- Email: [email protected]
Genotypes of hepatitis B virus among voluntary blood donors in northern Thailand
Hepatology Research 35 (2006) 263–266
Genotypes of hepatitis B virus among voluntary blood donors in northern Thailand Prapan Jutavijittum a,∗ , Yupa Jiviriyawat a , Amnat Yousukh a , Warunee Kunachiwa b , Kan Toriyama c b
a Department of Pathology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand Department of Clinical Immunology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand c Department of Pathology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan
Received 27 October 2005; received in revised form 24 April 2006; accepted 26 April 2006 Available online 30 May 2006
Abstract There are distinct ethnogeographic variations for the distribution of various hepatitis B virus (HBV) genotypes, and pathogenic and therapeutic differences are also observed. In general, genotype B infection has a relatively better prognosis than genotype C. In Thailand, genotypes C and B were reported as the major genotypes; however, there were no previous reports of HBV genotyping in the north of the country. From 1998 to 2000, 216 HBsAg-positive serum samples (164 males and 52 females, aged 16–52 years), were screened and collected from voluntary blood donors in four provinces of northern Thailand. The method of Naito et al. was employed in this study, with the multiplexPCR approach and genotype-specific primers to identify genotypes A–F. We found that the HBV genotype C was highly predominant in northern Thailand (89.3%), when compared with the previous reports of genotype C distribution among voluntary blood donors from other areas in the country (50–65%), followed by genotype B (7.4%), mixed infection of genotype B and C (1.9%) and genotype A (0.5%). Four samples (1.9%) were unclassifiable. There was no significant difference of genotype distribution among four northern Thai provinces or each age group. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Genotype; Hepatitis B virus; Epidemiology; Thailand
1. Introduction Hepatitis B virus (HBV) is endemic in many parts of the world, especially Asia and Africa. More than 2 billion people have had contact with the virus and more than 350 million are chronic carriers globally [1,2]. The natural course of chronic infection is variable, ranging from an inactive hepatitis B surface antigen (HBsAg) carrier state to a more or less progressive chronic hepatitis, potentially evolving to cirrhosis and hepatocellular carcinoma [3]. In areas of high endemicity, the most common route of transmission is perinatal or the infection can be acquired during the preschool years. In areas of intermediate endemicity, transmission is either perinatal or ∗
Corresponding author. Tel.: +66 53 945442; fax: +66 53 217144. E-mail address: [email protected] (P. Jutavijittum).
1386-6346/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.hepres.2006.04.010
horizontal. In areas of low endemicity, most HBV infections are acquired by horizontal transmission in early adult life, i.e. through intravenous drug use or unprotected sexual activities [2]. Currently, Thailand is classified as having an intermediate endemicity (3–6%) [4,5] and the seroprevalence of HBsAg among voluntary blood donors in the northern region is 8.7% [6]. Recently, a new classification system was introduced, based on a comparison of the complete genomic sequence. Eight HBV genotypes, A–H, are distinguishable, with each genotype differing by more than 8% at the nucleotide level when compared to each other [1]. The prevalence of HBV genotypes varies markedly in different parts of the world, as well as in different population subgroups. In brief, genotype B and C are prevalent in Asia, whereas genotype A and D prevail in Western countries and India. Genotype E
264
P. Jutavijittum et al. / Hepatology Research 35 (2006) 263–266
is restricted to Africa, and genotype F to Central and South America [7]. Single cases of HBV infection with genotype G have been reported in France and the USA. Genotype H was very recently identified, with a Central and South American origin anticipated [1]. Previous studies in Thailand showed that genotype C (54–73% of cases) was predominate over genotype B, A and D, but most data were reported from the whole group of asymptomatic HBV carriers and patients who developed liver disease, such as chronic hepatitis, liver cirrhosis and hepatocellular carcinoma [8–11]. A precise PCR genotyping system based on type-specific primers was developed by Naito et al. [12], and this approach allows an identification of genotypes A–F, which are the common genotypes. In this study, we employed this technique to determine the frequency of these A–F genotypes among HBsAg-positive voluntary blood donors residing in northern Thailand.
