HBV Genotype Research Articles

SAT-190-Specific genetic elements in HBsAg C-terminus profoundly affect HBsAg levels in vivo, hamper HBsAg secretion in vitro and alter HBsAg structural stability in HBeAg-negative chronic HBV genotype D infection

SAT-190-Specific genetic elements in HBsAg C-terminus profoundly affect HBsAg levels in vivo, hamper HBsAg secretion in vitro and alter HBsAg structural stability in HBeAg-negative chronic HBV genotype D infection

CMCdG, a Novel Nucleoside Analog with Favorable Safety Features, Exerts Potent Activity against Wild-Type and Entecavir-Resistant Hepatitis B Virus.

We designed, synthesized, and characterized a novel nucleoside analog, (1S,3S,5S)-3-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-5-hydroxy-1-(hydroxymethyl)-2-methylene-cyclopentanecarbonitrile, or 4'-cyano-methylenecarbocyclic-2'-deoxyguanosine (CMCdG), and evaluated its anti-hepatitis B virus (anti-HBV) activity, safety, and related features. CMCdG's in vitro activity was determined using quantitative PCR and Southern blotting assays, and its cytotoxicity was determined with a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, while its in vivo activity and safety were determined in human liver-chimeric mice infected with wild-type HBV genotype Ce (HBVWTCe) and an entecavir (ETV)-resistant HBV variant containing the amino acid substitutions L180M, S202G, and M204V (HBVETV-RL180M/S202G/M204V). CMCdG potently inhibited HBV production in HepG2.2.15 cells (50% inhibitory concentration [IC50], ∼30 nM) and HBVWTCe plasmid-transfected Huh7 cells (IC50, 206 nM) and efficiently suppressed ETV-resistant HBVETV-RL180M/S202G/M204V (IC50, 2,657 nM), while it showed no or little cytotoxicity (50% cytotoxic concentration, >500 μM in most hepatocytic cells examined). Two-week peroral administration of CMCdG (1 mg/kg of body weight/day once a day [q.d.]) to HBVWTCe-infected human liver-chimeric mice reduced the level of viremia by ∼2 logs. CMCdG also reduced the level of HBVETV-RL180M/S202G/M204V viremia by ∼1 log in HBVETV-RL180M/S202G/M204V-infected human liver-chimeric mice, while ETV (1 mg/kg/day q.d.) completely failed to reduce the viremia. None of the CMCdG-treated mice had significant drug-related changes in body weights or serum human albumin levels. Structural analyses using homology modeling, semiempirical quantum methods, and molecular dynamics revealed that although ETV triphosphate (TP) forms good van der Waals contacts with L180 and M204 of HBVWTCe reverse transcriptase (RT), its contacts with the M180 substitution are totally lost in the HBVETV-RL180M/S202G/M204V RT complex. However, CMCdG-TP retains good contacts with both the HBVWTCe RT and HBVETV-RL180M/S202G/M204V RT complexes. The present data warrant further studies toward the development of CMCdG as a potential therapeutic for patients infected with drug-resistant HBV and shed light on the further development of more potent and safer anti-HBV agents.

From the Editor’s desk…: April 2019

From the Editor’s desk…: April 2019

Patterns of hepatitis B virus S gene escape mutants and reverse transcriptase mutations among genotype D isolates in Jordan.

BackgroundHepatitis B virus (HBV) is an important infectious cause of morbidity and mortality in Jordan. HBV genotype D is the most prevalent in the country. Virus escape mutants in the HBV S region is an important public health problem halting preventive efforts. The aim of the current study was to investigate patterns of HBV escape and resistance mutations and to assess domestic transmission of the virus.MethodsPatients infected with HBV were recruited at Jordan University Hospital (n = 56) and were diagnosed during (1984–2012). A total of 37 partial HBV S sequences were generated using Sanger’s method. Mutation analysis was done using the HIV grade HBV drug resistance interpretation online tool and Geno2pheno (HBV) online tools. Domestic transmission of HBV was assessed using maximum likelihood phylogenetic inference with similar GenBank sequences.ResultsGenotyping revealed an exclusive presence of sub-genotype D1. Typical HBV escape mutants were identified in seven patients. These mutations included: L109R, Q129R, M133L, S143L and D144E with overall prevalence of 18.9% (95% CI [9.5–34.2]). Reverse transcriptase (RT) sequence analysis showed mutations in three patients with overall prevalence of 8.1% (95% CI [2.8–21.3]). RT mutations included: V173L, S202I, L180M, M204V and T184A. Transmission cluster analysis revealed a relatively high proportion of infections taking place as a result of domestic spread (29.7%).ConclusionsBased on our findings, RT mutation analysis appears to be of high value before the initiation of therapy in patients with chronic HBV infection in Jordan. Phylogenetic analyses revealed a considerable proportion of local spread in the country which should be considered in the preventive infection control efforts.

