Prevalence and genotype distribution of hepatitis C virus within hemodialysis units in Thailand: role of HCV core antigen in the assessment of viremia.

BACKGROUND: Individuals with end-stage renal disease have a higher risk of hepatitis C virus (HCV) acquisition during long-term hemodialysis (HD). Our report was designed to investigate HCV prevalence and genotype, in addition to the clinical use of HCV core antigen (HCVcAg), within multiple HD facilities in Thailand. METHODS: This cross-sectional report was investigated between January and June 2019. HCV infection was assessed by anti-HCV and confirmed active infection by measuring HCV RNA and HCVcAg. HCV genotype was determined by phylogenetic analysis using nucleotide sequences of NS5B region. RESULTS: Overall, 140 of 3,305 (4.2%) patients in 15 dialysis centers had anti-HCV positive. Among them, HCV RNA was further assessed in 93 patients and was detectable in 59 (63.4%) persons. Considering HCV viremia, HCVcAg measurement exhibited high accuracy (96.8%), sensitivity (94.9%) and specificity (100%) in comparison with HCV RNA testing. Moreover, individuals infected with HCV received a longer duration of dialysis vintage when compared to anti-HCV negative controls. The major sub-genotypes were 1a, 1b, 3a, 3b, 6f and 6n. Regarding phylogenetic analysis, there were 7 clusters of isolates with high sequence homology affecting 17 individuals, indicating possible HCV transmission within the same HD centers. CONCLUSIONS: HCV frequency and common sub-genotypes in HD centers were different from those found in the Thai general population. HCVcAg might be an alternate testing for viremia within resource-limited countries. Enhanced preventive practices, dialyzer reuse policy and better access to antiviral therapy are crucial for HCV micro-elimination within HD facilities.

Plasma B-cell activating factor levels and polymorphisms in hepatitis B-related hepatocellular carcinoma: Clinical correlation and prognosis.

BACKGROUND: B-cell activating factor (BAFF), an important cytokine for B lymphocyte activation, has been shown to be increased in chronic hepatitis B virus (HBV) infection. OBJECTIVE: This study aimed at evaluating clinical correlation and prognostic role of plasma BAFF and related polymorphisms in patients with HBV-related hepatocellular carcinoma (HCC). METHODS: Plasma BAFF levels were measured from 100 healthy controls and 490 patients with chronic HBV infection (200 with HCC and 290 without HCC). The rs9514828 and rs12583006 polymorphisms were determined by allelic discrimination. RESULTS: The HCC group had significantly higher BAFF levels compared with the non-HCC group and healthy controls. Among the non-HCC group, the HBeAg-positive subgroup had higher BAFF levels compared with the HBeAg-negative subgroup. In the HCC group, high BAFF levels at initial presentation significantly correlated with alpha-fetoprotein levels, Child-Pugh classification, tumor size and BCLC stage. Multivariate analyses showed that elevated BAFF concentration (± 1,100 pg/ml) was a significant and independent prognostic factor of overall survival in patients with HCC (OR = 2.28, 95%CI: 1.07-4.87; P = 0.034). HCC patients with high BAFF levels (± 1,100 pg/ml) had a poorer median survival than those with low levels (P < 0.001, log-rank test). Regarding BAFF polymorphisms, the frequency of rs9514828 CT + TT genotypes was higher distributed in patients with chronic HBV infection compared with healthy controls (58.0% vs. 46.0%, P = 0.029). CONCLUSIONS: Our data demonstrate for the first time that elevated plasma BAFF levels at baseline exhibit clinical correlation in terms of disease severity and overall survival in HCC patients. Thus, plasma BAFF at initial diagnosis could serve as a prognostic marker for HBV-related HCC.

Circulating BAFF and CXCL10 levels predict response to pegylated interferon in patients with HBeAg-positive chronic hepatitis B.

