Hepatitis B virus genotyping: current methods and clinical implications

International Journal of Infectious Diseases 14 (2010) e941–e953

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International Journal of Infectious Diseases journal homepage: www.elsevier.com/locate/ijid

Review

Hepatitis B virus genotyping: current methods and clinical implications Bassem S.S. Guirgis a, Radwa O. Abbas b, Hassan M.E. Azzazy a,b,* a b

Yousef Jameel Science and Technology Research Center, The American University in Cairo, New Cairo, Egypt Department of Chemistry, The American University in Cairo, AUC Avenue, PO Box 74, New Cairo 11835, Egypt

A R T I C L E I N F O

S U M M A R Y

Article history: Received 26 December 2008 Received in revised form 10 March 2010 Accepted 11 March 2010

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. ß 2010 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Corresponding Editor: Mark Holodniy, California, USA Keywords: HBV genotype Therapeutic response Prognosis Transplantation outcome HBV transmission Genotyping methods

1. Introduction Hepatitis B virus (HBV) infects nearly two billion people worldwide.1 Approximately 600,000 deaths occur every year as a result of the acute and chronic consequences of HBV infection.1 Disease chronicity rates are 5% in generally healthy infected adults and 80–90% in perinatally infected children.2 Chronic HBV affects nearly 350 million patients worldwide and may further progress to cirrhosis and/or hepatocellular carcinoma (HCC) in 15–40% of cases.3 Worldwide, HBV accounts for 30% of cirrhotic and 50% of HCC cases.4 The viral genome is a 3.2 kb circular partially duplex DNA molecule with the circularity maintained by 50 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

* Corresponding author. Tel.: +20 2 2615 2543; fax: +20 2 2795 7565. E-mail address: [email protected] (Hassan M.E. Azzazy).

transcriptional transactivator that codes for involved in the development of HCC4–6 (Figure 1). Due to the lack of proof-reading activity of DNA- and RNAdependent 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  105 per year.7,8 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%.9 The phylogenetic tree of complete HBV genomes showing the alignment of the different genotypes has been reviewed elsewhere.10 Table 1 shows the updated global distribution of HBV genotypes/subgenotypes10–22. 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 24,9,23–47. 2.1. Sequencing and phylogenetic analysis The gold standard method for HBV genotyping is whole genome sequencing followed by phylogenetic analysis.4,23 Although this technique is highly sensitive and allows the detection of new and recombinant genotypes, it is expensive, time-consuming, and

1201-9712/$36.00 – see front matter ß 2010 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijid.2010.03.020

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antigen (HBsAg) gene (s101–s237) and overlapping polymerase gene (rt99–rt280) using PCR.48 PCR amplicons are then added into four CLIPTM 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.48 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.49,50 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).50 2.2. INNO-LiPA

Figure 1. HBV genome (modified from Feitelson and Larkin6). The HBV genome (3.2 kb) is a circular partially duplex DNA molecule with the circularity maintained by 50 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.4,5

detects mainly the predominant genotype in genotype mixtures.4,23 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.23 The sensitivity of single gene sequencing depends on the degree of sequence homology as well as the sequence size.4,23 It is important to note that a commercial direct sequencing assay kit is available (TRUGENE HBV Genotyping Kit; Siemens Medical Solutions Diagnostics, NY, USA).48 This kit is a twoin-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

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.51 The amplified biotinylated PCR products are then hybridized to probes immobilized onto membrane strips that detect genotypespecific 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.51 This method can detect both single and mixed genotypes.4 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.24 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.24 2.3. Restriction fragment polymorphism (RFLP) RFLP depends on PCR amplification of the S gene (usually), restriction enzyme digestion, and separation of digested fragments

Table 1 Global distribution of HBV genotypes and subgenotypes10–22 Genotype

Distribution

Subgenotype

Distribution

A

Pandemic, but mostly prevalent in the USA and Northwest Europe

B

Northern and Southeast Asia

C

Asia and Pacific region

D

Mediterranean, the Middle East, North America, and India

E F

Africa and Tunisia Central and South America

G H

France, Germany, and USA Central and South America and Mexico

Aa/A1 Ae/A2 Bj/B1 Ba/B2 B3 B4 B5 Ce/C1 Cs/C2 C3 C4 D1 D2 D3 D4 D5 NA F1 F2 F3 F4 NA NA

Asia and Africa Europe and USA Japan China, Taiwan, and Vietnam Indonesia Vietnam Philippines East Asia South-east Asia Polynesia, Solomon Islands Northeast Australia India, Pakistan, Iran India, Russia, and the Baltic region India, Pakistan Solomon Islands, Oceania India NA Central America, Peru, Venezuela Venezuela Venezuela Bolivia NA NA

HBV, hepatitis B virus; NA, data not available.