Molecular epidemiology of hepatitis B virus infection in Norway

BackgroundHepatitis B virus (HBV) infection remains a serious global health challenge. The widespread distribution of HBV is highlighted by multiple HBV genotypes associated with different geographical origin and transmission patterns, as well as, clinical outcomes. Investigating population HBV genotype composition and origin is therefore highly warranted.MethodsIn this molecular epidemiological study we analysed 1157 HBV S-gene sequences collected from patients in Norway, primarily in the period 2004–2011, and linked them to epidemiological data from the Norwegian surveillance system for communicable diseases.ResultsOf the patients with reported country of infection (n = 909), 10% (n = 93) were infected in Norway, but the majority (n = 816; 90%) stated that they became infected outside of Norway. Of the patients infected outside of Norway, most became infected in Southeast and East Asia (n = 465; 51%) and Central, West, and North Africa (n = 254; 28%). The distribution of HBV genotypes in Norway is dominated by genotype D (32%) followed by genotype A (22%), B and C (18 and 18%, respectively), and E (7%). Genotype B, C and E were phylogenetically categorized by a majority of sequences originating from distinct geographical regions, either Asia or Africa, whereas genotype A and D originated from multiple geographic regions. However, within genotype A and D, our molecular epidemiology analysis indicated a geographical clustering of sequences depending on their geographical origin.ConclusionsThe majority of HBV patients in Norway became infected outside of Norway and were represented by most common genotypes. Patients stated to have been infected in Norway were found primarily within genotype A and D, and were phylogenetically characterized by both small local clusters and interspersed sequences that clustered with non-Norwegian sequences, indicating that a proportion of the patients assumed to have been infected in Norway likely became infected outside of Norway although assumed the contrary.

A rapid and sensitive recombinase aided amplification assay to detect hepatitis B virus without DNA extraction

BackgroundHepatitis B virus (HBV) infection is the major public health problem worldwide. In clinical practice, serological and molecular assays are the most commonly used diagnostic methods to detect HBV infection in clinical practices.MethodsHere we present a rapid and sensitive recombinase aided amplification assay (RAA) to detect HBV at 39.0 °C for 30 min without DNA extraction from serum samples. The analytical sensitivity of RAA assay was 100 copies per reaction and showed no cross reaction with human immunodeficiency virus (HIV) and hepatitis C virus (HCV). The universality of RAA assay was validated by testing of 41 archived serum samples with predefined HBV genotypes (B, C and D).ResultsA total of 130 archived suspected HBV infected serum samples were detected by commercial qPCR with DNA extraction and RAA assay without DNA extraction (heat-treatment). Compared with qPCR assay as a reference, the RAA assay obtained 95.7% sensitivity and 100% specificity and a kappa value of 0.818.ConclusionsWe developed a rapid, convenient, highly sensitive and specific method to detect HBV without DNA extraction in clinical samples. This RAA method of HBV detection is very suitable for clinical testing.

Correlations between cytokines produced by T cells and clinical-virological characteristics in untreated chronic hepatitis B patients

