Extrahepatic biliary atresia in a premature neonate with congenital cytomegalovirus infection

Biliary atresia (BA) is an obliterative cholangiopathy of infancy. The pathogenesis is unclear, but two pathological mechanisms have been proposed: (1) bile duct malformation during embryological and fetal development, and (2) disruption of already formed bile ducts by cholangio-destructive effects of a virus or by a virus-induced inflammatory reaction. Human cytomegalovirus (CMV) is a candidate virus, though the evidence supporting CMV as a causative agent of BA has thus far been circumstantial.1Averbukh L.D. Wu G.Y. Evidence for viral induction of biliary atresia: a review.J Clin Transl Hepatol. 2018; 6: 410-419Crossref PubMed Scopus (17) Google Scholar Herein, we present a case of a premature neonate with congenital disseminated CMV infection, extensive and preferential localisation of CMV inclusions in the bile duct epithelium, and progressive obliteration of the extrahepatic bile tract. A 33-year-old G4P2 woman, with adequate prenatal care, presented to the hospital at 26 2/7 weeks of gestational age (GA) with breech presentation and suspected placental abruption. She underwent caesarean section after receiving a dose of betamethasone and gave birth to a Caucasian baby girl. The newborn had a birthweight of 0.86 kg (50–90th percentile) and a head circumference of 22.5 cm (10–50th percentile). She had perinatal depression with APGARs of 2, 5, and 5 at 1, 5, and 10 minutes, respectively. The neonate was intubated and transferred to the neonatal intensive care unit. She was diagnosed with acute liver failure (Table 1). Prophylactic acyclovir was administered. Her condition unfortunately continued to deteriorate, and she was found to have cerebellar haemorrhage on imaging. Care was redirected towards comfort measures, and she died on day 5 of life. The newborn metabolic screen was unremarkable. Extensive infectious workup, including blood culture, serology (Epstein–Barr virus, hepatitis A/C, Toxoplasma gondii), and nucleic acid tests (NATs) (adenovirus, enterovirus, parechovirus, and parvovirus B19) were negative. CMV serology was not performed, but the urine CMV polymerase chain reaction (PCR) result was pending at the time of death. The mother consented to a full autopsy. The placenta was not available for pathological examination.Table 1Laboratory test resultsTestReference range (units)Day 1Day 2Day 3Day 4Day 5Bilirubin, total0–150 (μmol/L)207182153143205Bilirubin, direct0–20 (μmol/L)2847Aspartate aminotransferase10–65 (U/L)2704981963569Alanine aminotransferase≤34 (U/L)19610012695Alkaline phosphatase70–320 (U/L)264355Gamma glutamyl transferase20–200 (U/L)159134Ammonia; plasma12–47 (μmol/L)222134168INR0.9–1.12.02.21.91.6APTT28.0–38.0 (seconds)46.452.249.453.0Alpha 1 antitrypsin0.90–2.00 (g/L)1.52Abnormal results in bold.APTT, activated partial thromboplastin time; INR, international normalised ratio. Open table in a new tab Abnormal results in bold. APTT, activated partial thromboplastin time; INR, international normalised ratio. Postmortem examination revealed a jaundiced, premature female neonate with mild abdominal distension. Most organs had a yellow discoloration (Fig. 1A). The liver (84.6 g; expected 33.2±8.8 g) and spleen (6.82 g; expected 1.38±0.55 g) were enlarged (Fig. 1B). The gallbladder was normally formed and contained clear mucoid fluid devoid of bile pigments, also known as white bile, suggesting blockage in the extrahepatic biliary tract. The extrahepatic biliary tract appeared macroscopically normal. Cerebellar haemorrhage was identified. CMV immunohistochemical stains were performed on 4-μm sections of formalin-fixed, paraffin-embedded tissues using a Dako Omnis automated stainer (Dako Omnis ready-to-use CMV antibody, high pH, 20 min antibody, 20 min detection kit). CMV-immunopositive inclusions were present in multiple organs, indicative of disseminated CMV infection. CMV inclusions were frequently present in the intrahepatic bile duct epithelial cells and occasionally in the extrahepatic bile duct epithelial cells, causing bile duct injury (Fig. 1C–F). Additionally, the liver had massive hepatocellular necrosis and haemorrhage (Fig. 1G–I), though only rare CMV inclusions were found in the hepatocytes. There was no evidence of ductal plate