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Table 2 Comparison of common HBV genotyping methods Genotyping technique

Advantages

Disadvantages

Sequencing

 Gold standard, most reliable as it does not depend on a small nucleotide or amino acid sequence as other methods do23  Can detect new genotypes and recombination between genotypes23  Easier and cheaper than sequencing23  Overall success rate for the detection of all eight HBV genotypes was recently found to be 98%24  Highly specific31  Can detect mixed genotypes4,31  Simple and cost-effective method  Can be used in large population studies  Can determine subgenotypes42  Rapid, simple, and cost-effective method  Can be used in large population studies  Can determine subgenotypes25,44,45  Accuracy reported to be 93%26  Sensitive in detecting mixed genotypes (even if minor genotype is as low as 10%)26  No PCR required  Simple and cost-effective  Can be used in large population studies23,27,28  Sensitive  Determines genotype even at titers as low as 2000–5000 copies/ml30,31  Superior to sequencing in determining mixed genotypes30  Can detect mixed genotypes32  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/ml32  High sensitivity  Can also be used to detect YMDD mutations with higher sensitivity and lower detection limit (100 copies/ml33) compared to sequencing and can also detect mutations even in samples containing wild-type33–35  Extremely high sensitivity (detection threshold is 10 copies of HBV DNA/reaction36)  Can detect mixed genotypes even if the ratio between the two is 1000:1036  Low cross-contamination  Time saving  High throughput  High sensitivity 37,38  Superior than sequencing and oligonucleotide microarray chips in detecting mixed populations30

 Less efficient in detecting mixed genotypes23,31  Cumbersome (cannot be used in large-scale studies), expensive, and requires expertise23,31

INNO-LiPA

RFLP

Multiplex PCR

Serotyping

Oligonucleotide microarray chips

Reverse dot blot

RFMP

Invader assay

Real-time PCR

 Insensitive for HBV genomes with SNP or deletions in the sequences to which probes bind4  Relatively expensive compared to PCR-based and serological techniques23  May give indeterminate results (<6%)39,41,43  SNP at enzyme restriction sites may affect method sensitivity23  Indeterminate samples range from 2.9% to 4.5%9,46  SNP at primer site may affect method sensitivity23

 Indeterminate samples range from 1.4% to 23.4%29,40,47  SNP may affect process sensitivity23  More expensive than real-time PCR and sequencing30  Sensitivity may be affected by SNPs or deletions in the sequences to which the probes bind  Sensitivity may be affected by SNPs or deletions in the sequences to which the probes bind

 SNP at enzyme restriction sites may affect method sensitivity  Requires the availability of MALDI-TOF mass spectrometry (bulky, expensive, and requires trained operators)  Sensitivity may be affected by SNPs or deletions in the sequences to which the probes bind

 SNP at primer site may affect method sensitivity  Lower ability to distinguish between genotypes with close proximity between Tm37,38

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.

by electrophoresis.23,52 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.11 This method has been used to determine genotypes A– F.52,53 In 2004, Zeng et al. developed a modified RFLP technique based on the S gene allowing the detection of HBV genotypes A– H.54 In this method, two PCR rounds were undertaken prior to restriction enzyme digestion by five enzymes, namely StyI, BsrI, DpnI, HpaII, and EaeI.54 The method was compared with another RFLP method targeting the pre-S1 region and the results were concordant in 96.8% (181/187).54 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.54 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.55 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%.55

2.4. Multiplex PCR Naito et al. developed a multiplex nested PCR method for the detection of genotypes A–F.56 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.56 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.56 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.57 In 2007, Chen et al. modified the single round PCR to determine genotypes A–F in one reaction, as well as to

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subtype genotypes B and C into B1/B2 and C1/C2, respectively, in another reaction.25 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.26 Multiplex PCR had a higher accuracy (93.2%) compared to the RFLP method (87%) developed by Mizokami et al.52 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.26 This method can detect mixed genotypes with sensitivity for detecting minor species as low as 10%.26 2.5. Serological subtyping

it was 20  102 copies/ml. In contrast, oligonucleotide chip was able to determine genotype at all tested dilutions.30 When clinical samples were tested, 93% (117/126) of the results obtained by the three methods were concordant.30 In the nine discordant samples, sequencing only determined the predominant genotype, however both real-time PCR and oligonucleotide chip determined both genotypes.30 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.30 In 2008, Pas et al. reported the use of a prototype chip and compared the results with phylogenetic analysis.31 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.31

