Diagnosis of Neonatal and Infantile Cholestasis

➡️Definition and causes (1-4)

Neonatal and infantile cholestasis (NIC) is defined as impaired bile formation, flow, or both and usually manifests in the first year of life, particularly in the first three months. Its clinical presentation varies from self-limited illness to acute liver failure. It results in hepatic retention of bile and biliary substances, causing severe liver damage.  

The prevalence of NIC in infants is 1 out of 2,500. Its causes are due to either surgical or medical disorders. Such differentiation is crucial for the proper management and a better outcome. If biliary atresia (BA) is the cause, early referral for surgical intervention is the key to successful management. Timely diagnosis and management of treatable metabolic conditions, such as galactosemia, provide a better outcome.

Classically, infants present with prolonged jaundice (conjugated), dark urine, and pale stools. Prolonged jaundice, defined as jaundice lasting for more than two weeks of age, should raise the suspicion of cholestasis among health practitioners. Prolonged jaundice necessitating serum-conjugated bilirubin assessment, is observed in about 15% of infants. In the neonatal period, hyperbilirubinemia occurs in about 50%, thus the expected occurrence of physiological jaundice usually delays the diagnosis of cholestasis. Infants may rarely present with a recurrence of jaundice (conjugated), after the complete disappearance of the neonatal physiological jaundice.

When cholestasis is suspected, its definition in infants is as follows: a serum conjugated (direct) bilirubin of more than 1 mg/dl if the total bilirubin is less than 5 mg/dl, or a conjugated component of more than 20% of the total when total bilirubin exceeds 5 mg/dl.

As NIC has a diverse set of etiologies, some of which are treatable while others are not, the second step, after documenting cholestasis, is to recognize its etiology. Generally, causes are classified as either surgical or medical. Causes may be also classified according to their site, whether as extrahepatic (BA, choledochal malformation (CM), cholelithiasis, inspissated bile or mucous plug, or spontaneous bile duct perforation) or an intra-hepatic. 

Surgical causes of cholestasis include BA and non-biliary atresia or CM. BA represents the most common recognizable cause of NIC. Its incidence ranges from 0.55 to 1.3/10,000 live births. BA accounts for approximately 20–35 % of infantile cholestasis. Besides, it is frequently the leading cause of pediatric liver transplantation.

Pathologically BA is a rapid progressive inflammation and fibrosis of the bile ducts (both extrahepatic, and intrahepatic) leading to their partial or complete obliteration. The etiology of BA is likely multifactorial, including infections, malformations, vascular, autoimmune, and toxic factors, as well as genetic, and epigenetic causes.

BA is classified into types 1, 2 (a/b), and 3 according to the extent of extrahepatic bile duct involvement. Type 1 and 2 represent about 20% of BA cases, with their pathology limited to the distal extrahepatic bile ducts. Type 1 affects the CBD, and type 2 the common hepatic duct. These types carry a good prognosis after timely surgical correction. Type 3 is more common. It is characterized by gallbladder involvement, with fibrous tissue in most cases, mucocele in a few, and deep fibrous plates in the extrahepatic bile ducts. It involves atresia of the right and left hepatic ducts to the level of the porta hepatis. BA exists as an isolated pathology or in association with other malformations. Extrahepatic malformation is a common association, occurring in 20%. BA with splenic malformation (BASM) is a common extrahepatic malformation characterized by asplenia or polysplenia, cardiovascular malformations (dextrocardia, preduodenal portal vein, agenesis of retro-hepatic inferior vena cava, and anomalous superior vena cava), and laterality defects (intestinal malrotation and situs inversus). A score for diagnosis of BA based on clinical data, biochemical results, ultrasound and histological findings and having an accuracy of 98.83 has been proposed and validated.

Choledochal malformation (CM) is also termed congenital biliary dilatation. It is a malformation of the pancreaticobiliary system, characterized by a dilatated biliary tract associated commonly with pancreaticobiliary mal junction. It is classified into 5 types.

Timely surgical correction is mandatory to prevent progressive hepatic damage and biliary obstruction and their consequences, such as growth retardation, cholangitis, pancreatitis, hepatic fibrosis, and even cirrhosis and late malignant transformation in any biliary remnant. CM should be differentiated from cystic biliary atresia, liver cyst, and duodenal duplication.

