Hepatitis B virus genotyping: current methods and clinical implications

Open ArchivePublished:August 02, 2010DOI:https://doi.org/10.1016/j.ijid.2010.03.020

      Summary

      Hepatitis B virus (HBV) is a major cause of liver cirrhosis and hepatocellular carcinoma (HCC). Eight different HBV genotypes have been identified with distinct geographical distributions. Different genotyping methods exist including sequencing, INNO-LiPA, restriction fragment polymorphism (RFLP), multiplex PCR, serotyping, oligonucleotide microarray chips, reverse dot blot, restriction fragment mass polymorphism (RFMP), invader assay, and real-time PCR. Several investigators have studied the influence of HBV genotypes on clinical outcomes in chronic HBV patients. This review describes the current genotyping techniques, as well as their advantages and limitations. It also presents the clinical evidence that correlates HBV genotypes to clinical outcomes including disease severity, HCC development, response to therapy, disease chronicity, transplantation outcomes, and occult infection.

      Keywords

      1. Introduction

      Hepatitis B virus (HBV) infects nearly two billion people worldwide.

      World health Organization (WHO). Hepatitis B. Fact sheet N 204. Revised August 2008. http://www.who.int/mediacentre/factsheets/fs204/en/index.html [last accessed 15 July 2010].

      Approximately 600,000 deaths occur every year as a result of the acute and chronic consequences of HBV infection.

      World health Organization (WHO). Hepatitis B. Fact sheet N 204. Revised August 2008. http://www.who.int/mediacentre/factsheets/fs204/en/index.html [last accessed 15 July 2010].

      Disease chronicity rates are ≤5% in generally healthy infected adults and 80–90% in perinatally infected children.
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      Chronic HBV affects nearly 350 million patients worldwide and may further progress to cirrhosis and/or hepatocellular carcinoma (HCC) in 15–40% of cases.
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      The viral genome is a 3.2 kb circular partially duplex DNA molecule with the circularity maintained by 5′ cohesive ends. The negative DNA strand is responsible for mRNA transcription. The genome is made exclusively of condensed coding regions with four overlapping open reading frames (ORFs). The first, ORF P, codes for a terminal protein on the minus strand as well as viral polymerase (reverse transcriptase, RNase H, and DNA polymerase activity). ORF C codes for nucleocapsid structural protein as well as HBV e antigen (HBeAg), which is responsible for immunomodulation and replication inhibition functions. ORF S/pre-S codes for viral surface glycoproteins (HBsAg; hepatitis B surface antigen) that bind to cell receptors and facilitate viral entry. Finally, ORF X codes for a transcriptional transactivator that codes for involved in the development of HCC
      • Valsamakis A.
      Molecular testing in the diagnosis and management of chronic hepatitis B.

      World Health Organisation (WHO). The hepatitis B virus. WHO/CDS/CSR/LYO/2002.2: Hepatitis B. 2002. http://www.who.int/csr/disease/hepatitis/whocdscsrlyo20022/en/index2.html [last accessed 15 July 2010].

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      (Figure 1).
      Figure thumbnail gr1
      Figure 1HBV genome (modified from Feitelson and Larkin
      • Feitelson M.A.
      • Larkin J.D.
      New animal models of hepatitis B and C.
      ).
      The HBV genome (3.2 kb) is a circular partially duplex DNA molecule with the circularity maintained by 5′ cohesive ends. The genome is made exclusively of condensed coding regions with four overlapping open reading frames (ORFs), namely ORF P, C, S/pre-S, and X.
      • Valsamakis A.
      Molecular testing in the diagnosis and management of chronic hepatitis B.

      World Health Organisation (WHO). The hepatitis B virus. WHO/CDS/CSR/LYO/2002.2: Hepatitis B. 2002. http://www.who.int/csr/disease/hepatitis/whocdscsrlyo20022/en/index2.html [last accessed 15 July 2010].

      Due to the lack of proof-reading activity of DNA- and RNA-dependent DNA polymerase, mis-incorporations of nucleotides occur during viral replication. The rate of nucleotide substitution per site is estimated to be 1.4–3.2 × 10−5 per year.
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      • Naeem A.
      • et al.
      Common genotypes of hepatitis B virus prevalent in injecting drug abusers (addicts) of North West Frontier Province of Pakistan.
      • Orito E.
      • Mizokami M.
      • Ina Y.
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      • Yamamoto M.
      • et al.
      Host-independent evolution and a genetic classification of the hepadnavirus family based on nucleotide sequences.
      This has led to the emergence of HBV genotypes and subgenotypes. At least eight different genotypes (A–H) have been identified that differ in more than 8% of the genome, while subgenotypes differ by at least 4%.
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      • Mohamed N.I.
      • Hassan Z.K.
      • El-Sayed M.H.
      • Khaled M.M.
      • et al.
      Hepatitis B virus (HBV) genotypes in Egyptian pediatric cancer patients with acute and chronic active HBV infection.
      The phylogenetic tree of complete HBV genomes showing the alignment of the different genotypes has been reviewed elsewhere.
      • Schaefer S.
      Hepatitis B virus: significance of genotypes.
      Table 1 shows the updated global distribution of HBV genotypes/subgenotypes
      • Schaefer S.
      Hepatitis B virus: significance of genotypes.
      • Locarnini S.A.
      Clinical relevance of viral dynamics and genotypes in hepatitis B virus.
      • Liu C.
      • Kao J.
      Hepatitis B virus genotypes: epidemiology and therapeutic implications.
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      • Aal A.A.
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      • et al.
      Genotypes and phylogenetic characterization of hepatitis B and delta viruses in Egypt.
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      • Banerjee A.
      • Panigrahi R.
      • Chakrabarti S.
      • et al.
      Subgenotypes of hepatitis B virus genotype D (D1, D2, D3 and D5) in India: differential pattern of mutations, liver injury and occult HBV infection.
      • Baig S.
      • Siddiqui A.
      • Chakravarty R.
      • Moatter T.
      Hepatitis B virus subgenotypes D1 and D3 are prevalent in Pakistan.
      • Mohebbi S.R.
      • Amini-Bavil-Olyaee S.
      • Zali N.
      • Noorinayer B.
      • Derakhshan F.
      • Chiani M.
      • et al.
      Molecular epidemiology of hepatitis B virus in Iran.
      • Tallo T.
      • Tefanova V.
      • Priimägi L.
      • Schmidt J.
      • Katargina O.
      • Michailov M.
      • et al.
      D2: major subgenotype of hepatitis B virus in Russia and the Baltic region.
      • Devesa M.
      • Loureiro C.L.
      • Rivas Y.
      • Monsalve F.
      • Cardona N.
      • Duarte M.C.
      • et al.
      Subgenotype diversity of hepatitis B virus American genotype F in Amerindians from Venezuela and the general population of Colombia.
      • von Meltzer M.
      • Vásquez S.
      • Sun J.
      • Wendt U.C.
      • May A.
      • Gerlich W.H.
      • et al.
      A new clade of hepatitis B virus subgenotype F1 from Peru with unusual properties.
      • Huy T.T.
      • Ushijima H.
      • Sata T.
      • Abe K.
      Genomic characterization of HBV genotype F in Bolivia: genotype F subgenotypes correlate with geographic distribution and T(1858) variant.
      • Utsumi T.
      • Yano Y.
      • Truong B.X.
      • Tanaka Y.
      • Mizokami M.
      • Seo Y.
      • et al.
      Molecular epidemiological study of hepatitis B virus infection in two different ethnic populations from the Solomon Islands.
      • Norder H.
      • Couroucé A.M.
      • Coursaget P.
      • Echevarria J.M.
      • Lee S.D.
      • Mushahwar I.K.
      • et al.
      Genetic diversity of hepatitis B virus strains derived worldwide: genotypes, subgenotypes, and HBsAg subtypes.
      .
      Table 1Global distribution of HBV genotypes and subgenotypes
      • Schaefer S.
      Hepatitis B virus: significance of genotypes.
      • Locarnini S.A.
      Clinical relevance of viral dynamics and genotypes in hepatitis B virus.
      • Liu C.
      • Kao J.
      Hepatitis B virus genotypes: epidemiology and therapeutic implications.
      • Saudy N.
      • Sugauchi F.
      • Tanaka Y.
      • Suzuki S.
      • Aal A.A.
      • Zaid M.A.
      • et al.
      Genotypes and phylogenetic characterization of hepatitis B and delta viruses in Egypt.
      • Chandra P.K.
      • Biswas A.
      • Datta S.
      • Banerjee A.
      • Panigrahi R.
      • Chakrabarti S.
      • et al.
      Subgenotypes of hepatitis B virus genotype D (D1, D2, D3 and D5) in India: differential pattern of mutations, liver injury and occult HBV infection.
      • Baig S.
      • Siddiqui A.
      • Chakravarty R.
      • Moatter T.
      Hepatitis B virus subgenotypes D1 and D3 are prevalent in Pakistan.
      • Mohebbi S.R.
      • Amini-Bavil-Olyaee S.
      • Zali N.
      • Noorinayer B.
      • Derakhshan F.
      • Chiani M.
      • et al.
      Molecular epidemiology of hepatitis B virus in Iran.
      • Tallo T.
      • Tefanova V.
      • Priimägi L.
      • Schmidt J.
      • Katargina O.
      • Michailov M.
      • et al.
      D2: major subgenotype of hepatitis B virus in Russia and the Baltic region.
      • Devesa M.
      • Loureiro C.L.
      • Rivas Y.
      • Monsalve F.
      • Cardona N.
      • Duarte M.C.
      • et al.
      Subgenotype diversity of hepatitis B virus American genotype F in Amerindians from Venezuela and the general population of Colombia.
      • von Meltzer M.
      • Vásquez S.
      • Sun J.
      • Wendt U.C.
      • May A.
      • Gerlich W.H.
      • et al.
      A new clade of hepatitis B virus subgenotype F1 from Peru with unusual properties.
      • Huy T.T.
      • Ushijima H.
      • Sata T.
      • Abe K.
      Genomic characterization of HBV genotype F in Bolivia: genotype F subgenotypes correlate with geographic distribution and T(1858) variant.
      • Utsumi T.
      • Yano Y.
      • Truong B.X.
      • Tanaka Y.
      • Mizokami M.
      • Seo Y.
      • et al.
      Molecular epidemiological study of hepatitis B virus infection in two different ethnic populations from the Solomon Islands.
      • Norder H.
      • Couroucé A.M.
      • Coursaget P.
      • Echevarria J.M.
      • Lee S.D.
      • Mushahwar I.K.
      • et al.
      Genetic diversity of hepatitis B virus strains derived worldwide: genotypes, subgenotypes, and HBsAg subtypes.
      GenotypeDistributionSubgenotypeDistribution
      APandemic, but mostly prevalent in the USA and Northwest EuropeAa/A1Asia and Africa
      Ae/A2Europe and USA
      BNorthern and Southeast AsiaBj/B1Japan
      Ba/B2China, Taiwan, and Vietnam
      B3Indonesia
      B4Vietnam
      B5Philippines
      CAsia and Pacific regionCe/C1East Asia
      Cs/C2South-east Asia
      C3Polynesia, Solomon Islands
      C4Northeast Australia
      DMediterranean, the Middle East, North America, and IndiaD1India, Pakistan, Iran
      D2India, Russia, and the Baltic region
      D3India, Pakistan
      D4Solomon Islands, Oceania
      D5India
      EAfrica and TunisiaNANA
      FCentral and South AmericaF1Central America, Peru, Venezuela
      F2Venezuela
      F3Venezuela
      F4Bolivia
      GFrance, Germany, and USANANA
      HCentral and South America and MexicoNANA
      HBV, hepatitis B virus; NA, data not available.