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 adw4.10,27,28 Although PCR and sequencing have been used as the standard methods for genotyping, sequencing is not suitable for large epidemiological studies and population screening.27,28 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.23,27 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.27 However, it has been reported that the rate of indeterminate samples may reach up to 23.4%.29

Zhang et al. developed a FT-RDB for HBV genotyping based on a conserved region of the HBV S gene.32 First, PCR was performed using a 50 -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.32 This method is thus an inexpensive, accurate, and rapid method for HBV genotyping.32

2.6. Oligonucleotide microarray chips

2.8. Restriction fragment mass polymorphism (RFMP)

Song et al. developed a low density oligonucleotide microarray that can determine genotypes A–G.58 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).58 In 2007, Wang et al. compared oligonucleotide microarray chips to real-time PCR and sequencing.30 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%.30 Realtime 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%.30 The oligonucleotide chip method was found to be superior to both sequencing and real-time PCR with respect to sensitivity.30 Realtime PCR and sequencing were only able to determine the genotype when the titer was higher than 5.20  103 but not when

Lee et al. utilized RFMP for HBV genotyping based on genotypic variations in the S gene.59 Similar to RFLP, this method depends on restriction enzyme digestion of PCR products to produce genotypespecific oligonucleotide fragments. The mass of the produced fragments is then determined using matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectrometry.59 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.33–35 In such studies, the method was found to be superior to DNA sequencing since it has a lower detection limit (100 copies/ml33) and can also detect mutations in samples containing wild type.33–35

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

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.36 Amplification of the core/S region was first performed by multiplex PCR. This was followed by two invader reactions in a 384-well plate.36 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 50 end will occur secondary to the binding of both invader oligo and primary probes.36 This results in the release of what is known as 50 flap. In the second reaction, the released 50 flap products

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specifically bind to their generic FRET cassettes leading to their cleavage and the release of a specific fluorescent signal.36 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 1000:10.36 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%.36 The invader assay was proven to provide accurate results in nonconcordant samples as verified by sequencing.36 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.37,38,60 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. 37,38,60 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%.37 Also, Liu et al. found that results obtained with Tm analysis provided accuracy of 92.3%, which exceeded that of RFLP (87%).38 Unlike other methods for genotyping, real-time PCR does not allow for crosscontamination, is time-saving, has a high throughput, and is highly sensitive.37,38 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.37 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.38 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.12,61 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.62 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).63,64 Many studies (Table 3) have associated genotype C with HCC development.65,66 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).67 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

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stages including inactive carriers, chronic hepatitis patients, cirrhotic patients, and HCC patients with or without cirrhosis, was not significantly different.68 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).39,63,64,69,70 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).71 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.71 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.39,69 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).39 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).69 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).69 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.12 Ni et al. reported that out of 26 children with HCC resulting from HBV, 74% were infected with genotype B. 72 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.40 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).73 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).74 Lee et al. (475

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Table 3 Correlation between HBV genotypes and disease severity and HCC development. Study Ding et al. 2001

63

Chu et al. 200270

Patients and methods

Study findings

Conclusions

 n = 220 HBV DNA-positive (China; 95 AS; 46 CH; 35 LC; 44 HCC; B: 38; C: 179)  Method: RFLP

 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

 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

 65 genotype B and 102 genotype C HBeAg-positive patients  Method: INNO-LiPA and bidirectional automated sequencing

Sa´nchez-Tapias et al. 200241

 n = 244 (genotyped followed-up Spanish patients; A:131; D:94; F:19)  Method: RFLP

Yu et al. 200565

 146 HCC genotyped; 286 genotyped non-HCC

Kumar et al. 200575

 Method: MCA plus sequencing and phylogenetic analysis  n = 70

 Method: RFLP and sequencing and phylogenetic analysis

Toan et al. 200666

 86 AS; 92 LC; 84 HCC (Vietnamese)  Method: modified RFLP-PCR assay and sequencing

 28.9% and 48.6% of patients infected with genotype B and genotype C were HBeAg-positive, respectively (p < 0.05)  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)  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)  HBeAg seroconversion was 2.3 times higher in genotype B patients than in genotype C  8%, 4%, and 21% of patients infected with genotypes A, D, and F experienced hepatic decompensation, respectively (D vs. F: p = 0.02)  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)  HCC incidencesa 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 was more linked to HCC compared to other genotypes (adjusted OR 5.11; 95% CI 3.20–8.18)  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)  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)  AS: A 33.7%; B 4.6%; C 33.7%; D 5.8%; E 2.3%; F 2.3%; mixed 17.4%  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%  HCC: A 9.5%; B 11.9%; C 34.5%; D 19%; E 4.8%; F 2.4%; G 8.3%; mixed 9.6%