Medical causes of NIC include genetic, endocrine, infectious, hematologic, and toxic conditions, with many of them described as monogenic liver disorders. These conditions are categorized as intrahepatic causes of cholestasis.  

Genetic conditions include canalicular bile acid transport defects such as progressive familial intrahepatic cholestasis (PFIC), bile acid synthesis defects, lysosomal storage defects such as Niemann-Pick and Gaucher disease, amino acid metabolism defects such as tyrosinemia type 1, carbohydrate metabolism defects such as galactosemia and glycogen storage disease type IV, and urea cycle defect (Citrin deficiency), in addition to Alagille syndrome, cystic fibrosis, and alpha-1-antitrypsin deficiency.

Cholestasis may occur in association with endocrine disorders such as thyroid disorders, panhypopituitarism, and adrenal insufficiency or with infections such as cytomegalovirus, herpes simplex virus, parvovirus B19, human immunodeficiency virus (HIV), toxoplasma, syphilis, and congenital tuberculosis. It may be also a manifestation of hematologic conditions or an immune dysregulation such as hemophagocytic lymphohistiocytosis, congenital lupus, neonatal hemochromatosis (gestational alloimmune liver disease -GALD), and post-hemolytic conditions. In addition, cholestasis may occur secondary to certain conditions such as parenteral nutrition (parenteral nutrition associated cholestasis -PNALD), drugs, intestinal obstruction, cardiovascular disorders, neoplasia, and perinatal asphyxia.

Clinical manifestations (5-7)

History and physical examination are the initial steps in the evaluation and usually guide the diagnostic process. The report of acholic stools is a strong indicator of cholestasis. Dark urine is another nonspecific indicator of increased serum conjugated bilirubin levels.

Diagnosis of BA is time-critical and dependent on the identification of prolonged neonatal jaundice and pale or clay-colored stool. The identification of stool color may suffer from subjective reporting; an objective method of assessment, using a stool color card (SCC), may prove beneficial.

In Taiwan, screening infants with a stool color card was introduced in 2004. This markedly improved the sensitivity of detecting BA before 60 days, from 72.5% in 2004 to 97.5% in 2005. A recent study recommended using the stool color card as a mass neonatal screening tool, and including it in child health cards in low/middle-income countries such as Bangladesh for early pick-up of cholestatic cases.

Family history may suggest a genetic disorder such as cystic fibrosis, a-1-antitrypsin deficiency, or PFIC, or a disorder with an autosomal dominant inheritance pattern such as Alagille Syndrome.

Looking for bleeding or bruising due to a coagulopathy from vitamin K malabsorption or liver failure is essential in evaluating cholestatic infants.

Parameters of clinical interest in the history of the cholestatic infant

Family history

·       Consanguinity increases the risk of autosomal recessive disorders.

·       Neonatal cholestasis in parents or siblings may point to cystic fibrosis, a -1-antitrypsin deficiency, progressive familial intrahepatic cholestasis, and Alagille syndrome.

·   History of repeated fetal loss or early demise as GALD.

·   Spherocytosis and other hemolytic diseases which are known to aggravate conjugated hyperbilirubinemia

Prenatal history

·   Prenatal ultrasonographic findings may denote the presence of a CM or cholelithiasis.

·       Cholestasis of pregnancy may be seen in heterozygotes for PFIC gene mutations.

·       Maternal infections such as TORCH infections.

➡️Infant history

·       Gestational age: Prematurity is a risk factor for neonatal hepatitis.

·       Small for gestational age increases the risk of neonatal cholestasis and congenital infections.

·   Neonatal infections as in cases of urinary tract infection and sepsis-related cholestasis.

·       Sources of nutrition such as breast milk, formula, or parenteral nutrition, are important to suspect galactosemia, and PNALD.

·       Genetic and metabolic diseases may result in growth failure.

·       Persistent vomiting may point to metabolic diseases.

·       Delayed stooling may point to cystic fibrosis.

·       The presence of acholic stools suggests biliary obstruction.

·       Irritability, and lethargy suggest metabolic diseases, sepsis, or panhypopituitarism.

·       Hypoglycemia may suggest metabolic or endocrinal conditions.

➡️Physical findings in children with neonatal cholestasis

·       Assessment of general health: the finding of an ill-appearing infant may indicate that infections or metabolic diseases are the cause of cholestasis. Infants with biliary atresia typically appear generally thriving and well, apart from jaundice.

