Serum vascular endothelial growth factor per platelet count in patients with biliary atresia

Background: Biliary atresia (BA) is a progressive, sclerosing, inflammatory process resulting in complete obliteration of the extrahepatic bile ducts. The obstruction of bile flow engenders worsening cholestasis, hepatic fibrosis, and cirrhosis, which lead to portal hypertension and a decline in hepatic synthetic function. Hepatic stellate cells, which play roles in hepatic fibrogenesis, are an important source of various inflammatory mediators including vascular endothelial growth factor (VEGF) in the injured liver. Objectives: Investigate the level of serum VEGF and serum VEGF per platelet count in patients with BA and its relation to clinical characteristics. Methods: Peripheral blood samples were taken from 70 BA patients and 15 healthy control children. Serum VEGF was measured by enzyme-linked immunosorbent assay. We compared serum VEGF and serum VEGF per platelet count in BA patients with the respective results obtained in healthy control children. The relation of serum VEGF per platelet count with clinical variables of BA patients was investigated. Results: Serum VEGF levels and serum VEGF per platelet count in BA patients were not significantly different from those in normal controls (289.64±230.01 pg/mL vs. 312.36±189.05 pg/mL; p=0.72 and 1.72±1.21x106 vs. 1.57±0.97x106; p=0.66). Significant differences were observed among BA patients when VEGF per platelet count was categorized by the presence of esophageal varice (p=0.03). Only in BA patients was the serum level of VEGF correlated with the number of platelets (r=0.53, p<0.001). Conclusion: A high serum VEGF per platelet count is a useful marker for the development of portal hypertension in BA patients, especially for esophageal varice. Serum VEGF per platelet count may be useful for monitoring disease course in BA after hepatic portoenterostomy.

Differentiation of Brugia malayi and Brugia pahangi by PCR-RFLP of ITS1 and ITS2.

Lymphatic filariasis has been targeted by the World Health Organization for elimination by the year 2020. Malayan filariasis, caused by Brugia malayi, is endemic in southern Thailand where domestic cats serve as a major reservoir host. However, in nature, domestic cats also carry B. pahangi infection. In addition to chemotherapy and vector control, control in reservoir hosts is necessary to achieve the elimination of the disease. Therefore, differentiation between B. malayi and B. pahangi in the cat reservoir will help the lymphatic control program to monitor and evaluate the real disease situation. It is difficult to differentiate these two Brugia species by microscopic examination. The technique is also time-consuming and requires expertise. We employed the polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) technique of internal transcribed spacer regions, ITS1 and ITS2, of ribosomal DNA (rDNA) to differentiate B. malayi from B. pahangi species. Among the restriction enzymes tested, only the PCR product of ITS1 digested with Ase I could differentiate B. malayi from B. pahangi. This PCR-RFLP technique will be useful for lymphatic filariasis control programs for monitoring and evaluating animal reservoirs.

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.

Clinical and molecular characteristics of Thai patients with achondroplasia.

Achondroplasia is an autosomal dominant disorder characterized by disproportionately short stature, frontal bossing, rhizomelia, and trident hands. Most patients appear sporadically resulting from a de novo mutation associated with advanced paternal age. A glycine to arginine mutation at codon 380 (G380R) of the fibroblast growth factor receptor 3 gene (FGFR3) was found to be the most common cause of achondroplasia in various populations. We identified and clinically characterized 3 Thai patients with achondroplasia. In all of them, we also successfully identified the G380R mutation supporting the observation that this is the most common mutation in achondroplasia across different ethnic groups including Thai.

A case of Pfeiffer syndrome type 1 with an A344P mutation in the FGFR2 gene.

Pfeiffer syndrome, an autosomal dominant disorder, consists of craniosynostosis, broadening of the thumbs and great toes, and partial soft tissue syndactyly of the hands and feet. Three clinical subtypes have been classified mainly for the purpose of genetic counseling. Mutations in FGFR1 and FGFR2 are known to be associated with the syndrome. However, the correlation between genotype and phenotype is not well defined. Only one patient with Pfeiffer syndrome with no other clinical information has been reported to have had an A344P mutation of the FGFR2. Here we report a Thai male patient with sporadic Pfeiffer syndrome type 1 with impaired intelligence (IQ = 77). Mutation analysis revealed A344P in FGFR2. Identification of the clinical features and molecular defects in more patients is required to better correlate the genotype and phenotype of this complex syndrome.

Predominant human rotavirus genotype G1P[8] infection in infants and children in Bangkok, Thailand.

