Hepatitis E virus infection in Thai blood donors.

BACKGROUND: Hepatitis E virus (HEV) infection in several industrialized and developing countries is associated with the consumption of pork and other meat products, an exposure risk among the majority of blood donors. We aimed to evaluate the prevalence of HEV in plasma from healthy blood donors in Thailand. STUDY DESIGN AND METHODS: We screened blood samples collected between October and December 2015, from 30,115 individual blood donors in 5020 pools of six, for HEV RNA using in-house real-time reverse-transcription polymerase chain reaction (RT-PCR). Thrice-reactive samples were subjected to a commercial real-time RT-PCR (cobas HEV test) and evaluated for anti-HEV immunoglobulin M and immunoglobulin G antibodies. Genotyping using nested RT-PCR, nucleotide sequencing, and phylogenetic analysis was performed. RESULTS: Twenty-six donors were positive for HEV RNA by the in-house assay, nine of whom were also positive by cobas test. None of the latter were reactive for anti-HEV immunoglobulin M or immunoglobulin G antibodies. Six samples were successfully genotyped and found to be HEV genotype 3. Thus, the frequency of HEV infection among healthy Thai blood donors is 1 in 1158. CONCLUSION: The presence of HEV RNA in the Thai blood supply was comparable to the rates reported in western European countries, but higher than in North America and Australia.

Characterization of hepatitis B virus mutations in untreated patients co-infected with HIV and HBV based on complete genome sequencing.

Co-infection of HBV with HIV results in an accelerated course of HBV-associated chronic liver disease. Several studies have shown that viral mutations are related to disease progression in mono-infection with HBV. However, it is unclear whether HBV mutation patterns might differ between co-infected and mono-infected patients. To compare the frequencies and mutation patterns in the HBV genome between co-infection and mono-infection. Twenty-four treatment-naïve co-infected and 31 treatment-naïve mono-infected Thai patients were included. HBV mutations were characterized by whole genome sequencing of virus serum samples. The clinical features and frequency of known clinically significant mutations were compared between the two groups. No significant difference between the groups was found with respect to sex, age and HBeAg. However, HBV DNA levels were significantly higher in co-infected patients. The distribution of HBV genotypes was comparable between the two groups and restricted mostly to sub-genotypes C1 and B2. An isolate with recombinants of genotypes G/C1 was also identified in a patient with co-infection. There was no difference in the prevalence of mutations in the enhancer II/basal core promoter/precore region, pre-S/S and polymerase genes between the two groups. In conclusion, dual infections tend to engender increased HBV DNA levels. There was no major difference in the frequencies of common HBV mutations between co-infected and mono-infected patients. Thus, HBV mutations may not contribute to disease pathogenesis in Thai patients with co-infection.

Molecular analysis of hepatitis B virus associated with vaccine failure in infants and mothers: a case-control study in Thailand.

Perinatal transmission of hepatitis B virus (HBV) has been controlled incompletely despite adequate immunoprophylaxis in infants. The aim of this study was to characterize virological factors of HBV associated with vaccine failure in Thailand. Sera of 14 infected infants (13 HBeAg-positive and one HBeAg-negative) with vaccine failure and their respective mothers (group M1) were tested quantitatively for HBV DNA by real-time PCR, HBV genotypes and mutations were characterized by direct sequencing. Sera collected from 15 HBeAg-positive (group M2) and 15 HBeAg-negative (group M3) mothers whose infants had been vaccinated successfully served as controls. The results showed that group M1 and group M2 mothers had equal titers of HBV DNA but higher titers than group M3. All infected infants and their respective mothers had the same HBeAg status and HBV genotypes. DNA analysis in a pair of HBeAg-negative infant and mother revealed that both were infected with an HBV precore mutant (G1896A). Escape mutants in the "a" determinant region (residues 144 and 145) were detected in two (14%) infected infants. The prevalence of BCP mutations/deletions in groups M2 and M3 was higher significantly than in group M1 (P = 0.022 and P < 0.001, respectively). In conclusion, instead of the HBeAg status, a high titer of HBV DNA in mothers was the major contributor to perinatal transmission of HBV. Escape mutants might be associated with vaccine failure in some infants. BCP mutations/deletions in mothers might contribute to the prevention of mother-to-infant transmission of HBV.

The distribution of hepatitis B virus genotypes in Thailand.