2. Materials and methods 2.1. Samples Serum samples from voluntary blood donors in Chiang Mai, Chiang Rai, Lampang, and Lamphun provinces in northern Thailand (Fig. 1) were screened for blood-transmitted
pathogens at the 10th Regional Blood Center office in Chiang Mai. All blood donors were considered healthy by National Blood Center criteria. Testing for HBsAg was performed by a commercial ELISA kit, Enzygnost® HBsAg 5.0 (DadeBehring, Marburg Germany). From 1998 to 2000, samples with HBsAg-positive were collected and stored at −20 ◦ C. A total of 216 HBsAg-positive serum samples were obtained: 55 from Chiang Mai; 50 from Chiang Rai; 56 from Lampang; and 55 from Lamphun. All samples were coded by number; the identities of the patients were unknown to the research team. 2.2. Hepatitis B genotype determination We employed the method of Naito et al. [12]. DNA was extracted from 100 l of serum samples using a commercial nucleic acid extraction kit; High Pure Viral Nucleic Acid Kit (Roche Diagnostics, Mannheim, Germany). The resulting pellet was resuspended in RNase-free water and then subjected to nested PCR. The HBV genomes were amplified using the universal primers (P1 and S1-2) as outer primers, followed by two different mixtures (Mix A for genotypes A, B, and C; Mix B for genotypes D, E, and F) containing type-specific inner primers. Genotypes of HBV were determined by identifying genotype-specific DNA bands. The sizes of PCR products
Fig. 1. Map of northern Thailand and genotypic distribution of hepatitis B viral genotypes among voluntary blood donors with HBsAg in four provinces of northern Thailand.
P. Jutavijittum et al. / Hepatology Research 35 (2006) 263–266 Table 1 Genotypes of hepatitis B virus by gender among voluntary blood donors with HBsAg in northern Thailand HBV genotyping
Gender
Total (%)
Male (%)
Female (%)
C B B and C A Unclassified
144 (87.8) 14 (8.6) 2 (1.2) 1 (0.6) 3 (1.8)
49 (94.3) 2 (3.8) 0 0 1 (1.9)
193 (89.3) 16 (7.4) 2 (0.9) 1 (0.5) 4 (1.9)
were estimated according to the migration pattern of a 50bp DNA ladder. The final PCR products were sufficiently different in size to be distinguished by routine agarose gel electrophoresis. Positive controls for virus genotypes A–C were run. Negative controls for each PCR were run, including a no reagent control to check for contamination by viral DNA. All samples were run at least twice to ensure reproducibility. 2.3. Statistical analysis Chi-square and Student’s t-test were used where appropriate. A P-value of < 0.05 was considered statistically significant.
3. Results HBV genotypes were determined from 216 HBsAgpositive sera, derived from voluntary blood donors in four provinces of northern Thailand. These included 164 males and 52 females, aged 16–52 years, with a mean age of 25.4 (±8.3) years. The distribution of the HBV genotypes is shown in Table 1. Among the 216 HBsAg-positive blood donors, 89.3% were genotype C, followed by 7.4% for genotype B, 0.9% for mixed infection of genotype B and C, and 0.5% for genotype A; four samples (1.9%) were unclassifiable. The mean ages of the predominant genotype C and B were 25.0 (±7.9) and 28.2 (±10.0) years, respectively. There was no significant difference between the mean ages of genotype C and B, P = 0.136. Genotype C was found in 94.3% of females and 87.8% of males, while genotype B was detected in 8.6% of males and 3.8% of females. The distribution of HBV genotypes in four provinces of northern Thailand is shown in Fig. 1. There were no significant differences in distribution of the predominant genotype C and B among the four provinces, with P-values of 0.789 and 0.172, respectively.
