Symptomatic Neonatal Hepatitis a Disease From a Virus Variant Acquired in Utero

The hepatitis A virus (HAV) virus genome consists of ∼8000 nucleotides and is highly conserved.1 Transmission of HAV is almost exclusively horizontal by the fecal-oral route. Transmission is very rare during the neonatal period although outbreaks of severe hepatitis A have been reported in pediatric intensive care units2-7 attributed to transfusion of contaminated blood,2, 3 and vertical transmission,4 with horizontal spread by attendant health care personnel.3, 6 One case of vertical transmission followed maternal acute infection presenting with jaundice immediately after delivery at 33 weeks gestation.5 Transmission occurred presumably from fecal contamination around delivery because HAV RNA was not detected in cord blood. HAV RNA was documented in serum and feces from the baby at 32 (but not at 101) days after birth. Immunoglobulin was administered on Day 5, IgM anti-HAV remained undetectable and the baby remained symptomless.5 We present direct evidence of vertically transmitted hepatitis A infection that resulted in symptomatic disease in the baby. We identified the same virus variant in the mother who presented with symptomatic infection that persisted from the fourth month of pregnancy through to delivery at 36 weeks gestation. Case report. A 29-year-old Indian woman developed symptomatic hepatitis A infection during the fourth month of pregnancy after returning from a family trip to India. She was IgM anti-HAV-seropositive, with an aspartate aminotransferase (AST) value of 652 IU/l, serum total bilirubin of 44 μmol/l, international normalized ratio 1.1 and serum alkaline phosphatase of 1187 IU/l. Her other three children of 4, 5 and 8 years seroconverted but remained symptomless. The father was seronegative for IgM and IgG anti-HAV. No jaundice had been noted in the three previous pregnancies. She remained persistently jaundiced and HAV IgM-seropositive with elevated AST values (average, 103 IU/l) until spontaneous vaginal delivery at 36 weeks gestation. The baby weighed 1.82 kg, (−2 SD below the third percentile for gestational age). The mother became anicteric with normal liver tests within the first week postpartum. The baby was breast-fed for 10 days and the early postnatal period was uneventful. At Day 12 he became unwell with fever and was treated with a 5-day course of broad spectrum antibiotics. At 4 weeks fever recurred and dark urine, jaundice and mild hepatosplenomegaly were noted. Laboratory tests demonstrated an AST value of 109 IU/l, total bilirubin of 170 μmol/l (10 mg/dl); conjugated 106 μmol/l (6.2 mg/dl), alkaline phosphatase of 3193 IU/l, international normalized ratio 1.1, albumin 37 g/l, gamma-glutamyltranspeptidase value of 126 IU/l, total leukocyte count of 25.6 × 109/l. Total IgG, 7.58 g/l; IgA, 0.43 g/l; IgM, 0.57 g/l. He was seronegative for hepatitis B surface antigen, IgM anti-hepatitis B core antibodies and antibodies to hepatitis C and E viruses. HAV IgM and IgG antibodies (enzyme-linked immunosorbent assay) were positive in mother and baby. Liver biopsy at 8 weeks of age showed a severe inflammatory infiltrate of mononuclear cells in the portal tracts with marked cholestasis. HAV RNA was detected by reverse transcriptase (RT)-PCR in the baby's serum and liver (see Materials and methods). Other possible causes of conjugated hyperbilirubinemia in infancy were excluded by appropriate investigations. The baby made a full clinical and biochemical recovery. He remained well 4 years later. Materials and methods. Serial aliquots of stored serum (−20°C) were available from the mother (at 6 and 7 months of pregnancy and postpartum). Serum and a liver biopsy snap frozen (−70°C) at 4 weeks when the baby presented with hepatitis were available. RNA was extracted from 100 μl of serum (RNAID, La Jolla, CA) and from liver triturated under liquid nitrogen.8 First strand cDNA synthesis and first and second round PCRs were performed with primers to the 5′-untranslated region (5′UTR) of HAV RNA: first round (nt 41-60 and nt 290-309: HM175wt9 and second round [nucleotides (nt) 73 to 92 and nt 243 to 260], a gift of Dr. P. Simmonds, Edinburgh and to open reading frame 2 of HEV RNA.8 