: number of patients who received liver transplantation or died within follow-up period. There were no significant differences concerning the outcome between all groups.
Hepatotropic virus' impact on prognosis after Kasai procedure septal defect, cardiomyopathy, pyloric stenosis, liver cyst, polystystic kidneys, double kidney, and cleft palate in 1 patient each, respectively.
Survival with native liver Four patients (2, virus positive; 2, virus negative) were lost to follow-up. After a mean follow-up of 7.7 years (range, 4.6-16.1 years), 15 (23%) of 66 patients were still alive with their native liver, 6 of them virus positive and 9, virus negative. The remaining 51 patients (77%), 20 of them virus positive and 31 of them virus negative, received liver transplantation or died at a mean of 21 months (range, 2 months to 11 years) after the Kasai procedure. Of 66 patients, 12 (18%) received a liver transplant or died within 6 months after the Kasai procedure; the remaining 54 patients (82%) survived more than 6 months with their own liver. Of 26 virus-positive patients, 21 patients were detected positive for 1 virus, and 5 patients were detected positive for 2 different viruses, respectively (Table 1). All patients were negative for rotavirus, papillomavirus, parvovirus B19, herpex simplex virus, EBV, reovirus 3, and VZV. As shown in Fig. 1, there was no difference in the 3-year survival of patients with their native liver between viruspositive and virus-negative patients. The outcome of all patients is shown in Fig. 2.
Laboratory parameters There was no significant difference in liver enzymes, CRP, or bilirubin levels at any time point between viruspositive and virus-negative patients (Table 2). Table 2
1831
Discussion The role of hepatotropic virus infection in BA remains controversial [19-22]. Several aspects of this disease support the hypothesis that viral infection plays a role in the etiology. However, in a previous study, we tested 74 patients with BA at the time of their Kasai procedure for all common hepatotropic viruses. In that study, the 27 virus-positive patients (42%) were significantly older than those who were virus negative, suggesting that the presence of viral DNA or RNA could be a secondary phenomenon and not involved in the etiology of BA [16]. The impact of a potential virus infection of the biliary system on the progression of the disease, however, remains unclear. Nonetheless, it is clinically relevant information whether screening for hepatotropic viruses and potential antiviral treatment could be potentially beneficial. In a recent study, Shen et al [17] investigated CMV infection in 27 patients with BA. They observed that 22 showed some sort of CMV antibodies and that the rate of CMV-positive patients was higher than CMV-positive mothers. Moreover, they observed a significantly lower rate of clearance of jaundice at 3 months after the Kasai procedure in patients who were CMV positive compared with CMV-free patients (P b .05). This detrimental effect of the ongoing CMV infection in patients with BA could support use of aggressive antiviral therapy. In an initial study, Fischler et al [28] described successful treatment of CMV associated neonatal cholestasis with ganciclovir. In his series of 6 infants, there were 2 patients with BA, and one of them responded to the treatment. Based on this initial
Serum levels of different liver markers preoperatively and at 3 days, 7 days, 1 month, and 3 months after the Kasai procedure
CRP (mg/L) Virus positive (n = 26) Virus negative (n = 40) GGT (U/L) Virus positive Virus negative AST (U/L) Virus positive Virus negative ALT (U/L) Virus positive Virus negative CHE (kU/L) Virus positive Virus negative Bilirubine (μmol/L) Virus positive Virus negative
Preoperatively
3d
7d
1 mo
3 mo
Mean (range)
Mean (range)
Mean (range)
Mean (range)
Mean (range)
4 (1-11) 6 (1-50)
25 (5-73) 23 (1-59)
11 (2-86) 10 (1-55)
5 (2-14) 6 (1-20)
4 (1-16) 6 (1-39)
457 (42-1250) 401 (66-1097)
302 (44-652) 289 (22-620)
425 (59-1406) 416 (56-1515)
423 (21-890) 485 (131-1135)
268 (24-675) 343 (54-1467)
174 (18-520) 201 (26-2245)
479 (50-2284) 451 (37-3321)
149 (45-396) 143 (16-779)
139 (35-312) 160 (41-524)
146 (42-334) 158 (14-590)
137 (24-538) 143 (9-1363)
404 (60-1198) 393 (23-3367)
182 (58-376) 157 (14-831)
152 (27-394) 157 (43-696)
125 (43-270) 125 (19-455)
3.77 (2.47-6.16) 5.13 (2.53-9.22)
2.7 (1.41-3.88) 3.26 (1.53-7.28)
2.83 (1.57-4.6) 3.59 (0.93-7.9)
3.94 (2.42-5.88) 4.87 (0.92-10.26)
4.66 (2.53-7.28) 4.62 (1.94-7.27)
173 (107-291) 153 (63-256)
164 (73-284) 164 (91-300)
125 (39-199) 139 (9-306)
136 (23-417) 142 (32-392)
144 (5-395) 150 (4-515)
Data are given as median (range). Included are the results from 26 virus-positive patients and 40 virus-negative patients. AST indicates aspartate aminotransferase; ALT, alanine aminotransferase; GGT, γ-glutamyltransferase; CHE, choline esterase.
