Impact of HBV genotype and mutations on HBV DNA and qHBsAg levels in patients with HBeAg-negative chronic HBV infection.

BACKGROUND: HBV DNA and quantitative (q)HBsAg levels as prognostic markers for HBV-related disease are mostly validated in Asia and their significance in Western populations is uncertain. AIM: To analyse the impact of the HBV genotype and frequent mutations in precore (PC), basal core promoter (BCP) and preS on HBV DNA and qHBsAg levels. METHODS: HBV DNA and qHBsAg serum levels of 465 patients with HBeAg-negative chronic HBV infection were correlated with the HBV genotype and mutations in PC, BCP and preS. For a detailed analysis of the molecular virology, genotype A2 genomes harbouring these mutations were analysed for replication efficacy and HBsAg release in cell culture. RESULTS: While no impact of the HBV genotype on HBV DNA levels was observed, qHBsAg levels differed up to 1.4 log among the genotypes (P < 0.001), reflected by large differences regarding the 1000 IU/mL HBsAg cut-off. While PC mutations were associated with higher (P < 0.001), BCP mutations were associated with lower HBV DNA levels (P < 0.001). Higher qHBsAg levels were associated with preS and lower levels with PC mutations (P < 0.001 and P = 0.001, respectively). The cell culture experiments revealed a higher HBsAg release and shorter filaments in case of a HBV genome harbouring a preS deletion. In contrast, a perinuclear HBsAg accumulation was detected for the PC and BCP-variants, reflecting an impaired HBsAg release. CONCLUSIONS: qHBsAg serum levels depend on the HBV genotype and together with HBV DNA levels on frequent mutations in PC, BCP and preS in HBeAg-negative patients. qHBsAg cut-offs when used as prognostic markers require genotype-dependent validation.

High efficacy of sofosbuvir/velpatasvir and impact of baseline resistance-associated substitutions in hepatitis C genotype 3 infection.

BACKGROUND: Twelve weeks of the pangenotypic direct-acting antiviral (DAA) combination sofosbuvir/velpatasvir (SOF/VEL) was highly efficient in patients with hepatitis C virus (HCV) genotype 3 (GT3) infection in the ASTRAL-3 approval study. However, presence of resistance-associated substitutions (RASs) in the HCV nonstructural protein 5A (NS5A) was associated with lower treatment response. AIM: To assess the efficacy and safety of SOF/VEL ± ribavirin (RBV) and the impact of NS5A RASs and RBV use on treatment outcome in HCV GT3 infection in a real-world setting. METHODS: In this multicentre cohort study, GT3 patients from ten treatment centres across Germany were included. Sustained virological response was assessed 12 weeks after end-of-treatment (SVR12) in modified intention-to-treat (mITT) and per-protocol analysis (PP). NS5A RASs were tested by population-based sequencing. RESULTS: A total of 293 GT3 patients were included. The median age was 48 years, 70% were male, 25.3% were cirrhotic, 9.2% were HCV/HIV co-infected and 21.8% were treatment-experienced, including 4.1% with DAA experience. Baseline NS5A RASs (Y93H, A30K, L31M) were detected in 11.2%. RBV was added in 5% of noncirrhotic and 58.9% of cirrhotic patients, respectively. SVR12 rates for SOF/VEL±RBV were 95.9% (mITT) and 99.5% (PP), respectively. Only 1 virological relapse occurred in a cirrhotic patient previously treated with SOF/RBV. No treatment-related major adverse events occurred. CONCLUSION: Twelve weeks of SOL/VEL±RBV was safe and highly efficient in HCV GT3 across a diverse patient population. Baseline NS5A RASs were rarely observed and presence did not seem to impact SVR, regardless of the use of RBV.

No evidence of hepatitis B virus reactivation in patients with resolved infection treated with direct-acting antivirals for hepatitis C in a large real-world cohort.

