Phylogenetic analyses of HDV genomes in chronic HBsAg-positive patients in South Central Vietnam.

<p>(<b>a</b>) Representative HDV sequences of South Central Vietnamese HBV-HDV infection patients showing patient-specific HDV isolates. (<b>b</b>) Phylogenetic tree was inferred from distance analysis (Kimura 2 parameters model) and neighbor-joining reconstruction from HDV-region sequences. South Central Vietnamese HDV sequences are referred to as “letter/number”, i.e., “HM115”. The South Central Vietnamese HDV sequences were compared to HDV reference sequences, gathering the eight HDV clades (GenBank accession numbers are denoted in the figure). Phylogenetic analysis of HDV region nt 888 to nt 1122 showed that the HDV sequences were clustered in the Asian HDV-branches 1 and 5.</p

Primers used for HDV detection.

<p>Primers used for HDV detection.</p

Baseline data and clinical characteristics of the HBV patients.

<p>Baseline data and clinical characteristics of the HBV patients.</p

Distribution of HBV and HDV genotypes in HBV-HDV coinfected individuals.

<p>Distribution of HBV and HDV genotypes in HBV-HDV coinfected individuals.</p

Association with HDV genotypes and clinical parameters in 25 HBV-HDV coinfections.

<p>The distribution of HBV-DNA loads (<b>A</b>), ALT levels (<b>B</b>), and AST levels (<b>C</b>) according to HDV genotypes. <i>P</i> values were calculated by Mann-Whitney-Wilcoxon test. NS = not significant.</p

Phylogenetic analyses of HBV genomes in South Central Vietnamese HBsAg-positive patients.

<p>Phylogenetic tree was inferred from distance analysis (Kimura 2 parameters model) and neighbor-joining reconstruction from S gene region of HBV sequence (nt 455–786, numbering is referred to HM011485). South Central Vietnamese HBV sequences are referred to as “letter/number”, i.e., “HM115”. The South Central Vietnamese HBV sequences were compared to HBV reference sequences, gathering the seven HBV genotypes (GenBank accession numbers are denoted in the figure). Phylogenetic analysis of HBV region nt455 to nt 786 showed that the HDV sequences were clustered in the Asian HBV-genotype branches 1 and 4.</p

Nested HDV specific RT-PCR.

<p>(<b>a</b>) Primers for HDV-specific nested PCRs were selected in highly conserved regions of the HDV genome. For detecting HDV RNA genomes, primers HDV57-F and HDV60-R were used for first PCR round, HDV48-F and HDV54-R were used for nested PCR [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175304#pone.0175304.ref034" target="_blank">34</a>]. (<b>b</b>) For HDV genotyping, primers HDV04-F and HDV05-R were used for the first round, primers HDV06-F and HDV07-R were used for nested PCR [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175304#pone.0175304.ref031" target="_blank">31</a>]. The primers matched with reference sequences of eight prototype HDV genotypes retrieved from the NCBI-GenBank. The primer sequences target to two different regions of the HDV genome. The numbers 1 to 8 in (a) and (b) of each reference sequence indicate the respective HDV genotypes 1 to 8. (<b>c</b>) Schematic representation of the HDV genome and primer binding sites. R1, R2 = ribozyme domain; C = C-terminal amino acid extension; A = PolyA. HDAg: hepatitis delta antigen; E = RNA editing site (position at nt 1015) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175304#pone.0175304.ref038" target="_blank">38</a>]. Numbering is according to HDV strain NC1001653. (<b>d</b>) Representative agarose gel electrophoresis of amplified HDV products from nested-PCR using primers indicated in Fig 1B. The final PCR product length is 234 bp. HDV positive samples were identified in lanes 7 and 12. P, positive control was amplified from a full-length HDV plasmid (lane 18). N, negative control (lane 19). M, marker.</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

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