Role of a homozygous A(TA)7TAA promoter polymorphism and an exon 1 heterozygous frameshift mutation UGT1A1 in Crigler-Najjar syndrome type II in a Thai neonate.

Crigler-Najjar syndrome is a rare autosomal recessive disease caused by mutations in the UGT1A1 gene. These mutations result in the deficiency of UGT1A1, a hepatic enzyme essential for bilirubin conjugation. This report describes the case of a 4-month-old boy with the cardinal symptoms of Crigler-Najjar syndrome type II. Molecular genetic analysis showed a homozygous UGT1A1 promoter mutation [A(TA)7TAA] and a heterozygous insertion of 1 adenosine nucleotide between positions 353 and 354 in exon 1 of UGT1A1 that caused a frameshift with a premature stop codon.

Evidence of protection against clinical and chronic hepatitis B infection 20 years after infant vaccination in a high endemicity region.

Vaccination against hepatitis B virus (HBV) immediately after birth prevents neonatal infection by vertical transmission from HBV carrier mothers. There is an ongoing debate whether infant vaccination is sufficient to protect against infection when exposed to HBV later in life. We studied 222 Thai infants born to HBsAg -/+ and HBeAg -/+ mothers who were vaccinated with recombinant hepatitis B vaccine at 0-1-2-12 months of age. A subset of 100 subjects received a booster dose at age 5 years. Blood samples collected yearly for 20 years were examined for anti-HBs antibodies and serological markers of hepatitis B infection (anti-HBc, HBsAg, and in selected cases HBeAg, anti-HBe, HBV DNA). During the 20-year follow-up, no subject acquired new chronic HBV infection or clinical hepatitis B disease. During the first decade, possible subclinical breakthrough HBV infection (anti-HBc seroconversion) was only observed in subjects born to HBsAg +/HBeAg + mothers (6/49 [12.2%]). During the second decade, breakthrough HBV infections were detected in all groups (18/140 [12.8%]). Increases in anti-HBs concentrations that were unrelated to additional HBV vaccination or infection were detected in approximately 10% of subjects in each decade. Primary infant vaccination with a recombinant hepatitis B vaccine confers long-term protection against clinical disease and new chronic hepatitis B infection despite confirmed hepatitis B exposure.

The incidence of rotavirus a isolates of G genotype in Thailand in 2002-2004.

Rotaviruses are the leading cause of severe gastroenteritis among infants and young children worldwide. Between November 2002 and March 2004, 36 stool specimens of 108 children with acute diarrhea in Bangkok, Thailand were found positive for Rotavirus A (RV-A) by RT-PCR. The 36 isolates were subjected to genotyping by RFLP analysis and direct sequencing of a part of the gene for major outer capsid glycoprotein VP7. The sequences obtained were subjected to phylogenetic analysis. Among the isolates the genotypes G1 (5.6%), G2 (69.4 %) and G9 (25.0 %) were found. Comparison of these results with those of previous studies covering the period of 1982-1999 revealed a changing pattern of RV-A G genotypes and thus contributed to the understanding of RV-A epidemiology in Thailand. Any vaccine to be developed against this virus should target the G9 genotype as one of common global genotypes.

Molecular analysis of the a determinant of HBsAg in children of HBeAg-positive mothers upon failure of postexposure prophylaxis.

OBJECTIVE: To investigate the a determinant of hepatitis B virus (HBV) S gene for the presence of mutations responsible for vaccine failure. METHODS: The a determinant of HBV S gene was amplified in sera obtained from 11 HBV-positive infants and children born to asymptomatic HBeAg-positive mothers by nested polymerase chain reaction (PCR) and subsequently subjected to direct sequencing. The sequences obtained were translated into the corresponding amino acids and compared to amino acid sequences of HBV subtype adr. All infants under investigation had received recombinant hepatitis B vaccine within 24 hours after delivery and had completed the recommended vaccination course, consisting of three to four doses administered at defined intervals. RESULTS: The usual divergence regarding genotype and subtype was identified among the 11 samples tested. Only two exhibited a point mutation within the a determinant, one of which consisted of a substitution of glycine with alanine at position 145, and the other of a substitution of glutamine with arginine at position 129. CONCLUSION: Eleven neonates were positive for HBV infection, and two of them showed point mutations that might have rendered the virus resistant to the vaccine, possibly due to a change in the S protein's secondary structure. Yet, this remains a matter of speculation, since the other seven cases positive for hepatitis B viral DNA merely demonstrated the usual genotype and subtype. The presence of escape mutants of HBV can be considered rather negligible with respect to vaccination programs, especially as the vaccine has been shown to reduce hepatitis B, as well as hepatocellular carcinoma efficiently.

