Kathy Jackson

Serum hepatitis B surface antigen and hepatitis B e antigen titers: disease phase influences correlation with viral load and intrahepatic hepatitis B virus markers.

Although threshold levels for hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) titers have recently been proposed to guide therapy for chronic hepatitis B (CHB), their relationship to circulating hepatitis B virus (HBV) DNA and intrahepatic HBV replicative intermediates, and the significance of emerging viral variants, remains unclear. We therefore tested the hypothesis that HBsAg and HBeAg titers may vary independently of viral replication in vivo. In all, 149 treatment‐naïve CHB patients were recruited (HBeAg‐positive, n = 71; HBeAg‐negative, n = 78). Quantification of HBeAg and HBsAg was performed by enzyme immunoassay. Virological characterization included serum HBV DNA load, HBV genotype, basal core promoter (BCP)/precore (PC) sequence, and, in a subset (n = 44), measurement of intrahepatic covalently closed circular DNA (cccDNA) and total HBV DNA, as well as quantitative immunohistochemical (IHC) staining for HBsAg. In HBeAg‐positive CHB, HBsAg was positively correlated with serum HBV DNA and intrahepatic cccDNA and total HBV DNA (r = 0.69, 0.71, 0.76, P < 0.01). HBeAg correlated with serum HBV DNA (r = 0.60, P < 0.0001), although emerging BCP/PC variants reduced HBeAg titer independent of viral replication. In HBeAg‐negative CHB, HBsAg correlated poorly with serum HBV DNA (r = 0.28, P = 0.01) and did not correlate with intrahepatic cccDNA nor total HBV DNA. Quantitative IHC for hepatocyte HBsAg confirmed a relationship with viral replication only in HBeAg‐positive patients. Conclusion: The correlation between quantitative HBsAg titer and serum and intrahepatic markers of HBV replication differs between patients with HBeAg‐positive and HBeAg‐negative CHB. HBeAg titers may fall independent of viral replication as HBeAg‐defective variants emerge prior to HBeAg seroconversion. These findings provide new insights into viral pathogenesis and have practical implications for the use of quantitative serology as a clinical biomarker. (HEPATOLOGY 2010)

Efficacy and safety of tenofovir disoproxil fumarate in pregnancy to prevent perinatal transmission of hepatitis B virus

BACKGROUND & AIMSnPerinatal transmission of hepatitis B virus still occurs despite immunoprophylaxis in approximately 9% of children from highly viraemic mothers. Antiviral therapy in this setting has been suggested, however with limited evidence to direct agent choice.nnnMETHODSnWe conducted a multi-centre, prospective, opt-in observational study of antiviral safety and efficacy in pregnant women with high viral load (>7 log IU/ml); lamivudine was used from 2007 to 2010 and tenofovir disoproxil fumarate (TDF) from late 2010. Outcomes of treated and untreated cohorts were compared.nnnRESULTSn120 women with 130 pregnancies used TDF (58), lamivudine (52 including four who switched due to TDF intolerance) and no therapy (20). 96% were HBeAg positive, with baseline viral load mean 7.8 log IU/ml (±0.72) and ALT median 25 U/L (18.75-33). Duration of antiviral theraphy before birth was mean 58 days (±19) TDF and 53 (±14) lamivudine. Viral load declined by 3.64 log IU/ml (±0.9) TDF and 2.81 log IU/ml (±1.33) lamivudine. Virologic failure (birth viral load >7 IU/ml) occurred in 3% and 18% respectively. Congenital abnormality rate and neonatal growth centiles were similar across cohorts. Perinatal transmission reduced significantly to 2% and 0% in TDF and lamivudine cohorts, compared with 20% in untreated.nnnCONCLUSIONSnTDF in this setting is safe, effective and more potent than lamivudine. Antiviral therapy did not adversely impact obstetric or infant parameters. More TDF intolerance occurred than expected. Perinatal transmission was significantly reduced in antiviral therapy cohorts.

Short duration of lamivudine for the prevention of hepatitis B virus transmission in pregnancy: lack of potency and selection of resistance mutations