1832 observation, the same authors evaluated the effect of CMV infection on the course of BA. In this study, they reported no difference in long-term outcome between CMV-positive and CMV-negative patients with BA [23]. They included 28 patients with CMV infection, defined by either the detection of CMV–immunoglobin M or CMV in the urine. There were no data provided on CMV replication, and none of the patients received antiviral therapy. In our study, including 66 patients, we failed to detect any detrimental effect of hepatic viral infection on the prognosis of patients undergoing Kasai portoenterostomy for BA. In contrast to the previous reports, we did not restrict our investigation to individual viral strains but included all common hepatotropic viruses. Nonetheless, there was no detrimental effect detectable even looking at the individual strains. Thus, our data are in line with the findings of Fischler et al [23] but is contrary to the report of Shen et al [17]. It could be argued that there are regional differences in the prevalence of certain viral strains as well as difference between BA in Asian and Western countries. This hypothesis is supported by differences in incidence and outcome, that is, French, British, and Canadian groups report survival with native liver at 4 to 5 years to be 36% to 51%, whereas the figure from Japan is slightly higher [24-27]. As shown in Fig. 1, in our study, the 5-year survival with native liver after Kasai portoenterostomy is 31% and thus comparable with the figures of other Western countries. In our study, only liver tissue was assessed for viral RNA or DNA, whereas neither circulating viral antibodies nor peripheral viral RNA or DNA were investigated. Moreover, we cannot rule out that a viral infection initiated autoimmune mediated biliary duct damage and subsequent liver fibrogenesis in some patients, although the viruses were already cleared at the time of the Kasai procedure and thus speculated that virus-negative patients were actually operated on at a late time point. Nonetheless, our data strongly argue against the clinical relevance of an ongoing hepatic viral infection in patients with BA undergoing Kasai portoeneterostomy. Based on our results, we do not recommend routine screening of liver samples for hepatotropic viruses nor aggressive antiviral therapies in otherwise asymptomatic patients.
References [1] Petersen C. Pathogenesis and treatment opportunities for biliary atresia. Clin Liver Dis 2006;10:73-88. [2] Landing BH. Considerations of the pathogenesis of neonatal hepatitis, biliary atresia and choledochal cyst—the concept of infantile obstructive cholangiopathy. Prog Pediatr Surg 1974;6:113-39. [3] Riepenhoff-Talty M, Gouvea V, Evans MJ, et al. Detection of group C rotavirus in infants with extrahepatic biliary atresia. J Infect Dis 1996;174:8-15. [4] Bezerra JA, Tiao G, Ryckman FC, et al. Genetic induction of proinflammatory immunity in children with biliary atresia. Lancet 2002;360:1563-659.