 Genotype B is an independent factor for HBeAg seroconversion  Genotype B is associated with higher HBeAg loss

 Death resulting from liver complications was more common in genotype F patients than A and D

 Genotype C is highly linked to HCC

 Genotype A is more severe than genotype D

 Genotype A is highly prevalent in AS group (p < 0.0001), and D in LC (p < 0.0001)  Genotype C is associated with HCC (p = 0.023)

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.

chronic HBV patients; only 4 patients with genotype A; RFMP) reported that all studied genotype A patients had inactive liver disease with no HCC.59 Toan et al. (Table 3) also supported the finding that genotype D is more associated with liver cirrhosis and A with asymptomatic carrier.66 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).75 Baig et al. (71 asymptomatic and 14 liver cirrhosis/HCC) reported a linkage between genotype A and cirrhosis and disease severity.3 PCR with genotype-specific primers was used as the sole genotyping technique in this study.3 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.76 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.41,77 Sa´nchez-Tapias et al. (Table 3) reported that death resulting from liver complications was more common in genotype F patients than A and D.41 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).77 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).77 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. Tables 3 and 4 summarize the clinical implications and the correlation between HBV genotypes and disease severity.

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Table 4 Clinical implications of HBV genotypes to disease severity, response to IFN, disease chronicity, disease transmission, and transplantation outcomes Clinical implications

HBV genotype

Disease severity

 Prevalent in AS66

Response to IFNc

 

A

 Disease chronicity





B  Less prevalent in the LC and HCC group compared to AS63 (Table 3)  HCC recurrence and metastasis: 41%73 SR: 47%84,d  Response rates: 39–50%78–80,e Treatment duration:  Treatment duration: 4–15 months84 16–24 weeks78–80 Follow-up period: 12 months  Follow-up period: 48–52 post-treatment84 weeks78–80 Some studies report this genotype  Genotype B was prevalent in 28.6% of acute vs. 3% of in 26–39% of acute and chronic patients95 only 4–11.7% of chronic Others report in only 10% of acute patients 97,98 93 vs. 80% of chronic patients

C

D

 Highly prevalent in the LC and HCC group compared to AS63 (Table 3)  HCC recurrence and metastasis: 74%73  Response rate: 13–17%78–80,e  Treatment duration: 16–24 weeks78–80  Follow-up period: 48–52 weeks78–80  Found in 84.5% of chronic vs. 59.5% of acute hepatitis patients95

 Associated with disease severity (Child’s scoreb 2 and 3) and LC66,74,a

Sexual and vertical transmissiong

 52% of genotype A patients were infected through homosexual or bisexual contact and only 16% by heterosexual102

 67% of genotype B were infected through vertical route84

 55% of genotype C were infected through vertical route84

Transplant outcomesh

 Recurrence after transplantation: 20%110  Recurrence after: nearly 2.7 months110  ACR: 90%110

 Cumulative rates of viral  Recurrence after breakthrough after 3 years: 4%111 transplantation: 50–70%110  Recurrence after:  Recurrence is less common nearly 10 months110 with less severe graft damage compared to genotype C  ACR: 60%110  Cumulative rates of viral breakthrough after 3 years: 21%111

 SR: 23%84,d  Treatment duration: 4–15 months84  Follow-up period: 12 months post-treatment84  Some studies report this genotype in 80% of acute vs. 11% of chronic patients93  Others report 48% of chronic patients vs. 13.6% of acute patients9,f  15% of genotype D patients were infected through homosexual or bisexual contact and 42% by heterosexual conduct102  45% of genotype D were infected through vertical route84  Recurrence after transplantation: 80%110  Recurrence after: nearly 9.1 months110  ACR: 50%110

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.75 and Baig et al.3) 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.92 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. 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.84,99,101,107,108 h 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.