BackgroundHepatitis B virus (HBV) replicates non-cytopathically in the hepatocytes and HBV-related diseases are caused by immune-mediated inflammatory events. This study aimed to identify the relationship between clinical-virological characteristics and immunity in untreated chronic hepatitis B (CHB) patients.MethodsA total of 209 CHB patients were categorized into immune tolerant (IT, n = 17), inactive carrier (IC, n = 20), immune active (IA, n = 120), and gray zone (GZ, n = 72) phases. The quantitative hepatitis B surface antigen (qHBsAg), hepatitis B e antigen (HBeAg), anti-HBeAg (HBeAb), HBV genotype, viral mutant and frequencies of interleukin (IL)-4, IL-17, IL-10 and interferon-gamma (IFN-γ) produced by CD4+ and CD8+ T cells were tested. We also correlated these cytokines with clinical-virological characteristics using a linear regression model.ResultsCD8+ T cells frequency were significantly decreased in IT patients. Levels of CD4+ T cells IL-4+ or IL-10+ were strongly negatively associated with qHBsAg titers. The frequency of IFN-γ produced by CD4+ and CD8+ T cells showed significant positive association with age and alanine aminotransferase (ALT) level, while that had negative association with qHBsAg titers. Additionally, the ratios of mutations in the HBV precore (PC) stop codon and basal core promoter (BCP) and the combined mutations were 32.5, 27.2, and 11.3%, respectively. The frequency of CD4+ T cells IL-17+ was higher in patients with a PC mutation than that in patients carrying a wild-type sequence. Finally, little associations among T cell derived IL-4, IL-10, IL-17, and IFN-γ was observed in the current untreated CHB cohort.ConclusionsSeveral components of the immune system were correlated with HBV factors that influence an inflammatory process during CHB. Of particular relevance are the significant associations of between CD4+ T cells IL-4+ and qHBsAg level, and between CD4+ T cells IL-17+ and the presence of a mutation in PC.

Key mutations in HBsAg C-terminus correlate with lower HBsAg levels in vivo, hinder HBsAg release in vitro and affect HBsAg structural stability in HBeAg-negative chronic HBV genotype D infection

Key mutations in HBsAg C-terminus correlate with lower HBsAg levels in vivo, hinder HBsAg release in vitro and affect HBsAg structural stability in HBeAg-negative chronic HBV genotype D infection

Molecular Characterization of Coexistence of HBsAg and Anti-HBs in a Patients Infected with HBV Genotype I

Background: Reports on HBV genotype I in Vietnam, Laos, northeastern India, and China are limited and thus, no studies have examined HBV genotype I quasi species with coexistence of HBsAg and anti-HBs. Methods: The current study reports on an HBV genotype I quasi species isolated from a male patient (A02) showing coexistence of HBsAg and anti-HBs. The researchers identified a HBV strain isolated from a CHB patient from Zhaotong city, Yunnan province, China, who was infected with HBV genotype I and presented HBsAg and anti-HBs coexistence. In an effort to elucidate the mechanism underlying the coexistence of HBsAg and anti-HBs in this patient, the researchers performed full-length genome amplification, and the HBV strain was cloned to conduct a comprehensive analysis of mutations and to analyze quasi species variation in relation to the coexistence of HBsAg and anti-HBs. Results: The analysis indicated that the pre-core/core had the highest diversity, and HBsAg and MHBsAg accumulated more nonsynonymous mutations than synonymous mutations. Phylogenetic analyses and genotype identification suggest that A02 was infected with a putative genotype I strain. Unusual deletions of PreS (11 of 16 clones (69%) in PreS1) and amino acid variants of HBsAg (11 of 16 clones; 69%) were observed in the A02 quasi species that were associated with the coexistence of HBsAg and anti-HBs and other clinical features, thus conferring resistance to current antiviral therapies. Further functional studies are needed to assess the correlation between these variants and the coexistence of HBsAg and anti-HBs. Conclusions: The propensity for PreS1 deletions and the complexity of HBsAg variants in HBV isolates from a single patient infected with HBV genotype I could explain the coexistence of HBsAg and anti-HBs and the resistance to current antiviral therapies.

Analysis of serum hepatitis B virus RNA levels in a multiethnic cohort of pregnant chronic hepatitis B carriers