N. Schukfeh et al. [5] Davenport M, Gonde C, Redkar R, et al. Immunohistochemistry of the liver and biliary tree in extrahepatic biliary atresia. J Pediatr Surg 2001;36:1017-25. [6] Schreiber RA, Kleinman RE. Genetics, immunology, and biliary atresia: an opening or a diversion? J Pediatr Gastroenterol Nutr 1993;16:111-3. [7] Chang MH, Huang HH, Huang ES, et al. Polymerase chain reaction to detect human cytomegalovirus in livers of infants with neonatal hepatitis. Gastroenterology 1992;103:1022-5. [8] Domiati-Saad R, Dawson DB, Margraf LR, et al. Cytomegalovirus and human herpesvirus 6, but not human papillomavirus, are present in neonatal giant cell hepatitis and extrahepatic biliary atresia. Pediatric Dev Pathol 2000;3:367-437. [9] Fischler B, Ehrnst A, Forsgren M, et al. The viral association of neonatal cholestasis in Sweden: a possible link between cytomegalovirus infection and extrahepatic biliary atresia. J Pediatr Gastroenterol Nutr 1998;27:57-64. [10] Fjær RB, Bruu A-L, Nordbø SA. Extrahepatic bile duct atresia and viral involvement. Pediatr Transplant 2005;9:68-73. [11] Jevon GP, Dimmik JE. Biliary atresia and cytomegalovirus infection: a DNA study. Pediatr Dev Pathol 1999;2:11-4. [12] Brown WR, Sokol RJ, Levin MJ, et al. Lack of correlation between infection with reovirus 3 and extrahepatic biliary atresia or neonatal hepatitis. J Pediatr 1988;113:670-6. [13] Morecki R, Glaser JH, Cho S, et al. Biliary atresia and reovirus type 3 infection. N Engl J Med 1982;307:481-4. [14] Tyler KL, Sokol RJ, Oberhaus SM, et al. Detection of reovirus RNA in hepatobiliary tissues from patients with extrahepatic biliary atresia and choledochal cysts. Hepatology 1998;27:1475-82. [15] Bobo L, Ojeh C, Chiu D, et al. Lack of evidence for rotavirus by polymerase chain reaction/enzyme immunoassay of hepatobiliary samples from children with biliary atresia. Pediatr Res 1997;41:229-34. [16] Rauschenfels S, Krassmann M, Al-Masri AN, et al. Incidence of hepatotropic viruses in biliary atresia. Eur J Pediatr 2009;168:469-76. [17] Shen C, Zheng S, Wang W, et al. Relationship between prognosis of biliary atresia and infection of cytomegalovirus. World J Pediatr 2008;4:123-6. [18] Petersen C, Meier PN, Schneider A, et al. Endoscopic retrograde cholangiopancreaticography prior to explorative laparotomy avoids unnecessary surgery in patients suspected for biliary atresia. J Hepatol 2009;51:1055-60. [19] Sokol RJ, Mack C, Narkewicz MR, et al. Pathogenesis and outcome of biliary atresia: current concepts. J Pediatr Gastroenterol Nutr 2003;37: 4-21. [20] Selmi C, Vergani D, Mieli-Vergani G. Viruses and autoantibodies in biliary atresia. Gastroenterology 2010;139:1461-4. [21] A-Kader HH, Nowicki MJ, Kuramoto KI, et al. Evaluation of the role of hepatitis C virus in biliary atresia. Pediatr Infect Dis J 1994;13:657-9. [22] Riepenhoff-Talty M, Schaekel K, Clark HF, et al. Group A rotaviruses produce extrahepatic biliary obstruction in orally inoculated newborn mice. Pediatr Res 1993;33:394-9. [23] Fischler B, Svennsson JF, Nemeth A. Early cytomegalovirus infection and the long-term outcome of biliary atresia. Acta Pediatr 2009;98: 1600-2. [24] Chardot C, Carton M, Spire-Bendelac N, et al. Prognosis of biliary atresia in the era of liver transplantation: French national study from 1986 to 1996. Hepatology 1999;30:606-11. [25] McKiernan PJ, Baker AJ, Kelly DA. The frequency and outcome of biliary atresia in the UK and Ireland. Lancet 2000;355:25-9. [26] Schreiber RA, Barker CC, Roberts EA, et al. Biliary atresia: the Canadian experience. J Pediatr 2007;151:659-65. [27] Nio M, Ohi R, Miyano T, et al. Five and 10-year survival rates after surgery for biliary atresia: a report from the Japanese Biliary Atresia Registry. J Pediatr Surg 2003;38:997-1000. [28] Fischler B, Casswall TH, Malmborg P, et al. Ganciclovir treatment in infants with cytomegalovirus infection and cholestasis. J Pediatr Gastroenterol Nutr 2002;34:154-7.