BACKGROUND: Hepatitis B virus (HBV) reactivation has been observed following interferon (IFN)-based treatment in HBV/hepatitis C virus (HCV) co-infected patients. Recent reports suggest that reactivation may also occur in both hepatitis B surface antigen (HBsAg)-positive and HBsAg-negative patients during HCV treatment with direct-acting antivirals (DAAs). AIM: To investigate the rate of patients with HBV reactivation during IFN-based and IFN-free HCV treatment in a large real-world cohort. METHODS: A total of 848 patients with chronic hepatitis C were treated with different combinations of DAAs. Among patients with available outcome and HBV data, there were 272 patients hepatitis B core antibody (HBcAb)-positive (HBsAg-positive, n=9; HBsAg-negative, n=263), and 536 were HBcAb-negative. All HBcAb-positive patients were tested for HBV DNA at the end of DAA therapy and alanine transaminase (ALT) levels were frequently measured during therapy and follow-up. RESULTS: Seventy-three percent (n=192/263) of HBsAg-negative/HBcAb-positive patients had elevated ALT levels at baseline, which declined to normal values in all but 18 patients, and no HBV reactivation was observed. Eight patients had detectable but not quantifiable HBV DNA (<20 IU/mL) at end of treatment, but none were associated with elevated ALT. Five of nine HBsAg-positive/HBcAb-positive patients experienced transient or permanent HBV reactivation, three of whom required nucleos(t)ide treatment during (n=1) or after (n=2) DAA therapy. CONCLUSIONS: HBV reactivation was not observed in HBsAg-negative/HBcAb-positive patients but common in HBsAg-positive/HBcAb-positive patients treated with different combinations of DAAs for HCV.

Regression of fibrosis and portal hypertension in HCV-associated cirrhosis and sustained virologic response after interferon-free antiviral therapy.

It is still controversial, whether and to what amount cirrhosis and portal hypertension are reversible in patients with hepatitis C virus (HCV)-associated cirrhosis and sustained virologic response (SVR) after interferon-free antiviral therapy. In this study, we prospectively evaluated dynamics of liver and spleen stiffness in HCV-infected patients with advanced liver disease and SVR after interferon-free treatment. A total of 54 patients with HCV-associated cirrhosis and SVR were included. Liver and spleen stiffness was measured at therapy baseline (BL), end of treatment (EOT) and 24 weeks after EOT (FU24) by transient liver elastography (L-TE) as well as by acoustic radiation force impulse of the liver (L-ARFI) and spleen (S-ARFI), as well as biochemical, virologic and clinical data. Improvement of liver and spleen stiffness was found in 44 of 50 (88%), 31 of 54 (57%) and 25 of 54 (46%) of patients assessed by L-TE, L-ARFI and S-ARFI between baseline and FU24. Liver stiffness assessed by L-TE improved between BL [median (range), 32.5 (9.1-75) kPa] and EOT [median (range), 21.3 (6.7-73.5) kPa; (P<.0001)], and between BL and FU24 [median (range), 21.2 (5.4-70) kPa; (P<.0001)]. Liver stiffness assessed by L-ARFI improved between BL [median (range), 2.7 (1.2-4.1) m/s] and FU24 [median (range), 2.4 (1.2-3.9) m/s; P=.002), while spleen stiffness remained unchanged. Our data suggest that improvement of liver stiffness may be rather due to reduced necroinflammation and may be due to a less extent to regression of cirrhosis, as dynamics of liver stiffness improvement was more pronounced between BL and EOT than BL and FU24.

The efficacy and safety of direct acting antiviral treatment and clinical significance of drug-drug interactions in elderly patients with chronic hepatitis C virus infection.