BackgroundHepatitis B virus (HBV) flares have been reported due to alterations in the immune system during pregnancy. Recent studies in non-pregnant chronic hepatitis B (CHB) carriers have indicated that serum HBV RNA is a novel viral marker to assess treatment response and risk of disease flares. ObjectivesTo analyze serum HBV RNA levels in association with established HBV markers in pregnant and/or post-partum CHB carriers. Study designIn this prospective cohort study, serum and plasma were collected from 46 pregnant and/or post-partum CHB patients. Clinical data included demographics, hepatitis B e antigen (HBeAg) status (Abbott), quantitative hepatitis B s antigen (qHBsAg) levels (Abbott), HBV DNA (Abbott, sensitivity 10 IU/mL), alanine aminotransferase (ALT), liver stiffness measurement (LSM, post-partum), and treatment regime. Serum HBV total RNA and pre-genomic (pg)RNA were quantified using in-house assays, and HBV genotype was determined by direct population sequencing of HBV surface gene. Parametric and non-parametric statistical methods were used for analysis. ResultsIn this study, we found that serum HBV total RNA levels correlated with the HBeAg status, HBV DNA, qHBsAg, ALT, and LSM while serum HBV pgRNA levels did not (p < 0.05, N = 46). Additionally, HBV total RNA & pgRNA levels increased, HBV DNA levels decreased, and qHBsAg levels remained unchanged throughout tenofovir disoproxil fumarate (TDF) treatment (N = 2). ConclusionsThe associations between serum HBV total RNA with other validated markers indicates it may be a complementary HBV marker to monitor liver disease and HBV replication during pregnancy.

Ispitivanje markera krvlju prenosivih bolesti, algoritama testiranja i preventivnih mera

Introduction/Aim The most common blood borne diseases or transfusion transmitted infections (TTI) are hepatitis B, hepatitis C and human immunodeficiency virus (HIV). Numerous tests are used to prove the presence of these viruses indirectly (by detecting antibody presence - ELISA and CLIA) or directly (by detecting the presence of viral genome - NAT-PCR and LCR).To present a two-year analysis of testing for blood borne disease markers in pregnant women, patients preparing for surgery procedures and people with high-risk behavior (HBs Ag, anti-HCV and anti-HIV) at the Belgrade City Institute for Public Health and adopting measures for their prevention. Methodology During the two-year period, 11,983 pregnant women and 934 people with high-risk behavior were tested. Preliminary testing for HBs antigen, anti-HCV antibodies and anti-HIV antibodies was performed with 3rd generation ELISA tests ("Hepanostika HBsAg Uni-Form II", "Vironostika HIV Uni-Form II Ag / Ab" ; "The bioMérieux Clinical Diagnostics," manufactured in France; "Hepanostika HCV Ultra" is manufactured by "Beijing United Biomedical Co.Ltd.", China and the other two are manufactured in France). Primarily positive blood samples were tested again with the same test (in a duplicate procedure) on a new sample which was placed in a "dry" tube with no anticoagulant 10-14 days after being primarily positive for TTI markers. Those samples were also tested with a WB "Western Blot" confirmatory test - WB ("INNO-LIA HCV Ab III"; INNO-LIPA HBV Genotyping "and" INNO-LIA HIV I / II"). "Innogenetics NV ", Belgium is the manufacturer of all three confirmatory tests. Blood samples with positive results on the confirmatory tests for TTI were accepted as positive for HBs Ag, anti-HCV or anti-HIV. Results From 11,983 tested pregnant women, 152 (1.27%) were preliminarily reactive to HBsAg, 38 (0.32%) were confirmed positive for HBsAg and 114 (0.95%) were false reactive to HBsAg; 123 (1.03%) were preliminarily reactive to anti-HCV, 35 (0.29%) pregnant women were confirmed positive for antiHCV and 88 (0.74%) were false reactive for anti-HCV; and 15 (0.13%) pregnant women were preliminarily reactive to anti-HIV, 5 (0.04%) pregnant women were confirmed positive for anti-HIV and 10 (0.09%) were false reactive to anti-HIV. From 934 tested individuals with high-risk behavior 36 (3.85%) were preliminarily reactive to HBs Ag, 28 (2.99%) were confirmed positive for HBsAg and 8 (0.86%) were false reactive to HBsAg; 38 (4.07 %) were preliminarily reactive to anti-HCV, 33 (3.53%) were confirmed positive for anti-HCV and 5 (0.54%) false reactive to anti-HCV and 10 (1.07%) were preliminarily reactive to anti-HIV, 9 (0.96%) were confirmed anti-HIV positive and 1(0.11%) was false reactive to anti-HIV. Conclusion The results of our study showed a higher prevalence of HBsAg, anti-HCV and anti-HIV in people with high-risk behavior than in pregnant women. There are more false reactive results in pregnant women than in people with high-risk behavior. Preventive measures would include broader education on ways blood borne diseases are transmitted, testing blood and blood products with the latest generation of tests and correct execution of medical interventions with special attention on principles of asepsis and antisepsis.