4. Discussion A previous report described existence of a strong correlation between HBV genotypes and ethnicity, in that
265
HBV genotype B and C are common among indigenous populations of Asian countries [1]. We also found that genotype C was the predominant genotype, involving up to 93% of HBsAg-positive voluntary blood donors in northern Thailand. This result is higher than that from other areas in Thailand, in which genotype C was found in 50–65% of asymptomatic carriers [9–11]. Genotype B was the second most common type (7.4%) found in the north, but less frequent than in other areas that reported 20.6–45.5% of genotype B among asymptomatic carriers [9–11]. Although previous reports described a prevalence rate of 14.7% for genotype A and 5.0% for genotype D in other areas of Thailand, we found only one case of genotype A (0.5%) and no case of genotype D in northern Thailand. Moreover, we detected neither genotype E nor F in northern Thailand. In endemic areas, at medium endemic level of HBV infection (prevalence of HBsAg in the general population >2%), intrafamily transmission was the major mode by which infection spread. The pool of chronic HBV carriers is likely to have arisen from childhood infection [13]. Our study showed a unique genotypic distribution of HBV genotypes, of which genotype C is the most predominant. We found two cases (1.9%) of B and C mixed infection in our series and it has been reported that the multiple-PCR, which we employed, had superiority in detecting mixed infections [1]. The outcome of infection depends both on properties of the pathogenic organism and the host. It is suggested that HBV genotypes differ in their ability to induce chronic infection and also in their potential to induce acute hepatitis [1]. Many reports indicate that there are significant differences in the outcome of chronic hepatitis B in relation to the genotype of HBV. In particular, genotype C induces a more severe course of the disease with a high positive rate of HBeAg, greater necroinflammatory activity and fibrosis, and it shows a higher prevalence in the patients with cirrhosis and hepatocellular carcinoma [1,7,8,14]. The economic burden of HBV infection is substantial because of the high morbidity and mortality associated with end-stage liver disease such as cirrhosis, and hepatocellular carcinoma. The northern Thailand has been shown to be an endemic area of chronic HBV carriers with high predominant of genotype C. These subjects are at greater risk of more severe consequence of chronic liver disease and hepatocellular carcinoma. The long-term increase in morbidity and mortality rates is predicted in these sufferers. In conclusion, we found that HBV genotype C is a highly predominant type in northern Thailand. This finding corresponds with previous reports that HBV genotype C is the most common in Thailand and other Asian countries, and it raises concern that infected individuals will progress to severe chronic liver disease and cancer in the future. As perinatal transmission is the most likely mode of transmission in this area, this can be reduced by appropriate prophylaxis; vaccination of the infant at birth and administration of hepatitis B immune globulin. Efforts
266
P. Jutavijittum et al. / Hepatology Research 35 (2006) 263–266
should be attempted to increase compliance with hepatitis B vaccination.
Acknowledgements This study was supported by a Grant in Aid No. 13576002 (K.T.) from the Ministry of Education, Culture, Sports, Science and Technology and also supported in part by a Grant for International Health Cooperation Research No. 12C-4 (K.T.) from the Ministry of Health and Welfare, Japan. The authors gratefully acknowledge the Faculty of Medicine, Chiang Mai University, for its financial support in the presentation of these data at the International Meeting on the Molecular Biology of Hepatitis B Viruses in Heidelberg, Germany, during 18–21 September 2005.
References [1] Schaefer S. Review: hepatitis B virus: significance of genotypes. J Viral Hepat 2005;12:111–24. [2] Lavanchy D. Review: hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures. J Viral Hepat 2004;11:97–107. [3] Wang FS. Review: current status and prospects of studies on human genetic alleles associated with hepatitis B virus infection. World J Gastroenterol 2003;9:641–4.