Human rotavirus is the major etiologic agent of infantile diarrhea on a worldwide scale. In this study, rotaviruses were detected by reverse-transcription PCR in 42 of 83 stool specimens from children below the age of 3 years with acute diarrhea in Bangkok, Thailand, between November 1998 and August 1999. G and P types of all samples were characterized by restriction endonuclease analysis (REA) and multiplex PCR typing assay, respectively. Strain G1P[8] (76.1%) was the predominant type, followed by G1P[6] (2.4%). Strain G1 combined with mixed P[8]/P[6] was identified in 2 specimens (4.8%) and 7 untypeable G strains (16.7%) were observed. This information on the circulating G and P combinations should be useful for understanding the epidemiology of human rotavirus in Bangkok, Thailand.

Prevalence of video games among Thai children: impact evaluation.

The present study was performed in order to determine prevalence and favored types of video games among altogether 679 primary and secondary school children in Thailand. To that end, the authors distributed questionnaires comprising detailed questions as to demographic data, playing frequency, available location and preferred type of video games among the parents of the children and adolescents to be investigated. Consistent with the literature, our results showed an early onset of video game playing (7.6 years), a higher prevalence among boys compared with girls, and a predilection for games invoking some aggressive behavior. In conclusion, although health hazards created by video game playing have remained beyond proof we still recommend parents and teachers to play a more active part as to the choice of games and the time spent playing.

Clinical associations and prognostic significance of serum anti-p53 antibodies in Thai patients with hepatocellular carcinoma.

Mutations of the p53 gene have been reported to be of prognostic significance in hepatocellular carcinoma (HCC). However, the clinical associations and prognostic value of anti-p53 antibodies, known to be products of the host immune response to these mutations, have been controversial. Serum anti-p53 antibodies were measured in 121 Thai patients diagnosed with HCC using a specific enzyme-linked immunosorbent assay (ELISA) kit. The clinical/pathological characteristics of the patients were compared with respect to the presence of serum anti-p53 antibodies. Cox regression analysis was performed to assess factor interaction and association with survival. Anti-p53 antibodies were detected in 13.2% (16 of 121) of our patients. There were no differences between groups with regard to age, sex, viral markers (HBsAg or anti-HCV), severity of liver disease and tumor advancement. The median survival rates for patients positive and negative for anti-p53 antibodies were 4.0 and 3.0 months, respectively (p = 0.443, by log-rank test). Multivariate analysis demonstrated that an advanced Okuda stage, lack of therapy and presence of portal vein thrombosis were independent factors related to the prognosis of the patients. Nonetheless, the presence of anti-p53 antibodies did not constitute a predictive variable associated with a poorer prognosis. Serum assay of anti-p53 antibodies, although rapid and easily performed, may not be suitable as an alternative to molecular detection of mutations in assessing tumor advancement and prognosis of patients with HCC.

Increasing susceptibility to HAV among members of the young generation in Thailand.

The prevalence of antibodies to hepatitis A virus was studied in 961 children and adolescents, randomly selected from five different provinces in Thailand (Chonburi, Lopburi, Udonthani, Nakhon Si Thammarat and Lopburi). The highest prevalence was found in Nakhon Si Thammarat, with 32.1 percent of those aged 10-14 years and 57.1 percent of those aged 15-18 years showing evidence of protective immunity. However, this high rate could be explained by an outbreak of hepatitis A in 1992. In the remaining four provinces, the pattern was typically age-related in that all individuals showed between zero and 13 percent antibody prevalence until reaching the 15-to-18-year age group where it increased to between 5.6 and 22.7 percent. The overall sero-prevalence among all age groups was 7.9 percent. Thus, the majority of the younger generation is susceptible to hepatitis A virus infection thereby enhancing the impact, should an outbreak occur. Preventive measures that might be taken are education aimed at better hygiene and sanitation, as well as vaccination of susceptible individuals within high-risk populations.

Core promoter and precore mutants of hepatitis B virus: prevalence and clinical relevance in chronic hepatitis patients.

The present study was conducted to determine prevalence and exact type, as well as nucleotide position of the precore/core mutations of hepatitis B virus found in Thai patients diagnosed with chronic hepatitis and/or cirrhosis in relation to the clinical parameters established with the respective patients. To that end, 24 HBeAg-positive and 56 HBeAg-negative individuals were selected at random from a cohort of altogether 256 chronic liver disease patients. DNA was extracted from their blood sera, amplified by polymerase chain reaction using semi-nested primers and subjected to direct sequencing. Clinically, the HBeAg-positive chronic hepatitis patients displayed significantly higher transaminase levels than those negative for HBeAg. Our results showed 2 of the 7 (28.6%) PCR-positive HBeAg-positive sera displaying double mutations in the core promoter region at position 1762/64. The nucleotide sequences obtained from the 24 PCR-positive HBeAg-negative sera revealed 18 (75%) mutations in the core promoter region (1762/64), and/or 7 (29.2%) mutations at position 1753, and/or 6 (25%) mutations of the start codon (1814), and/or 8 of (33.3%) nucleotide 1896 turning codon 28 into a stop codon and one sample (4.2%) displaying a deletion between nucleotides 1758-1772. It is suggested that the mutations observed have an impact on the DNA secondary structure in such a way that successful transcription of the HBeAg gene is rendered impossible. To what extent this mutation influences the severity of chronic liver disease remains to be elucidated.