Characterization of small genomic regions of the hepatitis B virus should be performed with more caution

BackgroundHepatitis B virus is a hepatotropic DNA virus that reproduces via an RNA intermediate. It can lead to an increased risk of serious liver diseases such as hepatocellular carcinoma and is a serious threat to public health. Currently, the HBV are designated based on greater than 8% nucleotide variation along the whole genome. The recombination of HBV is very common, a large majority of which are recombinants between 2 genotypes. The current work aims to characterize a suspected recombinant involving 3 genotypes.MethodsFifty-seven HBV full-genome sequences were obtained from 57 patients co-infected with HBV and HIV-1 by amplification coupled with sequencing. JpHMM and RDP4 were used to perform recombination analysis respectively. The recombination results of a suspected 3-genotypic recombinant were further confirmed by both maximum likelihood phylogenetic tree and Mrbayes tree.ResultsJpHMM recombination analysis clearly indicated one 3-genotypic HBV recombinant composing of B/C/D. The genotype assignments are supported by significant posterior probabilities. The subsequent phylogenetic analysis of sub-regions derived from inferred breakpoints led to a disagreement on the assignment of D segment. Investigating the conflict, further exploration by RDP4 and phylogenies revealed that the jpHMM-derived 3-genotypic recombinant is actually a B/C genotypic recombinant with C fragment spanning 1899 to 2295 (jpHMM) or 1821 to 2199 (RDP4).ConclusionsThe whole analysis indicated that (i) determination of small genomic regions should be performed with more caution, (ii) combinations of various recombination detection approaches conduce to obtain impartial results, and (iii) a unified system of nomenclature of HBV genotypes is necessary.

Effects of tenofovir and telbivudine on HBV RNA in pregnant women with different genotypes of HBeAg-positive hepatitis B in Guizhou Province

Objective: To investigate whether HBV genotype influences HBV DNA and RNA responses to tenofovir(TDF) and telbivudine(LDT) in pregnant women with HBeAg-positive in Guizhou. Methods: This was a retrospective analysis of 75 pregnant women hepatitis B with HBsAg and HBeAg double-positive(19-38 years old, median age 26 years old), who were enrolled in the Department of Infectious Diseases and Obstetrics Clinic of the Affiliated Hospital of Guizhou Medical University from May 2016 to July 2017.Blood samples were collected at 12-24, 28-32 and 36-40 weeks of pregnancy for analyses of genotype, including hepatitis B surface antigen(HBsAg), hepatitis B e antigen (HBeAg), HBV DNA, HBV RNA and liver function, alanine transaminase(ALT), aspartate transaminase(AST), total bilirubin(TBiL), total bile acids(TBA), cholinesterase(CHE), alkaline phosphatase (ALP). Continuous variable was adopted by means of mean±standard deviation, and categorical variables were used for statistical analysis. Results: The HBV genotype was B in 64.0%(48/75)and C in 36.0%(27/75). The TDF and LDT groups showed no differences in demographic and clinical characteristics, including liver function tests, HBsAg, HBeAg, log(10)HBV DNA and log(10)HBV RNA.TDF groups, pre-treatment: HBV DNA (4.8±2.0), HBV RNA (6.4±1.1); at 4 weeks of treatment: HBV DNA (4.0±0.8), HBV RNA (6.0±0.9); at the end of treatment: HBV DNA (3.1±0.7), HBV RNA (5.5±0.8). LDT groups, pre-treatment HBV DNA (5.1±2.0), HBV RNA(6.5±0.9); at 4 weeks of treatment: HBV DNA (4.4±1.2), HBV RNA(6.5±0.8); at the end of treatment: HBV DNA(3.5±1.2), HBV RNA (6.1±0.7). Compared with pre-treatment (12-24 weeks), the TDF and LDT group showed significant reductions in log(10)(HBV DNA) and log(10)(HBV RNA) at 36-40 weeks ( P<0.05). Under the influence of excluding other variables, the genotype had a certain influence on the HBV RNA load.That was, HBV RNA in patients with the C genome decreased by 0.54 units(log(10)) at the end of the treatment compared to patients with the B genome, and the P value was less than 0.05. Conclusion: B and C genotypes are predominant in pregnant women with hepatitis B in Guizhou Province. B-type viruses are more easily controlled when different genotypes are treated with nucleotide analogues.