Phylogenetic analysis was performed on hepatitis B virus (HBV) strains obtained from 86 hepatitis B surface antigen (HBsAg) positive donors from Thailand originating throughout the country. Based on the S gene, 87.5% of strains were of genotype C while 10.5% were of genotype B, with all genotype B strains obtained from patients originating from the central or the south Thailand. No genotype B strains were found in the north of Thailand. Surprisingly, one patient was infected with a genotype H strain while another patient was infected with a genotype G strain. Complete genome sequencing and recombination analysis identified the latter as being a genotype G and C2 recombinant with the breakpoint around nucleotide position 700. The origin of the genotype G fragment was not identifiable while the genotype C2 fragment most likely came from strains circulating in Laos or Malaysia. The performance of different HBsAg diagnostic kits and HBV nucleic acid amplification technology (NAT) was evaluated. The genotype H and G/C2 recombination did not interfere with HBV detection.

Molecular epidemiological study of hepatitis B virus among migrant workers from Cambodia, Laos, and Myanmar to Thailand.

Although hepatitis B virus (HBV) infection is endemic in Southeast Asia, molecular epidemiological data on HBV circulating in some countries are limited. The aims of this study were to evaluate the seroprevalence of HBV and its genetic variability among migrant workers from Cambodia, Laos, and Myanmar in Thailand. Sera collected from 1,119 Cambodian, 787 Laotian, and 1,103 Myanmarese workers were tested for HBsAg. HBV DNA was amplified and the pre-S/S region was sequenced for genotyping and genetic mutation analysis. HBsAg was detected in 282 (9.4%). The prevalence of HBsAg among migrant workers from Cambodia, Laos, and Myanmar was 10.8%, 6.9%, and 9.7%, respectively. Of 224 subjects positive for HBV DNA, 86% were classified as genotype C (99% were sub-genotype C1) and 11.6% were genotype B (30.8%, 34.6%, and 30.8% were sub-genotypes B2, B3, and B4, respectively). Various point mutations in the "a" determinant region were detected in approximately 18% of these samples, of which Ile126Ser/Asn was the most frequent variant. Sequencing analysis showed that 19.1% of samples had pre-S mutations, with pre-S2 deletion as the most common mutant (7.7%) followed by pre-S2 start codon mutation (3.8%) and both pre-S2 deletion and start codon mutation (3.3%). High prevalence of HBV infection (approximately 7-11%) was found among migrant workers from Cambodia, Laos, and Myanmar, which may reflect the current seroprevalence in their respective countries. The data also demonstrated that HBV sub-genotype C1 was the predominant strain and various mutations of HBV occurring naturally were not uncommon among these populations.

Intrahepatic HBV DNA and covalently closed circular DNA (cccDNA) levels in patients positive for anti-HBc and negative for HBsAg.

Covalently closed circular DNA (cccDNA) is a unique episomal replicative intermediate molecule of hepatitis B virus (HBV) which plays a key role in viral persistence. The aim of this study was to prove cccDNA persistence in the liver tissue of patients negative for hepatitis B surface antigen (HBsAg) and positive for antibody to hepatitis core antigen (anti-HBc). Intrahepatic HBV DNA and cccDNA were determined using real-time and semi-nested PCR assays on the liver tissues of 35 patients who were negative for HBsAg and positive for anti-HBc with or without anti-HBs. HBV DNA was detected in the liver tissue of 4 out of 35 patients who were positive for anti-HBc. None of the samples harbored cccDNA. In this study population, which is of Asian origin, very low levels of HBV DNA were detected in a small percentage of patients with anti-HBc. Even using the highly sensitive semi-nested PCR assay, HBV cccDNA was not detectable in any anti-HBc positive patients either with or without anti-HBs.

Comparison of HBV ribonuclease H domain in naïve and drug resistant patients.

Nucleotide or nucleoside analog (NA) drug resistance has increasingly become a problem in HBV treatment. Due to the similarity between HBV polymerase and HIV-1 reverse transcriptase, knowledge obtained from HIV research might be applied to the treatment of HBV infection. A previous study has shown that HIV-1 ribonuclease H (RNase H) mutation may contribute to nucleoside reverse transcriptase inhibitor (NRTI) resistance. Therefore, we hypothesized that it might be possible to have a mutation in the HBV RNase H domain of HBV NA drug resistant patients. A one-year cross-sectional study was conducted at a single university hospital. Serum samples were collected from HBV infection treatment naive and suspected HBV NA drug resistant patients. To confirm HBV NA drug resistance, genotype specific resistance was examined. The HBV genotype and RNase H domain were sequenced and compared. In total, 37 HBV-infected patients were finally analyzed. Of these, 24 were considered sensitive to the drug and 13 resistant, as determined by the genotypic resistance method. Comparison between the two groups showed they had comparable baseline characteristics; no mutation in the HBV RNase H domain was detected. Possibly due to the small sample size, no significant mutations were found in the HBV RNase H domain of either group of HBV-infected patients. Further research of a larger patient group is needed to confirm these initial findings.