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Detection of hepatotropic viruses has no impact on the prognosis after Kasai procedure Nagoud Schukfeh a,⁎, Ali Al-Gamrah b , Claus Petersen a , Joachim Friedrich Kuebler a a
Department of Pediatric Surgery of Hannover Medical School, Hannover, Germany Department of Pediatric Surgery, University of Sana'a, Sana'a, Yemen
b
Received 18 December 2011; revised 2 April 2012; accepted 21 April 2012
Key words: Extrahepatic biliary atresia; Cholestasis; Risk factor; Liver; Portoenterostomy; Cytomegalovirus; Rotavirus; Epstein-Barr virus
Abstract Background/Purpose: A viral origin of biliary atresia (BA) is discussed, and several studies have demonstrated different viral strains in liver biopsies of patients undergoing Kasai portoenterostomy. We hypothesized that the presence of hepatotropic viruses in patients undergoing portoenterostomy contributes to the progression of the disease and negatively affect the outcome. Methods: Liver biopsies were prospectively taken from 70 patients undergoing portoenterostomy in our department from April 1996 to April 2004. Samples were screened by polymerase chain reaction for all common hepatic viruses. Primary outcome parameter was survival with the native liver. Secondary parameters were postoperative serum activity of liver enzymes and serum bilirubin levels at different time points. Patients underwent regular follow-up until October 2008. Results: Twenty-eight patients (40%) were positive for 1 or more hepatotropic viruses. Four patients were lost to follow-up. In the remaining 66 patients, there was no significant difference in survival with their native liver between virus-positive and virus-negative patients. After a mean follow-up of 7.7 years (range, 4.6-16.1 years), 15 (23%) of 66 patients still lived with their native liver. There was no difference in liver enzymes, C-reactive protein, or bilirubin at any time point between both groups. Conclusion: A significant number of our patients tested positive for hepatotropic viruses in liver biopsies at the time of the Kasai procedure, but the presence of virus had no influence on the course of BA. This suggests that the ongoing inflammatory process of BA leading to liver cirrhosis in most Kasaitreated patients is not affected by hepatotropic viruses. Our data question the necessity to aggressively screen for and treat viral infections in patients with BA. © 2012 Elsevier Inc. All rights reserved.
Biliary atresia (BA) is the most common neonatal cholestatic disorder, occurring in approximately 1 of 8000 (Asian countries) to 1 of 18,000 (European countries) live ⁎ Corresponding author. Department of Pediatric Surgery, Universitaetsklinikum Essen, Hufelandstr. 55, D-45147 Essen, Germany. Tel.: + 49 201 723 1110; fax: +49 201 723 1131. E-mail address: [email protected] (N. Schukfeh). 0022-3468/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jpedsurg.2012.04.024
births [1], with a female preponderance, characterized by complete fibrotic obliteration of the lumen of all or part of the extrahepatic biliary tree within 3 months of life. The etiology of BA remains unclear. Several factors have been suggested in the pathogenesis of extrahepatic BA. Since the findings of Landing [2] that indicated that BA could represent the result of a virally induced process of the liver and the hepatobiliary tree, several observations have pointed
Hepatotropic virus' impact on prognosis after Kasai procedure toward a potential association between viral infection and the development of BA. Riepenhoff-Talty et al [3] detected group C rotavirus in infants with extrahepatic BA, and a viral infection was considered as a potential initiator for the obliteration of the bile ducts caused by progressive fibrosing inflammation [4-6]. This hypothesis has been supported by findings of individual viral strains in patients with BA. In liver samples of patients undergoing the Kasai procedure, cytomegalovirus (CMV) [7-11], human papilloma virus [8], reovirus type 3 [12-14], and rotavirus [3,15] were observed. In a large retrospective study of 74 patients, we detected hepatotropic viruses in only 50% of the patients at the time of the Kasai procedure, and those patients with detectable viral RNA/DNA in their liver biopsies were significantly older than virus-free patients [16], suggesting that viral infection could be a secondary phenomenon. However, it remains disputed whether the presence of virus in livers of patients with BA at the time of the Kasai procedure could negatively affect the outcome [17,23]. If the presence of viruses at the time of the Kasai operation would influence the outcome, this would be a rational for screening for hepatotropic viruses and eventually using antiviral medication. This clinically relevant aspect was not sufficiently answered by our previous study looking at the viral incidence in Kasai patients. Therefore, in this study, we followed up these patients to investigate whether viral presence at the time of the Kasai procedure affects the outcome of patients with BA.