Chronic hepatitis B genotype E in African migrants: response to nucleos(t)ide treatment in real clinical practice

BackgroundHepatitis B virus (HBV) genotype E is a poorly studied genotype that almost exclusively occurs in African people. It seems to harbour intrinsic potential oncogenic activity and virological characteristics of immune scape but a paucity of information is available on clinical and virological characteristic of HBV genotype E-infected patients as well as on the efficacy of anti-HBV drugs for such patients. The increasing flow of migrants from high endemic HBV sub-Saharan Africa, where genotype E is the predominant one, to Western countries makes improving such knowledge critical in order to deliver proper medical care.MethodsProspective observational study of naïve patients of sub-Saharan origin treated for chronic HBV genotype E infection at a Tropical Medicine clinic sited in Spain from February 2004 to January 2018. The aim of the study was to describe the response of chronic HBV genotype E infection to nucleos(t)ide analogues (NA), entecavir or tenofovir, in real clinical practice.ResultsDuring the study period, 2209 sub-Saharan patients were assisted at our Tropical Medicine Unit and 609 (27.6%) had chronic HBV (CHB) infection. Genotype information was available for 55 naïve patients initiating treatment with NA (entecavir or tenofovir), 43 (84.3%) of them being genotype E, although 15 were excluded because they did not meet study inclusion criteria. Thus, a total of 28 CHB genotype E patients were included and followed for 24 months at least. Twenty-one patients were in HBeAg-negative chronic hepatitis phase and 7 patients in HBeAg-positive chronic hepatitis phase. After one year of treatment, among those with good adherence, 89.4% (17/19) of the HBeAg-negative patients and 80% of the HBeAg-positive ones had undetectable viral loads. Response rates reached 100% in both groups after 15–18 months of follow-up. Out of the 7 HBeAg-positive patients, 6 (85.7%) presented HBeAg loss in a median time of 31.8 months. Neither serious adverse effects nor hepatocarcinoma cases happened during the study period.ConclusionsHBV genotype may influence disease progression and antiviral response. Our study provides precious information on the efficacy and safety of NA treatment for CHB genotype E infection, a fairly unknown genotype with and increasing epidemiological impact.

Effectiveness of PCR primers for the detection of occult hepatitis B virus infection in Mexican patients.

BackgroundOccult hepatitis B infection (OBI) is defined as the presence of hepatitis B virus (HVB) DNA in the liver of HBsAg negative individuals with or without detectable viral DNA in serum. OBI is a diagnostic challenge as it is characterized by a very low viral load, intermittently detectable through time. Individuals with OBI can develop chronic hepatic disease, including liver cirrhosis and hepatocellular carcinoma. The aim of this work was to produce tools to improve OBI detection of the HVB genotypes prevalent in Mexico.MethodsWe designed and tested primers to detect OBI in serum samples by nested and real-time PCR. Conserved sites in the viral genome were determined by alignment of the most frequent HBV genotypes in Mexico (H, G/H, F and D) and primers spanning the entire viral genome were designed for first round and nested PCR. Primers were tested in serum samples of 45 patients not co-infected with hepatitis C virus or with HIV, out of a group of 116 HBsAg (-)/anti-HBc (+) individuals. Primers were also tested in a control group with chronic HBV. Nested PCR products obtained from HBsAg (-)/anti-HBc (+) were sequenced and used to design primers for real-time PCR (SYBR Green).ResultsThe most effective primer pairs to detect HBV products by nested PCR targeted ORF regions: PreS2/P, S/P, X/PreC, and C; while by real-time PCR they targeted ORF regions PreS2/P, S/P, X, and C. Out of the 45 HBsAg (-)/anti-HBc (+) patients tested, the viral genome was detected in 28 (62.2%) and 34 (75.5%), with nPCR and real-time PCR respectively.ConclusionPrimers designed for real-time PCR detected up to 75.5% of suspected OBI Mexican patients, with or without liver disease, which represents an improvement from previous PCR strategies.

High frequency of drug resistance mutations in the HBV genome in ART-experienced HIV-coinfected patients in southwestern Nigeria.

