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.

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.

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.

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.

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.

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.

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.

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.

Prevalence of core promotor and precore mutants of hepatitis B virus in thailand by RFLP and sequencing.

Hepatitis B virus has been known to frequently undergo mutations of its genome at various sites, mostly due to it employing a reverse transcriptase devoid of proofreading capacity in the course of its replication. The purpose of the present study has been to screen 257 HBsAg-positive chronic liver disease patients, more specifically 78 cases chosen at random out of those negative for HBeAg and 33 of the HBeAg-positive cases serving as controls for three discreet point mutations in the precore/core region of hepatitis B virus. To that end, HBV DNA extracted from sera was amplified by polymerase chain reaction (PCR) using semi-nested primers and subsequently subjected to restriction fragment length polymorphism (RFLP) analysis, 36 HBeAg-negative versus 30 HBeAg-positive sera, respectively, as well as to direct sequencing in some samples randomly selected to corroborate the RFLP results. Our results showed double mutations at positions 1762 and 1764 of the core promoter in between 25/36 (69.4%) and 19/25 (76%) of the sera tested, a missense mutation of the start codon in between 8/36 (22.2%), and 5/25 (20%) and a mutation turning codon 1896 into a stop codon in between 9/36 (25%) and 6/25 (24%) determined by RFLP and sequencing, respectively. These data indicate the double mutation at positions 1762 and 1764 to be the most prevalent among HBeAg-negative chronic hepatitis patients in Thailand whereas, in contrast to reports from other Asian countries, the mutation at nucleotide 1896 occurred in a mere 25%, while on the other hand the mutation abolishing the start of protein synthesis was observed to occur at a higher frequency than determined in several other geographical areas.

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.

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.

Hepatitis viruses and chronic liver disease.

We have investigated several groups of Thai patients diagnosed with chronic liver disease including chronic hepatitis, cirrhosis and hepatocellular carcinoma, as well as cholangiocarcinoma, for the prevalence of infection with either one of the hepatitis viruses B, C, G and the novel hepatitis virus TT (TTV). The 168 patients tested comprised 120 men and 48 women with their median age ranging from 42.3 to 62.3 years. Screening for antibodies to HBV and HCV was performed by a commercially available serological test kit, for the presence of HBV and TTV DNA by PCR, and of HCV and HGV RNA by RT-PCR, respectively. There was a clear two-fold higher prevalence of HBV (49%) over HCV (27%) infection and a four-fold higher frequency compared to HGV (13%) and TTV (11%) infection, respectively, in those individuals with chronic hepatitis, cirrhosis, and hepatocellular carcinoma, whereas all but one patient with cholangiocarcinoma the etiology of which has been ascribed to parasitic infestation, were free of all viral markers. In Thailand chronic HBV, and to a lesser extent, chronic HCV infection represent the two most common causes of hepatitis potentially proceeding to chronic liver disease, whereas the clinical significance pertinent to HGV and TTV remains to be elucidated.

Clearance of hepatitis TT virus infection among thalassemia children and IVDU.

The novel transfusion transmissible hepatitis virus TTV first isolated by a group from Japan has predominantly been detected in members of groups at high risk for contracting blood borne viruses. Aside from elevated liver enzymes, the symptoms associated with its infection have been reported to range from asymptomatic to hepatic failure. The purpose of the present study was to determine if and to what extent the host's immune response is capable of clearing TTV infection. Hence, we extracted DNA from sera obtained from altogether 201 intravenous drug users (IVDU) and 80 thalassemia children--both groups at high risk of parenteral exposure--and performed PCR using semi-nested primers. Those positive for TTV DNA were once again subjected to PCR after approximately one year in order to determine how many still harbored the virus. Our results showed TTV DNA to be absent in merely 20.6% of the formerly positive IVDU, whereas it was still present in all the thalassemia children who could be tested for the second time. Based on the small sample size and the high-risk environment, these results ought to be interpreted with caution and definitely merit further investigation.

TT virus infection in intravenous drug users.

Our group has investigated 201 intravenous drug users for the presence of TTV DNA by means of polymerase chain reaction (PCR). The majority of the individuals tested were male, their age ranging from 16 to 63 years, and the duration of intravenous drug use from one to 40 years. TTV DNA was present in 62 of the 201 IVDUs (30.8%) with its prevalence on the ascent between the age groups below 20 and those between 21 and 30 years, as well as between the groups below 60 and between 60 to 120 months' duration of drug intake, respectively. When tested again after 9 months, nine IVDU (23.7%) were found TTV negative by PCR hinting at potential immunological clearance. Our control group comprised 200 healthy blood donors, 7% of whom were found to harbor TTV DNA in an age-dependent fashion, as observed with the IVDU. From the liver function tests performed we could not detect any statistically significant difference regarding ALT elevation observed in TTV-positive compared with TTV-negative individuals. To date, TTV does not appear to cause any serious liver disease in the majority of cases examined.

Insufficient evidence of TT-viral etiology of post hepatitis aplastic anemia.

Post-hepatitis aplastic anemia is a rather rare pathologic condition of as yet unclear etiology especially as hepatitis viruses A to G have been excluded as the potential agents responsible. The novel TT virus, a single-stranded DNA virus first isolated from the serum of a patient with post-transfusion hepatitis in Japan might cause this condition. Therefore, our group subjected the sera of two children with post-hepatitis aplastic anemia to semi-nested PCR using primers specific for detection of TTV DNA. Although TTV DNA was not detectable in either sample it might be speculated that, like hepatitis viruses A to G, TTV could be found associated with this condition whereas it certainly does not constitute its sole etiologic agent.

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.