BACKGROUND: B-cell activating factor (BAFF), an essential cytokine for B lymphocytes activation, has been implicated in the pathogenesis of chronic viral hepatitis. However, the role of BAFF in patients with chronic hepatitis B (CHB) undergoing antiviral therapy is unknown. METHODS: Patients with HBeAg-positive CHB treated with 48-week pegylated interferon (PEG-IFN; n = 42), who had stored plasma samples during treatment were recruited. Serial plasma levels of BAFF and C-X-C motif chemokine 10 (CXCL10) during therapy were measured. RESULTS: Combined response (CR), defined as HBeAg seroconversion with HBV DNA < 2,000 IU/mL plus HBsAg decline ≥ 1 log10 IU/mL at 24 weeks post-treatment, was achieved in 11 (26.2%) patients. BAFF levels were elevated during treatment but decreased to pre-treatment levels after PEG-IFN cessation in both responders and non-responders. Low baseline BAFF (< 770 pg/ml) and high CXCL10 (≥ 320 pg/ml) levels were independently associated with CR in multivariate analysis. Baseline CXCL10/BAFF ratio of ≥ 0.45 was predictive of CR with positive and negative predictive values of 61.5 and 89.7%, respectively. CONCLUSIONS: In summary, low baseline BAFF and high CXCL10 levels were associated with treatment response to PEGIFN. The combined measurement of these immune markers may help individualized decision-making in patients with HBeAg-positive CHB.

Large transmission cluster of acute hepatitis C identified among HIV-positive men who have sex with men in Bangkok, Thailand.

A rapidly emerging and highly concentrated hepatitis C virus (HCV) outbreak has recently been observed among both acute and chronic HIV-positive men who have sex with men (MSM) in Bangkok, Thailand. NS5B regions of the HCV genome were amplified using nested PCR and sequenced. Phylogenetic inference was constructed by Maximum Likelihood methods and clusters were identified with support and genetic distance thresholds of 85% and of 4.5%. Forty-eight (25 acute HIV and 23 chronic HIV) MSM with incident HCV infection were included in the analysis. HCV genotype (GT) was 85% GT 1a and 15% GT 3a or 3b. Median age at HCV diagnosis was 34 (interquartile range, 28-41) years. 83.3% (40/48) had history of syphilis infection and 36% (16/44) reported crystal methamphetamine use. Only 2 (4%) reported ever injecting drugs, both crystal methamphetamine. In the phylogenetic clustering analysis, 83% belonged to one of two clusters: one large (75%) and one small (8%) cluster. All clusters were GT 1a. MSM with acute HIV infection were more likely to be in a cluster (92%) than those with chronic infection (74%). HCV screening should be regularly performed for MSM in ART clinics, and offering direct-acting antiviral agents to all MSM with HCV infection might contain the HCV epidemic from expanding further.

Decreased of BAFF-R expression and B cells maturation in patients with hepatitis B virus-related hepatocellular carcinoma.

BACKGROUND: Recent evidence has indicated the role of B cells and B cell-activating factor (BAFF) in the development of hepatocellular carcinoma (HCC). AIM: To characterize circulating BAFF receptor expression and B cell subpopulations in patients with hepatitis B virus (HBV)-related HCC. METHODS: Peripheral blood samples collected from 41 patients with chronic HBV infection (25 patients without HCC and 16 patients with HCC) and 9 healthy controls were assessed for BAFF receptors [BAFF-R(B cell-activating factor receptor), transmembrane activator and cyclophilin ligand interactor, B-cell maturation antigen] and B cell subpopulations by multicolor flow cytometry. RESULTS: The frequency of BAFF-R expressing B cells to total B cells was significantly lower in patients with HCC (3.39% ± 2.12%) compared with the non-HCC group (5.37% ± 1.90%) and healthy controls (6.23% ± 2.32%), whereas there was no difference in transmembrane activator and cyclophilin ligand interactor and B-cell maturation antigen. The frequencies of CD27+IgD+ memory B cells, CD27+IgD- class-switched memory B cells and plasmablasts were significantly lower in the patients with HCC compared to patients without HCC (1.23 ± 1.17 vs 3.09 ± 1.55, P = 0.001, 0.60 ± 0.44 vs 1.69 ± 0.86, P < 0.0001 and 0.16 ± 0.12 vs 0.37 ± 0.30, P = 0.014, respectively). However, the ratio of naïve and transitional B cell did not differ significantly between the three groups. In addition, decreased BAFF-R expression on B cells was significantly correlated with large tumor size and advanced tumor stage. CONCLUSION: Our data demonstrated BAFF-R expression was reduced in B cells that involved with the frequencies of B cells maturation in patients with HCC. The depletion of BAFF-R might play an important role in the development of HCC in patients with chronic HBV infection.