HBV and HIV infections are highly endemic in sub-Saharan Africa and Nigeria while HBV-HIV coinfection is not uncommon. Antiretroviral (ART)-treatment for HIV can affect HBV whereby antiviral resistance mutations in the HBV genome can be selected. Here, we determined the prevalence of resistance mutations among ART-experienced and ART-naive HIV-HBV-coinfected patients in southwestern Nigeria. A total of 81 serum samples from HBV-HIV-coinfected patients who were either ART-naive or received lamivudine (3TC)-containing ART-therapy and HBV-monoinfected patients were analysed. Hepatitis B surface antigen (HBsAg) was detected using ELISA. HBV-positive samples were confirmed by PCR amplification of the surface and polymerase regions. Mutations conferring drug resistance to HBV were analysed by direct sequencing. Phylogenetic analysis was performed to identify the HBV genotype. Of the 81 HBsAg-positive samples, 27 had detectable HBV DNA by real-time PCR with mean viral loads of 6.77 log IU/ml. Phylogenetic analyses showed a predominance of HBV genotype E. A high prevalence (22.2%; 6/27) of HBV resistance mutations among ART-experienced HBV-HIV-coinfected patients was detected. However, a relatively high selection rate of resistance mutations in drug-naive HIV-HBV-coinfected (3.7%; 1/27) and in HBV-monoinfected patients, potential drug resistance mutations (7.4%; 2/27) were also observed. HBV polymerase amino acid substitutions found included rtV173L, rtL180M, rtM204V, rtK212R, rtS213T, rtV214A, rtL229V and rtP237A/S. Drug resistant mutations were detected frequently in ART-experienced HIV-HBV patients. Well-coordinated antiviral therapy for HIV patients coinfected with HBV should include proper HBV diagnosis and resistance testing to minimize the emergence and spread of antiviral drug resistance.

Hepatitis B virus and hepatitis delta virus subtypes circulating in Algeria and seroprevalence of HDV infection.

Little is known about molecular characteristics of HBV strains circulating in Algeria and there are few data regarding HDV infection. The aim of this study is to describe the genetic diversity of HBV and HDV strains existing in Algeria and to determine the seroprevalence of HDV infection. Plasma samples from 134 patients were analyzed by enzyme immunoassay method for HBV and HDV serological markers. Genotyping of HBV and HDV strains were performed using direct sequencing followed by phylogenetic analyses of the PreS1 and R0 region of the HBV and HDV genome respectively. The PreS1 gene was successfully amplified in 119 patients (82 males and 37 females). Phylogenetic analysis of HBV strains revealed the presence of genotypes D (86.5%) and A2 (11.76%). The subgenotypes D are distributed as follows: HBV/D7 (43.5%), HBV/D3 (24.75%), HBV/D1 (16.8%) and HBV/D2 (14.85%). A recombinant between genotypes A, E and D was found. The seroprevalence of HDV infection among HBV carriers was less than 5.35%. Only one isolate of HDV genotype 1 was identified. Our data indicate the predominance of HBV subgenotype D7 and a low prevalence of HDV infection.

Correlations between serum hepatitis B surface antigen and hepatitis B core antibody titers and liver fibrosis in treatment-naïve CHB patients

BackgroundPrevious studies have revealed that quantitative hepatitis B surface antigen (HBsAg) or hepatitis B core antibody (qAnti-HBc) levels can be used as predictors of treatment response in both interferon-α and nucleoside analogue therapies. Few data have been published regarding the relationship between quantitative HBsAg or Anti-HBc levels and liver fibrosis stages in patients with chronic hepatitis B (CHB). MethodsWe conducted a cross-sectional study of treatment-naïve CHB patients. A total of 624 CHB patients were recruited. We assessed the serum HBsAg and qAnti-HBc levels, HBV DNA levels, HBV genotypes, BCP/PC mutations, histological fibrosis staging by Scheuer classification. ResultsIn HBeAg (+) patients, the S0-1 subjects had significantly higher serum HBsAg and lower qAnti-HBc levels than the S2-4 subjects (both p < 0.001). A moderate inverse correlation was present between serum HBsAg levels and fibrosis scores (r = −0.381, p < 0.001), and a moderate positive correlation was found between qAnti-HBc levels and fibrosis scores (r = 0.408, p < 0.001). In the HBeAg (−) patients, the S0-1 subjects also had significantly lower qAnti-HBc levels than the S2-4 subjects (p < 0.001); however, no significant difference in the HBsAg levels was observed between the S0-1 and S2-4 subjects (p > 0.05). Serum qAnti-HBc levels showed a moderate positive correlation with fibrosis scores (r = 0.383, p < 0.001), while serum HBsAg levels exhibited a low inverse correlation with fibrosis scores (r = −0.171, p < 0.001). Multiple logistic regression analysis showed that the parameters for predicting significant fibrosis (S ≥ 2) included age, PLT, qAnti-HBc levels, HBV genotype and BCP/PC mutations in HBeAg (+) group, and age, PLT, qAnti-HBc levels in HBeAg (−) group (all p < 0.05). The AUC of qAnti-HBc levels associated with the diagnosis of significant fibrosis abnormalities in HBeAg (+) and HBeAg (−) patients were 0.734 (95%CI 0.689 to 0.778) and 0.707 (95%CI 0.612 to 0.801), respectively. ConclusionOur study found an association between high serum qAnti-HBc levels and significant fibrosis in both HBeAg (+) and HBeAg (−) treatment-naïve CHB patients. However, low serum HBsAg levels were correlated with moderate to severe fibrosis in HBeAg (+) subjects only.

