A male two-month old infant was admitted for the first time to the Department of Paediatric Gastroenterology and Hepatology, CHCSM, for liver diagnostic work-up under suspicion of a congenital liver disease underlying severe clinical presentation of previously established CMV infection. The patient and his family members were Caucasians. The patient's sister (5.5 years) suffered from spondylo-meta-epiphyseal dysplasia, and other family members were healthy. The patient was born from the second pregnancy and normal delivery, body weight (BW) 2910 g, body length (BL) 49 cm and APGAR 10/10. On the second day of life he presented with jaundice and petechial haemorrhages of the skin and was admitted to the Neonatology Department in another hospital. Laboratory tests revealed hypoglycaemia, thrombocytopenia, conjugated hyperbilirubinemia, hypoalbuminemia, increased activities of aspartate aminotransferase and alanine aminotransferase and significantly prolonged prothrombine time, whereas the activity of the gamma-glutamyltransferase was in the reference interval (Table 1). The multisliced computerized tomography (MSCT) (Siemens Somatom Emotion 2-sliced, Germany) revealed a choledochal cyst type I or II. Abdominal magnetic resonance imaging (MRI) (Siemens MAGNETOM Harmony 1T, Germany) revealed more voluminous cholecyst, medially elongated, and no other formations of the cystic contents. Intrahepatic bile ducts were not dilated, and extrahepatic bile ducts have failed to show up. Brain ultrasound (USD) (Siemens Sonoline G 40 TM) and Hearing Screening (OAE) were normal.
Augmentation [fresh frozen plasma (FFP) 15 mL/kg and 5% albumin 5 mL/kg] and antimicrobial therapy comprising ampicilin (100 mg/kg) and gentamycin (3 mg/kg) combination were initiated, accompanied with combined parenteral and enteral feeding due to a poorly tolerated oral intake of amino-acid based infant formula. Because the findings were suggestive of perinatal liver failure the patient was transferred to the Neonatal Intensive Care Unit (NICU), and later the same day moved to the University Hospital for Infectious Diseases (UHID) due to positive PCR for CMV DNA (CMV real-time PCR assay; Cepheid, CA, USA), both in blood (452 copies/mL) and urine (393 4210 copies/mL), but not in the cerebrospinal fluid. Samples for PCR for CMV DNA were taken during hospitalisation in NICU, and laboratory diagnostics performed at UHID. Upon admission to UHID parenteral treatment with ganciclovir along with symptomatic and supportive measures was introduced. Initially, despite daily transfusions of FFP (20 mL/kg/day prepared by standard procedures of responsible manufacturer, Croatian Institute for Transfusion Medicine), vitamin K (0.03 mg/kg/day), prothrombin complex concentrate, a mixture of clotting factors II, VII, IX, X and protein C and S (Octaplex, Octapharma Austria) at a dose of 4 mL (100 Units) during four consecutive days and anti-thrombin III (Antithrombin III Immuno, Baxter, USA) at a dose of 300 Units/daily (six doses cumulatively), coagulation parameters remained low, although visible bleeding was not recorded. Conjugated hyperbilirubinemia persisted, and at discharge serum total bilirubin was 205.8, direct 78 mumol/L. Only after four weeks (5th week of life) a graduate normalization of coagulation factors and platelets occurred. Hospitalization was complicated by catheter-related sepsis caused by Methicillin-resistant Staphylococcus aureus (MRSA), which has been treated with vancomycin (45 mg/kg) for 14 days with good response, and mild flow rotavirus gastroenteritis (real-time PCR method) treated with standard hypoosmolar oral rehydration solution. Parenteral treatment with ganciclovir (at a dose of 2 x 6 mg/kg/daily) was carried out on daily basis during 3 weeks and continued with oral treatment with valganciclovir (at a dose of 2 x 18 mg/kg/day) until CMV PCR DNA analysis from blood negativization at the age of 3 months. Oral intake (standard infant formula) was gradually introduced and increased with initially good start in weight gain, but at the end weight become stagnant. Psychomotor development was satisfactory. Presence of the infection with other hepatotrophic viruses [Hepatitis A virus (HAV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Epstein-Barr virus (EBV)] was tested; Anti-HAV total (IgM/IgG), Anti-HAV IgM, HBsAg, anti-HBs, total anti-HBc, IgM anti-HBc, HBeAg, anti-HBe, DiaSorin, Italy; HCV Ag-Ab, Bio-Rad, France; HCV confirmation test immunoblot/western blot, Innogenetics, Belgium; however only positive was PCR for CMV DNA. The infant was vaccinated with ENGERIX-B [Hepatitis B Vaccine (Recombinant)] GlaxoSmithKline, while vaccine against tuberculosis BCG was not administered.