Successful direct-acting antiviral therapy improves circulating mucosal-associated invariant T cells in patients with chronic HCV infection.

OBJECTIVES: Mucosal-associated invariant T (MAIT) cells have been shown to contribute in the pathogenesis of various liver diseases, including chronic hepatitis C virus (HCV) infection. This study was aimed at investigating the frequency, phenotype, and function of circulating MAIT cells, as well as their alterations after successful direct-acting antivirals (DAAs) in HCV-infected patients with or without HIV infection. METHODS: A total 85 patients (51 HCV-monoinfection and 34 HCV/HIV-coinfection), who received elbasvir/grazoprevir from a clinical trial and 20 healthy controls were included. MAIT cells in blood were characterized using flow cytometry at baseline and 24 weeks post-treatment. RESULTS: HCV-monoinfected and HCV/HIV-coinfected patients achieved similar sustained virological response rates (SVR24, 94.1% vs. 97.1%). Circulating MAIT cells in the monoinfection and coinfection groups were presented at low frequencies in comparison with healthy controls (median, 1.1% vs. 1.1% vs. 2.4%, P<0.001) and exhibited features of chronic activation and impaired functional capacity. A negative correlation between circulating MAIT cell frequency and liver stiffness assessed by magnetic resonance elastography was observed. Compared with baseline, increased in circulating MAIT cells after successful DAA therapy was mainly detected in HCV-monoinfected patients compared with HCV/HIV-coinfected individuals. Moreover, MAIT cell restoration was predominantly observed among patients with significant fibrosis to cirrhosis (F2-F4). CONCLUSIONS: These data indicated that dysregulation of MAIT cells might play a role in the progression of chronic HCV infection. Partial restoration of MAIT cell frequency and function was observed after successful DAA therapy, particularly in HCV-monoinfected patients.

Independent and additive effects of PNPLA3 and TM6SF2 polymorphisms on the development of non-B, non-C hepatocellular carcinoma.

BACKGROUND: This study was aimed at evaluating the association between single nucleotide polymorphisms (SNPs) in the PNPLA3, NCAN, TM6SF2 and MBOAT7 and hepatocellular carcinoma (HCC) development in Thai patients according to underlying etiologies of liver disease. METHODS: These SNPs were determined by allelic discrimination in blood samples of 105 healthy controls and 530 patients with HCC [270 with hepatitis B virus (HBV-HCC), 131 with hepatitis C virus (HCV-HCC), and 129 with non-B, non-C HCC (NBNC-HCC) matched for age and gender]. RESULTS: G allele of PNPLA3 rs738409 variant was significantly higher in NBNC-HCC (49%) compared to healthy controls (32%), HBV-HCC (32%) and HCV-HCC (31%) (P < 0.001). T allele of TM6SF2 rs58542926 was more prevalent in NBNC-HCC (24%) than in healthy controls (8%), HBV-HCC (10%) and HCV-HCC (12%) (P < 0.001). The distribution of NCAN (rs2228603) and MBOAT7 (rs641738) was not different between groups. In multivariate logistic regression analysis, PNPLA3 rs738409 (OR 2.06, 95% CI 1.24-3.43; P = 0.005) and TM6SF2 rs58542926 (OR 2.22, 95% CI 1.34-3.65; P = 0.002) were independently associated with NBNC-HCC compared to viral-related HCC (VR-HCC). The proportion of patients with NBNC-HCC increased significantly along with the increase of the number of risk alleles. There was no association between these SNPs and overall survival in patients with HCC. CONCLUSIONS: These data showed that PNPLA3 and TM6SF2 polymorphisms were independently linked to NBNC-HCC but not HBV- or HCV-HCC in Thai populations. In addition, the risk genotypes might interact with each other through tumor development in patients with NBNC-HCC.

Association of vitamin-D-related genetic variations and treatment response to pegylated interferon in patients with chronic hepatitis B.