Changes in hepatitis A virus (HAV) seroprevalence in medical students in Bangkok, Thailand, from 1981 to 2016.

OBJECTIVE: This study aimed to determine the seroprevalence of anti-HAV IgG in Thai medical students in 2016 compared with the previous data and to demonstrate the cross-effective strategy to screen HAV seropositivity. RESULTS: Sera from 176 first-year medical students (age 19.07 ± 0.59 years; 50% female) at a university hospital in Thailand were tested for anti-HAV IgG. Data from HAV vaccination records and questionnaires were also collected. HAV seropositivity was unexpectedly high (62.5%, n = 110). 37.5% (n = 66) had an HAV vaccination record. Of these, 60.6% received the full HAV vaccination series, 4.5% received one HAV vaccination, 34.8% did not receive HAV vaccination, and 3.0% had natural HAV immunity. The long-term efficacy of HAV vaccination was at least 97.5% over a mean of 15.55 ± 2.44 years. There was a significant difference in immunity between students with (66.7%) and without (50.9%) vaccination records (P = 0.028). Most of the student's parents had a bachelor's degree or higher (87.9%; n = 272) and above average income (mean 17,000.76 ± 194.22 USD/person/year). Parental education and socioeconomic status influenced vaccination accessibility in these medical students. Screening of vaccination records instead of routine anti-HAV IgG testing is a cost-effective and reliable strategy to determine HAV immunity in medical students in Thailand.

Dynamics of HBV DNA levels, HBV mutations and biochemical parameters during antiviral therapy in a patient with HBeAg-negative chronic hepatitis B.

Chronic hepatitis B virus (HBV) infection leads to long-term sequelae such as cirrhosis and hepatocellular carcinoma. Antiviral therapy aims at controlling the viral replication and thus, decreasing the likelihood of such complications. In this study, we evaluated the dynamics of biochemical and virological parameters over 10 years of antiviral therapy in a Thai patient with chronic HBeAg-negative HBV infection, who had relapsed after two courses of interferon alfa treatment. Lamivudine administration initially led to a significant reduction in alanine aminotransferase (ALT) and HBV DNA levels, but a subsequent emergence of YIDD mutants caused an ALT flare and a virus breakthrough. A 4-log HBV DNA decrease and normalization of the ALT level were achieved within 3 months of adefovir monotherapy without any relapse during follow-up exceeding 20 months. Thus, careful monitoring during treatment and knowledge of cross-resistance to antiviral salvage therapy are crucial for the management of patients with chronic hepatitis B.

Myristica fragrans Houtt. methanolic extract induces apoptosis in a human leukemia cell line through SIRT1 mRNA downregulation.

BACKGROUND: Myristica fragrans Houtt. (nutmeg) contains antibacterial, antiviral and anti-cancer activities. However the mechanisms underlying those activities have not been clearly explained. OBJECTIVE: To study the effect of Myristica fragrans Houtt. methanolic extract on Jurkat human leukemia T cell line. MATERIAL AND METHOD: Methanol extract of Myristica fragrans Houtt. (Myristicaceae) was used to study the effect on Jurkat cell metabolic activity using an MTT assay and on apoptosis using annexin V staining. Expression of SIRT1 gene was determined by RT-PCR. RESULTS: At the concentrations 50 and 100 ig/mL, the methanol extract of Myristica fragrans Houtt significantly inhibited Jurkat cell proliferation and induced apoptosis as detected by annexin V staining. Downregulation of SIRT1 mRNA expression in Jurkat cells was observed even when the amount of methanol extract was 10 microg/mL. CONCLUSION: Methanol extract of Myristica fragrans Houtt induced apoptosis of Jurkat leukemia T cell line in a mechanisms involving SIRTI mRNA downregulation.

Analysis of connective tissue growth factor promoter polymorphism in Thai children with biliary atresia.