At the age of 2 months the patient was transferred to the Department of Paediatric Gastroenterology and Hepatology, CHCSM for further liver work-up. At admission was in good general condition, 54.5 cm length [13th centile (c)], 4200 g weight (11th c; BMI 14.4 kg/m2), and head circumference was 40 cm (76th c). Skin and sclera were icteric, with no signs of rash or bleeding, decreased fat tissue especially on the limbs, normal bone and joint systems. Milder abdominal distension with soft liver palpable for 2 cm and spleen palpable for 1 cm was present. There were no signs of peripheral oedema or deformity. Neurological status was mildly hypotonic, in accordance with age.
Laboratory testing revealed reduced A1AT serum concentration (0.2-0.3, reference values 0.9-2.0 g/L), which was an important cooperative factor apparently necessary for the development of unusually severe clinical presentation in the first days of life with signs of liver failure. Due to a decreased A1AT serum concentration, for the child and his parents A1AT genotyping was performed by using Light-Cycler  480 II (Roche Diagnostics Deutschland GmbH, Mannheim, Germany) real-time PCR method (LightMix  Kit Alpha1-Antitrypsin (AAT) Pi*S and Pi*Z, TIB MOLBIOL GmbH, Berlin, Germany). Genotyping was followed and confirmed with phenotyping (isoelectric focusing method followed by immunofixation on precasted agarose gel; semi-automated method on HYDRASYS Focusing system, Sebia, France), as it is our standard protocol for low A1AT serum values. The combination of genotyping and quantification, with a reflex to phenotyping, is the optimal strategy for the laboratory evaluation of A1AT deficiency. Genotype/phenotype results were concordant to each other: PiZZ phenotype for the child and PiMZ phenotype for both parents (mother A1AT serum concentration was 0.73, and father 0.71 g/L). A1AT genotyping was in concordance with phenotypes. Sweat test for cystic fibrosis screening, faecal elastase as well as thyroid hormone levels were normal. Abdominal USD (Siemens Sonoline G 40 TM, Germany) was unremarkable. Technetium 99m-hepatic iminodiacetic acid (HIDA) (Siemens Symbia T Germany) scintigraphy confirmed significant intrahepatic cholestasis with normal gallbladder function and the preserved passage into the intestine. Electrocardiographic and heart ultrasound (Siemens Sonoline G 40 TM) finding was normal. There were no signs that would raise suspicion of Alagille syndrome.
Therapy with ursodeoxycholic acid (UDCA; 10 mg/kg/daily), fat-soluble vitamin supplementation and medium-chain triglycerides (MCT) oil in meals was introduced along with hypercaloric feeding. UDCA may significantly improve clinical status and liver test results in some children with liver disease associated with PiZZ A1AT deficiency. High doses of vitamin D3 and other liposolubile vitamins (A, E, K) reimbursement continued as well as therapy with valganciclovir till the age of 3 months when CMV PCR DNA analysis from blood was negative. Coagulation parameters were corrected by daily FFP transfusions, vitamin K, prothrombin complex and antithrombin III supplementation. There was no need for vitamin K parenteral application. Hypercaloric diet (120% of the average daily caloric requirement) with extensively hydrolysed infant formula due to the higher MCT concentration was introduced and well tolerated.
Early introduced antiviral therapy and persistent supportive therapy in the first weeks of life bridged severe coagulation disorder, as well as hypoalbuminemia which were accompanied by all the expected consequences. In the further course liver disease was biochemically characterized by signs of moderate cholestasis with slowly but completely normalized synthetic and metabolic function. Despite the above mentioned events, the infant`s progress, somatic and psychomotor, was satisfactory. Within CMV infection there were no signs of the central nervous system impairment.
During the entire hospitalisation the patient was in good general condition, afebrile, vigorously crying, interestedly observing the surroundings, with good appetite and normal stools. He was discharged at the age of three months with a satisfactory weight gain (4750 g; 15th c) as well as somatic and motoric progress. Despite the high serum concentrations of bile acids (267.8; reference values < 10 micromol/L) there were no signs of pruritus.
The clinical course in the following 15 months showed excellent physical condition with normal weight gain and psychomotor development. At home, he was fed with frequent meals of extensively hydrolysed infant formula thickened with rice cereal and MCT oil added. The introduction of normal food was started at the age of 6 months. Vitamin D3 and multivitamin drops with docosahexaenoic acid (DHA) mL was continued and UDCA slowly tapering in accordance with the normalization of the liver function tests. At the age of 18 months, laboratory testing revealed no signs of cholestasys with normal values of serum bilirubin levels, bile acids (2.3 micromol/L) and vitamin D3 (170; reference values >= 75 nmol/L) levels, but with persistent signs of moderate hepatocellular lesion (Table 1).