BACKGROUND: Vitamin D, a potent immune-modulator, has been linked to the pathogenesis of chronic hepatitis B (CHB). This study was aimed at investigating the association between single nucleotide polymorphisms (SNPs) in vitamin-D-related genes and treatment response to pegylated interferon (PEG-IFN) in patients with CHB. METHODS: A total of 275 Thai patients (122 hepatitis B e antigen [HBeAg]-positive and 153 HBeAg-negative CHB) treated with 48-week PEG-IFN were recruited. Virological response (VR) at 48 weeks post-treatment was defined as HBeAg seroconversion plus HBV DNA <2,000 IU/ml for HBeAg-positive CHB and HBV DNA <2,000 IU/ml for HBeAg-negative CHB. The SNPs VDR (rs2228570), DBP (rs7041) and CYP27B1 (rs4646536) were analysed. RESULTS: The distribution of TT, CT and CC genotypes of rs4646536 in this cohort was 21.8%, 46.2% and 32.0%, respectively. There was no difference in its distribution according to HBeAg status. In HBeAg-positive CHB, patients with TT genotype, compared with non-TT genotype, achieved higher VR (53.3% versus 31.5%; P=0.032) and hepatitis B surface antigen (HBsAg) clearance (20.0% versus 5.4%; P=0.016). In HBeAg-negative CHB, the corresponding figures were 60.0% versus 30.9% (P=0.003) and 16.7% versus 5.7% (P=0.045), respectively. Patients with TT genotype had more rapid HBsAg decline than those with non-TT genotype. However, SNPs rs2228570 and rs7041were not associated with VR and HBsAg clearance. Logistic regression analysis demonstrated that SNP rs4646536 and baseline HBsAg level were independent predictors of VR in both HBeAg-positive and HBeAg-negative CHB. CONCLUSIONS: Our data suggest that SNP rs4646536 in the CYP27B1 gene is a predictive factor of response to PEG-IFN therapy in Thai patients with CHB.

Association of PINX1 but not TEP1 Polymorphisms with Progression to Hepatocellular Carcinoma in Thai Patients with Chronic Hepatitis B Virus Infection.

Hepatocellular carcinoma (HCC) is major health problem with high mortality rates, especially in patients with hepatitis B virus (HBV) infection. Telomerase function is one of common mechanisms affecting genome stability and cancer development. Recent studies demonstrated that genetic polymorphisms of telomerase associated genes such as telomerase associated protein 1 (TEP1) rs1713449 and PIN2/TERF1-interacting telomerase inhibitor 1 (PINX1) rs1469557 may be associated with risk of HCC and other cancers. In this study, 325 patients with HCC and 539 non-HCC groups [193 healthy controls, 80 patients with HBV-related liver cirrhosis (LC) and 266 patients with HBV-related chronic hepatitis (CH)] were enrolled to explore genetic polymorphisms of both SNPs using the allelic discrimination method based on MGB probe TaqMan real time PCR. We demonstrated that all genotypes of both genes were in Hardy-Wienberg equilibrium (>0.05). Moreover, there was no significant association between rs1713449 genotypes and HCC risk, HCC progression and overall survival (>0.05). Interestingly, we observed positive association of rs1469557 with risk of HCC when compared with the LC group under dominant (CC versus CT+TT, OR=1.89, 95% CI= 1.06-3.40, P=0.031) and allelic (C versus T alleles, OR=1.75, 95% CI=1.04-2.94, P=0.033) models, respectively. Moreover, overall survival of HCC patients with CC genotype of rs1469557 was significantly higher than non-CC genotype (Log-rank P=0.015). These findings suggest that PINX1 rs1469557 but not TEP1 rs1469557 might play a role in HCC progression in Thai patients with LC and be used as the prognosis marker to predict overall survival in HCC patients.

Association of interferon-gamma inducible protein 10 polymorphism with treatment response to pegylated interferon in HBeAg-positive chronic hepatitis B.