      2. Common HBV genotyping methods

      At least 10 different HBV genotyping methods have been developed which have variable sensitivity, specificity, turnaround time, and cost. A comparison of the 10 different genotyping techniques is presented in Table 2
      • Valsamakis A.
      Molecular testing in the diagnosis and management of chronic hepatitis B.
      • Zekri A.R.
      • Hafez M.M.
      • Mohamed N.I.
      • Hassan Z.K.
      • El-Sayed M.H.
      • Khaled M.M.
      • et al.
      Hepatitis B virus (HBV) genotypes in Egyptian pediatric cancer patients with acute and chronic active HBV infection.
      • Bartholomeusz A.
      • Schaefer S.
      Hepatitis B virus genotypes: comparison of genotyping methods.
      • Qutub M.O.
      • Germer J.J.
      • Rebers S.P.
      • Mandrekar J.N.
      • Beld M.G.
      • Yao J.D.
      Simplified PCR protocols for INNO-LiPA HBV Genotyping and INNO-LiPA HBV PreCore assays.
      • Chen J.
      • Yin J.
      • Tan X.
      • Zhang H.
      • Zhang H.
      • Chen B.
      • et al.
      Improved multiplex-PCR to identify hepatitis B virus genotypes A–F and subgenotypes B1, B2, C1 and C2.
      • Liu W.C.
      • Lindh M.
      • Buti M.
      • Phiet P.H.
      • Mizokami M.
      • Li H.H.
      • et al.
      Genotyping of hepatitis B virus genotypes A to G by multiplex polymerase chain reaction.
      • Laperche S.
      • Girault A.
      • Beaulieu M.J.
      • Bouchardeau F.
      • Couroucé A.M.
      Determination of hepatitis B virus subtypes by an enzyme immunoassay method using monoclonal antibodies to type-specific epitopes of HBsAg.
      • El Khouri M.
      • dos Santos V.A.
      Hepatitis B: epidemiological, immunological, and serological considerations emphasizing mutation.
      • Moriya T.
      • Kuramoto I.K.
      • Yoshizawa H.
      • Holland P.V.
      Distribution of hepatitis B virus genotypes among American blood donors determined with a PreS2 epitope enzyme-linked immunosorbent assay kit.
      • Wang Y.Z.
      • Wu G.X.
      • Luo L.B.
      • Chen M.
      • Ruan L.H.
      Oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus.
      • Pas S.D.
      • Tran N.
      • de Man R.A.
      • Burghoorn-Maas C.
      • Vernet G.
      • et al.
      Comparison of reverse hybridization, microarray, and sequence analysis for genotyping hepatitis B virus.
      • Zhang R.
      • Deng Y.
      • Muller C.P.
      • Ou Z.Y.
      • Ma L.
      • Wang M.
      • et al.
      Determination of hepatitis B virus genotype by flow-through reverse dot blot.
      • Hong S.P.
      • Kim N.K.
      • Hwang S.G.
      • Chung H.J.
      • Kim S.
      • Han J.H.
      • et al.
      Detection of hepatitis B virus YMDD variants using mass spectrometric analysis of oligonucleotide fragments.
      • Woo H.Y.
      • Park H.
      • Kim B.I.
      • Jeon W.K.
      • Cho Y.K.
      • Kim Y.J.
      Comparison of mass spectrometric analysis and TRUGENE HBV genotyping for monitoring lamivudine resistance in chronic hepatitis B patients.
      • Kim K.M.
      • Choi W.B.
      • Lim Y.S.
      • Lee H.C.
      • Chung Y.H.
      • Lee Y.S.
      • et al.
      Adefovir dipivoxil alone or in combination with ongoing lamivudine in patients with decompensated liver disease and lamivudine-resistant hepatitis B virus.
      • Tadokoro K.
      • Kobayashi M.
      • Yamaguchi T.
      • Suzuki F.
      • Miyauchi S.
      • Egashira T.
      • et al.
      Classification of hepatitis B virus genotypes by the PCR-Invader method with genotype-specific probes.
      • Payungporn S.
      • Tangkijvanich P.
      • Jantaradsamee P.
      • Theamboonlers A.
      • Poovorawan Y.
      Simultaneous quantitation and genotyping of hepatitis B virus by real-time PCR and melting curve analysis.
      • Liu W.C.
      • Mizokami M.
      • Buti M.
      • Lindh M.
      • Young K.C.
      • Sun K.T.
      • et al.
      Simultaneous quantification and genotyping of hepatitis B virus for genotypes A to G by real-time PCR and two-step melting curve analysis.
      • Orito E.
      • Ichida T.
      • Sakugawa H.
      • Sata M.
      • Horiike N.
      • Hino K.
      • et al.
      Geographic distribution of hepatitis B virus (HBV) genotype in patients with chronic HBV infection in Japan.
      • Yuan J.
      • Zhou B.
      • Tanaka Y.
      • Kurbanov F.
      • Orito E.
      • Gong Z.
      • et al.
      Hepatitis B virus (HBV) genotypes/subgenotypes in China: mutations in core promoter and precore/core and their clinical implications.
      • Sánchez-Tapias J.M.
      • Costa J.
      • Mas A.
      • Bruguera M.
      • Rodés J.
      Influence of hepatitis B virus genotype on the long-term outcome of chronic hepatitis B in Western patients.
      • Tanaka Y.
      • Orito E.
      • Yuen M.F.
      • Mukaide M.
      • Sugauchi F.
      • Ito K.
      • et al.
      Two subtypes (subgenotypes) of hepatitis B virus genotype C: a novel subtyping assay based on restriction fragment length polymorphism.
      • Kao J.H.
      • Chen P.J.
      • Lai M.Y.
      • Chen D.S.
      Hepatitis B genotypes correlate with clinical outcomes in patients with chronic hepatitis B.
      • Li Y.J.
      • Zhuang H.
      • Li J.
      • Dong Q.M.
      • Chen Y.J.
      • Niu J.Q.
      • et al.
      Distribution and clinical significance of hepatitis B virus (HBV) genotypes and subtypes in HBV-infected patients.
      • Wang J.
      • Gao J.W.
      • Li J.
      • Zhuang H.
      • Wang J.
      • Li Y.J.
      • et al.
      Establishment of a semi-nested PCR for identifying the sub-genotypes (Ba and Bj) of hepatitis B virus of genotype B.
      • Idrees M.
      • Khan S.
      • Riazuddin S.
      Common genotypes of hepatitis B virus.
      • Usuda S.
      • Okamoto H.
      • Iwanari H.
      • Baba K.
      • Tsuda F.
      • Miyakawa Y.
      • et al.
      Serological detection of hepatitis B virus genotypes by ELISA with monoclonal antibodies to type-specific epitopes in the preS2-region product.
      .
      Table 2Comparison of common HBV genotyping methods
      Genotyping techniqueAdvantagesDisadvantages
      Sequencing• Gold standard, most reliable as it does not depend on a small nucleotide or amino acid sequence as other methods do23• Less efficient in detecting mixed genotypes
      • Bartholomeusz A.
      • Schaefer S.
      Hepatitis B virus genotypes: comparison of genotyping methods.
      • Pas S.D.
      • Tran N.
      • de Man R.A.
      • Burghoorn-Maas C.
      • Vernet G.
      • et al.
      Comparison of reverse hybridization, microarray, and sequence analysis for genotyping hepatitis B virus.
      • Can detect new genotypes and recombination between genotypes
      • Bartholomeusz A.
      • Schaefer S.
      Hepatitis B virus genotypes: comparison of genotyping methods.
      • Cumbersome (cannot be used in large-scale studies), expensive, and requires expertise
      • Bartholomeusz A.
      • Schaefer S.
      Hepatitis B virus genotypes: comparison of genotyping methods.
      • Pas S.D.
      • Tran N.
      • de Man R.A.
      • Burghoorn-Maas C.
      • Vernet G.
      • et al.
      Comparison of reverse hybridization, microarray, and sequence analysis for genotyping hepatitis B virus.
      INNO-LiPA• Easier and cheaper than sequencing
      • Bartholomeusz A.
      • Schaefer S.
      Hepatitis B virus genotypes: comparison of genotyping methods.
      • Insensitive for HBV genomes with SNP or deletions in the sequences to which probes bind
      • Valsamakis A.
      Molecular testing in the diagnosis and management of chronic hepatitis B.
      • Overall success rate for the detection of all eight HBV genotypes was recently found to be 98%
      • Qutub M.O.
      • Germer J.J.
      • Rebers S.P.
      • Mandrekar J.N.
      • Beld M.G.
      • Yao J.D.
      Simplified PCR protocols for INNO-LiPA HBV Genotyping and INNO-LiPA HBV PreCore assays.
      • Relatively expensive compared to PCR-based and serological techniques
      • Bartholomeusz A.
      • Schaefer S.
      Hepatitis B virus genotypes: comparison of genotyping methods.
      • Highly specific
      • Pas S.D.
      • Tran N.
      • de Man R.A.
      • Burghoorn-Maas C.
      • Vernet G.
      • et al.
      Comparison of reverse hybridization, microarray, and sequence analysis for genotyping hepatitis B virus.
      • Can detect mixed genotypes
      • Valsamakis A.
      Molecular testing in the diagnosis and management of chronic hepatitis B.
      • Pas S.D.
      • Tran N.
      • de Man R.A.
      • Burghoorn-Maas C.
      • Vernet G.
      • et al.
      Comparison of reverse hybridization, microarray, and sequence analysis for genotyping hepatitis B virus.
      RFLP• Simple and cost-effective method• May give indeterminate results (<6%)
      • Orito E.
      • Ichida T.
      • Sakugawa H.
      • Sata M.
      • Horiike N.
      • Hino K.
      • et al.
      Geographic distribution of hepatitis B virus (HBV) genotype in patients with chronic HBV infection in Japan.
      • Sánchez-Tapias J.M.
      • Costa J.
      • Mas A.
      • Bruguera M.
      • Rodés J.
      Influence of hepatitis B virus genotype on the long-term outcome of chronic hepatitis B in Western patients.
      • Kao J.H.
      • Chen P.J.
      • Lai M.Y.
      • Chen D.S.
      Hepatitis B genotypes correlate with clinical outcomes in patients with chronic hepatitis B.
      • Can be used in large population studies• SNP at enzyme restriction sites may affect method sensitivity
      • Bartholomeusz A.
      • Schaefer S.
      Hepatitis B virus genotypes: comparison of genotyping methods.
      • Can determine subgenotypes
      • Tanaka Y.
      • Orito E.
      • Yuen M.F.
      • Mukaide M.
      • Sugauchi F.
      • Ito K.
      • et al.
      Two subtypes (subgenotypes) of hepatitis B virus genotype C: a novel subtyping assay based on restriction fragment length polymorphism.
      Multiplex PCR• Rapid, simple, and cost-effective method• Indeterminate samples range from 2.9% to 4.5%
      • Zekri A.R.
      • Hafez M.M.
      • Mohamed N.I.
      • Hassan Z.K.
      • El-Sayed M.H.
      • Khaled M.M.
      • et al.
      Hepatitis B virus (HBV) genotypes in Egyptian pediatric cancer patients with acute and chronic active HBV infection.
      • Idrees M.
      • Khan S.
      • Riazuddin S.
      Common genotypes of hepatitis B virus.
      • Can be used in large population studies• SNP at primer site may affect method sensitivity
      • Bartholomeusz A.
      • Schaefer S.
      Hepatitis B virus genotypes: comparison of genotyping methods.
      • Can determine subgenotypes
      • Chen J.
      • Yin J.
      • Tan X.
      • Zhang H.
      • Zhang H.
      • Chen B.
      • et al.
      Improved multiplex-PCR to identify hepatitis B virus genotypes A–F and subgenotypes B1, B2, C1 and C2.
      • Li Y.J.
      • Zhuang H.
      • Li J.
      • Dong Q.M.
      • Chen Y.J.
      • Niu J.Q.
      • et al.
      Distribution and clinical significance of hepatitis B virus (HBV) genotypes and subtypes in HBV-infected patients.
      • Wang J.
      • Gao J.W.
      • Li J.
      • Zhuang H.
      • Wang J.
      • Li Y.J.
      • et al.
      Establishment of a semi-nested PCR for identifying the sub-genotypes (Ba and Bj) of hepatitis B virus of genotype B.
      • Accuracy reported to be 93%
      • Liu W.C.
      • Lindh M.
      • Buti M.
      • Phiet P.H.
      • Mizokami M.
      • Li H.H.
      • et al.
      Genotyping of hepatitis B virus genotypes A to G by multiplex polymerase chain reaction.
      • Sensitive in detecting mixed genotypes (even if minor genotype is as low as 10%)
      • Liu W.C.
      • Lindh M.
      • Buti M.
      • Phiet P.H.
      • Mizokami M.
      • Li H.H.
      • et al.
      Genotyping of hepatitis B virus genotypes A to G by multiplex polymerase chain reaction.
      Serotyping• No PCR required• Indeterminate samples range from 1.4% to 23.4%
      • Moriya T.
      • Kuramoto I.K.
      • Yoshizawa H.
      • Holland P.V.
      Distribution of hepatitis B virus genotypes among American blood donors determined with a PreS2 epitope enzyme-linked immunosorbent assay kit.
      • Yuan J.
      • Zhou B.
      • Tanaka Y.
      • Kurbanov F.
      • Orito E.
      • Gong Z.
      • et al.
      Hepatitis B virus (HBV) genotypes/subgenotypes in China: mutations in core promoter and precore/core and their clinical implications.
      • Usuda S.
      • Okamoto H.
      • Iwanari H.
      • Baba K.
      • Tsuda F.
      • Miyakawa Y.
      • et al.
      Serological detection of hepatitis B virus genotypes by ELISA with monoclonal antibodies to type-specific epitopes in the preS2-region product.
      • Simple and cost-effective• SNP may affect process sensitivity
      • Bartholomeusz A.
      • Schaefer S.
      Hepatitis B virus genotypes: comparison of genotyping methods.
      • Can be used in large population studies
      • Bartholomeusz A.
      • Schaefer S.
      Hepatitis B virus genotypes: comparison of genotyping methods.
      • Laperche S.
      • Girault A.
      • Beaulieu M.J.
      • Bouchardeau F.
      • Couroucé A.M.
      Determination of hepatitis B virus subtypes by an enzyme immunoassay method using monoclonal antibodies to type-specific epitopes of HBsAg.
      • El Khouri M.
      • dos Santos V.A.
      Hepatitis B: epidemiological, immunological, and serological considerations emphasizing mutation.
      Oligonucleotide microarray chips• Sensitive• More expensive than real-time PCR and sequencing
      • Wang Y.Z.
      • Wu G.X.
      • Luo L.B.
      • Chen M.
      • Ruan L.H.
      Oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus.
      • Determines genotype even at titers as low as 2000–5000 copies/ml
      • Wang Y.Z.
      • Wu G.X.
      • Luo L.B.
      • Chen M.
      • Ruan L.H.
      Oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus.
      • Pas S.D.
      • Tran N.
      • de Man R.A.
      • Burghoorn-Maas C.
      • Vernet G.
      • et al.
      Comparison of reverse hybridization, microarray, and sequence analysis for genotyping hepatitis B virus.
      • Sensitivity may be affected by SNPs or deletions in the sequences to which the probes bind
      • Superior to sequencing in determining mixed genotypes
      • Wang Y.Z.
      • Wu G.X.
      • Luo L.B.
      • Chen M.
      • Ruan L.H.
      Oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus.
      Reverse dot blot• Can detect mixed genotypes
      • Zhang R.
      • Deng Y.
      • Muller C.P.
      • Ou Z.Y.
      • Ma L.
      • Wang M.
      • et al.
      Determination of hepatitis B virus genotype by flow-through reverse dot blot.
      • Sensitivity may be affected by SNPs or deletions in the sequences to which the probes bind
      • High sensitivity
      • Inexpensive, accurate, and rapid
      • Detection limit for genotypes B and D were found to be 102–103 and for A, C, and E were 103–104 copies/ml
      • Zhang R.
      • Deng Y.
      • Muller C.P.
      • Ou Z.Y.
      • Ma L.
      • Wang M.
      • et al.
      Determination of hepatitis B virus genotype by flow-through reverse dot blot.
      RFMP• High sensitivity• SNP at enzyme restriction sites may affect method sensitivity
      • Can also be used to detect YMDD mutations with higher sensitivity and lower detection limit (100 copies/ml
      • Hong S.P.
      • Kim N.K.
      • Hwang S.G.
      • Chung H.J.
      • Kim S.
      • Han J.H.
      • et al.
      Detection of hepatitis B virus YMDD variants using mass spectrometric analysis of oligonucleotide fragments.
      ) compared to sequencing and can also detect mutations even in samples containing wild-type
      • Hong S.P.
      • Kim N.K.
      • Hwang S.G.
      • Chung H.J.
      • Kim S.
      • Han J.H.
      • et al.
      Detection of hepatitis B virus YMDD variants using mass spectrometric analysis of oligonucleotide fragments.
      • Woo H.Y.
      • Park H.
      • Kim B.I.
      • Jeon W.K.
      • Cho Y.K.
      • Kim Y.J.
      Comparison of mass spectrometric analysis and TRUGENE HBV genotyping for monitoring lamivudine resistance in chronic hepatitis B patients.
      • Kim K.M.
      • Choi W.B.
      • Lim Y.S.
      • Lee H.C.
      • Chung Y.H.
      • Lee Y.S.
      • et al.
      Adefovir dipivoxil alone or in combination with ongoing lamivudine in patients with decompensated liver disease and lamivudine-resistant hepatitis B virus.
      • Requires the availability of MALDI-TOF mass spectrometry (bulky, expensive, and requires trained operators)
      Invader assay• Extremely high sensitivity (detection threshold is 10 copies of HBV DNA/reaction
      • Tadokoro K.
      • Kobayashi M.
      • Yamaguchi T.
      • Suzuki F.
      • Miyauchi S.
      • Egashira T.
      • et al.
      Classification of hepatitis B virus genotypes by the PCR-Invader method with genotype-specific probes.
      )
      • Sensitivity may be affected by SNPs or deletions in the sequences to which the probes bind
      • Can detect mixed genotypes even if the ratio between the two is 1000:10
      • Tadokoro K.
      • Kobayashi M.
      • Yamaguchi T.
      • Suzuki F.
      • Miyauchi S.
      • Egashira T.
      • et al.
      Classification of hepatitis B virus genotypes by the PCR-Invader method with genotype-specific probes.
      Real-time PCR• Low cross-contamination• SNP at primer site may affect method sensitivity
      • Time saving• Lower ability to distinguish between genotypes with close proximity between Tm
      • Payungporn S.
      • Tangkijvanich P.
      • Jantaradsamee P.
      • Theamboonlers A.
      • Poovorawan Y.
      Simultaneous quantitation and genotyping of hepatitis B virus by real-time PCR and melting curve analysis.
      • Liu W.C.
      • Mizokami M.
      • Buti M.
      • Lindh M.
      • Young K.C.
      • Sun K.T.
      • et al.
      Simultaneous quantification and genotyping of hepatitis B virus for genotypes A to G by real-time PCR and two-step melting curve analysis.
      • High throughput
      • High sensitivity
      • Payungporn S.
      • Tangkijvanich P.
      • Jantaradsamee P.
      • Theamboonlers A.
      • Poovorawan Y.
      Simultaneous quantitation and genotyping of hepatitis B virus by real-time PCR and melting curve analysis.
      • Liu W.C.
      • Mizokami M.
      • Buti M.
      • Lindh M.
      • Young K.C.
      • Sun K.T.
      • et al.
      Simultaneous quantification and genotyping of hepatitis B virus for genotypes A to G by real-time PCR and two-step melting curve analysis.
      • Superior than sequencing and oligonucleotide microarray chips in detecting mixed populations
      • Wang Y.Z.
      • Wu G.X.
      • Luo L.B.
      • Chen M.
      • Ruan L.H.
      Oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus.
      HBV, hepatitis B virus; MALDI-TOF, matrix-assisted laser desorption/ionization- time-of-flight; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; RFMP, restriction fragment mass polymorphism; SNP, single nucleotide polymorphisms; Tm, melting temperature; YMDD, tyrosine–methionine–aspartate–aspartate motif.

      2.1 Sequencing and phylogenetic analysis

      The gold standard method for HBV genotyping is whole genome sequencing followed by phylogenetic analysis.
      • Valsamakis A.
      Molecular testing in the diagnosis and management of chronic hepatitis B.
      • Bartholomeusz A.
      • Schaefer S.
      Hepatitis B virus genotypes: comparison of genotyping methods.
      Although this technique is highly sensitive and allows the detection of new and recombinant genotypes, it is expensive, time-consuming, and detects mainly the predominant genotype in genotype mixtures.
      • Valsamakis A.
      Molecular testing in the diagnosis and management of chronic hepatitis B.
      • Bartholomeusz A.
      • Schaefer S.
      Hepatitis B virus genotypes: comparison of genotyping methods.
      In order to sensitively detect the mixed genotypes, a greater clone number (more than 100) needs to be screened, further affecting cost and time requirements. An alternative to whole genome sequencing is single gene sequencing.
      • Bartholomeusz A.
      • Schaefer S.
      Hepatitis B virus genotypes: comparison of genotyping methods.
      The sensitivity of single gene sequencing depends on the degree of sequence homology as well as the sequence size.
      • Valsamakis A.
      Molecular testing in the diagnosis and management of chronic hepatitis B.
      • Bartholomeusz A.
      • Schaefer S.
      Hepatitis B virus genotypes: comparison of genotyping methods.
      It is important to note that a commercial direct sequencing assay kit is available (TRUGENE HBV Genotyping Kit; Siemens Medical Solutions Diagnostics, NY, USA).

      TRUGENE® HBV Genotyping Assay (RUO). Siemens Medical Solutions Diagnostics, Tarrytown, NY, USA. Information available at: http://www.medical.siemens.com (accessed June 2010).

      This kit is a two-in-one kit where, in about 8 h, both genotypes and HBV sequence mutations can be detected in plasma or serum specimens simultaneously. First, DNA extraction from plasma or serum is performed, followed by amplification of the hepatitis B surface antigen (HBsAg) gene (s101–s237) and overlapping polymerase gene (rt99–rt280) using PCR.

      TRUGENE® HBV Genotyping Assay (RUO). Siemens Medical Solutions Diagnostics, Tarrytown, NY, USA. Information available at: http://www.medical.siemens.com (accessed June 2010).