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.

Prevalence and clinical relevance of serum anti-p53 antibodies in patients with cholangiocarcinoma.

Cholangiocarcinoma (CCA) constitutes carcinoma of the bile duct found at a high prevalence in northeastern Thailand. In the present study, we examined the sera of altogether 82 Thai CCA patients for the presence of anti-p53 antibodies in order to investigate a role of the tumor suppressor gene, p53 in the carcinogenesis. Our results revealed anti-p53 antibodies in 7.3% of the cases tested, which conforms to the prevalence rate of p53 gene mutation recently reported at 5% among Thai patients. With limited number of the patients, anti-p53 antibodies were rapidly detected more frequently among patients with peripheral tumors than those with central tumors. However, further studies is required to establish significance and prognostic value of the antibodies in the context of CCA.

Parvovirus B19 antibodies in immunocompromized children in Thailand.

Parvovirus B19, a non-enveloped single stranded DNA virus is distributed worldwide. Sero-prevalence in adult populations amounts to approximately 50%. Clinical manifestations vary depending on the Immune status of the infected individuals and may include mild childhood Infection as well as hydrops fetalis due to intrauterine infection. To determine the prevalence of this infection among the immunocompromized individuals in Thailand, we determined, by indirect ELISA, levels of IgM and IgG antibodies to the parvovirus B19 in 106 immunocompromized children. These included 49 children who were on chemotherapy for treatment of malignancies, 18 who were receiving immunosuppressive drugs after organ transplantations, 14 who were under a regimen of corticosteroids and 25 who were positive for antibodies to HIV. The average prevalence of IgG antibodies in 106 children was 16.0%; the prevalence of antibodies was 33.3% in post-transplanted group, 16% in children positive for HIV, 12.2% in the group receiving chemotherapy for malignancies and 7.6% in the group treated with corticosteroids. All children were negative for IgM antibodies to parvovirus B19.

Serum interleukin-6 and interferon-gamma levels in patients with hepatitis B-associated chronic liver disease.

Hepatitis B virus (HBV) infection can elicit a variety of clinical sequelae ranging from acute self-limited hepatitis to hepatocellular carcinoma, which are not attributable to a direct cytopathic effect of the virus but rather to the individual host's immune response. Cytokines, low-molecular-weight proteins with a broad range of activity, have been shown to be involved in the regulation of hepatocyte functions, as well as in the pathogenesis leading to liver damage. In the present study, we investigated the correlation between serum interleukin 6 (IL-6) and interferon gamma (IFN-gamma) in altogether 75 patients chronically infected with HBV. They comprised 15 asymptomatic carriers, 15 chronic persistent hepatitis (CPH) and 15 chronic active hepatitis (CAH) patients, 15 cases of cirrhosis and 15 patients with hepatocellular carcinoma (HCC) previously diagnosed by serology and histology, respectively. IL-6 and IFN-gamma levels in their sera were determined using a commercially available kit. Our results showed various concentrations of serum IL-6 detectable in 6.7% of asymptomatic carriers, 13.3% of patients with CPH, 20% of patients with CAH, 33.3% in cirrhotic patients and 66.7% in HCC. In contrast, serum IFN-gamma was only found in 13.3% of asymptomatic carriers and CAH, but could not be detected in the other groups. Our data demonstrated a positive correlation between serum IL-6 and clinical severity of chronic HBV infection, whereas the IFN-gamma levels appeared not to be correlated. From this we conclude that among chronic hepatitis patients IFN-gamma is mostly not expressed at a level detectable by serology, whereas according to other authors it is involved in the immediate immune response triggered by acute hepatitis. IL-6 on the other hand, might rather be responsible for liver inflammation and regeneration in chronic liver disease.

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.

Prevalence of antibodies to parvovirus B 19 in Thailand.

