The Association Between Biliary Atresia and Cytomegalovirus Hepatitis

Address for correspondence: Hussam H lazim Microbiology Department, College of Medicine, Al-Nahrain University Kadhimiya, Baghdad, Iraq Tel: +9647801612798 E-mail: hussam_lazim@yahoo.com 1Hussam Hussain Lazim (M.Sc. Microbiology) College of Medicine, Al-Nahrain UniversityBaghdad, Iraq, 2Hala Sameh Arif (C.A.B.P. Pediatric) College of Medicine, Al-Nahrain UniversityBaghdad, Iraq, 3Haider Sabah Kadhim (Ph.D Microbiology) College of Medicine, Al-Nahrain University-Baghdad, Iraq, 4Khitam Razak AlKhafaji (F.I.C.M.S Pathology) College of Medicine, Baghdad University-Baghdad, Iraq, 5Kifah Hamdan Abdulghafour (Ph,D Pathology) College of Medicine, Baghdad University-Baghdad, Iraq Iraq. Abstract


Introduction
B iliary atresia (BA) is a disease characterized by a biliary obstruc on of unknown origin that presents in the neonatal period and it is the most frequent surgical cause of cholesta c jaundice in neonates, also it is a severe hepatobiliary disease in infancy characterized by a progressive, fi bro-oblitera ve process aff ec ng extrahepa c as well as intrahepa c bile ducts, leading to early liver cirrhosis 1 .The cause of BA remains unknown.Theories on pathogenesis include gene c predisposi on, abnormal morphogenesis, vascular abnormali es, exposure to environmental toxins, viral infec on, and autoimmune mediated bile duct destruc on 2 .
Mul ple viruses including hepa s B, human papillomavirus, Epstein-Barr virus, cytomegalovirus (CMV), rotavirus and reovirus have been proposed in the e ology of BA. 3,4,5,6 .Cytomegalovirus (CMV) is a member of the Herpesviridae 7 .CMV infec on is acquired either in the perinatal period and infancy or in adulthood through sexual contact, organ transplanta on or blood transfusion 8 .CMV considered as a one of agents that can infect the liver and cause hepa s 9 .The virus can replicate in both hepatocytes and cholangiocytes, it could directly induce injury in the liver and bile duct system, and induce immune damage in infected cells, revealing inclusion bodies in hepatocytes and vascular epithelial cells, especially in epithelial lining cells of bile duct 10 .This study aimed to determine the role of CMV in children with both chronic hepa s (nega ve for hepa s B and C) and have biliary atresia (extra and intra) in the same me.

Materials and Methods
A retrospec ve study done on 13 liver ssue paraffi n blocks of pa ents admi ed to Gastroenterology and Hepatology Teaching Hospital and Children Welfare Teaching Hospital (Medical City-Baghdad).Specimens collec on was done in the period from February 2014 to August 2014, for liver biopsies that were done during the period from 2008 to 2014 for children suff ering un explained hepa s (serologically nega ve for hepa s B and C) with biliary atresia (9 cases extra and 4 cases intra).Pa ents records were revised for: Age, sex, clinical manifesta ons.The diagnosis based on the presence of CMV protein (pp65) by using immunohistochemistry (IHC).Immunohistochemical staining was performed on paraffi n blocks a er sec oned (4 μm).We dewaxed the slides in xylene followed by rehydra on via a descending ethanol series (90%-50%).The slides were placed in the an gen retrival solu on (Sodium citrate buff er, PH 6.0) in water bath 95Cº for 5 min.Sec ons were blocked for endogenous peroxidase (3% H2O2, for 15 min at room temperature).Drops of protein blocks were added to slide s (15 min at room temperature).Monoclonal an body an CMV pp65 (dilu on 1:150, code number: ab49214, Abcam, USA) was added to slides and incubated at 37 Cº for 1 hr, then placed in 4Cº overnight.For visualiza on, we used a horseradish peroxidase detec on system.Finally, posi ve cells were visualized with the chromogendiaminobenzidine (DAB).A er counter staining them with hematoxylin.Dehydra on was done and sec ons were mounted with Disteren Plas cizer Xylene (DPX) and covered by coverslips and examined under light microscope.Posi ve control slide of Newcomer supply company (USA), this slide are posi ve for CMV proteins.Single experienced histopathologist revised the biopsies ssue for grading for infl amma on and staging for fi brosis.The grading of infl amma on and staging of fi brosis were determined according to Knodell histological ac vity index as follows; infl amma on : Nil (≤2), Mild (3-6), Moderate (7-11) and severe (≥12) while the fi brosis: Nil (1), Mild (2), Moderate (3) and Severe (≥4).