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-a), pegylated IFN-a (PEG-IFN-a), LAM (cytidine analog), adefovir dipivoxil (ADV; dATP analog that function as chain terminator), entecavir (20 -deoxyguanosine analog that inhibits polymerase priming activity and chain elongation), and telbivudine (dTTP analog).4 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).78–80 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.81 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).82 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. 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.83 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

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Table 5 Correlation between HBV genotypes and IFN/PEG-IFN response. Study

Patients and methods

Study findings

Conclusions

Chu et al. 200578

 n = 202 (Chinese; 126 CH; 76 normal control); B:38; C: 69; B + C:19  Method: multiplex PCR  Treatment protocola  78 genotype A (61 HBeAg-positive and 17 HBeAg-negative)  66 genotype D (38 HBeAg-positive, 28 HBeAg-negative)  Method: sequencing  Treatment protocol: standard IFN

 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

 SR-6s were 49% and 26% in HBeAg-positive and negative patients infected with genotypes A and D, respectively (p < 0.005)  SR-6s were 46% and 24% in HBeAg-positive patients infected with genotypes A and D, respectively (p < 0.03)  SR-6s were 59% and 29% in HBeAg-negative patients infected with genotypes A and D, respectively (p < 0.05)  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)  HBeAg loss was found to be influenced by HBV genotypes (p = 0.01)  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  Combined response rates, 24 weeks after end of PEG-IFN therapy for genotypes B, C, and D patients were 44%, 49%, and 16%, respectively  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)

 Genotype A was found to be an independent positive predictor of IFN SR (p < 0.009)

Erhardt et al. 200584

Janssen et al. 200586

Bonino et al. 200790

 n = 266 HBeAg-positive patients (A:90; B: 23; C:39; D:103 treated patients)  Method: INNO-LiPA  Treatment protocolb  518 HBeAg-negative CHB patients (Asian or Caucasian; infected with genotypes A, B, C or D)  Treatment protocolc

 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  HBV genotype was found to be a significant predictor for PEG-IFN with/without LAM response 24 weeks after treatment (p = 0.027)

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-a (3 MU) injected subcutaneously three times weekly for 24 weeks. b Patients were given PEG-IFN (100 mg/week) plus LAM (100 mg/day) or monotherapy PEG-IFN (100 mg/week) for 52 weeks. Between weeks 32 and 52 the dose of PEG-IFN was reduced to 50 mg/week in both treatment groups. c PEG-IFN 180 mg every week for 48 weeks plus placebo once daily; or PEG-IFN 180 mg every week for 48 weeks plus LAM 100 mg daily; or LAM 100 mg daily for 48 weeks.

compared to patients with genotype D, in both HBeAg-positive and negative patients.84 Genotype A was suggested to be an independent positive predictor of IFN sustained response (multivariate logistic regression analysis; p < 0.009).84 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.85 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).85 However, response to IFN therapy was independent of genotype in the case of prolonged therapy in this study.85 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.23 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.86 Genotype A patients were significantly more respon-

sive than D or C patients and genotype B patients were slightly more responsive than C.86 Flink et al. (n = 266; 52 weeks of treatment; follow up period 26 weeks; INNO-LiPA) reported that HBeAgpositive 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).87 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%).88 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 PEGIFN therapy.89 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%).89 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.90 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.10,12,91 Most recently, Wiegand et al. compiled data from

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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.92 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.12 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. Tables 4 and 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).93 This finding is also supported by Rodriguez-Frias et al. (Table 6).94 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.95 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. 94 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.96 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).9 Genotype A was slightly more prevalent in chronic

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active hepatitis than acute hepatitis.9 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.97,98 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.98 Zhang et al. (Table 6) reported that C2 was more prevalent in patients that progressed to chronic infection compared to B2.99 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.100 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. Tables 4 and 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.101 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.102 Thus genotype A appears to

Table 6 Correlation between HBV genotypes and disease chronicity. Study

Patients and methods

Study findings

Conclusions

Imamura et al. 200398

 61 AH; 531 CLD  Method: ELISA and RFLP

Wai et al. 200596

 530 CHB cases (matched = 75) compared with 25 ALF cases (USA)  Method: INNO-LiPA plus sequencing  A: 192; D: 234  Method: RFLP

 39% of acute forms of hepatitis and 12% of CLD were infected with genotype B (p < 0.001)  31% of acute self limited hepatitis and 62.5% of fulminant hepatitis cases were infected with genotype B (p = 0.027)  32% and 16% of ALF and CHB patients were infected with D, respectively (p = 0.007) after matching race and HBeAg

