The predominant genotypes of hepatitis B virus in Thailand

In Thailand, chronic liver disease (CLD) as a consequence of infection with hepatitis B virus (HBV) constitutes a public-health burden. Control and treatment are complicated by the virus exhibiting an unusually high mutation rate, with some genotypes apparently causing more severe disease than others. Restriction-fragment-length-polymorphism (RFLP) analysis of the pre-S region of the viral genome, amplified by PCR, was used to determine which genotypes were most prevalent among Thai patients chronically infected with the virus. The patients were chronic HBV carriers (40) or cases of chronic hepatitis (34), cirrhosis (14) or hepatocellular carcinoma (30). As indicated by the results of earlier studies on CLD patients in South-east Asia, genotype C (68.6%) was clearly predominant. RFLP patterns permitted the C1 (12.7%), C7 (45.7%), C8 (10.2%) and B1 (29.7%) subtypes to be identified. Two samples that could not be typed by RFLP were analysed by direct sequencing, categorized as type C, and tentatively designated as subtype C9. As comparison of the present data with those previously obtained by direct sequencing of PCR products indicates that RFLP analysis is as specific and reliable as sequencing and less expensive and time-consuming, RFLP analysis may be particularly useful for epidemiological studies.     

Simultaneous quantitation and genotyping of hepatitis B virus by real-time PCR and melting curve analysis

Hepatitis B virus (HBV) genotype and HBV DNA levels have been implicated in clinical evaluation and prognosis of patients with chronic HBV infection. The aim of the present study was to develop a rapid and sensitive method for simultaneous HBV DNA quantitation and differentiation between HBV genotypes B and C in a single-step reaction by real-time PCR and melting curve analysis using SYBR Green I fluorescent dye. The genotypes obtained by this method were compared with those examined by PCR-RFLP and direct sequencing on 52 serum samples of patients with chronic HBV infection. Using the results obtained by direct sequencing and phylogenetic analysis as the reference, the accuracy of HBV genotyping by PCR-RFLP and melting curve analysis was 90.38 and 92.31%, respectively. The geometric mean of HBV DNA levels was 3.42×10⁶, 2.10×10⁶, 1.19×10⁵ and 3.10×10⁴ copies/μl in asymptomatic carriers, patients with chronic hepatitis, cirrhosis and hepatocellular carcinoma, respectively. It is concluded that this method has the advantages of rapidity, reproducibility and accuracy, which would be feasible and attractive for large-scale analysis, particularly in regions where HBV genotypes B and C are prevalent.     

Variants within the "a" determinant of HBs gene in children and adolescents with and without hepatitis B vaccination as part of Thailand's Expanded Program on Immunization (EPI)

A total of 2229 children selected from five distinct areas of Thailand were screened for HBs antigen (HBsAg) by ELISA. Out of 51, forty-nine HBsAg-positive children were further examined for HBV-DNA by the polymerase chain reaction, utilizing the region of the hepatitis B Virus (HBV) genome encoding the major antigenic epitopes of hepatitis B surface antigen. Direct automated sequencing of the "a" determinant region revealed 11 of 49 children to display variable mutations. The vaccinated and nonvaccinated children had amino acid variants clustered between residues 120 and 160. Mutations between residues 120 and 160 were found at higher frequency in the vaccinated group (4/13; 30.8%) than in the nonvaccinated group (7/36; 19.4%), but this was not statistically significant. Infections with new HBV variants are contracted either vertically or horizontally within the group having received the vaccine, a finding confirmed by the presence of amino acid substitutions critical for immune escape. Hence, neither vaccine nor IgG has any apparent effect on those variants and the children turn into HBV carriers. However, the current vaccination program still efficiently protects perinatal transmission of HBV and unless long term studies lead us to conclude otherwise, inclusion of the variant strain(s) into a new vaccine formulation is not deemed necessary.     

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.     

Hepatitis TT virus infection in high-risk groups

Summary The novel hepatitis TT virus first described by a Japanese group has been reported to be parenterally transmitted and furthermore, to have been detected in patients with hepatitis of unknown etiology. Hence, in the present study its prevalence was investigated within groups at high risk for contracting blood-borne viruses, such as individuals with chronic liver disease, intravenous drug users and recipients of blood and blood products, as compared to voluntary blood donors and pregnant women. To that end, DNA was extracted from sera obtained from the respective patients and subjected to PCR using semi-nested primers. The frequency of TTV DNA detected within high risk groups, such as nine out of 50 patients with chronic non-A-to-G liver disease (18%), nine out of 98 hepatocellular carcinoma cases (9.2%), 17 out of 52 intravenous drug users (32.7%), 15 out of 80 thalassemia patients with multiple blood transfusions (18.8%) and three out of 31 prostitutes (9.7%) exceeded that among voluntary blood donors and pregnant women, which amounted to 14 out of 200 (7%) and seven out of 103 (6.8%), respectively. Additional molecular research should be performed in order to determine its short-, as well as long-term clinical significance.     

Molecular characterization of hepatitis-A-virus infections,in the context of two outbreaks in southern Thailand

As hepatitis A virus (HAV) is usually transmitted through the faecal-oral route, hepatitis A is a communicable disease. In countries of intermediate to low endemicity, sudden outbreaks of human infection with the virus may occur. Between September 2001 and April 2002, there were two outbreaks of HAV infection in the Ruso and Yeengor districts of Narathiwas province, in southern Thailand. Isolates of HAV were recovered during these outbreaks, from 14 in-patients with acute hepatitis in Ruso (12 positive for anti-HAV IgM and all positive for HAV RNA), 16 children with asymptomatic infection in Yeengor (14 positive for anti-HAV IgM and nine for HAV RNA), and four isolated cases in Bangkok (all positive for anti-HAV IgM). Molecular characterization of the VP1-P2A region of each isolate was followed by phylogenetic analysis. All of the isolates from Narathiwas province were found to be of genotype 1a, to have the same VP1 nucleotide sequence, and to show a high level of sequence homology (>/= 99.5%) with the isolates from Bangkok and with previous Thai isolates. These results should facilitate further research into HAV transmission and genotype identification in community outbreaks.     

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.