OBJECTIVE: Connective tissue growth factor (CTGF) has been proposed to play a key role in the pathogenesis of hepatic fibrosis in biliary atresia (BA). The aim of the present study was to determine the single nucleotide polymorphism (SNP) in the promoter region of CTGF gene in a Thai population, and to investigate the possible role of CTGF promoter polymorphism in the susceptibility of BA. MATERIAL AND METHOD: Genomic DNA was obtained from 84 patients with BA and 142 healthy controls. The -447 G/C and -132 C/G in CTGF promoter were amplified and examined by amplification-refractory mutation system (ARMs) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, respectively. The test of Hardy-Weinberg equilibrium (HWE) was performed using HWE program of SNPAnalyzer. Statistical analysis was carried out with SPSS and Epi Info. RESULT: According to the previous experiment, there were two SNPs, which were at position -447 and -132 on the promoter. However, there was only one SNP at the position -447 in the Thai population. No significant differences in genotype and allele frequency were observed between BA and controls or with BA subgroups. CONCLUSION: The present study demonstrated that CTGF polymorphism at -447 G/C was not associated with BA and the jaundice status of the postoperative BA patients.

Hepatitis E Virus in Pork and Variety Meats Sold in Fresh Markets.

Swine is an economically important livestock, yet pork consumption and close contact with pigs are associated with the risk of hepatitis E virus (HEV) infection. Limited data on the prevalence of HEV in Southeast Asia have mainly examined farm animals. To investigate the potential zoonotic transmission of HEV from dietary consumption of pork and variety meats (i.e., offal or organ meats), we obtained 1090 liver, 559 pork meat, and 556 intestine samples from fresh markets in the Bangkok metropolitan area between November 2014 and February 2015. The presence of HEV was assessed using reverse-transcription polymerase chain reaction. Concurrently, 720 bile and 553 fecal samples from a slaughterhouse were also examined. Overall, HEV RNA was found in 0.23 % of the market samples and 3.93 % of the slaughterhouse samples. Fecal and bile samples were more likely to test positive compared to liver, pork, and intestine samples (p < 0.001). Phylogenetic analysis showed that all HEV sequences obtained in this study formed a cluster closely related to genotype 3f. Pork and variety meats derived from pigs are commonly sold in fresh markets throughout Southeast Asia. Here, a relatively low HEV prevalence from pork and variety meats sold in Bangkok was found. Additional studies will be required to further assess potential dietary transmission of HEV elsewhere in the region.

Declining Trend of Hepatitis A Seroepidemiology in Association with Improved Public Health and Economic Status of Thailand.

Hepatitis A virus (HAV) is transmitted via the fecal-oral route from contaminated food or water. As part of the most recent survey of viral hepatitis burden in Thailand, we analyzed the current seroprevalence of HAV in the country and compared with data dating back to 1971. From March to October, 2014, a total of 4,260 individuals between one month and 71 years of age from different geographical regions (North = 961; Central = 1,125; Northeast = 1,109; South = 1,065) were screened for anti-HAV IgG antibody using an automated chemiluminescent microparticle immunoassay. Overall, 34.53% (1,471/4,260) possessed anti-HAV IgG antibody, and the age-standardized seroprevalence was 48.6%. Seroprevalence rates were 27.3% (North), 30.8% (Central), 33.8% (Northeast) and 45.8% (South) and were markedly lower than in the past studies especially among younger age groups. The overall trend showed an increase in the age by which 50% of the population were anti-HAV IgG antibody: 4.48 years (1971-1972), 6 (1976), 12.49 (1990), 36.02 (2004) and 42.03 (2014).This suggests that Thailand is transitioning from low to very low HAV endemicity. Lower prevalence of HAV correlated with improved healthcare system as measured by decreased infant mortality rate and improved national economy based on increased GDP per capita. The aging HAV immuno-naïve population may be rendered susceptible to potential HAV outbreaks similar to those in industrialized countries and may benefit from targeted vaccination of high-risk groups.

The Success of a Universal Hepatitis B Immunization Program as Part of Thailand's EPI after 22 Years' Implementation.