      PCR amplicons are then added into four CLIP™ sequencing reaction tubes. Bi-directional sequencing follows using two fluorescently-labeled DNA primers, thus enhancing process accuracy. The sequence obtained is compared to reference sequences for genotypes A–H to determine HBV genotype and to a universal mutation reporting reference sequence for mutational analysis.

      TRUGENE® HBV Genotyping Assay (RUO). Siemens Medical Solutions Diagnostics, Tarrytown, NY, USA. Information available at: http://www.medical.siemens.com (accessed June 2010).

      The detection limit reported by the manufacturer is about 2.0 × 103 HBV DNA copies/ml, which can be improved if the DNA extraction method is optimized.
      • Kessler H.H.
      • Stelzl E.
      • Marth E.
      • Stauber R.E.
      Detection of mutations in the hepatitis B virus polymerase gene.
      • Gintowt A.A.
      • Germer J.J.
      • Mitchell P.S.
      • Yao J.D.
      Evaluation of the MagNA Pure LC used with the TRUGENE HBV Genotyping Kit.
      When MagNA Pure LC rather than manual DNA extraction was used together with the TRUGENE Genotyping Kit, an analytical sensitivity of ≥200 IU/ml was achieved (1 IU/ml is about 5 copies/ml).
      • Gintowt A.A.
      • Germer J.J.
      • Mitchell P.S.
      • Yao J.D.
      Evaluation of the MagNA Pure LC used with the TRUGENE HBV Genotyping Kit.

      2.2 INNO-LiPA

      This is a reverse hybridization method that has been developed by Innogenetics and is commercially available as INNO-LiPA. First, HBV DNA is amplified by PCR using biotinylated primers complementary to a conserved sequence in the S/pre-S ORF.
      • Alavian S.M.
      • Keyvani H.
      • Rezai M.
      • Ashayeri N.
      • Sadeghi H.M.
      Preliminary report of hepatitis B virus genotype prevalence in Iran.
      The amplified biotinylated PCR products are then hybridized to probes immobilized onto membrane strips that detect genotype-specific differences in the HBV polymerase gene domains B to C. After washing, alkaline phosphatase (ALP)-labeled streptavidin is added, followed by substrate (BCIP/NBT chromogen) that gives a purple/brown precipitate in the presence of ALP.
      • Alavian S.M.
      • Keyvani H.
      • Rezai M.
      • Ashayeri N.
      • Sadeghi H.M.
      Preliminary report of hepatitis B virus genotype prevalence in Iran.
      This method can detect both single and mixed genotypes.
      • Valsamakis A.
      Molecular testing in the diagnosis and management of chronic hepatitis B.
      In 2006, Qutub et al. reported that the analytical sensitivity of a modified INNO-LiPA (using a standardized single-round INNO-LiPA and automated sample processing, and adding uracil N-glycosylase) was 100%, 90%, and only 10% at viral loads of 1000, 100, and 10 IU/ml, respectively.
      • Qutub M.O.
      • Germer J.J.
      • Rebers S.P.
      • Mandrekar J.N.
      • Beld M.G.
      • Yao J.D.
      Simplified PCR protocols for INNO-LiPA HBV Genotyping and INNO-LiPA HBV PreCore assays.
      The overall success rate for the detection of all eight HBV genotypes by this method was 98% using 100 clinical specimens with the majority having viral titers ranging from 105 to 107 IU/ml . However, it is important to note that genotypes A–C were the most commonly analyzed samples.
      • Qutub M.O.
      • Germer J.J.
      • Rebers S.P.
      • Mandrekar J.N.
      • Beld M.G.
      • Yao J.D.
      Simplified PCR protocols for INNO-LiPA HBV Genotyping and INNO-LiPA HBV PreCore assays.

      2.3 Restriction fragment polymorphism (RFLP)

      RFLP depends on PCR amplification of the S gene (usually), restriction enzyme digestion, and separation of digested fragments by electrophoresis.
      • Bartholomeusz A.
      • Schaefer S.
      Hepatitis B virus genotypes: comparison of genotyping methods.
      • Mizokami M.
      • Nakano T.
      • Orito E.
      • Tanaka Y.
      • Sakugawa H.
      • Mukaide M.
      • et al.
      Hepatitis B virus genotype assignment using restriction fragment length polymorphism patterns.
      A combination of different restriction enzymes has been used for RFLP, the choice of which has been determined according to the different HBV genotype sequences in GenBank.
      • Locarnini S.A.
      Clinical relevance of viral dynamics and genotypes in hepatitis B virus.
      This method has been used to determine genotypes A–F.
      • Mizokami M.
      • Nakano T.
      • Orito E.
      • Tanaka Y.
      • Sakugawa H.
      • Mukaide M.
      • et al.
      Hepatitis B virus genotype assignment using restriction fragment length polymorphism patterns.
      • Lindh M.
      • Andersson A.S.
      • Gusdal A.
      Genotypes, nt 1858 variants, and geographic origin of hepatitis B virus—large-scale analysis using a new genotyping method.
      In 2004, Zeng et al. developed a modified RFLP technique based on the S gene allowing the detection of HBV genotypes A–H.
      • Zeng G.B.
      • Wen S.J.
      • Wang Z.H.
      • Yan L.
      • Sun J.
      • Hou J.L.
      A novel hepatitis B virus genotyping system by using restriction fragment length polymorphism patterns of S gene amplicons.
      In this method, two PCR rounds were undertaken prior to restriction enzyme digestion by five enzymes, namely StyI, BsrI, DpnI, HpaII, and EaeI.
      • Zeng G.B.
      • Wen S.J.
      • Wang Z.H.
      • Yan L.
      • Sun J.
      • Hou J.L.
      A novel hepatitis B virus genotyping system by using restriction fragment length polymorphism patterns of S gene amplicons.
      The method was compared with another RFLP method targeting the pre-S1 region and the results were concordant in 96.8% (181/187).
      • Zeng G.B.
      • Wen S.J.
      • Wang Z.H.
      • Yan L.
      • Sun J.
      • Hou J.L.
      A novel hepatitis B virus genotyping system by using restriction fragment length polymorphism patterns of S gene amplicons.
      The remaining six samples were not typeable by the RFLP method based on pre-S1, but were typeable by the modified method after cloning and repeated RFLP analysis as verified by sequencing.
      • Zeng G.B.
      • Wen S.J.
      • Wang Z.H.
      • Yan L.
      • Sun J.
      • Hou J.L.
      A novel hepatitis B virus genotyping system by using restriction fragment length polymorphism patterns of S gene amplicons.
      Venegas et al. further modified this method by performing a single PCR round, followed by restriction enzyme digestion using Sau3A I, Bsr I, and Hpa II based on the S gene.
      • Venegas M.
      • Muñoz G.
      • Hurtado C.
      • Alvarez L.
      • Velasco M.
      • Villanueva R.A.
      • et al.
      Prevalence of hepatitis B virus genotypes in chronic carriers in Santiago, Chile.
      When the amplicons corresponding to genotypes A, B, C, D, and F were sequenced and compared with reference sequences, 99%, 97%, 98%, 97%, and 97% identity was reported. However the identity for genotypes E, G, and H was less than 95%.
      • Venegas M.
      • Muñoz G.
      • Hurtado C.
      • Alvarez L.
      • Velasco M.
      • Villanueva R.A.
      • et al.
      Prevalence of hepatitis B virus genotypes in chronic carriers in Santiago, Chile.

      2.4 Multiplex PCR

      Naito et al. developed a multiplex nested PCR method for the detection of genotypes A–F.
      • 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.
      In the first round, universal outer primers were used. For each sample, two second round PCR reactions were performed using universal inner primers plus genotype-specific primers – the first reaction to determine genotypes A–C and the second for genotypes D–F. The products of the second round were then run on an agarose gel giving genotype-specific band sizes. Universal outer primers (used in the first round) and inner primers (used in the second round) were designed according to the conserved nucleotide sequences between the pre-S1 and S gene.
      • 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.
      In contrast, genotype-specific primers were designed according to the nucleotide sequences in the region between the pre-S1 and S gene that were conserved within an HBV genotype but had poor homology with other genotypes.
      • 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.
      This method was modified by Kirschberg et al., where genotypes A–F were successfully detected using genotype-specific primer pairs that bind to different regions in the HBV genome in a single PCR round.
      • Kirschberg O.
      • Schüttler C.
      • Repp R.
      • Schaefer S.
      A multiplex-PCR to identify hepatitis B virus genotypes A–F.
      In 2007, Chen et al. modified the single round PCR to determine genotypes A–F in one reaction, as well as to subtype genotypes B and C into B1/B2 and C1/C2, respectively, in another reaction.
      • Chen J.
      • Yin J.
      • Tan X.
      • Zhang H.
      • Zhang H.
      • Chen B.
      • et al.
      Improved multiplex-PCR to identify hepatitis B virus genotypes A–F and subgenotypes B1, B2, C1 and C2.
      Recently, in 2008, Liu et al. developed a multiplex PCR method to identify genotypes A–G (as determined by phylogenetic analysis) using genotype-specific primers in two reactions based on core/surface/polymerase region.
      • Liu W.C.
      • Lindh M.
      • Buti M.
      • Phiet P.H.
      • Mizokami M.
      • Li H.H.
      • et al.
      Genotyping of hepatitis B virus genotypes A to G by multiplex polymerase chain reaction.
      Multiplex PCR had a higher accuracy (93.2%) compared to the RFLP method (87%) developed by Mizokami et al.
      • Mizokami M.
      • Nakano T.
      • Orito E.
      • Tanaka Y.
      • Sakugawa H.
      • Mukaide M.
      • et al.
      Hepatitis B virus genotype assignment using restriction fragment length polymorphism patterns.
      For genotypes A–G, the reported sensitivity of this method was 94.1%, 87.3%, 96.8%, 100%, 100%, 100%, and 100%, the specificity 92%, 97.5%, 88.9%, 91.7%, 92%, 92%, and 92% and the lowest copy number was 102, 103, 102, 103, 105, 104, and 103 copies/ml, respectively.
      • Liu W.C.
      • Lindh M.
      • Buti M.
      • Phiet P.H.
      • Mizokami M.
      • Li H.H.
      • et al.
      Genotyping of hepatitis B virus genotypes A to G by multiplex polymerase chain reaction.
      This method can detect mixed genotypes with sensitivity for detecting minor species as low as 10%.
      • Liu W.C.
      • Lindh M.
      • Buti M.
      • Phiet P.H.
      • Mizokami M.
      • Li H.H.
      • et al.
      Genotyping of hepatitis B virus genotypes A to G by multiplex polymerase chain reaction.

      2.5 Serological subtyping

      In this method, HBV serotypes or subtypes are recognized using antibodies against HBsAg. Serotypes are made up of a constant “a” determinant, plus two variable, mutually exclusive determinants d/y and w/r as well as other determinants. This results in nine serological subtypes, ayw1, ayw2, ayw3, ayw4, ayr, adw2, adw4, adrq+ and adrq. When molecular method results were matched to those obtained with serological assays, it was found that genotype A corresponded to adw2 (Europe, US, East Africa) and ayw1 (Africa), while B corresponded to adw2 (Asia) and ayw1 (Vietnam). Genotype C was matched with adr and ayr, D with ayw2 and ayw3, E with ayw4, F with adw4, G with adw2, and H with adw4.
      • Schaefer S.
      Hepatitis B virus: significance of genotypes.
      • Laperche S.
      • Girault A.
      • Beaulieu M.J.
      • Bouchardeau F.
      • Couroucé A.M.
      Determination of hepatitis B virus subtypes by an enzyme immunoassay method using monoclonal antibodies to type-specific epitopes of HBsAg.
      • El Khouri M.
      • dos Santos V.A.
      Hepatitis B: epidemiological, immunological, and serological considerations emphasizing mutation.
      Although PCR and sequencing have been used as the standard methods for genotyping, sequencing is not suitable for large epidemiological studies and population screening.
      • Laperche S.
      • Girault A.
      • Beaulieu M.J.
      • Bouchardeau F.
      • Couroucé A.M.
      Determination of hepatitis B virus subtypes by an enzyme immunoassay method using monoclonal antibodies to type-specific epitopes of HBsAg.
      • El Khouri M.
      • dos Santos V.A.
      Hepatitis B: epidemiological, immunological, and serological considerations emphasizing mutation.
      In contrast, serological assays using monoclonal anti-HBs antibodies with restricted subtype specificities in an enzyme immunoassay format are more cost-effective, easy to perform, and have higher throughput.
      • Bartholomeusz A.
      • Schaefer S.
      Hepatitis B virus genotypes: comparison of genotyping methods.
      • Laperche S.
      • Girault A.
      • Beaulieu M.J.
      • Bouchardeau F.
      • Couroucé A.M.
      Determination of hepatitis B virus subtypes by an enzyme immunoassay method using monoclonal antibodies to type-specific epitopes of HBsAg.
      Serological assays have also been found to be superior to molecular methods in typing HBV-PCR-negative carriers, who constituted 35% of the study population in the study of Laperche et al.
      • Laperche S.
      • Girault A.
      • Beaulieu M.J.
      • Bouchardeau F.
      • Couroucé A.M.
      Determination of hepatitis B virus subtypes by an enzyme immunoassay method using monoclonal antibodies to type-specific epitopes of HBsAg.
      However, it has been reported that the rate of indeterminate samples may reach up to 23.4%.
      • Moriya T.
      • Kuramoto I.K.
      • Yoshizawa H.
      • Holland P.V.
      Distribution of hepatitis B virus genotypes among American blood donors determined with a PreS2 epitope enzyme-linked immunosorbent assay kit.

      2.6 Oligonucleotide microarray chips

      Song et al. developed a low density oligonucleotide microarray that can determine genotypes A–G.
      • Song Y.
      • Dai E.
      • Wang J.
      • Liu H.
      • Zhai J.
      • Chen C.
      • et al.
      Genotyping of hepatitis B virus (HBV) by oligonucleotides microarray.
      First, PCR amplification of the pre-S region was performed with subsequent Cy5-dCTP labeling. The amplified products were heat denatured and added to silylated slides to which genotype-specific probes are immobilized. Fifteen probes were designed based on phylogenetic tree analysis and alignment of 228 pre-S region sequences. Following washing and drying, fluorescence signals were captured using a scanner. This method was found to be specific and was found to be sensitive to detect both single and mixed genotypes. Total concordance was achieved with RFLP (pre-S1; 37/37) and sequencing (24/24).
      • Song Y.
      • Dai E.
      • Wang J.
      • Liu H.
      • Zhai J.
      • Chen C.
      • et al.
      Genotyping of hepatitis B virus (HBV) by oligonucleotides microarray.
      In 2007, Wang et al. compared oligonucleotide microarray chips to real-time PCR and sequencing.
      • Wang Y.Z.
      • Wu G.X.
      • Luo L.B.
      • Chen M.
      • Ruan L.H.
      Oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus.
      They reported that the oligonucleotide chip detected both genotypes B and C if the mixture contained 50%, 10%, or even 1% of genotype B. However, when genotype B was only 0.1%, the method only detected genotype C. This was superior to sequencing, which detected both genotypes when the concentration of genotype B was 50%, but only genotype C when the concentration was 10%, 1%, or 0.1%.
      • Wang Y.Z.
      • Wu G.X.
      • Luo L.B.
      • Chen M.
      • Ruan L.H.
      Oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus.
      Real-time PCR was, however, more sensitive than both oligonucleotide chip and sequencing in detecting genotype mixtures. It managed to detect both genotypes even when genotype B was as low as 0.1%.
      • Wang Y.Z.
      • Wu G.X.
      • Luo L.B.
      • Chen M.
      • Ruan L.H.
      Oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus.
      The oligonucleotide chip method was found to be superior to both sequencing and real-time PCR with respect to sensitivity.
      • Wang Y.Z.
      • Wu G.X.
      • Luo L.B.
      • Chen M.
      • Ruan L.H.
      Oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus.
      Real-time PCR and sequencing were only able to determine the genotype when the titer was higher than 5.20 × 103 but not when it was 20 × 102 copies/ml. In contrast, oligonucleotide chip was able to determine genotype at all tested dilutions.
      • Wang Y.Z.
      • Wu G.X.
      • Luo L.B.
      • Chen M.
      • Ruan L.H.
      Oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus.
      When clinical samples were tested, 93% (117/126) of the results obtained by the three methods were concordant.
      • Wang Y.Z.
      • Wu G.X.
      • Luo L.B.
      • Chen M.
      • Ruan L.H.
      Oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus.
      In the nine discordant samples, sequencing only determined the predominant genotype, however both real-time PCR and oligonucleotide chip determined both genotypes.
      • Wang Y.Z.
      • Wu G.X.
      • Luo L.B.
      • Chen M.
      • Ruan L.H.
      Oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus.
      According to this study, the cost of analyzing a single sample was 120 Chinese Yuan for oligonucleotide chip, 70 Chinese Yuan for real-time PCR, and 100 Chinese Yuan for sequencing.
      • Wang Y.Z.
      • Wu G.X.
      • Luo L.B.
      • Chen M.
      • Ruan L.H.
      Oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus.
      In 2008, Pas et al. reported the use of a prototype chip and compared the results with phylogenetic analysis.
      • Pas S.D.
      • Tran N.
      • de Man R.A.
      • Burghoorn-Maas C.
      • Vernet G.
      • et al.
      Comparison of reverse hybridization, microarray, and sequence analysis for genotyping hepatitis B virus.
      First, PCR was performed, followed by biotin labeling, fragmenting, purifying, and hybridizing the amplicons with the chip. Samples were then stained with streptavidin–phycoerythrin conjugate and examined using a scanner. The detection limit of the DNA chip was nearly 5000 copies/ml and the sensitivity and specificity were 97.5% and 97.8%, respectively.
      • Pas S.D.
      • Tran N.
      • de Man R.A.
      • Burghoorn-Maas C.
      • Vernet G.
      • et al.
      Comparison of reverse hybridization, microarray, and sequence analysis for genotyping hepatitis B virus.