malformation. Following formalin fixation and paraffin embedding, the porta hepatis was sectioned at 50 μm intervals. Initial sections showed that the common hepatic duct was patent. However, on subsequent sections, the common hepatic duct was replaced by smaller bile ductules and was eventually obliterated, with surrounding chronic inflammation and fibrosis (Fig. 2A–C). There was no histological or immunohistochemical evidence of other infections (e.g., herpes simplex virus, adenovirus, parvovirus B19, Treponema pallidum). Bacterial cultures of blood, heart, and lung tissues showed no growth. CMV PCR performed on frozen postmortem tissues (thymus, heart, lung, liver, kidney) and premortem urine were positive. Of note, there was no histological evidence of neonatal haemochromatosis or haemophagocytic lymphohistiocytosis. Cytogenetics analysis by chromosomal microarray was negative. In light of the autopsy findings of disseminated CMV infection in the neonate, the mother's blood sample (collected at 26 2/7 weeks GA) was sent for CMV serology testing, and the result was IgG+/IgM–/high IgG avidity. The mother's frozen prenatal blood sample (collected at 9 5/7 weeks GA) was also tested, and the result was CMV IgG+/IgM+/low IgG avidity. The paired serological results indicated primary maternal CMV infection around the time of conception.2Prince H.E. Lapé-Nixon M. Role of cytomegalovirus (CMV) IgG avidity testing in diagnosing primary CMV infection during pregnancy.Clin Vaccine Immunol. 2014; 21: 1377-1384Crossref PubMed Scopus (131) Google Scholar This case was unique in that it simultaneously demonstrated the following: (1) CMV infection in the mother around the time of conception, (2) transmission of CMV from the mother to the fetus in utero, causing congenital CMV infection, (3) preferential and frequent CMV inclusions in the neonate's bile duct epithelium, and (4) obliteration of the extrahepatic bile duct by a fibroinflammatory process, likely triggered by CMV. In this case, the gallbladder, extrahepatic, and intrahepatic biliary systems were fully formed. The presence of a fully formed biliary tree suggested that CMV infection in early gestation did not cause extrahepatic biliary atresia (EHBA) by disturbing the embryological/fetal development of the biliary tract in this case. Rather, CMV damaged the biliary tract, after the latter was already formed, by infecting the bile duct epithelium and inducing a fibroinflammatory reaction which obliterated the extrahepatic bile duct, causing EHBA. In 1974, Benjamin Landing, a paediatrician, proposed that infantile obstructive cholangiopathies could have a viral aetiology.3Landing B.H. Considerations of the pathogenesis of neonatal hepatitis, biliary atresia and choledochal cyst--the concept of infantile obstructive cholangiopathy.Prog Pediatr Surg. 1974; 6: 113-139PubMed Google Scholar Since then, many case-control and cohort studies have examined the relationship between CMV and BA.1Averbukh L.D. Wu G.Y. Evidence for viral induction of biliary atresia: a review.J Clin Transl Hepatol. 2018; 6: 410-419Crossref PubMed Scopus (17) Google Scholar Results of these studies have been mixed. Only a few case reports with pathological descriptions linking congenital and infantile CMV to extrahepatic BA (EHBA) were available.4Stern H. Tucker S.M. Cytomegalovirus infection in the newborn and in early childhood. Three atypical cases.Lancet. 1965; 2: 1268-1271Abstract PubMed Scopus (27) Google Scholar, 5Lurie M. Elmalach I. Schuger L. Weintraub Z. Liver findings in infantile cytomegalovirus infection: similarity to extrahepatic biliary obstruction.Histopathology. 1987; 11: 1171-1180Crossref PubMed Scopus (19) Google Scholar, 6Hart M.H. Kaufman S.S. Vanderhoof J.A. et al.Neonatal hepatitis and extrahepatic biliary atresia associated with cytomegalovirus infection in twins.Am J Dis Child. 