Hepatitis B vaccination for newborns was introduced in two provinces in 1988 as part of Thailand's Expanded Program on Immunization (EPI), and extended to the whole country in 1992. Our previous studies showed that children and adolescents who were born after the implementation of this program had a carrier rate of less than 1%, compared with 5-6% before implementation. In 2014 we performed hepatitis B serosurveys among 5964 subjects in the different geographic regions of the country to evaluate the long-term immunogenicity and impact of universal hepatitis B vaccination in newborns as part of the 22-year EPI program, by assessing HBsAg, anti-HBc and anti-HBs seropositivity status. The number of HB virus (HBV) carriers, both children and young adults, who were born after universal HB vaccination was markedly reduced. The carrier rates among the age groups 6 months to 5 years, 5-10, 11-20, 21-30, 31-40, 41-50 and >50 years were respectively 0.1, 0.29, 0.69, 3.12, 3.78, 4.67 and 5.99%. The seropositivity rate for HBsAg in the post-EPI group was 0.6%, whereas in the pre-EPI group it was as high as 4.5% (p<0.001). HBV infection by means of detectable anti-HBc had also drastically declined in the population born after the HB vaccine was integrated into the EPI program. We estimated that the total number of HBV carriers amounted to 2.22 million, or 3.48% of the total population, most of whom are adults. The HB vaccine is the first vaccine shown to be effective in preventing the occurrence of chronic liver disease and hepatocellular carcinoma. Universal vaccination campaign will contribute to the eventual eradication of HBV-associated disease.

Hepatitis E virus infection: Epidemiology and treatment implications.

Hepatitis E virus (HEV) infection is now established as an emerging enteric viral hepatitis. Standard treatments in acute and chronic hepatitis E remain to be established. This study undertakes a review of the epidemiology, treatment implication and vaccine prevention from published literature. HEV infection is a worldwide public health problem and can cause acute and chronic hepatitis E. HEV genotypes 1 and 2 are primarily found in developing countries due to waterborne transmission, while the zoonotic potential of genotypes 3 and 4 affects mostly industrialized countries. An awareness of HEV transmission through blood donation, especially in the immunocompromised and solid organ transplant patients, merits an effective anti-viral therapy. There are currently no clear indications for the treatment of acute hepatitis E. Despite concerns for side effects, ribavirin monotherapy or in combination with pegylated interferon alpha for at least 3 mo appeared to show significant efficacy in the treatment of chronic hepatitis E. However, there are no available treatment options for specific patient population groups, such as women who are pregnant. Vaccination and screening of HEV in blood donors are currently a global priority in managing infection. New strategies for the treatment and control of hepatitis E are required for both acute and chronic infections, such as prophylactic use of medications, controlling large outbreaks, and finding acceptable antiviral therapy for pregnant women and other patient groups for whom the current options of treatment are not viable.

Swine is a possible source of hepatitis E virus infection by comparative study of hepatitis A and E seroprevalence in Thailand.

Hepatitis A virus (HAV) and hepatitis E virus (HEV) infection in developing countries are associated with contaminated food or water. Although Thailand is non-endemic for HEV, sporadic infections may occur from zoonotic transmission. Individuals between 7 months to 69 years (mean age = 32.8) from predominantly Islamic Narathiwat (n = 305) and swine farm-dense Lop Buri (n = 416) provinces were screened for anti-HEV and anti-HAV antibodies by commercial enzyme-linked immunosorbent assay and automated chemiluminescent microparticle immunoassay, respectively. Seroprevalence and relative antibody titers were analyzed according to age groups. HAV IgG antibody positive rates in Lop Buri and Narathiwat residents were 39.9% and 58%, respectively (p < 0.001). Greater than 90% of individuals >50 years old in both provinces possessed anti-HAV IgG. In contrast, seroprevalence for anti-HEV IgG was much higher in Lop Buri (37.3%) than in Narathiwat (8.9%) (p < 0.001). Highest anti-HEV IgG prevalence was found among 21-30 year-olds (50%) in Lop Buri and 41-50 year-olds (14.1%) in Narathiwat. In summary, fewer individuals possessed anti-HEV IgG in Narathiwat where most residents abstained from pork and fewer swine farms are present. Therefore, an increased anti-HEV IgG seroprevalence was associated with the density of swine farm and possibly pork consumption. Adults were more likely than children to have antibodies to both HEV and HAV.

Hepatitis B virus genetic variation and TP53 R249S mutation in patients with hepatocellular carcinoma in Thailand.

Chronic hepatitis B virus (HBV) infection and dietary exposure to aflatoxin B1 (AFB1) are major risk factors for hepatocellular carcinoma (HCC). The aim of this study was to evaluate the role of HBV genetic variation and the R249S mutation of the p53 gene, a marker of AFB1-induced HCC, in Thai patients chronically infected with HBV. Sixty-five patients with and 89 patients without HCC were included. Viral mutations and R249S mutation were characterized by direct sequencing and restriction fragment length polymorphism (RFLP) in serum samples, respectively. The prevalences of T1753C/A/G and A1762T/G1764A mutations in the basal core promotor (BCP) region were significantly higher in the HCC group compared to the non-HCC group. R249S mutation was detected in 6.2% and 3.4% of the HCC and non-HCC groups, respectively, which was not significantly different. By multiple logistic regression analysis, the presence of A1762T/G1764A mutations was independently associated with the risk of HCC in Thai patients.