      2.7 Flow-through reverse dot blot (FT-RDB)

      Zhang et al. developed a FT-RDB for HBV genotyping based on a conserved region of the HBV S gene.
      • Zhang R.
      • Deng Y.
      • Muller C.P.
      • Ou Z.Y.
      • Ma L.
      • Wang M.
      • et al.
      Determination of hepatitis B virus genotype by flow-through reverse dot blot.
      First, PCR was performed using a 5′-biotin labeled reverse primer. PCR amplicons were then denatured and rapidly cooled by ice prior to hybridization. The PCR products were then added to a nylon membrane to which genotype-specific probes were immobilized. After hybridization, detection was performed by the addition of streptavidin–horseradish peroxidase, followed by chromogen 3,3,5,5-tetramethylbenzidine (TMB). Any reagent added onto the nylon membrane was sucked through it using a hybridization machine that was equipped with a vacuum pump. Concordance between FT-RDB and sequencing was 96% (97/101). The four discordant samples were found to be mixed infection by FT-RDB but not by sequencing. The detection limit for examining genotypes B and D ranged between 102 and 103 copies/ml, and for A, C, and E ranged between 103 and 104 copies/ml.
      • Zhang R.
      • Deng Y.
      • Muller C.P.
      • Ou Z.Y.
      • Ma L.
      • Wang M.
      • et al.
      Determination of hepatitis B virus genotype by flow-through reverse dot blot.
      This method is thus an inexpensive, accurate, and rapid method for HBV genotyping.
      • Zhang R.
      • Deng Y.
      • Muller C.P.
      • Ou Z.Y.
      • Ma L.
      • Wang M.
      • et al.
      Determination of hepatitis B virus genotype by flow-through reverse dot blot.

      2.8 Restriction fragment mass polymorphism (RFMP)

      Lee et al. utilized RFMP for HBV genotyping based on genotypic variations in the S gene.
      • Lee J.M.
      • Ahn S.H.
      • Chang H.Y.
      • Shin J.E.
      • Kim D.Y.
      • Sim M.K.
      • et al.
      Reappraisal of HBV genotypes and clinical significance in Koreans using MALDI-TOF mass spectrometry.
      Similar to RFLP, this method depends on restriction enzyme digestion of PCR products to produce genotype-specific oligonucleotide fragments. The mass of the produced fragments is then determined using matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectrometry.
      • Lee J.M.
      • Ahn S.H.
      • Chang H.Y.
      • Shin J.E.
      • Kim D.Y.
      • Sim M.K.
      • et al.
      Reappraisal of HBV genotypes and clinical significance in Koreans using MALDI-TOF mass spectrometry.
      Other studies have reported the use of MALDI-TOF mass spectrometry for determination of YMDD (tyrosine–methionine–aspartate–aspartate motif) mutations, which are linked to lamivudine (LAM) drug resistance.
      • Hong S.P.
      • Kim N.K.
      • Hwang S.G.
      • Chung H.J.
      • Kim S.
      • Han J.H.
      • et al.
      Detection of hepatitis B virus YMDD variants using mass spectrometric analysis of oligonucleotide fragments.
      • Woo H.Y.
      • Park H.
      • Kim B.I.
      • Jeon W.K.
      • Cho Y.K.
      • Kim Y.J.
      Comparison of mass spectrometric analysis and TRUGENE HBV genotyping for monitoring lamivudine resistance in chronic hepatitis B patients.
      • Kim K.M.
      • Choi W.B.
      • Lim Y.S.
      • Lee H.C.
      • Chung Y.H.
      • Lee Y.S.
      • et al.
      Adefovir dipivoxil alone or in combination with ongoing lamivudine in patients with decompensated liver disease and lamivudine-resistant hepatitis B virus.
      In such studies, the method was found to be superior to DNA sequencing since it has a lower detection limit (100 copies/ml
      • Hong S.P.
      • Kim N.K.
      • Hwang S.G.
      • Chung H.J.
      • Kim S.
      • Han J.H.
      • et al.
      Detection of hepatitis B virus YMDD variants using mass spectrometric analysis of oligonucleotide fragments.
      ) and can also detect mutations in samples containing wild type.
      • Hong S.P.
      • Kim N.K.
      • Hwang S.G.
      • Chung H.J.
      • Kim S.
      • Han J.H.
      • et al.
      Detection of hepatitis B virus YMDD variants using mass spectrometric analysis of oligonucleotide fragments.
      • Woo H.Y.
      • Park H.
      • Kim B.I.
      • Jeon W.K.
      • Cho Y.K.
      • Kim Y.J.
      Comparison of mass spectrometric analysis and TRUGENE HBV genotyping for monitoring lamivudine resistance in chronic hepatitis B patients.
      • Kim K.M.
      • Choi W.B.
      • Lim Y.S.
      • Lee H.C.
      • Chung Y.H.
      • Lee Y.S.
      • et al.
      Adefovir dipivoxil alone or in combination with ongoing lamivudine in patients with decompensated liver disease and lamivudine-resistant hepatitis B virus.

      2.9 PCR invader assay

      Tadokoro et al. developed a PCR invader assay that successfully determined genotypes/subtypes Ae, Aa, Ba, Bj, and C–H.
      • Tadokoro K.
      • Kobayashi M.
      • Yamaguchi T.
      • Suzuki F.
      • Miyauchi S.
      • Egashira T.
      • et al.
      Classification of hepatitis B virus genotypes by the PCR-Invader method with genotype-specific probes.
      Amplification of the core/S region was first performed by multiplex PCR. This was followed by two invader reactions in a 384-well plate.
      • Tadokoro K.
      • Kobayashi M.
      • Yamaguchi T.
      • Suzuki F.
      • Miyauchi S.
      • Egashira T.
      • et al.
      Classification of hepatitis B virus genotypes by the PCR-Invader method with genotype-specific probes.
      The first reaction involves the binding of invader oligo as well as genotype-specific primary probes (P1 and P2) to the purified PCR amplicon. According to the HBV genotype, cleavage of either P1 or P2 at their 5′ end will occur secondary to the binding of both invader oligo and primary probes.
      • Tadokoro K.
      • Kobayashi M.
      • Yamaguchi T.
      • Suzuki F.
      • Miyauchi S.
      • Egashira T.
      • et al.
      Classification of hepatitis B virus genotypes by the PCR-Invader method with genotype-specific probes.
      This results in the release of what is known as 5′ flap. In the second reaction, the released 5′ flap products specifically bind to their generic FRET cassettes leading to their cleavage and the release of a specific fluorescent signal.
      • Tadokoro K.
      • Kobayashi M.
      • Yamaguchi T.
      • Suzuki F.
      • Miyauchi S.
      • Egashira T.
      • et al.
      Classification of hepatitis B virus genotypes by the PCR-Invader method with genotype-specific probes.
      The fluorescence signal produced thus corresponds to the HBV genotype and can be detected using a fluorescence plate reader. The detection threshold of this method is only 10 copies of HBV DNA/reaction and this method can detect mixed genotypes even if the ratio between the two genotypes is 1000:10.
      • Tadokoro K.
      • Kobayashi M.
      • Yamaguchi T.
      • Suzuki F.
      • Miyauchi S.
      • Egashira T.
      • et al.
      Classification of hepatitis B virus genotypes by the PCR-Invader method with genotype-specific probes.
      Concordance between PCR invader assay and enzyme-linked immunosorbent assay (ELISA) was 98.2% and between PCR invader assay and INNO-LiPA was 97.1%.
      • Tadokoro K.
      • Kobayashi M.
      • Yamaguchi T.
      • Suzuki F.
      • Miyauchi S.
      • Egashira T.
      • et al.
      Classification of hepatitis B virus genotypes by the PCR-Invader method with genotype-specific probes.
      The invader assay was proven to provide accurate results in non-concordant samples as verified by sequencing.
      • Tadokoro K.
      • Kobayashi M.
      • Yamaguchi T.
      • Suzuki F.
      • Miyauchi S.
      • Egashira T.
      • et al.
      Classification of hepatitis B virus genotypes by the PCR-Invader method with genotype-specific probes.

      2.10 Real-time PCR

      This method identifies both the HBV genotype and the HBV DNA level in serum, which aids in the prediction of therapeutic outcome.
      • Payungporn S.
      • Tangkijvanich P.
      • Jantaradsamee P.
      • Theamboonlers A.
      • Poovorawan Y.
      Simultaneous quantitation and genotyping of hepatitis B virus by real-time PCR and melting curve analysis.
      • Liu W.C.
      • Mizokami M.
      • Buti M.
      • Lindh M.
      • Young K.C.
      • Sun K.T.
      • et al.
      Simultaneous quantification and genotyping of hepatitis B virus for genotypes A to G by real-time PCR and two-step melting curve analysis.
      • Yeh S.H.
      • Tsai C.Y.
      • Kao J.H.
      • Liu C.J.
      • Kuo T.J.
      • Lin M.W.
      • et al.
      Quantification and genotyping of hepatitis B virus in a single reaction by real-time PCR and melting curve analysis.
      Real-time PCR using SYBR green I (a nucleic acid dye that binds to double-stranded DNA) or fluorescent probes allows the detection and quantification of HBV DNA, while melting curve analysis allows the determination of genotypes. Melting temperature(Tm) value differs between different genotypes depending on the complementarily between probe and target and/or GC content of the hybridization sequence.
      • Payungporn S.
      • Tangkijvanich P.
      • Jantaradsamee P.
      • Theamboonlers A.
      • Poovorawan Y.
      Simultaneous quantitation and genotyping of hepatitis B virus by real-time PCR and melting curve analysis.
      • Liu W.C.
      • Mizokami M.
      • Buti M.
      • Lindh M.
      • Young K.C.
      • Sun K.T.
      • et al.
      Simultaneous quantification and genotyping of hepatitis B virus for genotypes A to G by real-time PCR and two-step melting curve analysis.
      • Yeh S.H.
      • Tsai C.Y.
      • Kao J.H.
      • Liu C.J.
      • Kuo T.J.
      • Lin M.W.
      • et al.
      Quantification and genotyping of hepatitis B virus in a single reaction by real-time PCR and melting curve analysis.
      Payungporn et al. reported that the concordance between melting curve analysis and sequencing was 92.3%, while between RFLP and sequencing was only 90.4%.
      • Payungporn S.
      • Tangkijvanich P.
      • Jantaradsamee P.
      • Theamboonlers A.
      • Poovorawan Y.
      Simultaneous quantitation and genotyping of hepatitis B virus by real-time PCR and melting curve analysis.
      Also, Liu et al. found that results obtained with Tm analysis provided accuracy of 92.3%, which exceeded that of RFLP (87%).
      • Liu W.C.
      • Mizokami M.
      • Buti M.
      • Lindh M.
      • Young K.C.
      • Sun K.T.
      • et al.
      Simultaneous quantification and genotyping of hepatitis B virus for genotypes A to G by real-time PCR and two-step melting curve analysis.
      Unlike other methods for genotyping, real-time PCR does not allow for cross-contamination, is time-saving, has a high throughput, and is highly sensitive.
      • Payungporn S.
      • Tangkijvanich P.
      • Jantaradsamee P.
      • Theamboonlers A.
      • Poovorawan Y.
      Simultaneous quantitation and genotyping of hepatitis B virus by real-time PCR and melting curve analysis.
      • Liu W.C.
      • Mizokami M.
      • Buti M.
      • Lindh M.
      • Young K.C.
      • Sun K.T.
      • et al.
      Simultaneous quantification and genotyping of hepatitis B virus for genotypes A to G by real-time PCR and two-step melting curve analysis.
      The main disadvantage of this method is that it has a lower ability to distinguish between genotypes with close proximity between their Tm values, such as A and C, and thus could not be applied in certain countries such as North America which include different genotypes.
      • Payungporn S.
      • Tangkijvanich P.
      • Jantaradsamee P.
      • Theamboonlers A.
      • Poovorawan Y.
      Simultaneous quantitation and genotyping of hepatitis B virus by real-time PCR and melting curve analysis.
      However, this method could be modified through use of a stepwise approach, where first the genotype is differentiated into either ACDE or BFG groups, then the exact genotype is determined within the group.
      • Liu W.C.
      • Mizokami M.
      • Buti M.
      • Lindh M.
      • Young K.C.
      • Sun K.T.
      • et al.
      Simultaneous quantification and genotyping of hepatitis B virus for genotypes A to G by real-time PCR and two-step melting curve analysis.

      3. Clinical implications of HBV genotypes

      3.1 Disease severity and HCC development

      Disease severity has been found to be influenced by viral (including genotype), host, and environmental factors.
      • Liu C.
      • Kao J.
      Hepatitis B virus genotypes: epidemiology and therapeutic implications.
      • Girlanda R.
      • Mohsen A.H.
      • Smith H.
      • Sablon E.
      • Yuen M.F.
      • O’Grady J.
      • et al.
      Hepatitis B virus genotype A and D and clinical outcomes of liver transplantation for HBV-related disease.
      The incidence of HCC is influenced by HBV genotype, HBV DNA level, sex, age, pre-core A1896 mutation, and basal core promoter T1762/A1764 mutation.
      • Liu C.J.
      • Chen B.F.
      • Chen P.J.
      • Lai M.Y.
      • Huang W.L.
      • Kao J.H.
      • et al.
      Role of hepatitis B viral load and basal core promoter mutation in hepatocellular carcinoma in hepatitis B carriers.
      This article will only focus on the correlation between HBV genotypes and disease progression, including the development of liver cirrhosis and HCC.