1991; 145: 302-305PubMed Google Scholar, 7Kodo K. Sakamoto K. Imai T. et al.Cytomegalovirus-associated biliary atresia.J Pediatr Surg Case Rep. 2018; 35: 17-20Crossref Scopus (1) Google Scholar Some of these cases demonstrated: (1) CMV inclusions in the bile ducts and/or livers of patients who subsequently developed EHBA, or (2) detection of CMV in the blood and/or urine of patients with EHBA. A few case reports also demonstrated preferential localisation of CMV inclusions in the bile duct epithelium but not in the hepatocytes.8Kage M. Kosai K. Kojiro M. et al.Infantile cholestasis due to cytomegalovirus infection of the liver. A possible cause of paucity of interlobular bile ducts.Arch Pathol Lab Med. 1993; 117: 942-944PubMed Google Scholar,9Finegold M.J. Carpenter R.J. Obliterative cholangitis due to cytomegalovirus: a possible precursor of paucity of intrahepatic bile ducts.Hum Pathol. 1982; 13: 662-665Crossref PubMed Scopus (73) Google Scholar However, in these previously published case reports and studies, CMV inclusions in the bile duct epithelium and obliteration of the extrahepatic bile duct had not been simultaneously detected in the same patient. This case offers a few novel insights. For one, this is the first report that establishes the timing of maternal CMV infection in an infant with congenital CMV infection and EHBA. Our laboratory routinely stores prenatal blood samples, thereby allowing us to retrieve and test the first-trimester maternal blood sample and to determine that the mother acquired CMV infection around the time of conception. Furthermore, no prior studies have shown the co-occurrence of CMV inclusions in the bile duct epithelium and obliteration of extrahepatic bile ducts. In this case, we had robust histological and immunohistochemical evidence of extensive and preferential localisation of CMV inclusions in the bile duct epithelium and fibro-obliteration of the extrahepatic bile duct. Finally, due to the sectioning of the porta hepatitis at close intervals, we could observe the progression of a patent common hepatic duct into an obliterated one, associated with a fibroinflammatory response. The fibroinflammatory response was most likely triggered by CMV given that many other potential infectious and inflammatory aetiologies were ruled out. We acknowledge that the findings of this case could not indisputably demonstrate a cause-and-effect relationship between CMV and EHBA. However, this case provided some of the strongest supporting evidence to date that CMV has cholangio-destructive effects and can likely induce a secondary inflammatory reaction associated with obliteration of the extrahepatic bile duct. We also noted that in addition to EHBA, there was massive hepatic necrosis—a rare but well-described manifestation of CMV infection—which would explain the acute liver failure. The timing of primary maternal CMV infection around the time of conception could possibly explain the severe hepatic presentation in this infant. A recent meta-analysis showed that the rate of vertical transmission following primary maternal CMV infection increases with advancing gestation: lowest in the preconception period and highest in the third trimester. Yet the rate of fetal insults in CMV transmission is highest in the preconception period and decreases with advancing gestation, and severe fetal impairments typically occur with primary maternal CMV infection before or in the first trimester.10Chatzakis C. Ville Y. Makrydimas G. et al.Timing of primary maternal cytomegalovirus infection and rates of vertical transmission and fetal consequences.Am J Obstet Gynecol. 2020; 223: 870-883.e11Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar Additionally, it was unclear why previous studies did not identify CMV inclusions in the bile duct epithelium and EHBA simultaneously. A possible explanation may be that CMV inclusions were rapidly cleared before EHBA was diagnosed. The aetiology of EHBA is complex and likely involves both genetic and environmental factors.11Lendahl U. Lui V.C.H. Chung P.H.Y. et al.Biliary atresia - emerging diagnostic and therapy opportunities.EBioMedicine. 2021; 74103689Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar By reporting this case, we aim to raise awareness among healthcare providers of the importance of ruling out congenital CMV infection and the possibility of a secondary diagnosis, such as EHBA, in cases of neonatal acute hepatic failure. Additionally, we hope that the findings of this case add to our current understanding of CMV in the pathogenesis of EHBA. The mother in this report gave informed consent for publication of this case. The authors state that there are no conflicts of interest to disclose. No funding was secured for this study.