Controls included positive and negative sera and livers extracted concurrently from patients with acute, including fulminant, hepatitis A and E. Amplified PCR products were ligated into the pCRII vector and transformed (TA cloning; Invitrogen). Colonies (>5 per sample) were sequenced by the dideoxy sequencing method (Sequenase II; UBS). Multiple PCR reactions were performed separately on the three positive samples with different batches of Taq polymerase (AB Bioquote). Sequence consensus was confirmed with forward and reverse primers. Results. HAV RNA was detected in the serum of the mother (fourth month of pregnancy; subsequent sera were negative) and in the serum and liver of the baby at 4 weeks presentation with hepatitis. HEV RNA was not detected in any sample of serum or the liver biopsy. The HAV RNA in the liver and serum samples from the baby showed >99% homology with the maternal sequence. Separate reverse transcriptase-PCRs and sequence analyses of the 5′UTR for nt 93 to 243 showed an 11-nt deletion in the pY1 tract and two point deletions (at nt 103 or 104 and 119) in two major functional domains (nt 100 to 106 and nt 113 to 119). The three isolates showed 95% homology with 2 naturally occurring strains represented by the simian (captive owl monkey in Panama) isolate PA21 (genotype IIIa) and CF53 (genotype II) from France.1, 10 Two point deletions occurred at nt 103 or 104 and nt 119. A 3-nt deletion occurred in the 3′ end of the pY1 tract between nt 139 and nt 141. Comparable deletions of laboratory-adapted isolates have been reported to impair translation in vitro by interfering with the unfolding of RNA.11-13 Conversely the 2-nt deletion (UU) at nt 203 to 204 also seen here, as well as in CF53 and PA21, has been shown to be associated with enhanced growth in cell culture and virus replication by facilitating cap-independent translation in a specific cell line, derived from African green monkey cells.13 Five nucleotides were unique to the mother and baby compared with CF53, PA21 and HM175: nt105 and 139 as deletions; nt 112C; nt 177G and 180U. Discussion. A naturally occurring (nonpassaged) HAV variant originating in India was associated with symptomatic and protracted hepatitis A disease in a pregnant mother and subsequently in her baby born 4 months later. The intrauterine growth retardation observed may have reflected the severity and protracted illness in the mother. The three isolates showed 95% homology with the simian isolate PA21, representing genotype IIIa that is closely related to human isolates originating in Nepal and India.1, 10 The deletions found were similar to those reported to be involved in internal initiation of translation based on in vitro studies of laboratory-adapted isolates.11-13 Our observations show that infectious hepatitis A can persist >4 months in humans and result in vertical transmission and development of symptomatic hepatitis A in the neonate. Accordingly to prevent spread we recommend that neonates born after a maternal history of hepatitis A during pregnancy should be isolated from other babies after delivery. Immunoglobulin should be considered for other infants in the same ward especially if virus (or HAV RNA) is detectable in maternal or infant stools. Attendant health care personnel should be immune to hepatitis A. Transplacentally acquired maternal IgG antibodies usually have a protective role in perinatal infections, including hepatitis A. The baby did not receive immunoprophylaxis because transmission of hepatitis A was not anticipated after maternal infection presenting 4 months earlier. We conclude that hepatitis A virus can be implicated, albeit rarely, in the etiology of neonatal conjugated hyperbilirubinemia with potential for symptomatic illness and spread. Acknowledgments. Presented in part at the Triennial Symposium of Viral Hepatitis and Liver Disease, Rome, Italy, May, 1996. Elizabeth A. Fagan, M.D. Nedim Hadzic, M.D. Romil Saxena, M.D. Giorgina Mieli-Vergani, M.D. Section of Hepatology; Rush Presbyterian St. Luke's Medical Center; Chicago, IL (EAF) Department of Child Health; King's College Hospital; London, UK (NH, GMV) Department of Surgical Pathology; Yale University School of Medicine; New Haven, CT (RS)