Detection of HbsAg and HBV DNA in serum and saliva of HBV carriers.

Serum and saliva samples from 23 patients known to be HBsAg-positive HBV carriers and 17 healthy control subjects were analyzed for hepatitis B virus (HBV) by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). All serum samples of the HBV carriers were positive for HBsAg, with 21 also positive for HBV DNA. In comparison, 22 saliva samples of HBV carriers were positive for HBsAg whereas only 11 of the 23 tested were positive for HBV DNA. Based on these results we have arrived at the conclusion that the saliva of HBV carriers might be potentially infectious and also that saliva testing could serve as an alternative technique for identifying HBV carriers.

The declining pattern of seroepidemiology of hepatitis A virus infection among adolescents in Bangkok, Thailand.

Hepatitis A virus (HAV) is a health problem in countries where seroepidemiology shows changes from hyperendemicity to intermediate endemicity. Throughout the last decade, we studied, in Bangkok, the seroprevalence of hepatitis A virus antibody (anti-HAV) among adolescents of different age groups. In 1996, 245 serum specimens from children aged between 10 and 19 were tested for anti-HAV by ELISA method. The results were compared to those obtained in 1987 and 1993 from students of the same age and attending the same school. Anti-HAV was detected in 31.4%, 14.6% and 12.7% of school children in the years 1987, 1993 and 1996, respectively. Each year, it was found that an increasing prevalence of anti-HAV was related to an increasing age. From 1987 to 1996, the age specific prevalence of anti-HAV was markedly decreased in younger children. The surveillance of the epidemiological trend of HAV infection is important for implementing preventive measures and for controlling the disease.

Non-cirrhotic portal fibrosis associated with pulmonary arteriovenous communication and pulmonary arterial hypertension.

A case of non-cirrhotic portal fibrosis associated with pulmonary arteriovenous communication and pulmonary arterial hypertension is reported. The patient was a 7-year old boy who presented with hematemesis, cyanosis, hypoxemia and orthodeoxia. His liver pathology was compatible with non-cirrhotic portal fibrosis. His pulmonary angiography showed arteriovenous shunting and pulmonary arterial hypertension (mean pulmonary artery pressure 34 mmHg). His sister also had non-cirrhotic portal fibrosis with neither hypoxemia nor orthodeoxia. This report raises the possibility of non-cirrhotic portal fibrosis having a genetic etiology.

Is there evidence for intrauterine HBV infection in newborns of hepatitis B carrier mothers?

It has been proposed that some neonates infected with hepatitis B virus (HBV), acquire their infections in utero as demonstrated by HBV seromarkers in venous blood samples at birth. In this study, paired blood samples from 13 HBsAg-positive, 19 HBsAg- and HBeAg-positive, 2 HBsAg-negative mothers and 34 of their neonates, were drawn 24-72 hours after birth and tested for HBV-DNA in their peripheral blood mononuclear cells (PBMC). The presence of HBV-DNA in PBMC was detected in 69.2% (9/13) of HBsAg-positive mothers, 94.7% (18/19) of HBsAg- and HBeAg-positive mothers, and in none of their neonates. The conclusion from these results is that the evidence for hepatitis B infections occurring in neonates of hepatitis B carrier mothers in utero is uncommon.

Humoral immune response following hepatitis B vaccine booster dose in children with and without prior immunization.

One hundred and twenty-three children who had received no, incomplete and complete primary hepatitis B vaccination but had negative or very low anti-HBs titer were immunized with a single dose of recombinant hepatitis B vaccine. Blood tests for anti-HBs were obtained at 30 +/- 5 days after the booster immunization. Twelve of 18 (66.7%) children without prior immunization (group 1) seroconverted following the single dose Seroconversion rates in children who had undetectable anti-HBs with incomplete and complete primary immunization (group 2 and 3) were 83.34% and 94.5%, respectively. All children with complete 3- dose vaccination but who had low anti-HBs titer (group 4) also seroconverted. This study confirmed that immunological memory, allowing a protective anamnestic response, lasted at least 8 years in children who had received primary HB immunization with undetectable anti-HBs. Therefore, we conclude that the booster dose after complete vaccination is not necessary in healthy children.

High prevalence of antibody against hepatitis A virus in an institution for the mentally handicapped.