Sta s cal analysis:
The sta s cal analysis of this study performed with the sta s cal package for social sciences (SPSS) 19.0 and Microso Excel 2013.Categorical data formulated as count and percentage.Chi-square test was used to describe the associa on of these data.The lower level of accepted sta s cal signifi cant diff erence is below 0.05.

Results
Posi ve IHC for CMV pp65 was 10 cases (76.9%) as shown in Table 1 and Figure 1.The mean age of pa ents was (3.8) months with median (3) months (the age range from 2-14 months) and ten (76.9%) of thirteen were younger than 6 months of age.There was a ra o of nine males to four females (Table 1).
According to histopathological reports, there were grading for infl amma on and staging for fi brosis (Figure 3 and 4).There were 6 of 8 (75%) of cases with mild infl amma on were posi ve for CMV pp65IHC.There three out of six (50%) of cases with moderate fi brosis were posi ve for CMV IHC.There were 10 (76.9%) cases showed bile duct prolifera on (Figure 2) and 6 (46.1%) with giant cell hepa s (Figure 3).There were also 6 (46.1%) cases showing bile thrombi in their liver histology (Figure 4).

Discussion
CMV infec on is the most frequent congenital infec on worldwide and is various in its clinical manifesta ons 11 .Infants may acquire CMV infec on from the mother as a result of intrauterine infec on (congenital infec on), or through contact with infected genital secre ons during passage through the birth canal (perinatal infec on), or postpartum through breast feeding (postnatal infec on) 12 .
CMV has the ability to replicate in both hepatocytes and cholangiocytes.This virus could directly induce injury in the liver and bile duct system, and induce immune damage in infected cells, revealing inclusion bodies in hepatocytes and vascular epithelial cells, especially in epithelial lining cells of bile duct 10 (Figures 5 and 6).The swollen bile duct epithelium which is the cause of the unsmooth biliary fl ow could lead to intra-/ extra-hepa c cholestasis 13 .
In this study the detec on of CMV infec on was based on presence of virus protein pp65.This protein represents the largest component in virus structure 14 .The expression of this protein coincides with viral ly c replica on 15 , also the expression of this protein refl ect the ac ve viral infec on 16 .
Most of cases in this study were located in the age of less than 6 months; and this may be due to immaturity of immune system, congenital and perinatal infec on 17,18 .Similar age prevalence was reported also by Soomro et al 2011 19 .
The cases with mild histological infl amma on were the highest among others and the cases with moderate histological fi brosis were the highest among others.Regarding infl amma on, the explana on of this situa on may be due to that infl amma on is s ll in its beginning or infl amma on ended from long me with signifi cant fi brosis.While for the fi brosis, the result refl ects the extent of persistent or progressive hepa c injury and the small age of pa ents may be do not give a chance for fi brosis to develop into severe stage 20 .Ten cases with cases with bile duct prolifera on.The obstruc on of bile duct leads to lack of bile fl ow and as the result of the pressure of bile cause bile duct prolifera on 21 .Also six cases with giant cell hepa s, it's a common histological fi nding in infants with neonatal cholestasis 22 .

Conclusions
From current study, we conclude that CMV is one of the important viruses that can causes hepa s in infants (whom are nega ve for hepa s B and C), also this virus has signifi cant role in pathogenesis of biliary atresia.

Table 1 :
Characteris cs of pa ents with CMV pp65 IHC result

CMV pp65 IHC Type of BA Signs & symptoms Sex Age months Pa ents No.
M:male, F:female, HSM:hepatosplenomegaly, P. stool: Pale stool.

Table 2 :
Distribu on of CMV pp65 IHC according to infl amma on

Table 3 :
Distribu on of CMV pp65 IHC according to fi brosis