1829 Table 1 Summary of the different hepatotropic virus strains in patients with BA at the time of the Kasai procedure Detection of virus strains Reovirus positive CMV positive Adenovirus positive Enterovirus positive Virus positive
20/70 8/70 2/70 1/70 26/70
29% 11% 3% 1% 37%
atively. During the Kasai procedure, liver biopsies were taken in all patients. Specimens were fixed in 4% buffered formalin for immunostaining or immediately snap frozen in liquid N2 and stored at − 80°C for polymerase chain reaction (PCR) analysis. The samples were screened by PCR for common hepatic viruses: herpes simplex virus, Epstein-Barr virus (EBV), varicella zoster virus (VZV), CMV, adenovirus, parvovirus B19, enteroviruses, papillomavirus, rotavirus, and reovirus 3, as previously described [16], and detection of DNA viruses (herpes simplex virus, EBV, VZV, CMV, adenovirus, parvovirus B19, and human papillomavirus). The DNA of 50 mg of frozen liver tissue was extracted using a commercial kit (DNeasy Tissue Kit; Qiagen, Duesseldorf, Germany), and the resuspended DNA was stored at −20°C. A LightCycler (Roche Molecular Systems, Pleasanton, USA) was used for real-time quantitative PCR. Detection of RNA viruses (enterovirus, rotavirus, and reovirus 3); RNA was extracted using TRIzol reagent (Gibco BRL, San Francisco, CA, USA) using 25 to 50 mg of frozen liver tissue.
Patients and methods 1,0
The study design was reviewed and approved by the local ethics committee.
Virus positive patients Virus negative patients
0,8
Biopsies from 70 patients with BA (30 male and 40 female) treated at the Hannover Medical School, Germany, between April 1996 and April 2004 were prospectively included in the study. Biliary atresia was diagnosed after the exclusion of other cholestatic diseases of the newborn by preoperative endoscopic retrograde cholangiopancreatography, as described previously [18]. The diagnosis was confirmed by intraoperative cholangiography in doubtful cases. All patients underwent our in-house workup for neonatal cholestasis including blood samples for direct and indirect serum bilirubin, liver enzymes, blood count, coagulation parameters, α-1–antitrypsin, hepatitis, and TORCH (Toxoplasmosis, Others, Rubella, Cytomegalovirus, Herpes Simplex) serology. Abdominal and cardiac sonography was performed to exclude associated malformations. Biliary atresia was then confirmed by performing endoscopic retrograde cholangiopancreatography in all patients preoper-
Survival rate (%)
Patient samples 0,6
0,4
0,2
0,0 0
2,5
5
7,5
10
Time (years)
Fig. 1 Kaplan-Meier survival curve. Displayed is the time of survival of patients with BA with their native liver in years after the Kasai procedure. The points indicate the end of follow-up. Log-rank test, P = .907; there is no statistically significant difference.
1830
N. Schukfeh et al.
Outcome assessment
Results
Survival with the native liver was determined as a primary end point. The course of serum bilirubin, aspartate aminotransferase, alanine aminotransferase, γglutamyltransferase, and C-reactive protein (CRP) as secondary end points was monitored at the time of the Kasai procedure and at 3 days, 7 days, 1 month, and 3 months after the procedure and at last follow-up. The mean follow-up after Kasai procedure was 7.7 years (range, 4.6-16.1 years).
Patient characteristics In the 70 patients who underwent a Kasai procedure, 28 (40%) were positive (11 male and 17 female) and 42 (60%) were negative (19 male and 23 female) for different hepatotropic viruses. At the time of the Kasai procedure, the mean age of the patients was 55 days (range, 19-122 days) for all patients, 64 days (range, 19-122 days) for viruspositive and 51 days (19-91 days) for virus-negative patients, respectively. The difference between both groups with an older age of virus-positive patients was statistically significant (Student's t test, P = .037). Five patients underwent treatment with intravenous antibiotics owing to the postoperative development of cholangitis. Eleven patients (15.7%; 3, virus positive; 8, virus negative) had syndromatic BA with associated nonhepatic malformations. Associated malformations included atrial septal defect in 3 patients and ventricular
Statistical analysis Significance was assessed by twin-tailed unpaired Student's t test, with significance assumed at P b .05. Survival rates were compared by log-rank analysis based on KaplanMeier survival curves. Descriptive analyses were performed using the χ2 test or Fisher's Exact test, respectively.