[4] Andre F. Review: hepatitis B epidemiology in Asia, the Middle East and Africa. Vaccine 2000;18(Suppl. 1):S20–2. [5] Mohamed R, Desmond P, Suh DJ, et al. Review: practical difficulties in the management of hepatitis B in the Asia-Pacific region. J Gastroenterol Hepatol 2004;19:958–69. [6] Jutavijittum P, Jiviriyawat Y, Yousukh A, et al. A seroepidemiological study on hepatitis B virus, hepatitis C virus and human immunodeficiency virus infection in northern Thailand. Jpn J Trop Med Hyg 1999;27:13–7. [7] Kao JH, Chen DS. Review: global control of hepatitis B virus infection. Lancet Infect Dis 2002;2:395–403. [8] Tangkijvanich P, Mahachai V, Komolmit P, et al. Hepatitis B virus genotypes and hepatocellular carcinoma in Thailand. World J Gastroenterol 2005;11:2238–43. [9] Theamboonlers A, Jantaradsamee P, Kaew-In N, Tangkijvanich P, Hirsch P, Poovorawan Y. The predominant genotypes of hepatitis B virus in Thailand. Ann Trop Med Parasitol 1999;93:737–43. [10] Theamboonlers A, Tangkijvanich P, Pramoolsinsap C, Poovorawan Y. Genotypes and subtypes of hepatitis B virus in Thailand. Southeast Asian J Trop Med Public Health 1998;29:786–91. [11] Sugauchi F, Chutaputti A, Orito E, et al. Hepatitis B virus genotypes and clinical manifestation among hepatitis B carriers in Thailand. J Gastroenterol Hepatol 2002;17:671–6. [12] Naito H, Hayashi S, Abe K. Rapid and specific genotyping system for hepatitis B virus corresponding to six major genotypes by PCR using type-specific primers. J Clin Microbiol 2001;39:362–4. Erratum in: J Clin Microbiol 2001;39:1686. [13] Stroffolini T. The changing pattern of hepatitis B virus infection over the past three decades in Italy. Dig Liver Dis 2005;37:622–7. [14] Kao JH, Chen PJ, Lai MY, Chen DS. Clinical and virological aspects of blood donors infected with hepatitis B virus genotypes B and C. J Clin Microbiol 2002;40:22–5.
Genotypes of hepatitis B virus among voluntary blood donors in northern Thailand Prapan Jutavijittum a,∗ , Yupa Jiviriyawat a , Amnat Yousukh a , Warunee Kunachiwa b , Kan Toriyama c b
a Department of Pathology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand Department of Clinical Immunology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand c Department of Pathology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan
Received 27 October 2005; received in revised form 24 April 2006; accepted 26 April 2006 Available online 30 May 2006
Abstract There are distinct ethnogeographic variations for the distribution of various hepatitis B virus (HBV) genotypes, and pathogenic and therapeutic differences are also observed. In general, genotype B infection has a relatively better prognosis than genotype C. In Thailand, genotypes C and B were reported as the major genotypes; however, there were no previous reports of HBV genotyping in the north of the country. From 1998 to 2000, 216 HBsAg-positive serum samples (164 males and 52 females, aged 16–52 years), were screened and collected from voluntary blood donors in four provinces of northern Thailand. The method of Naito et al. was employed in this study, with the multiplexPCR approach and genotype-specific primers to identify genotypes A–F. We found that the HBV genotype C was highly predominant in northern Thailand (89.3%), when compared with the previous reports of genotype C distribution among voluntary blood donors from other areas in the country (50–65%), followed by genotype B (7.4%), mixed infection of genotype B and C (1.9%) and genotype A (0.5%). Four samples (1.9%) were unclassifiable. There was no significant difference of genotype distribution among four northern Thai provinces or each age group. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Genotype; Hepatitis B virus; Epidemiology; Thailand
1. Introduction Hepatitis B virus (HBV) is endemic in many parts of the world, especially Asia and Africa. More than 2 billion people have had contact with the virus and more than 350 million are chronic carriers globally [1,2]. The natural course of chronic infection is variable, ranging from an inactive hepatitis B surface antigen (HBsAg) carrier state to a more or less progressive chronic hepatitis, potentially evolving to cirrhosis and hepatocellular carcinoma [3]. In areas of high endemicity, the most common route of transmission is perinatal or the infection can be acquired during the preschool years. In areas of intermediate endemicity, transmission is either perinatal or ∗
Corresponding author. Tel.: +66 53 945442; fax: +66 53 217144. E-mail address: [email protected] (P. Jutavijittum).