      3.1.1 Genotype C is associated with faster liver damage than genotype B

      Most but not all studies have concluded that HBV genotype C predicts a worse prognosis (including cirrhosis and HCC) compared to B. Correlation between genotype C and the development of liver cirrhosis has been reported in numerous studies, including Ding et al. (Table 3) and You et al. (n = 122 genotyped chronic HBV Chinese patients; B: 48; C: 69; using reverse dot blot; p = 0.002).
      • Ding X.
      • Mizokami M.
      • Yao G.
      • Xu B.
      • Orito E.
      • Ueda R.
      • et al.
      Hepatitis B virus genotype distribution among chronic hepatitis B virus carriers in Shanghai, China.
      • You J.
      • Sriplung H.
      • Chongsuvivatwong V.
      • Geater A.
      • Zhuang L.
      • Huang J.H.
      • et al.
      Profile, spectrum and significance of hepatitis B virus genotypes in chronic HBV-infected patients in Yunnan, China.
      Many studies (Table 3) have associated genotype C with HCC development.
      • Yu M.W.
      • Yeh S.H.
      • Chen P.J.
      • Liaw Y.F.
      • Lin C.L.
      • Liu C.J.
      • et al.
      Hepatitis B virus genotype and DNA level and hepatocellular carcinoma: a prospective study in men.
      • Toan N.L.
      • Song le H.
      • Kremsner P.G.
      • Duy D.N.
      • Binh V.Q.
      • Koeberlein B.
      • et al.
      Impact of the hepatitis B virus genotype and genotype mixtures on the course of liver disease in Vietnam.
      Interestingly, Chan et al. (n = 86 HCC patients; sequencing) reported that subgenotype Ce is associated with higher HCC risk followed by Cs then B (p < 0.0001 for Ce vs. B; p = 0.02 for Cs vs. B).
      • Chan H.L.
      • Tse C.H.
      • Mo F.
      • Koh J.
      • Wong V.W.
      • Wong G.L.
      • et al.
      High viral load and hepatitis B virus subgenotype Ce are associated with increased risk of hepatocellular carcinoma.
      On the other hand, Tseng et al. (n = 242 genotype C HBV carriers; Cs: 23; Ce: 219; subgenotyping performed by detection of single nucleotide polymorphism at nucleotide position 1858) reported that the distribution of genotype C subgenotypes (Ce and Cs) among different disease stages including inactive carriers, chronic hepatitis patients, cirrhotic patients, and HCC patients with or without cirrhosis, was not significantly different.
      • Tseng T.C.
      • Liu C.J.
      • Chen P.J.
      • Lai M.Y.
      • Lin C.L.
      • Kao J.H.
      • et al.
      Subgenotypes of hepatitis B virus genotype C do not correlate with disease progression of chronic hepatitis B in Taiwan.
      Table 3Correlation between HBV genotypes and disease severity and HCC development.
      StudyPatients and methodsStudy findingsConclusions
      Ding et al. 2001
      • Ding X.
      • Mizokami M.
      • Yao G.
      • Xu B.
      • Orito E.
      • Ueda R.
      • et al.
      Hepatitis B virus genotype distribution among chronic hepatitis B virus carriers in Shanghai, China.
      • n = 220 HBV DNA-positive (China; 95 AS; 46 CH; 35 LC; 44 HCC; B: 38; C: 179)• 27.4%, 17%, 8.6%, and 2.3% of AS, CH, LC, and HCC patients were infected with genotype B, respectively (p < 0.01)• Genotype C is associated with HBeAg positivity
      • Method: RFLP• 69.5%, 83%, 91.4%, and 97.7% of AS, CH, LC, and HCC patients were infected with genotype C, respectively (p < 0.01)• Prevalence of genotype C increases from AS, CH, LC to HCC, in contrast to genotype B
      • 28.9% and 48.6% of patients infected with genotype B and genotype C were HBeAg-positive, respectively (p < 0.05)
      Chu et al. 2002
      • Chu C.J.
      • Hussain M.
      • Lok A.S.
      Hepatitis B virus genotype B is associated with earlier HBeAg seroconversion compared with hepatitis B virus genotype C.
      • 65 genotype B and 102 genotype C HBeAg-positive patients• Cumulative rates of HBeAg loss were 8%, 30%, and 50% in genotype B patients, and 0%, 23%, and 38% in genotype C patients at years 1, 3, and 5, respectively (p < 0.05)• Genotype B is an independent factor for HBeAg seroconversion
      • Method: INNO-LiPA and bidirectional automated sequencing• Cumulative rates of spontaneous HBeAg seroconversion were 6%, 24%, and 46% in genotype B patients and only 0%, 18%, and 34% in C patients at years 1, 3, and 5, respectively (p < 0.05)• Genotype B is associated with higher HBeAg loss
      • HBeAg seroconversion was 2.3 times higher in genotype B patients than in genotype C
      Sánchez-Tapias et al. 2002
      • Sánchez-Tapias J.M.
      • Costa J.
      • Mas A.
      • Bruguera M.
      • Rodés J.
      Influence of hepatitis B virus genotype on the long-term outcome of chronic hepatitis B in Western patients.
      • n = 244 (genotyped followed-up Spanish patients; A:131; D:94; F:19)• 8%, 4%, and 21% of patients infected with genotypes A, D, and F experienced hepatic decompensation, respectively (D vs. F: p = 0.02)• Death resulting from liver complications was more common in genotype F patients than A and D
      • Method: RFLP• 2%, 1%, and 5% of patients infected with genotypes A, D, and F experienced HCC, respectively
      • 5%, 1%, and 21% of patients infected with genotypes A, D, and F died due to a liver disease, respectively (A vs. F: p = 0.02; D vs. F: p = 0.002)
      • 55%, 32%, and 26% of A, D, and F achieved sustained remission, respectively (A vs. F; p = 0.01)
      Yu et al. 2005
      • Yu M.W.
      • Yeh S.H.
      • Chen P.J.
      • Liaw Y.F.
      • Lin C.L.
      • Liu C.J.
      • et al.
      Hepatitis B virus genotype and DNA level and hepatocellular carcinoma: a prospective study in men.
      • 146 HCC genotyped; 286 genotyped non-HCC• HCC incidences
      HCC incidence: measured as the ratio of the number of case patients to the number of control subjects.
      were 8/81 and 11/15 in genotype non-C and C patients (having baseline HBV DNA level of ≤4.22 log10 copies/ml), respectively
      • Genotype C is highly linked to HCC
      • Method: MCA plus sequencing and phylogenetic analysis• Genotype C was more linked to HCC compared to other genotypes (adjusted OR 5.11; 95% CI 3.20–8.18)
      Kumar et al. 2005
      • Kumar A.
      • Kumar S.I.
      • Pandey R.
      • Naik S.
      • Aggarwal R.
      Hepatitis B virus genotype A is more often associated with severe liver disease in northern India than is genotype D.
      • n = 70• ALT levels higher than 1.5 times normal occurred in 81% of genotype A patients but only in 58% of genotype D (p < 0.05)• Genotype A is more severe than genotype D
      • Method: RFLP and sequencing and phylogenetic analysis• HBeAg was positive in 86% of genotype A but only in 62% of genotype D (p < 0.05)
      • Negative anti-HBe occurred in 92% of genotype A but only in 72% of genotype D (p < 0.05)
      • In patients older than 25 years, 82% of genotype A patients and 57% of genotype D had cirrhosis (p < 0.05)
      Toan et al. 2006
      • Toan N.L.
      • Song le H.
      • Kremsner P.G.
      • Duy D.N.
      • Binh V.Q.
      • Koeberlein B.
      • et al.
      Impact of the hepatitis B virus genotype and genotype mixtures on the course of liver disease in Vietnam.
      • 86 AS; 92 LC; 84 HCC (Vietnamese)• AS: A 33.7%; B 4.6%; C 33.7%; D 5.8%; E 2.3%; F 2.3%; mixed 17.4%• Genotype A is highly prevalent in AS group (p < 0.0001), and D in LC (p < 0.0001)
      • Method: modified RFLP-PCR assay and sequencing• LC: A 10.8%; B 8.7%;C 14.1%; D 44.6%; E 2.2%; F 3.3%; G 3.3%; mixed 13.1%• Genotype C is associated with HCC (p = 0.023)
      • HCC: A 9.5%; B 11.9%; C 34.5%; D 19%; E 4.8%; F 2.4%; G 8.3%; mixed 9.6%
      ALT, alanine transaminase; anti-HBe, antibody to HBV e antigen; AS, asymptomatic; CH, chronic hepatitis; CI, confidence interval; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; LC, liver cirrhosis; MCA, melting curve analysis; OR, odds ratio; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism.
      a HCC incidence: measured as the ratio of the number of case patients to the number of control subjects.
      Several studies have reported that genotype B is associated with less HBeAg positivity and higher HBeAg clearance and seroconversion compared to C, including the studies of Orito et al. (n = 720 genotyped patients; B: 88; C: 610; RFLP plus sequencing and phylogenetic analysis; p < 0.01 for HBeAg positivity), Ding et al. (Table 3), Chu et al. (Table 3), Orito et al. (296 HCC patients; Ba:13; Bj:22; C:256; RFLP plus sequencing and phylogenetic analysis; p < 0.01 for Bj vs. C in HBeAg positivity), and You et al. (p = 0.05 for B vs. C in HBeAg positivity).
      • Orito E.
      • Ichida T.
      • Sakugawa H.
      • Sata M.
      • Horiike N.
      • Hino K.
      • et al.
      Geographic distribution of hepatitis B virus (HBV) genotype in patients with chronic HBV infection in Japan.
      • Ding X.
      • Mizokami M.
      • Yao G.
      • Xu B.
      • Orito E.
      • Ueda R.
      • et al.
      Hepatitis B virus genotype distribution among chronic hepatitis B virus carriers in Shanghai, China.
      • You J.
      • Sriplung H.
      • Chongsuvivatwong V.
      • Geater A.
      • Zhuang L.
      • Huang J.H.
      • et al.
      Profile, spectrum and significance of hepatitis B virus genotypes in chronic HBV-infected patients in Yunnan, China.
      • Orito E.
      • Sugauchi F.
      • Tanaka Y.
      • Ichida T.
      • Sata M.
      • Tanaka E.
      • et al.
      Differences of hepatocellular carcinoma patients with hepatitis B virus genotypes of Ba, Bj or C in Japan.
      • Chu C.J.
      • Hussain M.
      • Lok A.S.
      Hepatitis B virus genotype B is associated with earlier HBeAg seroconversion compared with hepatitis B virus genotype C.
      These studies further support the fact that infection with genotype B is associated with less severe liver damage compared to C.
      Sumi et al. reported that HBeAg negativity (p < 0.01) and seroconversion (p = 0.022) was higher in genotype B than C and that genotype B is an independent factor associated with HBe seroconversion (n = 585; genotyped patients; B: 30; C: 224 biopsy proven chronic liver disease; 74 HCC; ELISA and RFLP).
      • Sumi H.
      • Yokosuka O.
      • Seki N.
      • Arai M.
      • Imazeki F.
      • Kurihara T.
      • et al.
      Influence of hepatitis B virus genotypes on the progression of chronic type B liver disease.
      Also advanced fibrosis was significantly less common in genotype B compared to genotype C patients aged ≤45 years (p = 0.02). However, the association between genotype C and advanced fibrosis was lost when patients older than 45 years were compared. Thus they concluded that genotype B patients seem to develop advanced liver fibrosis slower than genotype C patients, but that the lifelong risk of advanced fibrosis and HCC development appears to be similar between genotype B and C patients.
      • Sumi H.
      • Yokosuka O.
      • Seki N.
      • Arai M.
      • Imazeki F.
      • Kurihara T.
      • et al.
      Influence of hepatitis B virus genotypes on the progression of chronic type B liver disease.

      3.1.2 HCC mean age, metastasis, and recurrence in genotype B and C patients

      Several studies have reported that genotype C is associated with younger HCC mean age compared to B.
      • Orito E.
      • Ichida T.
      • Sakugawa H.
      • Sata M.
      • Horiike N.
      • Hino K.
      • et al.
      Geographic distribution of hepatitis B virus (HBV) genotype in patients with chronic HBV infection in Japan.
      • Orito E.
      • Sugauchi F.
      • Tanaka Y.
      • Ichida T.
      • Sata M.
      • Tanaka E.
      • et al.
      Differences of hepatocellular carcinoma patients with hepatitis B virus genotypes of Ba, Bj or C in Japan.
      Orito et al. (B: 15; C: 100 HCC patients) reported that the mean ages of genotype B and C patients with HCC were 70.1 ± 9.2 years and 55.2 ± 9.7 years, respectively (p < 0.01).
      • Orito E.
      • Ichida T.
      • Sakugawa H.
      • Sata M.
      • Horiike N.
      • Hino K.
      • et al.
      Geographic distribution of hepatitis B virus (HBV) genotype in patients with chronic HBV infection in Japan.
      Orito et al. (Ba:13; Bj:22; C:256) reported that subgenotype Bj is associated with HCC in the elderly, while genotype C is associated with those of a younger age (mean age difference p < 0.01).
      • Orito E.
      • Sugauchi F.
      • Tanaka Y.
      • Ichida T.
      • Sata M.
      • Tanaka E.
      • et al.
      Differences of hepatocellular carcinoma patients with hepatitis B virus genotypes of Ba, Bj or C in Japan.
      Interestingly in this study, the mean age for HCC patients was also significantly higher in Bj-infected cases than the Ba-infected cases (p < 0.01).
      • Orito E.
      • Sugauchi F.
      • Tanaka Y.
      • Ichida T.
      • Sata M.
      • Tanaka E.
      • et al.
      Differences of hepatocellular carcinoma patients with hepatitis B virus genotypes of Ba, Bj or C in Japan.
      In contrast, other studies have concluded that genotype B is associated with HCC development at a young age and genotype C at an older age. It was reported that genotype B is linked with HCC development in young non cirrhotic patients.
      • Liu C.
      • Kao J.
      Hepatitis B virus genotypes: epidemiology and therapeutic implications.
      Ni et al. reported that out of 26 children with HCC resulting from HBV, 74% were infected with genotype B.
      • Ni Y.H.
      • Chang M.H.
      • Wang K.J.
      • Hsu H.Y.
      • Chen H.L.
      • Kao J.H.
      • et al.
      Clinical relevance of hepatitis B virus genotype in children with chronic infection and hepatocellular carcinoma.
      Similarly, Yuan et al. (n = 34 HCC patients; B: 21; C1: 9; C2: 4; ELISA and RFLP) reported that all HCC patients aged ≤35 years were infected with subgenotype Ba, while all subgenotype C2 HCC patients were older than 50 years.
      • Yuan J.
      • Zhou B.
      • Tanaka Y.
      • Kurbanov F.
      • Orito E.
      • Gong Z.
      • et al.
      Hepatitis B virus (HBV) genotypes/subgenotypes in China: mutations in core promoter and precore/core and their clinical implications.
      The contradictory findings reported by the different studies regarding correlation of genotypes B and C to HCC development age may be explained by differences in subgenotypes. Further studies that correlate HBV subgenotypes with HCC mean age are still required.
      HCC recurrence and metastasis were found more common in genotype C patients compared to B. Huang et al. (69 cases with recurrence or metastasis) reported that 74% and 41% of patients infected with genotypes C and B, respectively, suffered from HCC metastasis or recurrence (p = 0.009).
      • Huang Y.
      • Wang Z.
      • An S.
      • Zhou B.
      • Zhou Y.
      • Chan H.L.
      • et al.
      Role of hepatitis B virus genotypes and quantitative HBV DNA in metastasis and recurrence of hepatocellular carcinoma.

      3.1.3 Is genotype D more severe than A?

      Genotype D has been reported to be more severe as compared to A in some studies. Thakur et al. (130 Indian HBV patients; A: 60; D: 63; RFLP plus gene sequencing) reported that genotype D was more strongly associated with disease severity (Child's score B or C) compared to A (p < 0.05 for genotype D vs. A).
      • Thakur V.
      • Guptan R.C.
      • Kazim S.N.
      • Malhotra V.
      • Sarin S.K.
      Profile, spectrum and significance of HBV genotypes in chronic liver disease patients in the Indian subcontinent.
      Lee et al. (475 chronic HBV patients; only 4 patients with genotype A; RFMP) reported that all studied genotype A patients had inactive liver disease with no HCC.
      • Lee J.M.
      • Ahn S.H.
      • Chang H.Y.
      • Shin J.E.
      • Kim D.Y.
      • Sim M.K.
      • et al.
      Reappraisal of HBV genotypes and clinical significance in Koreans using MALDI-TOF mass spectrometry.
      Toan et al. (Table 3) also supported the finding that genotype D is more associated with liver cirrhosis and A with asymptomatic carrier.
      • Toan N.L.
      • Song le H.
      • Kremsner P.G.
      • Duy D.N.
      • Binh V.Q.
      • Koeberlein B.
      • et al.
      Impact of the hepatitis B virus genotype and genotype mixtures on the course of liver disease in Vietnam.
      In contrast, Kumar et al. (Table 3) reported that genotype A was more severe than D, being significantly more associated with high alanine transaminase levels, HBeAg positivity, anti-HBe negativity, and cirrhosis (cirrhosis only significant in patients older than 25 years).
      • Kumar A.
      • Kumar S.I.
      • Pandey R.
      • Naik S.
      • Aggarwal R.
      Hepatitis B virus genotype A is more often associated with severe liver disease in northern India than is genotype D.
      Baig et al. (71 asymptomatic and 14 liver cirrhosis/HCC) reported a linkage between genotype A and cirrhosis and disease severity.
      • Baig S.
      • Siddiqui A.A.
      • Ahmed W.
      • Qureshi H.
      • Arif A.
      The association of complex liver disorders with HBV genotypes prevalent in Pakistan.
      PCR with genotype-specific primers was used as the sole genotyping technique in this study.
      • Baig S.
      • Siddiqui A.A.
      • Ahmed W.
      • Qureshi H.
      • Arif A.
      The association of complex liver disorders with HBV genotypes prevalent in Pakistan.
      Recently, Madan et al. (n = 237 genotyped patients; D: 162; A: 61; ELISA and RFLP) reported that genotypes A and D do not influence disease severity and chronicity.
      • Madan K.
      • Batra Y.
      • Sreenivas V.
      • Mizokami M.
      • Tanaka Y.
      • Chalamalasetty S.B.
      • et al.
      HBV genotypes in India: do they influence disease severity?.
      Long-term studies with large homogeneous populations are thus required to understand the influence of HBV genotypes A and D on disease severity. Such studies should implement a common genotyping method verified by sequencing.