Total n = 66
Virus positive n = 26 (39%)
Bilirubin 3m <20µmol/l n = 13 (50%)
Own liver Mean: 8 years n=4 (31%)
LTX/Death n=9 (69%)
Virus negative n = 40 (61%)
Bilirubin 3m >20µmol/l n = 13 (50%)
Own liver Mean: 6 years n=2 (15%)
LTX/Death n = 11 (85%)
Bilirubin 3m <20µmol/l n = 10 (25%)
Own liver Mean: 7 years n=3 (30%)
LTX/Death n=7 (70%)
Bilirubin 3m >20µmol/l n = 30 (75%)
Own liver Mean: 7 years n=6 (20%)
LTX/Death n = 24 (80%)
Fig. 2 Flowchart. Outcome of all patients. Bilirubin 3 m: serum bilirubin level at 3 months after the Kasai procedure. Own liver: number and mean survival in years of patients still living with their native liver. LTX/Death: number of patients who received liver transplantation or died within follow-up period. There were no significant differences concerning the outcome between all groups.
Hepatotropic virus' impact on prognosis after Kasai procedure septal defect, cardiomyopathy, pyloric stenosis, liver cyst, polystystic kidneys, double kidney, and cleft palate in 1 patient each, respectively.
Survival with native liver Four patients (2, virus positive; 2, virus negative) were lost to follow-up. After a mean follow-up of 7.7 years (range, 4.6-16.1 years), 15 (23%) of 66 patients were still alive with their native liver, 6 of them virus positive and 9, virus negative. The remaining 51 patients (77%), 20 of them virus positive and 31 of them virus negative, received liver transplantation or died at a mean of 21 months (range, 2 months to 11 years) after the Kasai procedure. Of 66 patients, 12 (18%) received a liver transplant or died within 6 months after the Kasai procedure; the remaining 54 patients (82%) survived more than 6 months with their own liver. Of 26 virus-positive patients, 21 patients were detected positive for 1 virus, and 5 patients were detected positive for 2 different viruses, respectively (Table 1). All patients were negative for rotavirus, papillomavirus, parvovirus B19, herpex simplex virus, EBV, reovirus 3, and VZV. As shown in Fig. 1, there was no difference in the 3-year survival of patients with their native liver between viruspositive and virus-negative patients. The outcome of all patients is shown in Fig. 2.
Laboratory parameters There was no significant difference in liver enzymes, CRP, or bilirubin levels at any time point between viruspositive and virus-negative patients (Table 2). Table 2
1831
Discussion The role of hepatotropic virus infection in BA remains controversial [19-22]. Several aspects of this disease support the hypothesis that viral infection plays a role in the etiology. However, in a previous study, we tested 74 patients with BA at the time of their Kasai procedure for all common hepatotropic viruses. In that study, the 27 virus-positive patients (42%) were significantly older than those who were virus negative, suggesting that the presence of viral DNA or RNA could be a secondary phenomenon and not involved in the etiology of BA [16]. The impact of a potential virus infection of the biliary system on the progression of the disease, however, remains unclear. Nonetheless, it is clinically relevant information whether screening for hepatotropic viruses and potential antiviral treatment could be potentially beneficial. In a recent study, Shen et al [17] investigated CMV infection in 27 patients with BA. They observed that 22 showed some sort of CMV antibodies and that the rate of CMV-positive patients was higher than CMV-positive mothers. Moreover, they observed a significantly lower rate of clearance of jaundice at 3 months after the Kasai procedure in patients who were CMV positive compared with CMV-free patients (P b .05). This detrimental effect of the ongoing CMV infection in patients with BA could support use of aggressive antiviral therapy. In an initial study, Fischler et al [28] described successful treatment of CMV associated neonatal cholestasis with ganciclovir. In his series of 6 infants, there were 2 patients with BA, and one of them responded to the treatment. Based on this initial