 Genotype B is more prevalent in acute forms of hepatitis than CLD  Genotype B is more associated with fulminant hepatitis than acute self limited hepatitis  Genotype D is more prevalent in ALF than CHB

Rodriguez-Frias et al. 200694 Zekri et al. 20079

Zhang et al. 200899

Kobayashi et al. 200895

 n = 68 (genotyped Egyptian pediatric cancer patients; 46 CAH; 22 AH)  Method: multiplex PCR  n = 294 acute hepatitis B  Method: multiplex PCR and sequencing and phylogenetic analysis  n = 4430 (Japan; 153 AH; 4277 CH)  Method: ELISA

   

In acute infection: genotype A:D ratio = 0.40 In chronic HBV infection: genotype ratio A:D = 0.83 p = 0.03 AH: A 9%; B 45.5%; C 13.6%; D 13.6%; mixed 18.2%

 CAH: A 10.4%; B 16.7%; C 6.3%; D 47.9%; mixed 14.6%  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)  AH: A 28.6%; B 10.3%; C 59.5%  CH: A 3%; B 12.3%; C 84.5%

 Genotype D is more associated with acute infection compared to A  D is more prevalent in CAH than AH (p < 0.05)

 HBV C2 is an independent factor for chronicity development as determined by multivariate analysis (AOR 6.97 (95% CI 1.59–30.63))  Distribution of genotypes among acute and chronic groups was significantly different (p < 0.001)

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.

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Table 7 Correlation between HBV genotypes and routes of transmission Study Erhardt et al. 2005

84

Lin et al. 2005109

Halfon et al. 2006101

Inui et al. 2007105

Patients and methods

Study findings

Conclusions

 n = 165 CH (A: 78;B: 6; C: 11 ;D: 66)  Method: sequencing  Eight families with HBsAg-positive mothers  Seven families with HBsAg-positive fathers  Method: RFLP  n = 262 (A:64; D:70 CH)  Method: INNO-LiPA and sequencing

 20%, 67%, 55% and 45% of genotypes A, B, C, and D patients were infected through perinatal route, respectively  Transmission from HBsAg-positive mothers (vertical): the rate of HBsAg positivity in children was 85.7%  From HBsAg-positive fathers (horizontal): the rate was 65.4%  p = 0.16 (non-significant difference)  5%, 3%, 31%, and 5% of genotype A patients were transmitted through drug use, transfusion, sexual, and vertical routes, respectively  3%, 23%, 9%, 7%, and 14% of genotype D patients were transmitted through drug use, transfusion, sexual, vertical, and transplantation routes, respectively  Genotypes found: C 86%; B 9%; D 2.5%; A 1.0%  97% and 49% of children infected through the vertical route and by other routes (such as father to child or through family members excluding fathers and mothers) were infected with C, respectively  Genotype C children who were maternally infected are much greater in number compared to those with genotypes B, D, or A (p < 0.01)

 Genotypes B, C, and D are more associated with perinatal transmission than A (p < 0.03)  Genotyping with phylogenic analysis could be used to study intrafamilial transmission

 n = 118 (Japanese; 116 CH; 2 AH)  Method: sequencing

 Genotype A is associated with patients infected through sexual contact (p < 0.001), while genotype D through BT (p < 0.001)

 C was found to be more strongly associated with mother-to-child transmission compared to other genotypes

AH, acute hepatitis; BT, blood transfusion; CH, chronic hepatitis; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; RFLP, restriction fragment length polymorphism.

be associated with homosexual or bisexual contact.102 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.101,102 Sa´nchez et al. (n = 67; G:7; sequencing and phylogenetic analysis) reported a significant difference in genotype distribution between two patient groups, the first comprising chronic or acute hepatitis patients and the other comprising men who have sex with men (MSM) (p < 0.001).103 Genotype G was only found

in the MSM group, always co-infected with other genotypes A or H.103 3.4.2. Vertical transmission The correlation between HBV genotypes and perinatal (vertical; mother-to-child) transmission has also been reported. Guo et al. (RFLP and sequencing) reported that 3/7 (42.9%), 3/7 (42.9%), and 1/7 (14%) of the babies infected through vertical transmission were infected with B, C, and D, respectively.104 Erhardt et al. (Table 7) reported that genotypes B, C, and D were more associated with perinatal

