AIMTo detect hyper-conserved regions in the hepatitis B virus (HBV) X gene (HBX) 5’ region that could be candidates for gene therapy.METHODSThe study included 27 chronic hepatitis B treatment-naive patients in various clinical stages (from chronic infection to cirrhosis and hepatocellular carcinoma, both HBeAg-negative and HBeAg-positive), and infected with HBV genotypes A-F and H. In a serum sample from each patient with viremia > 3.5 log IU/mL, the HBX 5’ end region [nucleotide (nt) 1255-1611] was PCR-amplified and submitted to next-generation sequencing (NGS). We assessed genotype variants by phylogenetic analysis, and evaluated conservation of this region by calculating the information content of each nucleotide position in a multiple alignment of all unique sequences (haplotypes) obtained by NGS. Conservation at the HBx protein amino acid (aa) level was also analyzed.RESULTSNGS yielded 1333069 sequences from the 27 samples, with a median of 4578 sequences/sample (2487-9279, IQR 2817). In 14/27 patients (51.8%), phylogenetic analysis of viral nucleotide haplotypes showed a complex mixture of genotypic variants. Analysis of the information content in the haplotype multiple alignments detected 2 hyper-conserved nucleotide regions, one in the HBX upstream non-coding region (nt 1255-1286) and the other in the 5’ end coding region (nt 1519-1603). This last region coded for a conserved amino acid region (aa 63-76) that partially overlaps a Kunitz-like domain.CONCLUSIONTwo hyper-conserved regions detected in the HBX 5’ end may be of value for targeted gene therapy, regardless of the patients’ clinical stage or HBV genotype.

Background: Spontaneous HDV-RNA fluctuations, assessed by nonstandardised inhouse assays, have been reported during the course of chronic hepatitis delta (CHD).Aims: To evaluate changes in serum HDV-RNA concentrations in untreated CHD patients and correlate these changes with other HBV markers.Methods: A total of 323 consecutive serum samples from 56 CHD patients (detectable HDV-RNA) followed for >3 years were retested for HDV-RNA levels by a sensitive technique using the first WHO international HDV-RNA standard. Quantitative HBsAg, HBV-DNA, and HBV-RNA were also determined.Results: Most participants were male, middle-aged, white European, and HBeAgnegative (82%). Almost half had liver cirrhosis and 64% were receiving nucleos(t)ide analogues. At inclusion, median-HDV-RNA was 5.3 (4.2-6.5) log 10 IU/mL, HBsAg 4.0 (3.5-4.3) log 10 IU/mL, and HBV-DNA 1.6 (1.0-2.6) log 10 IU/mL; ALT values were normal in 13 (23%). During a mean follow-up of 5.6 (3-16) years, 14 (25%) showed ≥2log 10 HDV-RNA decline, including 11 (20%) who spontaneously achieved undetectable HDV-RNA. Four patients (7%) lost HBsAg, with undetectable HDV-RNA. The remaining 42 (75%) had persistently detectable HDV-RNA. During follow-up, patients with a ≥2log 10 HDV-RNA decline showed a greater HBsAg drop (−0.7 ± 1.1 vs −0.09 ± 0.9 log IU/mL; P = 0.039) than those with a <2 log 10 HDV-RNA decline. Overall, ALT and HBV-DNA levels decreased over time. There were no differences in clinical outcomes between groups.Conclusions: One-quarter of untreated CHD patients showed a ≥2log 10 decline in HDV-RNA and 20% reached HDV-RNA undetectability during a mean follow-up of | 463 PALOM et AL. How to cite this article: Palom A, Sopena S, Riveiro-Barciela M, et al. One-quarter of chronic hepatitis D patients reach HDV-RNA decline or undetectability during the natural course of the disease.

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