      3.1.4 Genotype F is highly associated with severe disease

      Genotype F was found to be associated with severe infection and young HCC development.
      • Sánchez-Tapias J.M.
      • Costa J.
      • Mas A.
      • Bruguera M.
      • Rodés J.
      Influence of hepatitis B virus genotype on the long-term outcome of chronic hepatitis B in Western patients.
      • Livingston S.E.
      • Simonetti J.P.
      • McMahon B.J.
      • Bulkow L.R.
      • Hurlburt K.J.
      • Homan C.E.
      • et al.
      Hepatitis B virus genotypes in Alaska Native people with hepatocellular carcinoma: preponderance of genotype F.
      Sánchez-Tapias et al. (Table 3) reported that death resulting from liver complications was more common in genotype F patients than A and D.
      • Sánchez-Tapias J.M.
      • Costa J.
      • Mas A.
      • Bruguera M.
      • Rodés J.
      Influence of hepatitis B virus genotype on the long-term outcome of chronic hepatitis B in Western patients.
      Similarly, Livingston et al. (n = 1129 non HCC; 47 HCC patients; F:32 HCC; sequencing) reported a strong link between genotype F and HCC development (p < 0.001).
      • Livingston S.E.
      • Simonetti J.P.
      • McMahon B.J.
      • Bulkow L.R.
      • Hurlburt K.J.
      • Homan C.E.
      • et al.
      Hepatitis B virus genotypes in Alaska Native people with hepatocellular carcinoma: preponderance of genotype F.
      The median age of HCC diagnosis in genotype F patients was only 22.5 years compared to 60 years for other genotypes (p = 0.002).
      • Livingston S.E.
      • Simonetti J.P.
      • McMahon B.J.
      • Bulkow L.R.
      • Hurlburt K.J.
      • Homan C.E.
      • et al.
      Hepatitis B virus genotypes in Alaska Native people with hepatocellular carcinoma: preponderance of genotype F.
      In summary, genotype C is more associated with rapid progression to advanced liver fibrosis, higher rate of HCC development, recurrence and metastasis, higher HBeAg positivity, and lower HBeAg clearance and seroconversion compared to genotype B. Genotype D may be more associated with severe disease and liver cirrhosis compared to A. Finally, genotype F was found to be more associated with higher mortality rates compared to other genotypes. Table 3, Table 4 summarize the clinical implications and the correlation between HBV genotypes and disease severity.
      Table 4Clinical implications of HBV genotypes to disease severity, response to IFN, disease chronicity, disease transmission, and transplantation outcomes
      Clinical implicationsHBV genotype
      ABCD
      Disease severity• Prevalent in AS
      • Toan N.L.
      • Song le H.
      • Kremsner P.G.
      • Duy D.N.
      • Binh V.Q.
      • Koeberlein B.
      • et al.
      Impact of the hepatitis B virus genotype and genotype mixtures on the course of liver disease in Vietnam.
      • Less prevalent in the LC and HCC group compared to AS
      • Ding X.
      • Mizokami M.
      • Yao G.
      • Xu B.
      • Orito E.
      • Ueda R.
      • et al.
      Hepatitis B virus genotype distribution among chronic hepatitis B virus carriers in Shanghai, China.
      (Table 3)
      • Highly prevalent in the LC and HCC group compared to AS
      • Ding X.
      • Mizokami M.
      • Yao G.
      • Xu B.
      • Orito E.
      • Ueda R.
      • et al.
      Hepatitis B virus genotype distribution among chronic hepatitis B virus carriers in Shanghai, China.
      (Table 3)
      • Associated with disease severity (Child's score
      Child's score: a scoring system that allows prediction of disease prognosis by measuring bilirubin, serum albumin, international normalized ratio, ascites, and hepatic encephalopathy.
      2 and 3) and LC
      • Toan N.L.
      • Song le H.
      • Kremsner P.G.
      • Duy D.N.
      • Binh V.Q.
      • Koeberlein B.
      • et al.
      Impact of the hepatitis B virus genotype and genotype mixtures on the course of liver disease in Vietnam.
      • Thakur V.
      • Guptan R.C.
      • Kazim S.N.
      • Malhotra V.
      • Sarin S.K.
      Profile, spectrum and significance of HBV genotypes in chronic liver disease patients in the Indian subcontinent.
      ,
      Other studies (Kumar et al.75 and Baig et al.3) reported that genotype A was more severe than genotype D.
      • HCC recurrence and metastasis: 41%
      • Huang Y.
      • Wang Z.
      • An S.
      • Zhou B.
      • Zhou Y.
      • Chan H.L.
      • et al.
      Role of hepatitis B virus genotypes and quantitative HBV DNA in metastasis and recurrence of hepatocellular carcinoma.
      • HCC recurrence and metastasis: 74%
      • Huang Y.
      • Wang Z.
      • An S.
      • Zhou B.
      • Zhou Y.
      • Chan H.L.
      • et al.
      Role of hepatitis B virus genotypes and quantitative HBV DNA in metastasis and recurrence of hepatocellular carcinoma.
      Response to IFN
      Response to nucleoside/nucleotide analogues is suggested not to be significantly influenced by HBV genotypes.92
      • SR: 47%
      • Erhardt A.
      • Blondin D.
      • Hauck K.
      • Sagir A.
      • Kohnle T.
      • Heintges T.
      • et al.
      Response to interferon alfa is hepatitis B virus genotype dependent: genotype A is more sensitive to interferon than genotype D.
      ,
      The patient population included both HBeAg-positive and negative patients; the table does not include the study of Hou et al.85 which reported that response after prolonged therapy was independent of HBV genotype.
      • Response rates: 39–50%
      • Chu R.H.
      • Ma L.X.
      • Wang G.
      • Shao L.H.
      Influence of HLA-DRB1 alleles and HBV genotypes on interferon-alpha therapy for chronic hepatitis B.
      • Kao J.H.
      • Wu N.H.
      • Chen P.J.
      • Lai M.Y.
      • Chen D.S.
      Hepatitis B genotypes and the response to interferon therapy.
      • Wai C.T.
      • Chu C.J.
      • Hussain M.
      • Lok A.S.
      HBV genotype B is associated with better response to interferon therapy in HBeAg(+) chronic hepatitis than genotype C.
      ,
      HBeAg-positive.
      • Response rate: 13–17%
      • Chu R.H.
      • Ma L.X.
      • Wang G.
      • Shao L.H.
      Influence of HLA-DRB1 alleles and HBV genotypes on interferon-alpha therapy for chronic hepatitis B.
      • Kao J.H.
      • Wu N.H.
      • Chen P.J.
      • Lai M.Y.
      • Chen D.S.
      Hepatitis B genotypes and the response to interferon therapy.
      • Wai C.T.
      • Chu C.J.
      • Hussain M.
      • Lok A.S.
      HBV genotype B is associated with better response to interferon therapy in HBeAg(+) chronic hepatitis than genotype C.
      ,
      HBeAg-positive.
      • SR: 23%
      • Erhardt A.
      • Blondin D.
      • Hauck K.
      • Sagir A.
      • Kohnle T.
      • Heintges T.
      • et al.
      Response to interferon alfa is hepatitis B virus genotype dependent: genotype A is more sensitive to interferon than genotype D.
      ,
      The patient population included both HBeAg-positive and negative patients; the table does not include the study of Hou et al.85 which reported that response after prolonged therapy was independent of HBV genotype.
      • Treatment duration: 4–15 months
      • Erhardt A.
      • Blondin D.
      • Hauck K.
      • Sagir A.
      • Kohnle T.
      • Heintges T.
      • et al.
      Response to interferon alfa is hepatitis B virus genotype dependent: genotype A is more sensitive to interferon than genotype D.
      • Treatment duration: 16–24 weeks
      • Chu R.H.
      • Ma L.X.
      • Wang G.
      • Shao L.H.
      Influence of HLA-DRB1 alleles and HBV genotypes on interferon-alpha therapy for chronic hepatitis B.
      • Kao J.H.
      • Wu N.H.
      • Chen P.J.
      • Lai M.Y.
      • Chen D.S.
      Hepatitis B genotypes and the response to interferon therapy.
      • Wai C.T.
      • Chu C.J.
      • Hussain M.
      • Lok A.S.
      HBV genotype B is associated with better response to interferon therapy in HBeAg(+) chronic hepatitis than genotype C.
      • Treatment duration: 16–24 weeks
      • Chu R.H.
      • Ma L.X.
      • Wang G.
      • Shao L.H.
      Influence of HLA-DRB1 alleles and HBV genotypes on interferon-alpha therapy for chronic hepatitis B.
      • Kao J.H.
      • Wu N.H.
      • Chen P.J.
      • Lai M.Y.
      • Chen D.S.
      Hepatitis B genotypes and the response to interferon therapy.
      • Wai C.T.
      • Chu C.J.
      • Hussain M.
      • Lok A.S.
      HBV genotype B is associated with better response to interferon therapy in HBeAg(+) chronic hepatitis than genotype C.
      • Treatment duration: 4–15 months
      • Erhardt A.
      • Blondin D.
      • Hauck K.
      • Sagir A.
      • Kohnle T.
      • Heintges T.
      • et al.
      Response to interferon alfa is hepatitis B virus genotype dependent: genotype A is more sensitive to interferon than genotype D.
      • Follow-up period: 12 months post-treatment
      • Erhardt A.
      • Blondin D.
      • Hauck K.
      • Sagir A.
      • Kohnle T.
      • Heintges T.
      • et al.
      Response to interferon alfa is hepatitis B virus genotype dependent: genotype A is more sensitive to interferon than genotype D.
      • Follow-up period: 48–52 weeks
      • Chu R.H.
      • Ma L.X.
      • Wang G.
      • Shao L.H.
      Influence of HLA-DRB1 alleles and HBV genotypes on interferon-alpha therapy for chronic hepatitis B.
      • Kao J.H.
      • Wu N.H.
      • Chen P.J.
      • Lai M.Y.
      • Chen D.S.
      Hepatitis B genotypes and the response to interferon therapy.
      • Wai C.T.
      • Chu C.J.
      • Hussain M.
      • Lok A.S.
      HBV genotype B is associated with better response to interferon therapy in HBeAg(+) chronic hepatitis than genotype C.
      • Follow-up period: 48–52 weeks
      • Chu R.H.
      • Ma L.X.
      • Wang G.
      • Shao L.H.
      Influence of HLA-DRB1 alleles and HBV genotypes on interferon-alpha therapy for chronic hepatitis B.
      • Kao J.H.
      • Wu N.H.
      • Chen P.J.
      • Lai M.Y.
      • Chen D.S.
      Hepatitis B genotypes and the response to interferon therapy.
      • Wai C.T.
      • Chu C.J.
      • Hussain M.
      • Lok A.S.
      HBV genotype B is associated with better response to interferon therapy in HBeAg(+) chronic hepatitis than genotype C.
      • Follow-up period: 12 months post-treatment
      • Erhardt A.
      • Blondin D.
      • Hauck K.
      • Sagir A.
      • Kohnle T.
      • Heintges T.
      • et al.
      Response to interferon alfa is hepatitis B virus genotype dependent: genotype A is more sensitive to interferon than genotype D.
      Disease chronicity• Some studies report this genotype in 28.6% of acute vs. 3% of chronic patients
      • Kobayashi M.
      • Ikeda K.
      • Arase Y.
      • Suzuki F.
      • Akuta N.
      • Hosaka T.
      • et al.
      Change of hepatitis B virus genotypes in acute and chronic infections in Japan.
      • Genotype B was prevalent in 26–39% of acute and only 4–11.7% of chronic patients
      • Sakai T.
      • Shiraki K.
      • Sugimoto K.
      • Ohmori S.
      • Murata K.
      • Takase K.
      • et al.
      Hepatitis B genotypes in patients with acute hepatitis B virus infection.
      • Imamura T.
      • Yokosuka O.
      • Kurihara T.
      • Kanda T.
      • Fukai K.
      • Imazeki F.
      • et al.
      Distribution of hepatitis B viral genotypes and mutations in the core promoter and precore regions in acute forms of liver disease in patients from Chiba, Japan.
      • Found in 84.5% of chronic vs. 59.5% of acute hepatitis patients
      • Kobayashi M.
      • Ikeda K.
      • Arase Y.
      • Suzuki F.
      • Akuta N.
      • Hosaka T.
      • et al.
      Change of hepatitis B virus genotypes in acute and chronic infections in Japan.
      • Some studies report this genotype in 80% of acute vs. 11% of chronic patients
      • Mayerat C.
      • Mantegani A.
      • Frei P.C.
      Does hepatitis B virus (HBV) genotype influence the clinical outcome of HBV infection?.
      • Others report in only 10% of acute vs. 80% of chronic patients
      • Mayerat C.
      • Mantegani A.
      • Frei P.C.
      Does hepatitis B virus (HBV) genotype influence the clinical outcome of HBV infection?.
      • Others report 48% of chronic patients vs. 13.6% of acute patients
      • Zekri A.R.
      • Hafez M.M.
      • Mohamed N.I.
      • Hassan Z.K.
      • El-Sayed M.H.
      • Khaled M.M.
      • et al.
      Hepatitis B virus (HBV) genotypes in Egyptian pediatric cancer patients with acute and chronic active HBV infection.
      ,
      Patient population: Egyptian pediatric cancer patients.
      Sexual and vertical transmission
      Genotypes B and C can be transmitted sexually and genotype A via vertical transmission. Genotype E can also be transmitted through sexual and vertical routes.84,99,101,107,108
      • 52% of genotype A patients were infected through homosexual or bisexual contact and only 16% by heterosexual
      • Koedijk F.D.
      • van Houdt R.
      • Op de Coul E.L.
      • Dukers N.H.
      • Niesters H.G.
      • Mostert M.C.
      • et al.
      Hepatitis B virus transmission patterns in the Netherlands, 2004.
      • 67% of genotype B were infected through vertical route
      • Erhardt A.
      • Blondin D.
      • Hauck K.
      • Sagir A.
      • Kohnle T.
      • Heintges T.
      • et al.
      Response to interferon alfa is hepatitis B virus genotype dependent: genotype A is more sensitive to interferon than genotype D.
      • 55% of genotype C were infected through vertical route
      • Erhardt A.
      • Blondin D.
      • Hauck K.
      • Sagir A.
      • Kohnle T.
      • Heintges T.
      • et al.
      Response to interferon alfa is hepatitis B virus genotype dependent: genotype A is more sensitive to interferon than genotype D.
      • 15% of genotype D patients were infected through homosexual or bisexual contact and 42% by heterosexual conduct
      • Koedijk F.D.
      • van Houdt R.
      • Op de Coul E.L.
      • Dukers N.H.
      • Niesters H.G.
      • Mostert M.C.
      • et al.
      Hepatitis B virus transmission patterns in the Netherlands, 2004.
      • 45% of genotype D were infected through vertical route
      • Erhardt A.
      • Blondin D.
      • Hauck K.
      • Sagir A.
      • Kohnle T.
      • Heintges T.
      • et al.
      Response to interferon alfa is hepatitis B virus genotype dependent: genotype A is more sensitive to interferon than genotype D.
      Transplant outcomes
      The study of Ben-Ari et al. 2004112 is not presented in the table; it reports no significant difference between genotype prevalence in patients undergoing liver transplantation and stable patients. Also Girlanda et al.61 reported that genotypes A and D did not significantly influence the outcome of liver transplant.
      • Recurrence after transplantation: 20%
      • Devarbhavi H.C.
      • Cohen A.J.
      • Patel R.
      • Wiesner R.H.
      • Dickson R.C.
      • Ishitani M.B.
      Preliminary results: outcome of liver transplantation for hepatitis B virus varies by hepatitis B virus genotype.
      • Cumulative rates of viral breakthrough after 3 years: 4%
      • Lo C.M.
      • Cheung C.K.
      • Lau G.K.
      • Yuen M.F.
      • Liu C.L.
      • Chan S.C.
      • et al.
      Significance of hepatitis B virus genotype in liver transplantation for chronic hepatitis B.
      • Recurrence after transplantation: 50–70%
      • Devarbhavi H.C.
      • Cohen A.J.
      • Patel R.
      • Wiesner R.H.
      • Dickson R.C.
      • Ishitani M.B.
      Preliminary results: outcome of liver transplantation for hepatitis B virus varies by hepatitis B virus genotype.
      • Recurrence after transplantation: 80%
      • Devarbhavi H.C.
      • Cohen A.J.
      • Patel R.
      • Wiesner R.H.
      • Dickson R.C.
      • Ishitani M.B.
      Preliminary results: outcome of liver transplantation for hepatitis B virus varies by hepatitis B virus genotype.
      • Recurrence after: nearly 2.7 months
      • Devarbhavi H.C.
      • Cohen A.J.
      • Patel R.
      • Wiesner R.H.
      • Dickson R.C.
      • Ishitani M.B.
      Preliminary results: outcome of liver transplantation for hepatitis B virus varies by hepatitis B virus genotype.
      • Recurrence is less common with less severe graft damage compared to genotype C• Recurrence after: nearly 10 months
      • Devarbhavi H.C.
      • Cohen A.J.
      • Patel R.
      • Wiesner R.H.
      • Dickson R.C.
      • Ishitani M.B.
      Preliminary results: outcome of liver transplantation for hepatitis B virus varies by hepatitis B virus genotype.
      • Recurrence after: nearly 9.1 months
      • Devarbhavi H.C.
      • Cohen A.J.
      • Patel R.
      • Wiesner R.H.
      • Dickson R.C.
      • Ishitani M.B.
      Preliminary results: outcome of liver transplantation for hepatitis B virus varies by hepatitis B virus genotype.
      • ACR: 90%
      • Devarbhavi H.C.
      • Cohen A.J.
      • Patel R.
      • Wiesner R.H.
      • Dickson R.C.
      • Ishitani M.B.
      Preliminary results: outcome of liver transplantation for hepatitis B virus varies by hepatitis B virus genotype.
      • ACR: 60%
      • Devarbhavi H.C.
      • Cohen A.J.
      • Patel R.
      • Wiesner R.H.
      • Dickson R.C.
      • Ishitani M.B.
      Preliminary results: outcome of liver transplantation for hepatitis B virus varies by hepatitis B virus genotype.
      • ACR: 50%
      • Devarbhavi H.C.
      • Cohen A.J.
      • Patel R.
      • Wiesner R.H.
      • Dickson R.C.
      • Ishitani M.B.
      Preliminary results: outcome of liver transplantation for hepatitis B virus varies by hepatitis B virus genotype.
      • Cumulative rates of viral breakthrough after 3 years: 21%
      • Lo C.M.
      • Cheung C.K.
      • Lau G.K.
      • Yuen M.F.
      • Liu C.L.
      • Chan S.C.
      • et al.
      Significance of hepatitis B virus genotype in liver transplantation for chronic hepatitis B.
      ACR, acute cellular rejection; ALF, acute liver failure; AS, asymptomatic; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; IFN, interferon; LC, liver cirrhosis; SR, sustained responder.
      a Other studies (Kumar et al.
      • Kumar A.
      • Kumar S.I.
      • Pandey R.
      • Naik S.
      • Aggarwal R.
      Hepatitis B virus genotype A is more often associated with severe liver disease in northern India than is genotype D.
      and Baig et al.
      • Baig S.
      • Siddiqui A.A.
      • Ahmed W.
      • Qureshi H.
      • Arif A.
      The association of complex liver disorders with HBV genotypes prevalent in Pakistan.
      ) reported that genotype A was more severe than genotype D.
      b Child's score: a scoring system that allows prediction of disease prognosis by measuring bilirubin, serum albumin, international normalized ratio, ascites, and hepatic encephalopathy.
      c Response to nucleoside/nucleotide analogues is suggested not to be significantly influenced by HBV genotypes.
      • Wiegand J.
      • Hasenclever D.
      • Tillmann H.L.
      Should treatment of hepatitis B depend on hepatitis B virus genotypes? A hypothesis generated from an explorative analysis of published evidence.
      d The patient population included both HBeAg-positive and negative patients; the table does not include the study of Hou et al.
      • Hou J.
      • Schilling R.
      • Janssen H.L.
      • Hansen B.E.
      • Heijtink R.
      • Sablon E.
      • et al.
      Genetic characteristics of hepatitis B virus genotypes as a factor for interferon-induced HBeAg clearance.
      which reported that response after prolonged therapy was independent of HBV genotype.
      e HBeAg-positive.
      f Patient population: Egyptian pediatric cancer patients.
      g Genotypes B and C can be transmitted sexually and genotype A via vertical transmission. Genotype E can also be transmitted through sexual and vertical routes.
      • Erhardt A.
      • Blondin D.
      • Hauck K.
      • Sagir A.
      • Kohnle T.
      • Heintges T.
      • et al.
      Response to interferon alfa is hepatitis B virus genotype dependent: genotype A is more sensitive to interferon than genotype D.
      • Zhang H.W.
      • Yin J.H.
      • Li Y.T.
      • Li C.Z.
      • Ren H.
      • Gu C.Y.
      • et al.
      Risk factors for acute hepatitis B and its progression to chronic hepatitis in Shanghai, China.
      • Halfon P.
      • Bourlière M.
      • Pol S.
      • Benhamou Y.
      • Ouzan D.
      • Rotily M.
      • et al.
      Multicentre study of hepatitis B virus genotypes in France: correlation with liver fibrosis and hepatitis B e antigen status.
      • Candotti D.
      • Danso K.
      • Allain J.P.
      Maternofetal transmission of hepatitis B virus genotype E in Ghana, West Africa.
      • Michitaka K.
      • Horiike N.
      • Chen Y.
      • Yatsuhashi H.
      • Yano M.
      • Kojima N.
      • et al.
      Infectious source factors affecting the severity of sexually transmitted acute hepatitis due to hepatitis B virus genotype C.
      h The study of Ben-Ari et al. 2004
      • Ben-Ari Z.
      • Ashur Y.
      • Daudi N.
      • Shmilovitz-Wiess H.
      • Brown M.
      • Sulkes J.
      • et al.
      Genotype prevalence, viral load and outcome of hepatitis B virus precore mutant infection in stable patients and in patients after liver transplantation.
      is not presented in the table; it reports no significant difference between genotype prevalence in patients undergoing liver transplantation and stable patients. Also Girlanda et al.
      • Girlanda R.
      • Mohsen A.H.
      • Smith H.
      • Sablon E.
      • Yuen M.F.
      • O’Grady J.
      • et al.
      Hepatitis B virus genotype A and D and clinical outcomes of liver transplantation for HBV-related disease.
      reported that genotypes A and D did not significantly influence the outcome of liver transplant.