BACKGROUND: Interferon-gamma inducible protein 10 (IP-10) plays an important role in the clinical outcome of chronic hepatitis B (CHB). This study aimed to investigate the association between single nucleotide polymorphisms (SNPs) G-201A of the IP-10 gene and treatment response to pegylated interferon (PEG-IFN) in patients with hepatitis B e antigen (HBeAg)-positive CHB. METHODS: We retrospectively analysed data of patients with HBeAg-positive CHB treated with PEG-IFN for 48 weeks. Virological response (VR) was defined as HBeAg clearance and HBV DNA <2,000 IU/ml at 24 weeks post-treatment. The SNPs G-201A, IFNL3 (rs12979860) and HLA-DPA1 (rs3077) were assessed. RESULTS: Among 107 patients, VR was achieved in 45 (42.1%) patients. Hepatitis B surface antigen (HBsAg) clearance and decline (<100 IU/ml) were observed in 10 (9.3%) and 22 (20.6%) patients, respectively. The distribution of GG, GA and AA genotypes of G-201A was 76.6%, 19.6% and 3.7%, respectively. Patients with GG genotype, compared to those with non-GG genotype, achieved higher VR rate (48.8% versus 19.2%; P=0.011), decreased HBsAg (25.6% versus 4.0%; P=0.019), and demonstrated a trend in HBsAg clearance (11.0% versus 4%; P=0.294). Patients with GG genotype had more rapid HBsAg decline and higher baseline serum IP-10 levels than those with non-GG genotype (432.2 ±339.0 versus 257.3 ±145.7 pg/ml; P=0.028). SNPs rs12979860 and rs3077 were not associated with VR. Logistic regression analysis suggested that SNP G-201A was an independent predictor of VR (odds ratio 3.81, 95% CI 1.31, 11.12; P=0.014). CONCLUSIONS: Data from this study demonstrated for the first time that IP-10 polymorphism is independently associated with treatment response to PEG-IFN in patients with HBeAg-positive CHB.

Association of PNPLA3 Polymorphism with Hepatocellular Carcinoma Development and Prognosis in Viral and Non-Viral Chronic Liver Diseases.

BACKGROUND: The aim of this study was to evaluate any association between a single nucleotide polymorphism (SNP) in the patatin-like phospholipase domain containing 3 (PNPLA3) (rs738409, C>G) and the development and prognosis in patients with hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Two hundred heathy controls and 388 HCC cases were included: 211 with HBV, 98 patients with HCV, 29 with alcoholic steatohepatitis (ASH) and 52 with non-alcoholic steatohepatitis (NASH). The SNP was determined by real-time PCR based on TaqMan assays. RESULTS: The prevalence of rs738409 genotypes CC, CG and GG in controls was 91 (45.5%), 88 (44.0%), and 21 (10.5%), respectively, while the corresponding genotypes in all patients with HCC was 158 (40.7%), 178 (45.9%), and 52 (13.4%). The GG genotype had significantly higher distribution in patients with ASH/NASH-related HCC compared with controls (OR=2.34, 95% CI=1.16-4.71, P=0.018), and viral-related HCC cases (OR=2.15, 95% CI=1.13-4.08, P=0.020). However, the frequency of the GG genotype was similar between controls and patients with viral-related HCC. At initial diagnosis, HBV-related HCC were larger and at more advanced BCLC stage than the other HCC groups. There were no significant differences between the GG and non-GG groups regarding clinical characteristics, tumor stage and overall survival. CONCLUSIONS: These data suggest an influence of the PNPLA3 polymorphism on the occurrence of HCC in patients with ASH/NASH but not among those with chronic viral hepatitis. However, the polymorphism was not associated with the prognosis of HCC.

Diagnostic and Prognostic Roles of Serum Osteopontin and Osteopontin Promoter Polymorphisms in Hepatitis B-related Hepatocellular Carcinoma.