This study sought to assess the antiviral efficacy of lamivudine (LMV) administered during third trimester to reduce maternal viraemia and to identify the emergence of LMV resistance. A prospective observational analysis was performed on 26 mothers with high viral load (>107 IU/mL). Twenty‐one women received LMV (treated group) for an average of 53 days (range 22–88 days), and the remaining five formed the untreated control group. Serum samples from two time points were used to measure HBV DNA levels and antiviral drug resistance. The LMV‐treated women achieved a median HBV DNA reduction of 2.6‐log10 IU/mL. Although end‐of‐treatment (EOT) HBV DNA in four (18%) LMV‐treated women remained at >107 IU/mL (±0.5 log IU/mL), no mother‐to‐baby transmission was observed. In contrast, a baby from the untreated mother was HBsAg positive at 9 months postpartum. Four technologies were used for drug resistance testing. Only ultra‐deep pyrosequencing (UDPS) was sufficiently sensitive to detect minor viral variants down to <1%. UDPS showed that LMV therapy resulted in increased viral quasispecies diversity and positive selection of HBV variants with reverse transcriptase amino acid substitutions at sites associated with primary LMV resistance (rtM204I/V and rtA181T) in four (19%) women. These viral variants were detected mostly at low frequencies (0.63–5.92%) at EOT, but one LMV‐treated mother had an rtA181T variant that increased from 2.2% pretherapy to 25.59% at EOT. This mother was also infected with the vaccine escape variant (sG145R), which was inhibited by LMV treatment. LMV therapy during late pregnancy only reduced maternal viraemia moderately, and drug‐resistant viral variants emerged.

Anti-viral therapy for prevention of perinatal HBV transmission: extending therapy beyond birth does not protect against post-partum flare

Antepartum anti‐viral therapy (AVT) is often administered to prevent perinatal transmission of hepatitis B virus (HBV) infection. Little is known about the effect of AVT on post‐partum flare rates and severity.

Molecular virology of hepatitis B virus, sub‐genotype C4 in northern Australian Indigenous populations

Indigenous Australians experience a significant health burden from chronic hepatitis B infection; however, the strain of hepatitis B virus (HBV) found among Indigenous Australians has not been well characterized. Blood samples were collected from 65 Indigenous Australians with chronic HBV infection from across the Top End of Australias Northern Territory. Phylogenetic analysis of HBV from these samples revealed that 100% of the isolates were genotype C, sub‐genotype C4, expressing the serotype ayw3. This strain is a divergent group within the HBV/C genotype, and has only been described in Indigenous Australians. Evidence of recombination was suggested by discordant phylogenetic clustering of the C4 sequences when comparing the full genome to the surface region and confirmed by recombination analysis which showed the surface gene region to be most closely related to genotype J, while the remaining regions of the genome were most similar to genotype C sequences. Mutational analysis revealed the presence of multiple mutations that have been linked with more rapid liver disease progression and an increased risk of hepatocellular carcinoma. These mutations were detected in the majority of sequences examined. Variants associated with vaccine failure were detected as the predominant viral quasi‐species in 3/35 samples. In summary, the HBV C4 variant found in this population has a high potential to cause advanced liver disease and to escape vaccination programs. Further in vitro functional and natural history studies are warranted in order to determine the clinical and public health consequences of infection with the HBV C4 variant in these communities. J. Med. Virol. 86:695–706, 2014.

In Vitro Studies Show that Sequence Variability Contributes to Marked Variation in Hepatitis B Virus Replication, Protein Expression, and Function Observed across Genotypes.

ABSTRACT The hepatitis B virus (HBV) exists as 9 major genotypes (A to I), one minor strain (designated J) and multiple subtypes. Marked differences in HBV natural history, disease progression and treatment response are exhibited by many of these genotypes and subtypes. For example, HBV genotype C is associated with later hepatitis B e antigen (HBeAg) seroconversion and high rates of liver cancer compared to other HBV genotypes, whereas genotype A2 is rarely associated with HBeAg-negative disease or liver cancer. The reasons for these and other differences in HBV natural history are yet to be determined but could in part be due to sequence differences in the HBV genome that alter replicative capacity and/or gene expression. Direct comparative studies on HBV replication and protein expression have been limited to date due largely to the absence of infectious HBV cDNA clones for each of the HBV genotypes present in the same genetic arrangement. We have produced replication-competent infectious cDNA clones of the most common subtypes of genotypes A to D, namely, A2, B2, C2, D3, and the minor strain J, and compared their HBV replication phenotype using transient-transfection models. We identified striking differences in HBV replicative capacity as well as HBeAg and surface (HBsAg) protein expression across genotypes, which may in part be due to sequence variability in regulatory regions of the HBV genome. Functional analysis showed that sequence differences in the major upstream regulatory region across genotypes impacted promoter activity. IMPORTANCE There have been very few studies directly comparing the replication phenotype of different HBV genotypes, for which there are marked differences in natural history and disease progression worldwide. We have generated replication-competent 1.3-mer cDNA clones of the major genotypes A2, B2, C2, and D3, as well as a recently identified strain J, and identified striking differences in replicative capacity and protein expression that may contribute to some of the observed differences in HBV natural history observed globally.