Serum levels of different liver markers preoperatively and at 3 days, 7 days, 1 month, and 3 months after the Kasai procedure
CRP (mg/L) Virus positive (n = 26) Virus negative (n = 40) GGT (U/L) Virus positive Virus negative AST (U/L) Virus positive Virus negative ALT (U/L) Virus positive Virus negative CHE (kU/L) Virus positive Virus negative Bilirubine (μmol/L) Virus positive Virus negative
Preoperatively
3d
7d
1 mo
3 mo
Mean (range)
Mean (range)
Mean (range)
Mean (range)
Mean (range)
4 (1-11) 6 (1-50)
25 (5-73) 23 (1-59)
11 (2-86) 10 (1-55)
5 (2-14) 6 (1-20)
4 (1-16) 6 (1-39)
457 (42-1250) 401 (66-1097)
302 (44-652) 289 (22-620)
425 (59-1406) 416 (56-1515)
423 (21-890) 485 (131-1135)
268 (24-675) 343 (54-1467)
174 (18-520) 201 (26-2245)
479 (50-2284) 451 (37-3321)
149 (45-396) 143 (16-779)
139 (35-312) 160 (41-524)
146 (42-334) 158 (14-590)
137 (24-538) 143 (9-1363)
404 (60-1198) 393 (23-3367)
182 (58-376) 157 (14-831)
152 (27-394) 157 (43-696)
125 (43-270) 125 (19-455)
3.77 (2.47-6.16) 5.13 (2.53-9.22)
2.7 (1.41-3.88) 3.26 (1.53-7.28)
2.83 (1.57-4.6) 3.59 (0.93-7.9)
3.94 (2.42-5.88) 4.87 (0.92-10.26)
4.66 (2.53-7.28) 4.62 (1.94-7.27)
173 (107-291) 153 (63-256)
164 (73-284) 164 (91-300)
125 (39-199) 139 (9-306)
136 (23-417) 142 (32-392)
144 (5-395) 150 (4-515)
Data are given as median (range). Included are the results from 26 virus-positive patients and 40 virus-negative patients. AST indicates aspartate aminotransferase; ALT, alanine aminotransferase; GGT, γ-glutamyltransferase; CHE, choline esterase.
1832 observation, the same authors evaluated the effect of CMV infection on the course of BA. In this study, they reported no difference in long-term outcome between CMV-positive and CMV-negative patients with BA [23]. They included 28 patients with CMV infection, defined by either the detection of CMV–immunoglobin M or CMV in the urine. There were no data provided on CMV replication, and none of the patients received antiviral therapy. In our study, including 66 patients, we failed to detect any detrimental effect of hepatic viral infection on the prognosis of patients undergoing Kasai portoenterostomy for BA. In contrast to the previous reports, we did not restrict our investigation to individual viral strains but included all common hepatotropic viruses. Nonetheless, there was no detrimental effect detectable even looking at the individual strains. Thus, our data are in line with the findings of Fischler et al [23] but is contrary to the report of Shen et al [17]. It could be argued that there are regional differences in the prevalence of certain viral strains as well as difference between BA in Asian and Western countries. This hypothesis is supported by differences in incidence and outcome, that is, French, British, and Canadian groups report survival with native liver at 4 to 5 years to be 36% to 51%, whereas the figure from Japan is slightly higher [24-27]. As shown in Fig. 1, in our study, the 5-year survival with native liver after Kasai portoenterostomy is 31% and thus comparable with the figures of other Western countries. In our study, only liver tissue was assessed for viral RNA or DNA, whereas neither circulating viral antibodies nor peripheral viral RNA or DNA were investigated. Moreover, we cannot rule out that a viral infection initiated autoimmune mediated biliary duct damage and subsequent liver fibrogenesis in some patients, although the viruses were already cleared at the time of the Kasai procedure and thus speculated that virus-negative patients were actually operated on at a late time point. Nonetheless, our data strongly argue against the clinical relevance of an ongoing hepatic viral infection in patients with BA undergoing Kasai portoeneterostomy. Based on our results, we do not recommend routine screening of liver samples for hepatotropic viruses nor aggressive antiviral therapies in otherwise asymptomatic patients.
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