Hepatitis A virus infection constitutes a world-wide public health problem, predominantly in developing countries. Mentally handicapped children, due to their incapacity for looking after themselves, comprise one of the high risk groups for hepatitis A virus infection. The aim of the present study was to determine the prevalence of hepatitis A virus antibody (anti-HAV) among the children and adults in the Institute for the Mentally Handicapped located in Nonthaburi, Thailand. The prevalence of anti-HAV IgG antibody was 92%. Immunity acquired against HAV was shown to increase in direct proportion to the age. To prevent future outbreaks of hepatitis A, water supply, sanitary conditions and personal hygiene should be improved at this and similar institutions. Furthermore, persons new to the institution (patients and staff) should be screened for anti-HAV and vaccinated with hepatitis A vaccine if nonimmune.

Hepatocellular carcinoma: significance of HBV vertical transmission.

In two cases of childhood hepatocellular carcinoma in Thailand, we established vertical transmission of hepatitis B virus infection as the underlying cause. With the first patient, the family history of HBV carriage became evident and a pedigree could be devised which demonstrated the high prevalence among the family members and hence evidence of vertical transmission. In the case of the second patient, we performed PCR and subsequent direct sequencing of HBV DNA isolated from his HBsAg-positive mother's, as well as from his serum, comparing the nucleotide sequences with those of a pregnant woman diagnosed as an asymptomatic HBV carrier, of another asymptomatic HBV carrier and of a reference strain, respectively, all belonging to the same genotype and subtype as the samples tested. Our results clearly indicate the necessity for nation-wide hepatitis B vaccination starting at birth, at least in hyperendemic areas like the Far East, in order to forestall HBV carriage and ensuing cirrhosis and/or HCC by preventing vertical transmission.

Alpha 1-antitrypsin phenotype of children with liver diseases in Thailand.

Alpha1-antitrypsin deficiency (PiZZ) constitutes not only the most common hereditary cause of liver diseases, but also of the most prevalent metabolic diseases in need of liver transplantation. It is a codominantly inherited disorder which predisposes to chronic liver disease, usually beginning in early infancy. The purpose of the present study has been to investigate alpha 1-antitrypsin phenotype in pediatric patients with various liver diseases. Phenotypic identification of alpha 1-antitrypsin variants has been carried out in 69 children with various liver diseases and 100 healthy controls using isoelectric focusing on polyacrylamide gel slabs. PIMM represents the most common phenotype detected in both groups (92% in the group with liver diseases and 88% in normal controls). We could detect PiZZ in only one healthy child but in none of those with liver diseases. Consequently alpha 1-antitrypsin deficiency does not appear to be a common cause for liver disease among children in Thailand. Further studies are necessary to elucidate the frequency of various alpha 1-antitrypsin variants and the clinical relevance with respect to liver diseases in Thailand.

Immunogenicity and adverse effects of live attenuated varicella vaccine (Oka-strain) in children with chronic liver disease.

Varicella infection may cause significant morbidity and mortality especially in immunocompromised persons. Children with chronic liver disease who undergo liver transplantation and need long term immunosuppressive therapy are at risk to acquire the infection. Twenty-nine children (aged 1-12 years) with chronic liver disease were enrolled to receive one dose of live attenuated varicella vaccine (Oka-strain). During the 16-week follow-up period, no vaccine-related serious adverse events were reported. Seroconversion rates at 8 weeks post vaccination were 100%. Geometric mean titer (GMT) values and seropositive rates at 16 weeks tended to relate to the clinical severity of liver disease. This study demonstrates that varicella vaccine is safe and Immunogenic in children with chronic liver disease.

Comparison study of combined DTPw-HB vaccines and separate administration of DTPw and HB vaccines in Thai children.

The safety, immunogenicity and tolerability of two different DTPw-HBV combination vaccines, containing 5 and 10 microg of HBsAg; were investigated in comparison with separate administration of DTPw and HBV (10 microg of HBsAg). A three dose primary vaccination course at 2, 4 and 6 months of age was followed by a booster dose at 18 months. All vaccines were safe and well tolerated. The DTPw-HBV combination vaccine containing 10 microg of HBsAg elicited significantly higher anti-HBs titres than the other two vaccines after the primary and booster vaccination course. All vaccines elicited a high response against the other components. Based on these results, DTPw-HBV (10 microg HBsAg) was the most effective vaccine at this schedule.

Problems and prevention of viral hepatitis in Thailand.