Table 8 Correlation between HBV genotypes and transplantation outcomes Study

Patients and methods

Devarbhavi et al. 2002110

 A: 10; C: 6; D: 5 (HBV-related  20%, 50%, and 80% of patients infected with genotypes transplant patients) A, C, and D, respectively, developed recurrent HBV after transplantation  Method: ELISA  Survival rates for patients infected with genotypes A, C, and D were 90%, 83%, and 60%, respectively  HBV recurrence developed after a mean time of 2.7, 10, and 9.1 months post-transplantation for patients infected with genotypes A, C, and D, respectively  90%, 60%, and 50% of patients infected with genotypes A, C, and D, respectively, suffered ACR  n = 81 (Israel; subgroupa)  OLT patients: D 96%; A 4%  Method: RFLP  Stable patients: D 90.3%; A 3.2%; C 6.5%  All patients who suffered recurrence after transplantation were infected with genotype D (NS)  15 genotype A, 13 D, 12 A + D  Genotype D patients were four-folds more likely to die than genotype A patients (NS in multivariate analysis)  Method: INNO-LiPA plus  27%, 54%, and 33% of genotype A, D, and A/D patients, clonal sequencing followed respectively, suffered recurrence (A vs. D, p = 0.14; NS according by phylogenetic analysis to multivariable analysis)b  43 LT genotype B; 74 LT  In genotype B patients, cumulative rates of viral breakthrough 1, 2, genotype C (Hong Kong) and 3 years after transplantation were 0%, 0%, and 4%, respectively, while in genotype C patients were 12%, 18%, and 21%,  Method: sequencing and respectively (p = 0.017) phylogenetic analysis  Liver biopsies after viral breakthrough taken from two genotype B patients showed no histological evidence of recurrent hepatitis and were negative to immunostaining with HBcAg or HBsAg  7/10 genotype C patients had histological recurrence, including two patients with fibrosing cholestatic hepatitis

Ben-Ari et al. 2004112

Girlanda et al. 200462

Lo et al. 2005111

Study findings

Conclusions  Genotype D was associated with higher HBV recurrence and mortality after transplantation compared to genotypes A and C  Genotype A was associated with the most rapid HBV recurrence and a higher ACR compared to genotypes C and D

 No statistically significant difference exists between genotype prevalence in patients who underwent OLT and stable patients  Genotypes A and D do not significantly influence the outcome of LT

 Genotype C is associated with high recurrence rates and severe graft damage due to the emergence of lamivudine-resistant mutants

ACR, acute cellular rejection; ELISA, enzyme linked immunosorbent assay; HBcAg, hepatitis B core antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; LT, liver transplantation ; NS, non-significant; OLT, orthotopic liver transplantation; RFLP, restriction fragment length polymorphism. a Subgroup: 50 OLT patients with HBV DNA negative at the time of transplantation; 31 stable patients with anti-HBe positive. b The only factor that significantly predicted HBV recurrence in this study was HBV replication at the time of liver transplantation (p = 0.03).

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transmission than A.84 Inui et al. (Table 7) also reported a high association between genotype C and vertical HBV transmission.105 3.4.3. Other routes of transmission Recently, Panessa et al. (n = 70 acute hepatitis; D: 44; sequencing and phylogenetic analysis) reported that genotype D was significantly prevalent in injection drug users (p = 0.0025) and previously incarcerated patients (p = 0.0067).106 It is important to note that the link between genotypes and certain transmission routes does not mean that they cannot be transmitted through other routes. 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 In summary, genotype A seems to be linked to bisexual or homosexual contact, genotype G to homosexual (MSM), and D to heterosexual. Genotypes B, C, and probably D have been found to be associated with vertical transmission. Tables 4 and 7 summarize the clinical implications and the correlation between HBV genotypes and modes of transmission. Genotyping together with phylogenetic analysis may also allow a better understanding and tracing of intrafamilial transmission. Lin et al. (Table 7) reported that children infected through close contact with infected fathers had a comparable HBsAg positivity to those infected through vertical transmission.109 This illustrates the high possibility of horizontal transmission between HBsAgpositive fathers and their children, underscoring the importance of child vaccination. 3.5. Liver transplantation A few studies with conflicting results have been published regarding the correlation between HBV genotypes and transplantation outcomes. Devarbhavi et al. (Table 8) reported that genotype D is associated with higher HBV recurrence and mortality after transplantation compared to genotypes A and C.110 Genotype A was associated with the most rapid HBV recurrence rate and a higher acute cellular rejection rate compared to genotypes C and D.110 Lo et al. (Table 8) reported that genotype C was associated with higher recurrence and more severe graft damage compared to genotype B.111 In contrast, other studies have concluded that HBV genotypes do not influence the need for transplantation nor its outcome. BenAri et al. (Table 8) reported no statistically significant difference in genotype prevalence in patients undergoing liver transplantation and stable patients.112 Girlanda et al. (Table 8) reported that genotypes A and D were not significant predictors of transplantation outcomes.61 Tables 4 and 8 summarize the clinical implications and correlation between HBV genotypes and liver transplantation outcomes. 3.6. Occult HBV Occult HBV infection is a special form of hepatitis B infection where patients are negative for HBsAg, while at the same time being positive for HBV DNA. Over the past few years, few studies have investigated the association between HBV genotypes and occult infection. Raffa et al. (n = 18 occult HBV genotyped patients; sequencing) reported that genotypes A and D are highly prevalent in occult HBV infection in HIV patients.113 Pinarbasi et al. (n = 13; occult HBV patients) reported that all HBV occult patients were infected with genotype D.114 4. Conclusions At least 10 HBV genotyping techniques currently exist, of which sequencing is still considered the gold standard method. INNO-