·       Dysmorphic features. Neonates with Alagille syndrome may exhibit characteristic facial appearance with a broad nasal bridge, triangular facies, and deep-set eyes.

·       Vision and slit lamp examination: may show cataracts (e.g., galactosemia- congenital rubella), posterior embryotoxon (e.g., Alagille syndrome), or septo-optic dysplasia.

·       Hearing assessment: is helpful in congenital infections, and storage disease.

·       Cardiac examination: murmur, signs of heart failure, and congenital heart disease are common in Alagille syndrome, BA, and BASM.

·       Abdominal examination: looking for the presence of ascites, abdominal wall veins, liver size and consistency, spleen size and consistency, and umbilical hernia.

·       A stool examination is crucial, and the primary physician should make every effort to view by himself the stool color does not depend on caregivers’ description. Acholic or hypopigmented stool suggest cholestasis or biliary obstruction.

·       Neurologic examination: Note overall vigor, tone and neonatal reflexes.

➡️Extended Stepwise Diagnostic Approach for Cholestasis (2,8,9):

Step 1: Recognize cholestasis

-        Timeframe: Evaluate any infant jaundiced beyond 2 weeks of age or 3 weeks for breastfed infants, with fractionated serum bilirubin to detect conjugated hyperbilirubinemia. The early recognition of cholestasis is crucial to prevent progressive liver damage.

-        Initial Test:

-   Measure total and direct serum bilirubin.

-   Direct bilirubin (conjugated bilirubin) of more than 1 mg/dl, if the total bilirubin is less than 5 mg/dl, or a conjugated component of more than 20% of the total, when total bilirubin exceeds 5 mg/dl.

Step 2: Identify Red Flags for Cholestasis

-        Acholic stools (pale or white stools).

-        Dark urine.

-        Failure to thrive, poor feeding, or irritability.

-        Family history of liver disease, consanguinity, or genetic disorders.

-        Dysmorphic features as in Alagille syndrome, and Down syndrome.

-        Noticeable bleeding, e.g., that observed during circumcision.

-        Extrahepatic features, e.g., heart murmurs, splenomegaly, or kidney abnormalities.

-        The history of meconium ileus, and failure to pass stool early in life suggests cystic fibrosis.

Step 3: Comprehensive Physical Examination

-        Abdominal examination for hepatomegaly, splenomegaly, and ascites.

-        Assessment of dysmorphic features associated with genetic syndromes, e.g., Alagille syndrome.

-        Panhypopituitarism: hypoplastic genitalia, poor growth, and hypoglycemia.

-        Cardiac examination for murmurs or signs of heart failure.

-        Neurological examination for poor muscle tone or developmental delay as in septo-optic dysplasia or Niemann-Pick disease type C.

Step 4: Initial Laboratory Workup

-        Fractionated bilirubin: total and direct serum bilirubin.

-        Liver function tests: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), albumin, and coagulation profile (prothrombin time and INR). Severe coagulopathy unresponsive to vitamin K may indicate a genetic or metabolic disorder. Low GGT suggests PFIC types 1,2,4,5,6 or bile acid synthesis defects.

-        Complete blood count

-        Metabolic screen:

-   Glucose check for hypoglycemia, which may indicate panhypopituitarism, galactosemia, or tyrosinemia.

-   Thyroid functions: Thyroid stimulating hormone (TSH) and free T4 for hypothyroidism.

-   Urine for non-glucose reducing substance.

Step 5: Diagnostic Imaging

-        Abdominal ultrasound is first-line imaging. It evaluates liver anatomy and bile duct structure. It can assess:

-   Gallbladder abnormalities such as absent gallbladder in biliary atresia.

-   CM or biliary tree dilation.

-   Signs of advanced liver disease such as coarse liver texture, splenomegaly or ascites.

Step 6: Second tier tests for genetic/ metabolic condition 

If the initial workup is inconclusive or if the infant presents with syndromic features, move to a more detailed genetic and metabolic investigation.

·       Genetic Disorders:

-        Alagille Syndrome:

o   GGT: Often disproportionately elevated.

o   An echocardiogram to evaluate associated cardiac anomalies.

o   Genetic testing for JAG1 or NOTCH2 mutations.

-        Cystic Fibrosis:

o   Sweat chloride test or CFTR genetic testing.