1386-6346/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.hepres.2006.04.010
horizontal. In areas of low endemicity, most HBV infections are acquired by horizontal transmission in early adult life, i.e. through intravenous drug use or unprotected sexual activities [2]. Currently, Thailand is classified as having an intermediate endemicity (3–6%) [4,5] and the seroprevalence of HBsAg among voluntary blood donors in the northern region is 8.7% [6]. Recently, a new classification system was introduced, based on a comparison of the complete genomic sequence. Eight HBV genotypes, A–H, are distinguishable, with each genotype differing by more than 8% at the nucleotide level when compared to each other [1]. The prevalence of HBV genotypes varies markedly in different parts of the world, as well as in different population subgroups. In brief, genotype B and C are prevalent in Asia, whereas genotype A and D prevail in Western countries and India. Genotype E
264
P. Jutavijittum et al. / Hepatology Research 35 (2006) 263–266
is restricted to Africa, and genotype F to Central and South America [7]. Single cases of HBV infection with genotype G have been reported in France and the USA. Genotype H was very recently identified, with a Central and South American origin anticipated [1]. Previous studies in Thailand showed that genotype C (54–73% of cases) was predominate over genotype B, A and D, but most data were reported from the whole group of asymptomatic HBV carriers and patients who developed liver disease, such as chronic hepatitis, liver cirrhosis and hepatocellular carcinoma [8–11]. A precise PCR genotyping system based on type-specific primers was developed by Naito et al. [12], and this approach allows an identification of genotypes A–F, which are the common genotypes. In this study, we employed this technique to determine the frequency of these A–F genotypes among HBsAg-positive voluntary blood donors residing in northern Thailand.
2. Materials and methods 2.1. Samples Serum samples from voluntary blood donors in Chiang Mai, Chiang Rai, Lampang, and Lamphun provinces in northern Thailand (Fig. 1) were screened for blood-transmitted
pathogens at the 10th Regional Blood Center office in Chiang Mai. All blood donors were considered healthy by National Blood Center criteria. Testing for HBsAg was performed by a commercial ELISA kit, Enzygnost® HBsAg 5.0 (DadeBehring, Marburg Germany). From 1998 to 2000, samples with HBsAg-positive were collected and stored at −20 ◦ C. A total of 216 HBsAg-positive serum samples were obtained: 55 from Chiang Mai; 50 from Chiang Rai; 56 from Lampang; and 55 from Lamphun. All samples were coded by number; the identities of the patients were unknown to the research team. 2.2. Hepatitis B genotype determination We employed the method of Naito et al. [12]. DNA was extracted from 100 l of serum samples using a commercial nucleic acid extraction kit; High Pure Viral Nucleic Acid Kit (Roche Diagnostics, Mannheim, Germany). The resulting pellet was resuspended in RNase-free water and then subjected to nested PCR. The HBV genomes were amplified using the universal primers (P1 and S1-2) as outer primers, followed by two different mixtures (Mix A for genotypes A, B, and C; Mix B for genotypes D, E, and F) containing type-specific inner primers. Genotypes of HBV were determined by identifying genotype-specific DNA bands. The sizes of PCR products
Fig. 1. Map of northern Thailand and genotypic distribution of hepatitis B viral genotypes among voluntary blood donors with HBsAg in four provinces of northern Thailand.