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LiPA may be a suitable alternative, but it is expensive compared to other methods such as multiplex PCR, RFLP, and serotyping. Other genotyping techniques have been developed including oligonucleotide microarray chips, reverse dot blot, RFMP, invader assay, and real-time PCR, and have been found to be highly sensitive. Numerous studies have investigated the influence of HBV genotypes on clinical outcomes. Genotype C is associated with rapid fibrosis development, high HCC development rate, recurrence, and metastasis compared to genotype B. It appears that genotype D may be associated with more severe disease compared to A and that genotype F is linked to high mortality rates. Although HBV genotypes do not significantly influence response to nucleoside/nucleotide antiviral therapy, responsiveness to IFN is clearly affected by genotype. HBeAg-positive patients infected with genotype B have a better response compared to those with genotype C, and genotype A responds better than genotype D, especially during short term therapy. Genotype A also responds well to PEG-IFN. It is still unclear whether genotypes A and D are more prevalent in acute or chronic hepatitis, however genotypes B and C appear to be more prevalent in acute and chronic infection, respectively. Genotype A appears to be linked to bisexual or homosexual contact, genotype G to homosexual (MSM), and D to heterosexual. Genotypes B, C, and probably D are linked to vertical transmission. Very few studies have reported the influence of HBV genotypes on transplantation outcome and occult HBV infection. Studies showing the influence of genotypes E–H and HBV subgenotypes on clinical outcomes are scarce. The Asian-Pacific Association for the Study of the Liver (APASL), European Association for the Study of the Liver (EASL), and American Association for the Study of Liver Diseases (AASLD) guidelines have recommended further studies on the correlation between HBV genotypes and clinical outcomes before using HBV genotyping as a routine test in disease management.115–117 Studies using large well-characterized patient populations that implement a sensitive genotyping technique verified by sequencing are clearly needed to support existing evidence and resolve contradictory findings. Acknowledgements This review was funded by a grant from Yousef Jameel Science & Technology Research Center at AUC to H. Azzazy. We are grateful to Sarah H. Radwan from the Chemistry Department for drawing Figure 1. Conflict of interest: No conflict of interest to declare. References [1] 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]. [2] Hyams KC. Risks of chronicity following acute hepatitis B virus infection: a review. Clin Infect Dis 1995;20:992–1000. [3] Baig S, Siddiqui AA, Ahmed W, Qureshi H, Arif A. The association of complex liver disorders with HBV genotypes prevalent in Pakistan. Virol J 2007;4: 128. [4] Valsamakis A. Molecular testing in the diagnosis and management of chronic hepatitis B. Clin Microbiol Rev 2007;20:426–39. [5] 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]. [6] Feitelson MA, Larkin JD. New animal models of hepatitis B and C. ILAR J 2001;42:127–38. [7] Alam MM, Zaidi SZ, Shaukat S, Sharif S, Angez M, Naeem A, et al. Common genotypes of hepatitis B virus prevalent in injecting drug abusers (addicts) of North West Frontier Province of Pakistan. Virol J 2007;4:63. [8] Orito E, Mizokami M, Ina Y, Moriyama EN, Kameshima N, Yamamoto M, et al. Host-independent evolution and a genetic classification of the hepadnavirus family based on nucleotide sequences. Proc Natl Acad Sci U S A 1989;86: 7059–62.

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