BACKGROUND: Direct antiviral therapies for chronic hepatitis C virus (HCV) infection have expanded treatment options for neglected patient populations, including elderly patients who are ineligible/intolerant to receive interferon (IFN)-based therapy. AIM: To investigate the efficacy, tolerability and potential for drug-drug interactions (DDIs) of IFN-free treatment in patients aged ≥65 years in a large real-world cohort. METHODS: A total of 541 patients were treated with different combinations of direct antiviral agents (DAAs: ledipasvir/sofosbuvir ±ribavirin; daclatasvir/sofosbuvir ±ribavirin; paritaprevir/ombitasvir ±dasabuvir ±ribavirin or simeprevir/sofosbuvir ±ribavirin in genotype 1/4, and daclatasvir/sofosbuvir ±ribavirin or sofosbuvir/ribavirin in genotype 2/3). Efficacy, safety and potential DDIs were analysed and compared between patients aged <65 years (n = 404) and patients aged ≥65 years (n = 137) of whom 41 patients were ≥75 years. RESULTS: Sustained virological response rates were 98% and 91% in patients aged ≥65 years and <65 years, respectively. Elderly patients took significantly more concomitant medications (79% vs. 51%; P < 0.0001). The number of concomitant drugs per patient was highest in patients ≥65 years with cirrhosis (median, three per patient; range, 0-10). Based on the hep-druginteractions database, the proportion of predicted clinically significant DDIs was significantly higher in elderly patients (54% vs. 28%; P < 0.0001). The number of patients who experienced treatment-associated adverse events was similar between the two age groups (63% vs. 65%; P = n.s.). CONCLUSIONS: Elderly patients are at increased risk for significant DDIs when treated with DAAs for chronic HCV infection. However, with careful pre-treatment assessment of concomitant medications, on-treatment monitoring or dose-modifications, significant DDIs and associated adverse events can be avoided.

Taking the next step forward - Diagnosing inherited infantile cholestatic disorders with next generation sequencing.

Identifying rare genetic forms of infantile cholestasis is challenging due to their similar clinical presentation and their diverse etiology. After exclusion of common non-genetic causes a huge list of rare differential diagnosis remains to be solved. More than 90 genes are associated with monogenic forms of infantile cholestasis, thus preventing routine genetic workup by Sanger sequencing. Here we demonstrate a next generation sequencing approach to discover the underlying cause in clinically well characterized patients in whom common causes of infantile cholestasis have been excluded. After validation of the analytical sensitivity massive parallel sequencing was performed for 93 genes in six prospectively studied patients. Six novel mutations (PKHD1: p.Thr777Met, p.Tyr2260Cys; ABCB11: p.Val1112Phe, c.611+1G > A, p.Gly628Trpfs*3 and NPC1: p.Glu391Lys) and two known pathogenic mutations were detected proving our multi gene panel for infantile cholestasis to be a sensitive and specific method overcoming the complexity of the phenotype-based, candidate gene approach. Three exemplary clinical cases of infants with cholestasis are presented and discussed in the context of their genetic and histopathological findings (autosomal recessive polycystic kidney disease, atypical PFIC and Niemann-Pick syndrome type C1). These case reports highlight the critical impact of integrating clinical, histopathological and genetic data during the process of multi gene panel testing to ultimately pinpoint rare genetic diagnoses.

Soluble CD163 is an indicator of liver inflammation and fibrosis in patients chronically infected with the hepatitis B virus.

Soluble CD163 (sCD163), a marker for macrophage activation, was found to be associated with the severity of liver cirrhosis. The aim of the current study was to investigate whether serum sCD163 levels correlate with liver inflammation and fibrosis in patients with chronic hepatitis B virus (HBV) infection. In a retrospective cohort study, serum sCD163 levels were assessed by ELISA together with clinical and laboratory data in 186 patients with chronic HBV infection and 15 healthy controls. The relation between parameters for liver fibrosis and necroinflammation and sCD163 levels was analysed. Additionally, sCD163 was quantified in a subset of follow-up serum samples after initiation of antiviral treatment. sCD163 levels differed among phases of chronic HBV infection (P < 0.0001), and sCD163 concentrations were associated with inflammatory activity and fibrosis in the liver. sCD163 levels ≥ 1961 ng/l had a high specificity in the identification of subjects with substantial fibrosis (F ≥ 2). sCD163 concentrations decreased significantly after initiation of antiviral treatment. The correlation of sCD163 levels with necroinflammation and fibrosis and the sCD163 decline under treatment indicates that macrophage activation plays a role in HBV-related liver pathogenesis.