      3.2 Antiviral therapy

      The US Food and Drug Administration (FDA) has approved six drugs for the treatment of HBV infections, namely interferon-alpha (IFN-α), pegylated IFN-α (PEG-IFN-α), LAM (cytidine analog), adefovir dipivoxil (ADV; dATP analog that function as chain terminator), entecavir (2′-deoxyguanosine analog that inhibits polymerase priming activity and chain elongation), and telbivudine (dTTP analog).
      • Valsamakis A.
      Molecular testing in the diagnosis and management of chronic hepatitis B.
      In the coming sections, the influence of HBV genotypes on response to therapy will be discussed.

      3.2.1 HBeAg-positive genotype B patients are more responsive to IFN compared to genotype C patients

      Many studies have reported that HBeAg-positive genotype B patients are more responsive to IFN compared to genotype C patients, including Chu et al. (Table 5), Kao et al. (n = 58; B:32; C:26; RFLP; p = 0.045), and Wai et al. (B: 31; C: 42 treated patients; INNO-LiPA plus direct sequencing; p = 0.03).
      • Chu R.H.
      • Ma L.X.
      • Wang G.
      • Shao L.H.
      Influence of HLA-DRB1 alleles and HBV genotypes on interferon-alpha therapy for chronic hepatitis B.
      • Kao J.H.
      • Wu N.H.
      • Chen P.J.
      • Lai M.Y.
      • Chen D.S.
      Hepatitis B genotypes and the response to interferon therapy.
      • Wai C.T.
      • Chu C.J.
      • Hussain M.
      • Lok A.S.
      HBV genotype B is associated with better response to interferon therapy in HBeAg(+) chronic hepatitis than genotype C.
      Furusyo et al. (n = 158 HBV Japanese patients; ELISA) reported that genotype C was associated with a continuation of HBV replication even after HBeAg clearance by IFN therapy, indicating a poor prognosis.
      • Furusyo N.
      • Nakashima H.
      • Kashiwagi K.
      • Kubo N.
      • Hayashida K.
      • Usuda S.
      • et al.
      Clinical outcomes of hepatitis B virus (HBV) genotypes B and C in Japanese patients with chronic HBV infection.
      It is important to note that in the study of Luo et al. (n = 261 patients followed up for 3 years; RFLP and phylogenetic analysis), there was no significant difference in IFN response after a 3-year follow-up period, between genotype C and recombinant B (recombination with the pre-C/C gene of genotype C).
      • Luo K.
      • He H.
      • Liu Z.
      • Zhu Y.
      • Mao Q.
      • Liang W.
      No significant differences in histology and response to interferon treatment in hepatitis B carriers of genotypes C and recombinant B.
      This suggests that different genotype B strains may have a wide range of different responses to IFN therapy, including those that are comparable to that of genotype C.
      Table 5Correlation between HBV genotypes and IFN/PEG-IFN response.
      StudyPatients and methodsStudy findingsConclusions
      Chu et al. 2005
      • Chu R.H.
      • Ma L.X.
      • Wang G.
      • Shao L.H.
      Influence of HLA-DRB1 alleles and HBV genotypes on interferon-alpha therapy for chronic hepatitis B.
      • n = 202 (Chinese; 126 CH; 76 normal control); B:38; C: 69; B + C:19• 50%, 13%, and 26% of patients infected with genotypes B, C, and B + C responded to IFN therapy, respectively (p < 0.005)• Genotype C patients are more resistant to IFN therapy compared to B or B + C
      • Method: multiplex PCR
      • Treatment protocol
      IFN-α (3 MU) injected subcutaneously three times weekly for 24 weeks.
      Erhardt et al. 2005
      • Erhardt A.
      • Blondin D.
      • Hauck K.
      • Sagir A.
      • Kohnle T.
      • Heintges T.
      • et al.
      Response to interferon alfa is hepatitis B virus genotype dependent: genotype A is more sensitive to interferon than genotype D.
      • 78 genotype A (61 HBeAg-positive and 17 HBeAg-negative)• SR-6s were 49% and 26% in HBeAg-positive and negative patients infected with genotypes A and D, respectively (p < 0.005)• Genotype A was found to be an independent positive predictor of IFN SR (p < 0.009)
      • 66 genotype D (38 HBeAg-positive, 28 HBeAg-negative)• SR-6s were 46% and 24% in HBeAg-positive patients infected with genotypes A and D, respectively (p < 0.03)
      • Method: sequencing• SR-6s were 59% and 29% in HBeAg-negative patients infected with genotypes A and D, respectively (p < 0.05)
      • Treatment protocol: standard IFN• SR-12s were 47% and 23% in HBeAg-positive and negative patients infected with genotypes A and D, respectively (p < 0.002)
      • SR-12s were 44% and 24% in HBeAg-positive patients infected with genotypes A and D, respectively (p < 0.04)
      • SR-12s were 59% and 21% in HBeAg-negative patients infected with genotypes A and D, respectively (p < 0.01)
      Janssen et al. 2005
      • Janssen H.L.
      • van Zonneveld M.
      • Senturk H.
      • Zeuzem S.
      • Akarca U.S.
      • Cakaloglu Y.
      • et al.
      Pegylated interferon alfa-2b alone or in combination with lamivudine for HBeAg-positive chronic hepatitis B: a randomised trial.
      • n = 266 HBeAg-positive patients (A:90; B: 23; C:39; D:103 treated patients)• HBeAg loss was found to be influenced by HBV genotypes (p = 0.01)• Genotype A patients were significantly more responsive than D (p = 0.01) or C (p = 0.006) and genotype B patients were slightly more responsive than C
      • Method: INNO-LiPA• HBeAg loss was achieved in 47%, 44%, 28%, and 25% of genotypes A, B, C, and D, 26 weeks following PEG-IFN with or without lamivudine, therapy, respectively
      • Treatment protocol
      Patients were given PEG-IFN (100μg/week) plus LAM (100mg/day) or monotherapy PEG-IFN (100μg/week) for 52 weeks. Between weeks 32 and 52 the dose of PEG-IFN was reduced to 50μg/week in both treatment groups.
      Bonino et al. 2007
      • Bonino F.
      • Marcellin P.
      • Lau G.K.
      • Hadziyannis S.
      • Jin R.
      • Piratvisuth T.
      • et al.
      Peginterferon Alfa-2a HBeAg-Negative Chronic Hepatitis B Study Group. Predicting response to peginterferon alpha-2a, lamivudine and the two combined for HBeAg-negative chronic hepatitis B.
      • 518 HBeAg-negative CHB patients (Asian or Caucasian; infected with genotypes A, B, C or D)• Combined response rates, 24 weeks after end of PEG-IFN therapy for genotypes B, C, and D patients were 44%, 49%, and 16%, respectively• HBV genotype was found to be a significant predictor for PEG-IFN with/without LAM response 24 weeks after treatment (p = 0.027)
      • Treatment protocol
      PEG-IFN 180μg every week for 48 weeks plus placebo once daily; or PEG-IFN 180μg every week for 48 weeks plus LAM 100mg daily; or LAM 100mg daily for 48 weeks.
      • SVR was more linked to genotype C in patients treated with PEG-IFN with/without LAM compared to D (p < 0.001)
      • Genotype B (p = 0.0033) and C (p < 0.0001) achieved combined response more than D, one year after end of therapy (PEG-IFN monotherapy or LAM monotherapy or PEG-IFN plus LAM)
      CH, chronic hepatitis; CHB; chronic hepatitis B patients; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus; IFN, interferon; LAM, lamivudine; PCR, polymerase chain reaction; PEG-IFN, pegylated interferon; SR, Sustained response; SR-6, sustained response after 6 months; SR-12, sustained response after 12 months; SVR, Sustained virological response.
      a IFN-α (3 MU) injected subcutaneously three times weekly for 24 weeks.
      b Patients were given PEG-IFN (100 μg/week) plus LAM (100 mg/day) or monotherapy PEG-IFN (100 μg/week) for 52 weeks. Between weeks 32 and 52 the dose of PEG-IFN was reduced to 50 μg/week in both treatment groups.
      c PEG-IFN 180 μg every week for 48 weeks plus placebo once daily; or PEG-IFN 180 μg every week for 48 weeks plus LAM 100 mg daily; or LAM 100 mg daily for 48 weeks.
      Ma et al. (94 HBeAg-positive patients B: 25; C: 43; D: 16; PCR microplate hybridization ELISA; 6 months of IFN; 6 months follow up) reported that genotype B was associated with a better response compared to C and D. Only genotype B patients in this study experienced a complete response.
      • Ma J.C.
      • Wang L.W.
      • Li X.J.
      • Liao Y.F.
      • Hu X.Y.
      • Gong Z.J.
      Relationship between HBV genotypes and anti-viral therapeutic efficacy of interferon-alpha.

      3.2.2 Genotype A patients are more responsive to IFN compared to genotype D patients

      Some studies have arrived at the conclusion that genotype A patients have a better response to IFN compared to those infected with other genotypes. Importantly, Erhardt et al. (Table 5) reported that patients with genotype A responded better to therapy compared to patients with genotype D, in both HBeAg-positive and negative patients.
      • Erhardt A.
      • Blondin D.
      • Hauck K.
      • Sagir A.
      • Kohnle T.
      • Heintges T.
      • et al.
      Response to interferon alfa is hepatitis B virus genotype dependent: genotype A is more sensitive to interferon than genotype D.
      Genotype A was suggested to be an independent positive predictor of IFN sustained response (multivariate logistic regression analysis; p < 0.009).
      • Erhardt A.
      • Blondin D.
      • Hauck K.
      • Sagir A.
      • Kohnle T.
      • Heintges T.
      • et al.
      Response to interferon alfa is hepatitis B virus genotype dependent: genotype A is more sensitive to interferon than genotype D.
      Hou et al. (103 HBeAg-positive chronic hepatitis B (CHB) patients; A: 46; INNO-LiPA and cloning and sequencing; follow up 6 months post-treatment) reported that genotype A patients have a faster response to IFN compared to those with other genotypes.
      • Hou J.
      • Schilling R.
      • Janssen H.L.
      • Hansen B.E.
      • Heijtink R.
      • Sablon E.
      • et al.
      Genetic characteristics of hepatitis B virus genotypes as a factor for interferon-induced HBeAg clearance.
      They reported that the rate of HBeAg clearance was significantly higher in genotype A compared to other genotypes (including genotype D) after 16 weeks of IFN therapy (p = 0.014 for A vs. other genotypes; p = 0.05 for A vs. D). Logistic regression analysis revealed that genotype A is a positive predictor of IFN responsiveness (16 weeks of treatment; p = 0.001).
      • Hou J.
      • Schilling R.
      • Janssen H.L.
      • Hansen B.E.
      • Heijtink R.
      • Sablon E.
      • et al.
      Genetic characteristics of hepatitis B virus genotypes as a factor for interferon-induced HBeAg clearance.
      However, response to IFN therapy was independent of genotype in the case of prolonged therapy in this study.
      • Hou J.
      • Schilling R.
      • Janssen H.L.
      • Hansen B.E.
      • Heijtink R.
      • Sablon E.
      • et al.
      Genetic characteristics of hepatitis B virus genotypes as a factor for interferon-induced HBeAg clearance.
      The better responsiveness of genotypes A and B to IFN compared to D and C may be partially due to the faster development of mutations in the basal core promoter region in genotypes D and C than in A and B.
      • Bartholomeusz A.
      • Schaefer S.
      Hepatitis B virus genotypes: comparison of genotyping methods.