To this day, viral hepatitis remains a major public health problem in Thailand. Chronic infection with hepatitis B and C viruses are the leading causes of chronic liver diseases, including cirrhosis and hepatocellular carcinoma (HCC). Outbreaks of hepatitis A virus continue to occur in Thailand, even after several years of consistently declining prevalence rates. Also, the reduction in prevalence of hepatitis D virus infection has been observed among intravenous drug users over the past decade. Hepatitis E virus constitutes a rather unusual cause of sporadic acute hepatitis in Thailand. Highly effective vaccines are currently available for prevention of hepatitis A and B, however, as yet no effective vaccine for hepatitis C is imminent. Following rapid progress in the development of molecular techniques, several new hepatitis viruses have been identified. Among these, Hepatitis G, TT and SEN viruses have recently been described but their significance as to causation of human liver disease has yet to be established. This article reviews the current epidemiology, molecular biology, and strategies aimed at prevention and control of hepatitis virus infection in Thailand emphasizing new developments and recent data obtained from our research studies.

Extrahepatic biliary atresia in twins: zygosity determination by short tandem repeat loci.

Extrahepatic biliary atresia (EHBA) is an infantile obstructive cholangiopathy of unknown etiology. This condition is generally thought to be an acquired disease without familial tendency. We reported eight pairs of discordant twins in a series of 143 patients with operatively established EHBA, One pair was dizygotic twin from sex discrimination. Six were identical ABO blood groups and 2 were also the same minor blood groups (Dce, MM and Le a-b-). The last 2 sets were monzygotic twins by common DNA polymorphisms short tandem repeat loci. Our findings support the hypothesis that, EHBA is an acquired rather than a hereditary disease.

Elevated serum IL-18 and interferon-gamma in medium-term survivors of biliary atresia.

INTRODUCTION: Biliary atresia (BA) is a fatal disease in children. Its main pathological feature is progressive immune-mediated cholangiopathy. Interleukin (IL)-12, IL-18, and interferon-gamma (IFN-gamma) play important roles in various immunological diseases. THE OBJECTIVE: was to investigate whether these serum markers were associated with clinical outcome in BA. METHODS: Serum levels of IL-12, IL-18, and IFN-gamma were determined using enzyme-linked immunosorbent assay from 46 BA patients (median age of 9 years) and 19 normal controls. The BA patients were then categorized into three groups according to their outcome: jaundice-free (29 cases), mild to moderate jaundice (10 cases), and marked jaundice (7 cases). The comparisons of serum IL-12, IL-18, and IFN-gamma levels among groups of the patients were performed using one-way analysis of variance with post-hoc tests. Data are expressed as mean + standard deviation. RESULTS: Serum IL-18 and IFN-gamma in BA patients were higher than the normal controls (IL-18: 113.3 + 82.6 vs. 80.5 + 9.9 pg/mL, p = 0.011 and IFN-gamma: 41.7 + 5.1 vs. 38.0 + 1.9 pg/mL, p < 0.001). There was no difference in serum IL-12 between BA and controls. Further analysis demonstrated that, in BA patients, only serum IL-18 levels significantly increased with the degree of jaundice (test for trend, p = 0.004). CONCLUSIONS: Serum IL-18 and IFN-gamma levels were increased in medium-term survivors of BA. The elevated serum IL-18 in BA patients was associated with worse clinical outcome. These results suggest that IL-18 and IFN-gamma play roles in the pathophysiology of BA. Additionally, IL-18 is likely to be involved in the disease progression.

Elevation of serum galectin-3 and liver stiffness measured by transient elastography in biliary atresia.

