HDV-genotype distribution in Vietnamese HBsAg-positive patients.

<p>(<b>A</b>) Representative HDV sequences of the HBsAg-positive patients aligned with HDV reference sequence NC001653. HDV sequences spanning the region from nt 335 to nt 635 showing patient-specific HDV isolates. (<b>B</b>) Phylogenetic analysis inferred from distance analysis (Kimura 2 parameters model) and neighbor-joining reconstruction from HDV-sequences (nt 888 to nt 1122) of the Vietnamese HDV isolates and the corresponding region of the reference sequences showing that the Vietnamese HDV isolates clustered mainly in the Asian HDV-genotype 1 branch and two isolates (C03 and C06) in the HDV-genotype 2 branch. Vietnamese HDV sequences are referred to as “letter/number”, i.e., “B127”. The Vietnamese HDV sequences were compared to HDV reference sequences, gathering the 8 HDV genotypes which are denoted at the right in brackets (NCBI-Genbank accession numbers are denoted in the figure). The numbers at the nodes indicate bootstrapping values. The bar represents nucleotide substitutions per position.</p

HBV replication of different HBV-genotypes in HBV/HDV coinfection.

<p>(<b>A</b>) Determination of HBV loads of the predominant HBV-genotype A and HBV-genotype C samples in the Vietnamese HDV-positive patients (*denotes p<0.05). (<b>B</b>) Comparison of HBV loads in the HBV-genotype A samples according to the status of HDV-coinfection. The HBV load was closely significantly lower in HDV-positive patients than HDV-negative patients (p = 0.053). p<0.05 is statistically significant.</p

Strategy of primer design and RT-PCR schema.

<p>(<b>A</b>) Primer design for HDV-specific nested RT-PCR located in a highly conserved region of the HDV genome. HDV-specific primers HDV-04 and HDV-05 for the first PCR round, HDV-06 and HDV-07 for the nested PCR were designed for HDV detection and genotyping. The primers were matched and aligned with 38 reference sequences of the eight HDV genotypes available in the NCBI-GenBank. The primer sequences target to the ribozyme and HDAg domains of the HDV genome. The numbers 1 to 8 of each reference sequence code for the respective HDV-genotype. R1, R2 = ribozyme domain; C = C-terminal amino acid extension. HDAg: Hepatitis Delta antigen; position marked at nt 1015 indicates RNA editing site. (<b>B</b>) Schematic representation of primer binding sites. The primers HDV-06 and HDV-07 of the nested PCR used for HDV detection and HDV-genotyping span the region from nt 888 to nt 1122. The HDV-fragment from the position nt 316 to nt 691 is generated by the primer pair HDV-19 and HDV-20. Numbering is according to HDV strain NC1001653. (<b>C</b>) Representative example of 1.5% agarose gelelectrophoresis of amplified HDV products. HDV specific nRT-PCR amplicons of 235 bp are shown. HDV positive samples could be identified in lanes 3, 5, 13, 14, 22, 30, and 32. Positive control (PC) was a HDV full-length plasmid. NC = negative control. Marker (M) is 100bp DNA ladder. </p

Optimisation of quantitative miRNA panels to consolidate the diagnostic surveillance of HBV-related hepatocellular carcinoma

<div><p>Background</p><p>Circulating microRNAs (miRNA) are biomarkers for several neoplastic diseases, including hepatocellular carcinoma (HCC). We performed a literature search, followed by experimental screening and validation in order to establish a miRNA panel in combination with the assessment of alpha-fetoprotein (AFP) levels and to evaluate its performance in HCC diagnostics.</p><p>Methods</p><p>Expression of miRNAs was quantified by quantitative PCR (qPCR) in 406 serum samples from 118 Vietnamese patients with hepatitis B (HBV)-related HCC, 69 patients with HBV-related liver cirrhosis (LC), 100 chronic hepatitis B (CHB) patients and 119 healthy controls (HC).</p><p>Results</p><p>Three miRNAs (mir-21, mir-122, mir-192) were expressed differentially among the studied subgroups and positively correlated with AFP levels. The individual miRNAs mir-21, mir-122, mir192 or the triplex miRNA panel showed high diagnostic accuracy for HCC (HCC vs. CHB, AUC = 0.906; HCC vs. CHB+LC, AUC = 0.81; HCC vs. CHB+LC+HC, AUC = 0.854). When AFP levels were ≤20ng/ml, the triplex miRNA panel still was accurate in distinguishing HCC from the other conditions (CHB, AUC = 0.922; CHB+LC, AUC = 0.836; CHB+LC+HC, AUC = 0.862). When AFP levels were used in combination with the triplex miRNA panel, the diagnostic performance was significantly improved in discriminating HCC from the other groups (LC, AUC = 0.887; CHB, AUC = 0.948; CHB+LC, AUC = 0.887).</p><p>Conclusions</p><p>The three miRNAs mir-21, mir-122, mir-192, together with AFP, are biomarkers that may be applied to improve diagnostics of HCC in HBV patients, especially in HBV-related LC patients with normal AFP levels or HCC patients with small tumor sizes.</p></div

Characteristics of study paticipants according to clinical presentation.

<p>Characteristics of study paticipants according to clinical presentation.</p

Reconstructed <i>CR1</i> haplotype distribution among world populations.

<p>Reconstructed <i>CR1</i> haplotype distribution among world populations.</p

Diagnostic performance of miRNA panels in combination with alpha-fetoprotein in differentiating HCC against control subjects.

<p>Diagnostic performance of miRNA panels in combination with alpha-fetoprotein in differentiating HCC against control subjects.</p

Diagnostics performance of triplex miRNA panel in patients with normal AFP level.

<p>Diagnostic performance of the panel Mir@AFP based on the miRNAs miR-21, miR-122, miR-192 and AFP in differentiating hepatocellular carcinoma with normal AFP levels from other conditions (<20 ng/l). (A): HCC vs. CHB; (B): HCC vs. LC; (C): HCC vs. LC+CHB and (D): HCC vs. (LC+CHB+HC).</p

Differential expression miRNAs and AFP levels in different groups.

<p>Differential expression of miR-21 (A), miR-122 (B) and miR-192 (C) and alpha-fetoprotein (D) in different groups of HBV-related liver diseases including hepatocellular carcinoma (HCC), liver cirrhosis (LC), chronic hepatitis B (CHB) and healthy controls (HC). Numbers in brackets = n of individuals tested. <i>P</i> values were calculated by non-parametric Mann-Whitney U-test for pair-wise comparisons between groups.</p

Diagnostics performance of miRNA panels and in combination with AFP levels.

<p>Diagnostic performance of the triplex miRNA panel based on the miRNAs miR-21, miR-122 and miR-192 levels in differentiating hepatocellular carcinoma from other conditions. (A): HCC vs. CHB; (B): HCC vs. LC; (C): HCC vs. LC+CHB and (D): HCC vs. (LC+CHB+HC).</p