-        PFIC:

o   Check for low GGT in PFIC types 1, 2, 4, 5, and 6.

o   Genetic testing: ATP8B1 (PFIC1), ABCB11 (PFIC2), ABCB4 (PFIC3), tight junction protein-2 (PFIC4), NR1H4 gene (PFIC5), and MYO5B (PFIC6).

-        Alpha-1 Antitrypsin Deficiency:

o   Alpha-1 antitrypsin phenotype and serum levels.

o   Liver biopsy may show hepatocellular injury, cholestasis, or PAS-positive, diastase-resistant granules.

-        Bile Acid Synthesis Defects:

o   Serum bile acids are typically low with no pruritus despite severe conjugated jaundice.

o   Urinary bile acid profiling via fast atom bombardment mass spectrometry.

o   Genetic testing for enzyme deficiencies involved in bile acid synthesis.

·       Metabolic Disorders:

-        Galactosemia: enzyme assay for GALT deficiency, and genetic testing for GALT gene.

-        Tyrosinemia: Check serum or urinary succinyl acetone, and AFP is usually >50.000.

-        Urea cycle disorders: elevated serum citrulline in neonatal citrin deficiency.

-        Biomarkers, enzyme assay or genetic mutations for diagnosis for Niemann-Pick disease type C and Gaucher disease.

Step 7: Infectious Causes

-        Cytomegalovirus (CMV): polymerase chain reaction testing saliva, urine, or blood for CMV DNA. CMV IgM antibodies, though less sensitive.

-        TORCH infections: Test for Toxoplasma, Rubella, Syphilis, and herpes simplex virus type 1 and 2 if there is maternal history or early onset jaundice with systemic illness.

-        Urinary Tract Infections: Perform urine culture to rule out sepsis-related cholestasis.

Step 8: Endocrine Disorders

-        Panhypopituitarism:

o   Measure early morning cortisol, TSH, free T4, and perform MRI-brain imaging.

o   Look for associated anomalies like septo-optic dysplasia.

Step 9: Advanced Diagnostic Techniques

-        Liver biopsy:

o   Consider if diagnostic uncertainty remains.

o   Bile duct proliferation, and fibrosis are typical features in biliary atresia, while giant cell transformation is characteristic for neonatal hepatitis syndromes.

-        Laparoscopic or intraoperative cholangiography is the gold standard for confirming BA by direct visualizing of the bile ducts.

-        Magnetic Resonance Cholangio-Pancreatography (MRCP) is not reliable as the normal biliary tract is not consistently visualized in children under 3 months of age; false negative results are thus common.

-        Hepatic iminodiacetic acid (HIDA) radionucloide scan is not readily available, time consuming and is not recommended anymore.

Step 10: Management Plan Based on Diagnosis

·       BA: Early referral for Kasai portoenterostomy within the first 60 days is crucial.

·       Genetic/metabolic disorders: Initiate targeted therapies, e.g., cholic acid for some bile acid synthesis defects, dietary management for galactosemia, fructosemia, and specific treatment for tyrosinemia.

·       Infectious causes: Treat with appropriate antivirals for herpes simplex and CMV or antibiotics (bacterial infections).

·       Endocrine causes: Treat with hormone replacement (e.g., cortisol, thyroxine) if panhypopituitarism or hypothyroidism is diagnosed.

Step 11: Multidisciplinary Management and Follow-Up

Consult specialists: Pediatric surgeons, gastroenterologist, geneticist, infectious disease expert, endocrinologist, metabolic diseases specialist, nutritionist or transplantation experts based on the underlying cause.

Regular monitoring of liver functions, growth, and response to treatment.

➡️Purpose and Scope

·   This guideline has been developed to standardize the diagnosis of neonatal and infantile cholestasis (NIC). It provides an evidence-based guidance for healthcare personnel, such as general practitioners, general pediatricians, family medicine physicians, pediatric hepatologists, pediatric surgeons, neonatologists, nurse practitioners, physician’s assistants and pediatric gastroenterologists to help in early diagnosis of neonates and infants with cholestasis in Egypt.

· The guideline aims to provide recommendations to support: a) pediatricians working in primary and secondary care for the initial assessments of NIC; b) neonatologists and pediatric gastroenterologists working in a tertiary referral center for the extensive diagnostic work-up.

·  The guideline focuses on timely detection of cholestasis (as its detection is often late in our health care settings), and identification of its causes (particularly treatable ones), which is crucial for the best patient outcome.