P. Jutavijittum et al. / Hepatology Research 35 (2006) 263–266 Table 1 Genotypes of hepatitis B virus by gender among voluntary blood donors with HBsAg in northern Thailand HBV genotyping
Gender
Total (%)
Male (%)
Female (%)
C B B and C A Unclassified
144 (87.8) 14 (8.6) 2 (1.2) 1 (0.6) 3 (1.8)
49 (94.3) 2 (3.8) 0 0 1 (1.9)
193 (89.3) 16 (7.4) 2 (0.9) 1 (0.5) 4 (1.9)
were estimated according to the migration pattern of a 50bp DNA ladder. The final PCR products were sufficiently different in size to be distinguished by routine agarose gel electrophoresis. Positive controls for virus genotypes A–C were run. Negative controls for each PCR were run, including a no reagent control to check for contamination by viral DNA. All samples were run at least twice to ensure reproducibility. 2.3. Statistical analysis Chi-square and Student’s t-test were used where appropriate. A P-value of < 0.05 was considered statistically significant.
3. Results HBV genotypes were determined from 216 HBsAgpositive sera, derived from voluntary blood donors in four provinces of northern Thailand. These included 164 males and 52 females, aged 16–52 years, with a mean age of 25.4 (±8.3) years. The distribution of the HBV genotypes is shown in Table 1. Among the 216 HBsAg-positive blood donors, 89.3% were genotype C, followed by 7.4% for genotype B, 0.9% for mixed infection of genotype B and C, and 0.5% for genotype A; four samples (1.9%) were unclassifiable. The mean ages of the predominant genotype C and B were 25.0 (±7.9) and 28.2 (±10.0) years, respectively. There was no significant difference between the mean ages of genotype C and B, P = 0.136. Genotype C was found in 94.3% of females and 87.8% of males, while genotype B was detected in 8.6% of males and 3.8% of females. The distribution of HBV genotypes in four provinces of northern Thailand is shown in Fig. 1. There were no significant differences in distribution of the predominant genotype C and B among the four provinces, with P-values of 0.789 and 0.172, respectively.
4. Discussion A previous report described existence of a strong correlation between HBV genotypes and ethnicity, in that
265
HBV genotype B and C are common among indigenous populations of Asian countries [1]. We also found that genotype C was the predominant genotype, involving up to 93% of HBsAg-positive voluntary blood donors in northern Thailand. This result is higher than that from other areas in Thailand, in which genotype C was found in 50–65% of asymptomatic carriers [9–11]. Genotype B was the second most common type (7.4%) found in the north, but less frequent than in other areas that reported 20.6–45.5% of genotype B among asymptomatic carriers [9–11]. Although previous reports described a prevalence rate of 14.7% for genotype A and 5.0% for genotype D in other areas of Thailand, we found only one case of genotype A (0.5%) and no case of genotype D in northern Thailand. Moreover, we detected neither genotype E nor F in northern Thailand. In endemic areas, at medium endemic level of HBV infection (prevalence of HBsAg in the general population >2%), intrafamily transmission was the major mode by which infection spread. The pool of chronic HBV carriers is likely to have arisen from childhood infection [13]. Our study showed a unique genotypic distribution of HBV genotypes, of which genotype C is the most predominant. We found two cases (1.9%) of B and C mixed infection in our series and it has been reported that the multiple-PCR, which we employed, had superiority in detecting mixed infections [1]. The outcome of infection depends both on properties of the pathogenic organism and the host. It is suggested that HBV genotypes differ in their ability to induce chronic infection and also in their potential to induce acute hepatitis [1]. Many reports indicate that there are significant differences in the outcome of chronic hepatitis B in relation to the genotype of HBV. In particular, genotype C induces a more severe course of the disease with a high positive rate of HBeAg, greater necroinflammatory activity and fibrosis, and it shows a higher prevalence in the patients with cirrhosis and hepatocellular carcinoma [1,7,8,14]. The economic burden of HBV infection is substantial because of the high morbidity and mortality associated with end-stage liver disease such as cirrhosis, and hepatocellular carcinoma. The northern Thailand has been shown to be an endemic area of chronic HBV carriers with high predominant of genotype C. These subjects are at greater risk of more severe consequence of chronic liver disease and hepatocellular carcinoma. The long-term increase in morbidity and mortality rates is predicted in these sufferers. In conclusion, we found that HBV genotype C is a highly predominant type in northern Thailand. This finding corresponds with previous reports that HBV genotype C is the most common in Thailand and other Asian countries, and it raises concern that infected individuals will progress to severe chronic liver disease and cancer in the future. As perinatal transmission is the most likely mode of transmission in this area, this can be reduced by appropriate prophylaxis; vaccination of the infant at birth and administration of hepatitis B immune globulin. Efforts
266
P. Jutavijittum et al. / Hepatology Research 35 (2006) 263–266
should be attempted to increase compliance with hepatitis B vaccination.