BACKGROUND: The aims of this study were to evaluate the diagnostic and prognostic roles of serum osteopontin (OPN) and single nucleotide polymorphisms (SNPs) in the OPN promoter in patients with hepatitis B-related hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Four groups were studied, which included 157 patients with HCC, 73 with liver cirrhosis (LC) and 97 with chronic hepatitis (CH), along with 80 healthy subjects. Serum OPN and alpha-fetoprotein (AFP) levels were measured. The SNPs -66 T/G, -156 G/δG and -433 C/T within the OPN promoter were determined by direct sequencing. RESULTS: Serum OPN levels were significantly higher in patients with HCC than in the other groups. Area under receiver operating characteristics curves in distinguishing HCC from chronic liver disease (CLD; CH and LC) were 0.782 (95% CI; 0.729-0.834) for OPN and 0.888 (95% CI; 0.850-0.927) for AFP. Using the optimal cut-off value (70 ng/mL), OPN had sensitivity and specificity of 72% and 71%, respectively. Serum OPN was superior to AFP in detecting early-stage HCC (68% vs. 46%). A combination of both markers yielded an improved sensitivity for detecting early HCC to 82%. A high OPN level was significantly correlated with advanced BCLC stage and was an independent prognostic factor for HCC. The SNPs -156 and -443 were associated with susceptibility to HCC, but were not related to overall survival. CONCLUSIONS: Serum OPN is a useful diagnostic and prognostic marker for HCC. The combined use of serum OPN and AFP improved the diagnosis of early HCC. Genetic variation in the OPN promoter is associated with the risk, but not the prognosis of HCC.

Human miR-5193 Triggers Gene Silencing in Multiple Genotypes of Hepatitis B Virus.

Hepatitis B virus (HBV) infection can lead to various disease states including asymptomatic, acute hepatitis, chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC), which remain a major health problem worldwide. Previous studies demonstrated that microRNA (miRNAs) plays an important role in viral replication. This study aimed to predict and evaluate human miRNAs targeting multiple genotypes of HBV. Candidate human miRNAs were analyzed by data obtained from miRBase and RNAhybrid. Then miRNAs were selected based on hybridization patterns and minimum free energy (MFE). The silencing effect of miRNA was evaluated by real-time PCR, the luciferase reporter assay and the ELISA assay. Five human miRNAs including miR- 142-5p, miR-384, miR-500b, miR-4731-5p and miR-5193 were found to target several HBV genotypes. Interestingly, miR-5193 was found to be the most potent miRNA that could target against all HBV transcripts in almost all HBV genotypes with a highly stable hybridization pattern (5' canonical with MFE lower than -35 kcal/mol). Moreover, miR-5193 caused significant silencing in luciferase activity (53% reduction), luciferase transcript (60% reduction) and HBV surface antigen (HBsAg) production (20-40% reduction depending on genotypes). Therefore, miR-5193 might be useful and have a vital role for inhibition of HBV replication in the future.

Increased Oxidative Stress and RUNX3 Hypermethylation in Patients with Hepatitis B Virus-Associated Hepatocellular Carcinoma (HCC) and Induction of RUNX3 Hypermethylation by Reactive Oxygen Species in HCC Cells.

Promoter hypermethylation of the runt-related transcription factor 3 (RUNX3) gene is associated with increased risk of hepatocellular carcinoma (HCC). Oxidative stress plays a vital role in both carcinogenesis and progression of HCC. However, whether oxidative stress and RUNX3 hypermethylation in HCC have a cause- and-effect relationship is not known. In this study, plasma protein carbonyl and total antioxidant capacity (TAC) in patients with hepatitis B virus (HBV)-associated HCC (n=60) and age-matched healthy subjects (n=80) was determined. RUNX3 methylation in peripheral blood mononuclear cells (PBMC) of subjects was measured by methylation-specific PCR. Effect of reactive oxygen species (ROS) on induction of RUNX3 hypermethylation in HCC cells was investigated. Plasma protein carbonyl content was significantly higher, whereas plasma TAC was significantly lower, in HCC patients than healthy controls. Based on logistic regression, increased plasma protein carbonyl and decreased plasma TAC were independently associated with increased risk for HCC. PBMC RUNX3 methylation in the patient group was significantly greater than in the healthy group. RUNX3 methylation in hydrogen peroxide (H2O2)-treated HepG2 cells was significantly higher than in untreated control cells. In conclusion, increase in oxidative stress in Thai patients with HBV-associated HCC was demonstrated. This oxidative increment was independently associated with an increased risk for HCC development. RUNX3 in PBMC was found to be hypermethylated in the HCC patients. In vitro, RUNX3 hypermethylation was experimentally induced by H2O2. Our findings suggest that oxidative stress is a cause of RUNX3 promoter hypermethylation in HCC cells.