HIV and Hepatitis B coinfection among perinatally HIV-infected Thai adolescents.

OBJECTIVES: This study aimed to determine the prevalence of hepatitis B virus (HBV) coinfection and HBV seropositivity in perinatally HIV-infected adolescents. A secondary objective was to describe the clinical characteristics of adolescents with chronic HBV/HIV coinfection. MATERIALS AND METHODS: Multicenter cross-sectional study of perinatally HIV-infected adolescents aged 12-25 years. HBV surface antigen, surface antibody (anti-HBs) and core antibody (anti-HBc) were measured. Coinfection was defined as having persistently positive HBV surface antigen. Seroprotective antibody from immunization was defined as having anti-HBs ≥10 mIU/mL with negative anti-HBc. HBV DNA quantitation and rtM204V/I mutation analysis (lamivudine resistance-associated mutation) were performed in adolescents with chronic HBV infection. RESULTS: From November 2010 to March 2011, 521 patients were enrolled. Mean (SD) of CD4 lymphocyte count was 685 (324) cells/µL. The prevalence of HBV/HIV coinfection was 3.3% (95% confidence interval: 1.9-5.2%). Protective antibody against HBV was found in 18% of population, and this was significantly higher among adolescents who received than those who did not receive HBV revaccination after receiving antiretroviral therapy (93% versus 6%, P < 0.01). Among adolescents with chronic HBV infection, 88% have received lamivudine; however, 69% have HBV DNA >10 copies/mL and 75% had the rtM204V/I mutation. CONCLUSIONS: The prevalence of HBV coinfection in HIV-infected Thai adolescents was 3.3%. Most HIV-infected adolescents had no HBV protective antibody; therefore, revaccination with HBV vaccine is encouraged. The high prevalence of HBV-lamivudine resistance underscores the importance of HBV screening prior to antiretroviral therapy initiation to guide the selection of optimal regimen for coinfected children.

Cross-species transmission of gibbon and orangutan hepatitis B virus to uPA/SCID mice with human hepatocytes.

To investigate the potential of cross-species transmission of non-human primate HBV to humans, severe combined immunodeficiency mice transgenic for urokinase-type plasminogen activator, in which the mouse liver has been engrafted with human hepatocytes, were inoculated with non-human primate HBV. HBV-DNA positive serum samples from a gibbon or orangutan were inoculated into 6 chimeric mice. HBV-DNA, hepatitis B surface antigen (HBsAg), and HB core-related antigen in sera and HBV cccDNA in liver were detectable in 2 of 3 mice each from the gibbon and orangutan. Likewise, applying immunofluorescence HBV core protein was only found in human hepatocytes expressing human albumin. The HBV sequences from mouse sera were identical to those from orangutan and gibbon sera determined prior to inoculation. In conclusion, human hepatocytes have been infected with gibbon/orangutan HBV.

A case-control study on sequence variations in the enhancer II/core promoter/precore and X genes of hepatitis B virus in patients with hepatocellular carcinoma.

PURPOSE: To evaluate the sequence variations in the enhancer II (EnhII)/basal core promotor (BCP)/precore (PC) and X genes of hepatitis B virus (HBV) in Thai patients with hepatocellular carcinoma (HCC) by conducting a cross-sectional case-control study. METHODS: As much as 60 patients with HCC and 60 patients without HCC, who were matched for sex, age, hepatitis B e antigen (HBeAg) status, and HBV genotype, were included. Viral mutations in the EnhII/BCP/PC and X regions were characterized by direct sequencing in serum samples. RESULTS: The prevalence of T1753C/A, A1762T/G1764A and G1899A mutations were significantly higher in the HCC group compared to the non-HCC group (43.3 vs. 23.3%, P = 0.02; 88.3 vs. 53.0%, P < 0.001; and 35.0 vs. 8.3%, P = 0.001, respectively). No significant difference between groups was found with respect to G1613A, C1653T, C1766T/T1768A, A1846T/C, T1858C, and G1896A mutations. By multiple logistic regression analysis, the presence of cirrhosis, A1762T/G1764A and G1899A mutations were independently associated with the risk of HCC. CONCLUSION: These data suggested that A1762T/G1764A and G1899A mutations were associated with the development of HCC in Thai patients.