Acknowledgements This study was supported by a Grant in Aid No. 13576002 (K.T.) from the Ministry of Education, Culture, Sports, Science and Technology and also supported in part by a Grant for International Health Cooperation Research No. 12C-4 (K.T.) from the Ministry of Health and Welfare, Japan. The authors gratefully acknowledge the Faculty of Medicine, Chiang Mai University, for its financial support in the presentation of these data at the International Meeting on the Molecular Biology of Hepatitis B Viruses in Heidelberg, Germany, during 18–21 September 2005.
References [1] Schaefer S. Review: hepatitis B virus: significance of genotypes. J Viral Hepat 2005;12:111–24. [2] Lavanchy D. Review: hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures. J Viral Hepat 2004;11:97–107. [3] Wang FS. Review: current status and prospects of studies on human genetic alleles associated with hepatitis B virus infection. World J Gastroenterol 2003;9:641–4.
[4] Andre F. Review: hepatitis B epidemiology in Asia, the Middle East and Africa. Vaccine 2000;18(Suppl. 1):S20–2. [5] Mohamed R, Desmond P, Suh DJ, et al. Review: practical difficulties in the management of hepatitis B in the Asia-Pacific region. J Gastroenterol Hepatol 2004;19:958–69. [6] Jutavijittum P, Jiviriyawat Y, Yousukh A, et al. A seroepidemiological study on hepatitis B virus, hepatitis C virus and human immunodeficiency virus infection in northern Thailand. Jpn J Trop Med Hyg 1999;27:13–7. [7] Kao JH, Chen DS. Review: global control of hepatitis B virus infection. Lancet Infect Dis 2002;2:395–403. [8] Tangkijvanich P, Mahachai V, Komolmit P, et al. Hepatitis B virus genotypes and hepatocellular carcinoma in Thailand. World J Gastroenterol 2005;11:2238–43. [9] Theamboonlers A, Jantaradsamee P, Kaew-In N, Tangkijvanich P, Hirsch P, Poovorawan Y. The predominant genotypes of hepatitis B virus in Thailand. Ann Trop Med Parasitol 1999;93:737–43. [10] Theamboonlers A, Tangkijvanich P, Pramoolsinsap C, Poovorawan Y. Genotypes and subtypes of hepatitis B virus in Thailand. Southeast Asian J Trop Med Public Health 1998;29:786–91. [11] Sugauchi F, Chutaputti A, Orito E, et al. Hepatitis B virus genotypes and clinical manifestation among hepatitis B carriers in Thailand. J Gastroenterol Hepatol 2002;17:671–6. [12] Naito H, Hayashi S, Abe K. Rapid and specific genotyping system for hepatitis B virus corresponding to six major genotypes by PCR using type-specific primers. J Clin Microbiol 2001;39:362–4. Erratum in: J Clin Microbiol 2001;39:1686. [13] Stroffolini T. The changing pattern of hepatitis B virus infection over the past three decades in Italy. Dig Liver Dis 2005;37:622–7. [14] Kao JH, Chen PJ, Lai MY, Chen DS. Clinical and virological aspects of blood donors infected with hepatitis B virus genotypes B and C. J Clin Microbiol 2002;40:22–5.