BACKGROUND AND AIM: Biliary atresia (BA) is an intractable neonatal liver disease characterized by progressive fibrosclerotic obliteration of the extrahepatic biliary tree. The aim of this study was to evaluate serum galectin-3 in postoperative BA patients and the association between galectin-3, clinical outcome and liver stiffness score. METHODS: 58 BA patients post Kasai operation and 20 controls were enrolled. None of the patients had undergone liver transplantation. BA patients were classified into 2 groups according to their serum total bilirubin (TB) levels (TB<2 mg/dL, no jaundice vs. TB≥2 mg/dL, persistent jaundice) and alanine aminotransferase (ALT) levels (ALT<45 IU/L, normal ALT vs. ALT≥45 IU/L, elevated ALT). Serum galectin-3 levels were determined by enzyme-linked immunosorbent assay. Liver stiffness scores were measured by transient elastography (FibroScan). RESULTS: BA patients had higher serum galectin-3 levels (5.1±0.3 vs. 3.8±0.4 ng/mL, p=0.01) and greater liver stiffness values than healthy controls (29.7±3.0 vs. 5.1±0.5 kPa, p<0.001). Serum galectin-3 levels were markedly elevated in BA patients with jaundice compared to those without jaundice (6.4±0.5 vs. 4.4±0.3 ng/mL, p=0.001). Furthermore, BA patients with elevated ALT displayed significantly higher levels of serum galectin-3 than those with normal ALT (5.9±0.4 vs. 3.8±0.3 ng/mL, p=0.001). Additionally, BA patients with portal hypertension had considerably higher serum galectin-3 levels than those without portal hypertension (6.1±0.4 vs. 3.7±0.3 ng/mL, p<0.001). CONCLUSIONS: Increased serum galectin-3 is associated with a poor outcome in postoperative BA patients. Serum galectin-3 could be used as a biochemical parameter reflecting the deterioration of liver function and the severity of liver fibrosis in postoperative BA.

Correlation of circulating endoglin with clinical outcome in biliary atresia.

BACKGROUND AND AIM: Biliary atresia (BA) is a chronic progressive inflammatory disorder of the extrahepatic and intrahepatic biliary system in children. The aim of the present study was to investigate circulating endoglin levels in BA patients compared with healthy controls and to determine the relationship between plasma endoglin levels and outcome parameters of BA patients after Kasai operation. METHODS: Fifty-five postoperative BA patients and 14 healthy controls were recruited. The patients were divided into two groups based on their serum total bilirubin levels (TB<34.2, no jaundice vs. TB>or=34.2 micromol/L, persistent jaundice) and serum alanine aminotransferase (ALT<45, normal ALT vs. ALT>or=45 IU/L, high ALT). Circulating endoglin levels were analyzed by enzyme-linked immunosorbent assay. RESULTS: Average levels of plasma endoglin were significantly higher in BA patients compared to healthy controls (7.8+/-0.4 vs. 6.5+/-0.4 ng/mL; P=0.02). BA patients with persistent jaundice had higher plasma endoglin levels than those without jaundice (9.2+/-0.8 vs. 6.9+/-0.3 ng/mL; P=0.006). Furthermore, the concentrations of plasma endoglin in BA patients with high ALT were significantly higher compared to those with normal ALT (8.5+/-0.5 vs. 6.3+/-0.5 ng/mL, P=0.003). In addition, BA patients with portal hypertension had more elevated plasma endoglin levels than those without portal hypertension (8.8+/-0.6 vs. 6.1+/-0.3 ng/mL, P=0.001). Plasma endoglin was positively correlated with serum ALT (r=0.36, P=0.007) and serum GGT (r=0.44, P=0.001). CONCLUSION: High circulating endoglin correlated with a poor outcome for BA. Plasma endoglin can be utilized as a potential biomarker reflecting the severity of ongoing liver injury and biliary obstruction in BA patients after Kasai procedure.

Cyclooxygenase-2 overexpression is associated with clinical outcome in biliary atresia.

OBJECTIVE: The purpose of this study was to investigate the association between cyclooxygenase-2 (COX-2) expression and clinical outcome in biliary atresia (BA) patients. METHODS: Six months after surgery, twenty-eight BA patients were divided into three groups according to their liver function tests: group A with satisfactory liver function (n=11), group B with moderate liver dysfunction (n=8), and group C with severe liver dysfunction (n=9). COX-2 expression was determined by immunohistochemistry. Choledochal cysts (n=5) and normal liver samples (n=4) served as controls. RESULTS: Our data have shown that the intrahepatic biliary epithelium in BA specimens expressed COX-2. The mean immunoreactive score of COX-2 in BA patients was significantly higher than that in choledochal cyst and normal liver (4.0+/-0.6, 0.9+/-0.3, and 0.7+/-0.3, respectively, p<0.002). Strong expression of COX-2 was observed in BA patients with severe liver dysfunction. Subgroup analysis showed that the mean COX-2 immunoreactive scores of patients in group A, B, and C were 2.1+/-0.6, 3.6+/-1.1, and 5.9+/-0.9, respectively. The COX-2 immunoreactive score in BA patients with severe liver dysfunction was higher than in patients with satisfactory liver function (p<0.005). CONCLUSION: Increased COX-2 expression of biliary epithelial cells at the time of Kasai operation was associated with an adverse therapeutic outcome in BA, suggesting that COX-2 could play a plausible role in the liver pathology of BA.