      3.2.3 Influence of HBV genotypes on PEG-IFN response

      The response to PEG-IFN has been reported to be influenced by HBV genotypes in many studies. Janssen et al. (Table 5) reported that HBeAg loss as a response to PEG-IFN monotherapy or PEG-IFN plus LAM was influenced by HBV genotype and that genotypes A and B had a better response compared to C and D after 52 weeks of treatment.
      • Janssen H.L.
      • van Zonneveld M.
      • Senturk H.
      • Zeuzem S.
      • Akarca U.S.
      • Cakaloglu Y.
      • et al.
      Pegylated interferon alfa-2b alone or in combination with lamivudine for HBeAg-positive chronic hepatitis B: a randomised trial.
      Genotype A patients were significantly more responsive than D or C patients and genotype B patients were slightly more responsive than C.
      • Janssen H.L.
      • van Zonneveld M.
      • Senturk H.
      • Zeuzem S.
      • Akarca U.S.
      • Cakaloglu Y.
      • et al.
      Pegylated interferon alfa-2b alone or in combination with lamivudine for HBeAg-positive chronic hepatitis B: a randomised trial.
      Flink et al. (n = 266; 52 weeks of treatment; follow up period 26 weeks; INNO-LiPA) reported that HBeAg-positive CHB patients infected with genotype A experienced a better PEG-IFN response (with or without LAM) compared to other genotypes (loss of HBsAg in A vs. D; p = 0.006).
      • Flink H.J.
      • van Zonneveld M.
      • Hansen B.E.
      • de Man R.A.
      • Schalm S.W.
      • Janssen H.L.
      • et al.
      Treatment with peg-interferon alpha-2b for HBeAg-positive chronic hepatitis B: HBsAg loss is associated with HBV genotype.
      Hadziyannis et al. (n = 448; PEG-IFN plus placebo for 48 weeks) reported that genotype A patients had higher HBsAg seroconversion in both HBeAg-positive and negative patients at 24 weeks post-treatment (HBeAg-positive: A 22%, B 0%, C 2%, D 0%; HBeAg-negative: A 18%, B 2%, C 3%, D 0%).
      • Hadziyannis S.
      • Lau G.
      • Marcellin P.
      • Piratvisuth T.
      • Cooksley G.
      • Bonino F.
      • et al.
      Sustained HBsAg seroconversion in patients with chronic hepatitis B treated with peginterferon alpha-2a (40kd) (pegasys).
      In contrast, Lau et al. (271 genotyped patients treated with PEG-IFN plus placebo for 48 weeks; A: 23; B: 76; C: 162; D: 9; INNO-LiPA) reported no significant difference between different genotypes in HBeAg seroconversion 24 weeks after PEG-IFN therapy.
      • Lau G.K.
      • Piratvisuth T.
      • Luo K.X.
      • Marcellin P.
      • Thongsawat S.
      • Cooksley G.
      • et al.
      Peginterferon Alfa-2a HBeAg-Positive Chronic Hepatitis B Study Group. Peginterferon alfa-2a, lamivudine, and the combination for HBeAg-positive chronic hepatitis B.
      It is important to note that genotype A tended to have a relatively higher response to PEG-IFN compared to other genotypes (A 52%, B 30%, C 31%, D 22%).
      • Lau G.K.
      • Piratvisuth T.
      • Luo K.X.
      • Marcellin P.
      • Thongsawat S.
      • Cooksley G.
      • et al.
      Peginterferon Alfa-2a HBeAg-Positive Chronic Hepatitis B Study Group. Peginterferon alfa-2a, lamivudine, and the combination for HBeAg-positive chronic hepatitis B.
      It is thus clear that PEG-IFN should be a first-line therapy for genotype A patients.
      Another study by Bonino et al. (Table 5) reported that HBeAg negative patients infected with genotypes B and C responded better to PEG-IFN compared to genotype D.
      • Bonino F.
      • Marcellin P.
      • Lau G.K.
      • Hadziyannis S.
      • Jin R.
      • Piratvisuth T.
      • et al.
      Peginterferon Alfa-2a HBeAg-Negative Chronic Hepatitis B Study Group. Predicting response to peginterferon alpha-2a, lamivudine and the two combined for HBeAg-negative chronic hepatitis B.

      3.2.4 Influence of HBV genotypes on nucleoside/nucleotide response and resistance

      Contradicting studies have been reported on whether HBV genotypes influence the responsiveness to nucleoside/nucleotide analogues.
      • Schaefer S.
      Hepatitis B virus: significance of genotypes.
      • Liu C.
      • Kao J.
      Hepatitis B virus genotypes: epidemiology and therapeutic implications.
      • Akuta N.
      • Kumada H.
      Influence of hepatitis B virus genotypes on the response to antiviral therapies.
      Most recently, Wiegand et al. compiled data from 20 studies and arrived at the conclusion that responsiveness to nucleoside/nucleotide analogues (LAM, ADV, entecavir, and telbivudine) is not significantly influenced by HBV genotype.
      • Wiegand J.
      • Hasenclever D.
      • Tillmann H.L.
      Should treatment of hepatitis B depend on hepatitis B virus genotypes? A hypothesis generated from an explorative analysis of published evidence.
      Important to mention is that resistance against LAM (measured as mutations in the YMDD conserved region) appears to emerge earlier in genotype A patients compared to genotype D patients.
      • Liu C.
      • Kao J.
      Hepatitis B virus genotypes: epidemiology and therapeutic implications.
      In summary, HBeAg-positive genotype B patients have been found to be more responsive to IFN therapy compared to genotype C patients; and A gives a better response compared to other genotypes (including D), especially during short-term therapy. Patients infected with genotype A seem to have a better response to PEG-IFN compared to those with other genotypes. HBV genotypes do not seem to influence response to nucleoside/nucleotide antiviral therapy. Table 4, Table 5 summarize the clinical implications and correlation between HBV genotypes and response to IFN therapy.

      3.3 Disease chronicity

      3.3.1 Prevalence of genotype A in acute and chronic hepatitis patients

      Few studies have reported that genotype A is more prevalent in chronic than acute disease. Mayerat et al. (n = 65; A: 31; D: 28; length polymorphism analysis and direct cycle sequencing) reported that genotype A is more prevalent in chronic infection and genotype D in acute disease in European countries (p < 0.0001).
      • Mayerat C.
      • Mantegani A.
      • Frei P.C.
      Does hepatitis B virus (HBV) genotype influence the clinical outcome of HBV infection?.
      This finding is also supported by Rodriguez-Frias et al. (Table 6).
      • Rodriguez-Frias F.
      • Jardi R.
      • Buti M.
      • Schaper M.
      • Hermosilla E.
      • Valdes A.
      • et al.
      Hepatitis B virus genotypes and G1896A precore mutation in 486 Spanish patients with acute and chronic HBV infection.
      In contrast, Kobayashi et al. (Table 6), in a study on a large population (n = 4430), reported that genotype A is highly prevalent in acute infection while C is prevalent in chronic infection.
      • Kobayashi M.
      • Ikeda K.
      • Arase Y.
      • Suzuki F.
      • Akuta N.
      • Hosaka T.
      • et al.
      Change of hepatitis B virus genotypes in acute and chronic infections in Japan.
      Table 6Correlation between HBV genotypes and disease chronicity.
      StudyPatients and methodsStudy findingsConclusions
      Imamura et al. 2003
      • Imamura T.
      • Yokosuka O.
      • Kurihara T.
      • Kanda T.
      • Fukai K.
      • Imazeki F.
      • et al.
      Distribution of hepatitis B viral genotypes and mutations in the core promoter and precore regions in acute forms of liver disease in patients from Chiba, Japan.
      • 61 AH; 531 CLD• 39% of acute forms of hepatitis and 12% of CLD were infected with genotype B (p < 0.001)• Genotype B is more prevalent in acute forms of hepatitis than CLD
      • 31% of acute self limited hepatitis and 62.5% of fulminant hepatitis cases were infected with genotype B (p = 0.027)• Genotype B is more associated with fulminant hepatitis than acute self limited hepatitis
      • Method: ELISA and RFLP
      Wai et al. 2005
      • Wai C.T.
      • Fontana R.J.
      • Polson J.
      • Hussain M.
      • Shakil A.O.
      • Han S.H.
      • et al.
      Clinical outcome and virological characteristics of hepatitis B-related acute liver failure in the United States.
      • 530 CHB cases (matched = 75) compared with 25 ALF cases (USA)• 32% and 16% of ALF and CHB patients were infected with D, respectively (p = 0.007) after matching race and HBeAg• Genotype D is more prevalent in ALF than CHB
      • Method: INNO-LiPA plus sequencing
      Rodriguez-Frias et al. 2006
      • Rodriguez-Frias F.
      • Jardi R.
      • Buti M.
      • Schaper M.
      • Hermosilla E.
      • Valdes A.
      • et al.
      Hepatitis B virus genotypes and G1896A precore mutation in 486 Spanish patients with acute and chronic HBV infection.
      • A: 192; D: 234• In acute infection: genotype A:D ratio = 0.40• Genotype D is more associated with acute infection compared to A
      • Method: RFLP• In chronic HBV infection: genotype ratio A:D = 0.83
      • p = 0.03
      Zekri et al. 2007
      • Zekri A.R.
      • Hafez M.M.
      • Mohamed N.I.
      • Hassan Z.K.
      • El-Sayed M.H.
      • Khaled M.M.
      • et al.
      Hepatitis B virus (HBV) genotypes in Egyptian pediatric cancer patients with acute and chronic active HBV infection.
      • n = 68 (genotyped Egyptian pediatric cancer patients; 46 CAH; 22 AH)• AH: A 9%; B 45.5%; C 13.6%; D 13.6%; mixed 18.2%• D is more prevalent in CAH than AH (p < 0.05)
      • Method: multiplex PCR• CAH: A 10.4%; B 16.7%; C 6.3%; D 47.9%; mixed 14.6%
      Zhang et al. 2008
      • Zhang H.W.
      • Yin J.H.
      • Li Y.T.
      • Li C.Z.
      • Ren H.
      • Gu C.Y.
      • et al.
      Risk factors for acute hepatitis B and its progression to chronic hepatitis in Shanghai, China.
      • n = 294 acute hepatitis B• 20/25 (80%) and 5/25 (20%) of patients who progressed to chronic were infected with genotypes C2 and B2, respectively (p = 0.013: C2 vs. B2)• HBV C2 is an independent factor for chronicity development as determined by multivariate analysis (AOR 6.97 (95% CI 1.59–30.63))
      • Method: multiplex PCR and sequencing and phylogenetic analysis
      Kobayashi et al. 2008
      • Kobayashi M.
      • Ikeda K.
      • Arase Y.
      • Suzuki F.
      • Akuta N.
      • Hosaka T.
      • et al.
      Change of hepatitis B virus genotypes in acute and chronic infections in Japan.
      • n = 4430 (Japan; 153 AH; 4277 CH)• AH: A 28.6%; B 10.3%; C 59.5%• Distribution of genotypes among acute and chronic groups was significantly different (p < 0.001)
      • Method: ELISA• CH: A 3%; B 12.3%; C 84.5%
      AH, acute hepatitis; ALF, acute liver failure; AOR, adjusted odds ratio; CAH, chronic active hepatitis; CH, chronic hepatitis; CHB, chronic hepatitis B; CI, confidence interval; CLD, chronic liver disease; ELISA, enzyme-linked immunosorbent assay; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism.

      3.3.2 Prevalence of genotype D in acute and chronic hepatitis patients

      Wai et al. (Table 6) supported the finding of Rodriguez-Frias et al.
      • Rodriguez-Frias F.
      • Jardi R.
      • Buti M.
      • Schaper M.
      • Hermosilla E.
      • Valdes A.
      • et al.
      Hepatitis B virus genotypes and G1896A precore mutation in 486 Spanish patients with acute and chronic HBV infection.
      that genotype D is more prevalent in acute disease, and reported a significant association between genotype D and acute liver failure compared to chronic hepatitis.
      • Wai C.T.
      • Fontana R.J.
      • Polson J.
      • Hussain M.
      • Shakil A.O.
      • Han S.H.
      • et al.
      Clinical outcome and virological characteristics of hepatitis B-related acute liver failure in the United States.
      In contrast, Zekri et al. (Table 6) reported that genotype D was significantly more prevalent in chronic active hepatitis than acute hepatitis (p < 0.05).
      • Zekri A.R.
      • Hafez M.M.
      • Mohamed N.I.
      • Hassan Z.K.
      • El-Sayed M.H.
      • Khaled M.M.
      • et al.
      Hepatitis B virus (HBV) genotypes in Egyptian pediatric cancer patients with acute and chronic active HBV infection.
      Genotype A was slightly more prevalent in chronic active hepatitis than acute hepatitis.
      • Zekri A.R.
      • Hafez M.M.
      • Mohamed N.I.
      • Hassan Z.K.
      • El-Sayed M.H.
      • Khaled M.M.
      • et al.
      Hepatitis B virus (HBV) genotypes in Egyptian pediatric cancer patients with acute and chronic active HBV infection.
      The patient population in the study of Zekri et al., Egyptian pediatric cancer patients, may be a limiting factor to the reported findings.

      3.3.3 High prevalence of genotype B in acute and genotype C in chronic hepatitis

      Sakai et al. (n = 203; B: 14; RFLP; p < 0.001) and Imamura et al. (Table 6; n = 592; ELISA; p < 0.001) reported that genotype B is more prevalent in acute infection.
      • Sakai T.
      • Shiraki K.
      • Sugimoto K.
      • Ohmori S.
      • Murata K.
      • Takase K.
      • et al.
      Hepatitis B genotypes in patients with acute hepatitis B virus infection.
      • Imamura T.
      • Yokosuka O.
      • Kurihara T.
      • Kanda T.
      • Fukai K.
      • Imazeki F.
      • et al.
      Distribution of hepatitis B viral genotypes and mutations in the core promoter and precore regions in acute forms of liver disease in patients from Chiba, Japan.
      Imamura et al. reported that genotype B was linked to more severe acute disease being significantly more prevalent in fulminant disease than acute self limited hepatitis.
      • Imamura T.
      • Yokosuka O.
      • Kurihara T.
      • Kanda T.
      • Fukai K.
      • Imazeki F.
      • et al.
      Distribution of hepatitis B viral genotypes and mutations in the core promoter and precore regions in acute forms of liver disease in patients from Chiba, Japan.
      Zhang et al. (Table 6) reported that C2 was more prevalent in patients that progressed to chronic infection compared to B2.
      • Zhang H.W.
      • Yin J.H.
      • Li Y.T.
      • Li C.Z.
      • Ren H.
      • Gu C.Y.
      • et al.
      Risk factors for acute hepatitis B and its progression to chronic hepatitis in Shanghai, China.
      Other studies have reported no correlation between HBV genotypes and disease chronicity rates. For example, Krekulova et al. (n = 45; A: 33; D: 12; sequencing) reported no correlation between genotypes A and D and disease chronicity.
      • Krekulova L.
      • Rehak V.
      • da Silva Filho H.P.
      • Zavoral M.
      • Riley L.W.
      Genotypic distribution of hepatitis B virus in the Czech Republic: a possible association with modes of transmission and clinical outcome.
      It is still unclear whether genotypes A and D are more prevalent in acute or chronic hepatitis patients; however, the current findings suggest that genotypes B and C are more prevalent in acute and chronic infections, respectively. More studies with large homogeneous populations using the same genotyping method that is verified by sequencing are needed to reach consistent conclusions regarding the influence of genotype on disease chronicity. Table 4, Table 6 summarize the clinical implications and the influence of HBV genotypes on disease chronicity.

      3.4 HBV transmission

      3.4.1 Sexual transmission

      Halfon et al. (Table 7) reported that genotype A was associated with patients infected through sexual contact, while genotype D through blood transfusion.
      • Halfon P.
      • Bourlière M.
      • Pol S.
      • Benhamou Y.
      • Ouzan D.
      • Rotily M.
      • et al.
      Multicentre study of hepatitis B virus genotypes in France: correlation with liver fibrosis and hepatitis B e antigen status.
      Koedijk et al. (158 genotyped acute HBV; sequencing and phylogenetic analysis) reported that 52% and only 15% of genotype A and D patients, respectively, were infected via homosexual or bisexual contact, while 16% and 42% were infected via heterosexual contact.
      • Koedijk F.D.
      • van Houdt R.
      • Op de Coul E.L.
      • Dukers N.H.
      • Niesters H.G.
      • Mostert M.C.
      • et al.
      Hepatitis B virus transmission patterns in the Netherlands, 2004.
      Thus genotype A appears to be associated with homosexual or bisexual contact.
      • Koedijk F.D.
      • van Houdt R.
      • Op de Coul E.L.
      • Dukers N.H.
      • Niesters H.G.
      • Mostert M.C.
      • et al.
      Hepatitis B virus transmission patterns in the Netherlands, 2004.
      Understanding the association between HBV genotypes and route of transmission is essential since a recent increase in genotype A, for example, may indicate a recent lack of proper safe sex practices.
      • Halfon P.
      • Bourlière M.
      • Pol S.
      • Benhamou Y.
      • Ouzan D.
      • Rotily M.
      • et al.
      Multicentre study of hepatitis B virus genotypes in France: correlation with liver fibrosis and hepatitis B e antigen status.
      • Koedijk F.D.
      • van Houdt R.
      • Op de Coul E.L.
      • Dukers N.H.
      • Niesters H.G.
      • Mostert M.C.
      • et al.
      Hepatitis B virus transmission patterns in the Netherlands, 2004.
      Table 7Correlation between HBV genotypes and routes of transmission.
      StudyPatients and methodsStudy findingsConclusions
      Erhardt et al. 2005
      • Erhardt A.
      • Blondin D.
      • Hauck K.
      • Sagir A.
      • Kohnle T.
      • Heintges T.
      • et al.
      Response to interferon alfa is hepatitis B virus genotype dependent: genotype A is more sensitive to interferon than genotype D.
      • n = 165 CH (A: 78;B: 6; C: 11 ;D: 66)• 20%, 67%, 55% and 45% of genotypes A, B, C, and D patients were infected through perinatal route, respectively• Genotypes B, C, and D are more associated with perinatal transmission than A (p < 0.03)
      • Method: sequencing
      Lin et al. 2005
      • Lin C.L.
      • Kao J.H.
      • Chen B.F.
      • Chen P.J.
      • Lai M.Y.
      • Chen D.S.