Infection with human parvovirus B 19, a single-stranded non-enveloped DNA virus of worldwide distribution, is rather common and displays a broad spectrum of clinical manifestations of varying severity, depending on the patient's immune response. As the target of infection are the erythroid precursor cells, patients can experience an aplastic crisis. Usually, at least in immunocompetent individuals, viremia ceases with the appearance of virus-specific antibodies in the patient's serum whereupon the patients retain lifelong immunity to reinfection. Since data as to the prevalence of this agent has not been established for Thailand, the purpose of the present study was to investigate its frequency among 3 distinct groups, comprising 30 healthy children. 64 children with acute unrelated illness, and 35 voluntary blood donors, respectively, by means of enzyme linked immunosorbent assay. Our results have shown that, as reported for other countries, anti-parvovirus IgG increases in an age-dependent manner and is established at an overall prevalence of 20.16%, inviting the conclusion that the local population is infected by this agent as frequently as those of other countries in the Far East. Further studies need to be undertaken in order to elucidate its prevalence among members of high-risk groups.

Coinfections with hepatitis g and/or c virus in hepatitis B-related chronic liver disease.

Concurrent infections with HGV and/or HCV (HGV/HCV) were investigated in 196 patients with HBV-related chronic liver disease (115 chronic hepatitis, 31 liver cirrhosis, 50 hepatocellular carcinoma), and in 100 HBsAg carriers. Coinfections were detected in 18 (9.2%) patients with HGV (10) or HCV (5) or both agents (3), but in none of the HBsAg carriers. Patients with coinfection were more frequently exposed to blood transfusions (55.6% vs 5.6%) and also were more commonly anti-HBe positive. Serum levels of HBV-DNA were lower in patients with HCV coinfection than in those coinfected with HGV. Interferon was administered to 39 patients with chronic active hepatitis including 7 patients with HGV/HCV coinfection. Sustained clearance of HBV-DNA was observed in 10 (25.6%) patients who were solely infected with HBV. These patients were significantly younger and had much lower histological scores than non-responders. Patients with HCV coinfection had significantly higher pre-treatment histological scores than those without HCV. After interferon treatment, a significant reduction in histological scores was observed in all patients except those coinfected with HGV/HCV. None of the 7 patients with coinfection had sustained clearance of HBV-DNA or HCV-RNA, and only one had cleared HGV-RNA. These results suggest that parenteral exposure is a risk factor for HGV/HCV coinfection in chronic HBV infection. HGV infection shows no significant impact on chronic HBV infection. HCV coinfection appears to inhibit HBV replication, but causes more severe chronic hepatitis and increases resistance to interferon therapy.

Comparison of the reactogenicity and immunogenicity of two different dose levels of hepatitis A vaccine in healthy children and adolescents.

An open study was performed to compare the reactogenicity and immunogenicity of an inactivated hepatitis A vaccine administered in two different doses and schedules to 460 healthy volunteers aged 3-18 years. Participants were randomized to two groups to receive either two doses of 720 ELISA Units (EL.U) inactivated hepatitis A per 0.5 ml dose according to a 0, 6-month schedule, or three doses of 360 EL.U according to a 0, 1, 6-month schedule. Transient local injection soreness was the most commonly reported symptom in almost half of both groups with no serious adverse events. One month after the primary course (one dose of 720 EL.U and two doses of 360 EL.U), 99% of 720 EL.U vaccinees had seroconverted, compared with 100% seroconversion in the 360 EL.U group. All vaccinees were seropositive after the booster dose of both vaccines with geometric mean anti-HAV titers of 2,359 and 2,967 mIU/ml in the 720 EL.U and 360 EL.U groups, respectively. The vaccine containing 720 EL.U of antigen per dose offers the advantage of convenience and acceptance of immunization afforded by a two-dose course of vaccination accompanied by a comparable antibody response with that achieved after three doses of vaccine containing 360 EL.U of antigen per dose.

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.

Cytomegalovirus infection in immunocompromised children in Thailand.

Cytomegalovirus (CMV) constitutes the most widespread cause of congenital and perinatal viral infections on a global scale, exceeding a 90%-prevalence among blood donors in Thailand. The present study was aimed at determining the prevalence of CMV in the sera of 113 immunocompromised children by means of serology, as well as polymerase chain reaction using nested primers. Our results showed anti-CMV IgG, i.e. latent infection, prevalent in an age-dependent manner irrespective of the disorder underlying the children's immunocompromised condition, whereas anti-CMV IgM was equally prevalent throughout all age groups and disease patterns and, therefore, unreliable as a marker. Detection of serum CMV DNA by PCR represented the most exact diagnostic parameter by far, indicating active infection long before clinical symptoms may appear. In conclusion, based on the high prevalence of latent CMV infection among the general population in Thailand, we recommend especially the sera of immunocompromised patients be examined for the presence of viral DNA by means of PCR in order to provide clinical guidelines for their proper management.