Correlation between serum HBV DNA level and HBsAg titer in HBeAg-positive pregnant women and impact of genomic variability of hepatitis B virus pre S/S regions on their correlations

Objective: To analyze the correlation between serum HBV DNA level and HBsAg titer in hepatitis B e antigen positive pregnant women without antiviral therapy, and investigate the impact of genomic variability of preS/S regions on their correlations. Methods: Prenatal serum samples from 882 pregnant women with chronic HBV infection who were positive for HBsAg, HBeAg and HBV DNA and were not on antiviral therapy were included in the analysis. The Abbott i2000 and m2000 systems were used to qualitatively or quantitatively detect HBsAg, HBeAg and HBV DNA levels, respectively. HBV genotyping was performed using a type-specific primer nested polymerase chain reaction (nPCR). In addition, serum samples of pregnant women with HBV DNA levels correlated with HBsAg titer and HBV DNA levels higher than HBsAg titers were used to perform preS/S region amplification by nPCR method. PCR products were directly sequenced and mutation sites were analyzed by MEGA6.0 stasticial software. Mann-Whitney U test was used for the measurement data, and 2-test test for count data. Correlations between variables were analyzed using Spearman's rank correlation. Results: Serum HBsAg titer of HBeAg-positive pregnant women was positively correlated with HBV DNA level (r = 0.754, P < 0.01). Compared with the control group, mutation sites A60V (100% vs. 15.38%, χ(2) = 7.61, P < 0.01), V90A (100% vs. 30.77%, χ(2) = 4.43, P < 0.05) and I161T of HBV preS/S region (80.00% vs. 0, χ(2) = 9.14, P < 0.01) showed a significant decrease in HBsAg titer. Conclusion: Serum HBV DNA levels were positively correlated with HBsAg titer in HBeAg-positive pregnant women. Therefore, serum HBsAg titer may be used as a surrogate marker of serum HBV DNA. Single or multiple amino acid mutations sites A60V, V90A, and I161T in preS/S region may be one of the reasons that lead to a significant drop in HBsAg titer and affect its correlation with HBV DNA levels.

Clinical Features of Hepatitis B e-antigen Positive Chronic Hepatitis B Patients co-existing Precore and/or Basal Core Promoter Mutations

Aim: Characterize the clinical features of treatment-naive hepatitis B e-antigen (HBeAg)-positive chronic hepatitis B (CHB) patients harboring either precore (PC) or basal core promoter (BCP; PC/BCP) mutations. Methods: Alanine aminotransferase, HBsAg, HBV-DNA, HBV genotypes, PC/BCP mutations and liver stiffness measurement were assessed for 186 treatment-naive HBeAg-positive CHB patients. Results: PC/BCP mutations were found in 40.31% of the treatment-naive HBeAg-positive CHB patients. Patients with PC/BCP mutations showed significantly higher alanine aminotransferase and liver stiffness measurement levels. The serum HBsAg and HBV DNA levels in patients with BCP mutations were significantly lower than those in patients with wild-type virus. PC/BCP mutations were more prevalent in patients in immune clearance phase than those in immune tolerance phase. Conclusion: The emergences of PC/BCP mutations possibly indicate that HBeAg positive patients are experiencing the phase of immune clearance.