Molecular epidemiology of hepatitis delta virus in the Western Pacific region

BACKGROUNDnHepatitis delta virus (HDV) is a defective RNA virus requiring the presence of the hepatitis B virus (HBV) for the completion of its life cycle. Active replication of HDV can lead to severe hepatitis, and although present worldwide has an irregular geographical distribution, especially in the Asian Pacific region.nnnOBJECTIVESnThe aim of this study was to determine the prevalence and molecular epidemiology of HDV isolates in Oceania following the 1998 evaluation of the hepatitis B vaccine program.nnnSTUDY DESIGNnSera collected from 184 hepatitis B surface antigen (HBsAg) positive Pacific Islanders living in Micronesia, Polynesia and Melanesia were tested for HDV RNA.nnnRESULTSnTwenty of 54 patients with chronic hepatitis B (CHB) from Kiribati were positive for serum HDV RNA (37%), whilst sera from patients with CHB from Tonga (59), Fiji (42) and Vanuatu (29) were negative. The mean HDV RNA load for the 20 samples was 7.00log10copies/mL. Phylogenetic analysis revealed that the Kiribati HDV isolates were of genotype 1 and clustered with a previously published isolate from Nauru forming a distinct clade of Pacific HDV. All Micronesian isolates contained a serine at codon 202 of large hepatitis delta antigen (L-HDAg) demonstrating possible relatedness to strains of HDV-1 of African origin.nnnCONCLUSIONSnThis study has confirmed endemic HDV infection in Micronesia and identified Kiribati as having amongst the highest prevalence for HDV viraemia in patients with CHB. Further investigations are ongoing into the origins of this unique HDV Pacific strain, and its inter-relationship with HBV.

Clinical significance of hepatitis B virion and SVP productivity: relationships between intrahepatic and serum markers in chronic hepatitis B patients

Background Clinical use of hepatitis B viral (HBV) quantitative seromarkers remains questionable since it is not precisely known whether they represent intrahepatic viral replication. Covalently closed circular DNA (cccDNA), relaxed circular DNA (rcDNA), and pregenomic RNA (pgRNA) are more likely to represent active HBV replication and their measurement can be used to derive virion productivity (VP; rcDNA/cccDNA), subviral particle (SVP) productivity (quantitative HBsAg/cccDNA), and replicative activity (RA; pgRNA/cccDNA). These can be used to compare relative HBV replication between HBeAg-negative and -positive patients. Objective To study the clinical significance of intrahepatic HBV replication phenomenon between HBeAg-negative and -positive patients and its correlation with quantitative HBV seromarkers. Method This was a prospective study between January 2010 and December 2011. Study subjects were naive chronic hepatitis B patients from Cipto Mangunkusumo and Medistra Hospitals. All patient samples underwent liver biochemistry and HBV seromarkers testing (HBeAg, quantitative HBsAg and HBV DNA levels), and patients underwent liver biopsy. Stored liver specimens were analysed for intrahepatic rcDNA, cccDNA, and pgRNA with quantification performed by real-time PCR. Comparison of HBV markers between HBsAg-positive and -negative patients was carried out using the Mann–Whitney U-test. Pearson’s correlation test was performed among HBV intrahepatic and seromarkers using their log-transformed values. Results A total of 104 patients were enrolled in this study; 54 (51.9%) were male. Patients’ mean age was 41.9u2009±u200911.63 years (range 19–70 years). Sixty-one patients (58.7%) were HBeAg-negative. All HBV markers were significantly higher in HBeAg-positive than HBeAg-negative patients, except for SVP productivity and RA. Serum HBV DNA was strongly correlated with intrahepatic total HBV DNA (ru2009=u20090.771), cccDNA (ru2009=u20090.774), and rcDNA (ru2009=u20090.780) while serum quantitative HBsAg showed only moderate correlation with intrahepatic total DNA (ru2009=u20090.671), cccDNA (ru2009=u20090.632), rcDNA (ru2009=u20090.675), and SVP productivity (ru2009=u20090.557). Conclusions Serum HBV DNA concentration and quantitative HBsAg might not accurately predict intrahepatic viral activity. Virion and SVP production do not occur in parallel with replicative activity.

HBeAg levels at week 24 predict response to 8 years of tenofovir in HBeAg‐positive chronic hepatitis B patients

Hepatitis B e antigen (HBeAg) seroconversion is a treatment endpoint for HBeAg‐positive CHB, and a necessary precursor to HBsAg loss. Biomarkers that predict serological outcomes would be useful.

Mother to Child Transmission of Hepatitis B: Examining Viral Cut Offs, Maternal HBsAg Serology and Infant Testing

Antipartum antiviral therapy in the setting of high viral load is recommended to prevent mother‐to‐child transmission of hepatitis B although recommended viral load cut‐offs vary. Quantitative HBsAg has been proposed as an alternative screening strategy to identify high viral load in this setting. Guidelines suggest testing all infants for vaccine response and infection. We set out to re‐examine viral load cut‐offs; the predictive value of quantitative HBsAg and the need for follow‐up infant testing in our cohort.