Cytochrome P450 epoxygenase dependence of opioid analgesia: fluconazole does not interfere with remifentanil-mediated analgesia in human subjects.

Cytochrome P450 (CYP) inhibitors may reduce opioid analgesia by inhibiting CYP activity-dependent post-opioid receptor signaling pathways in the brain. This suggestion was predicated on observations of highly attenuated morphine antinociception in rodents after intracerebroventricular injection of fluconazole or carrying a neuron-specific deletion of the cytochrome P450 reductase. However, based on assessments of thermal and electrical pain tolerance, respiratory function, and side effects in 21 healthy volunteers, before and during steady-state concentrations of 1.5 and 3.0 ng/ml of remifentanil at the effect site (viz., the central nervous system), administration of 400 mg/day fluconazole for 8 days in a double-blind, placebo-controlled manner failed to attenuate opioid effects. Although CYP inhibitors such as fluconazole are unlikely to attenuate remifentanil analgesia in humans, extrapolation of the findings to other opioids is premature because differences among opioid effects, such as ligand-selective biased signaling at opioid receptors, leave the possibility that CYP-dependent opioid signaling in the brain might be limited to morphine and may not extend to remifentanil.

[Non-invasive methods for the evaluation of liver fibrosis in clinical practice]. / Nicht-invasive Verfahren zur Bestimmung der Leberfibrose in der klinischen Praxis.

Staging of the degree of liver fibrosis is important for the estimation of prognosis, surveillance and treatment decision in patients with chronic liver diseases. At present, liver biopsy is still the reference standard for the assessment of liver fibrosis. However, it is an invasive method with respective complications and limitations. Thus, non-invasive methods such as blood fibrosis markers and ultrasound-based elastography methods have been intensively evaluated for the assessment of liver fibrosis. The aim of the present article is to give an overview of research and clinical applicability of non-invasive methods in chronic liver disease.

Mutations selected in the hepatitis C virus NS3 protease domain during sequential treatment with boceprevir with and without pegylated interferon alfa-2b.

Treatment with hepatitis C virus (HCV)-NS3-protease inhibitors lead to the selection of resistant variants. Viral kinetics and resistance profiles in patients who are re-treated with the same protease inhibitor are unknown. Viral kinetics and NS3-resistance mutations obtained by clonal sequencing of the NS3-protease were analyzed in nine HCV-genotype-1-infected nonresponder patients who were sequentially treated with boceprevir (400 mg t.i.d.) for 1 week, peginterferon-alfa-2b for 2 weeks and combination of the two for 2 weeks in varying order. In addition to predominant wild-type isolates, previously described boceprevir-resistant mutations (V36, T54, R155, A156, V170) were observed. Furthermore, two resistant mutations (Q41, F43) were detected for the first time in vivo. In three patients, mutations selected after initial treatment with boceprevir were re-selected during subsequent boceprevir exposure. However, mutational patterns after the first and second exposure to boceprevir were different in five patients. In one patient, a viral variant (V55A) known to reduce susceptibility to boceprevir was the predominant variant observed at baseline and throughout treatment and was associated with a shallow viral decline. Different resistance mutations were selected during treatment with boceprevir ± peginterferon. Sequential short-term dosing of boceprevir was not associated with accumulation of resistant variants but pre-existing variants may impair virologic response.

New HCV therapies on the horizon.

Improved understanding of the hepatitis C virus (HCV) life cycle has led to the discovery of numerous potential targets for antiviral therapy. HCV polyprotein processing and replication have been identified as the most promising viral targets. However, viral entry and fusion, RNA translation, virus assembly and release and several host cell factors may provide alternative attractive targets for future anti-HCV therapies. Inhibitors of the HCV NS3/4A protease are currently the most advanced in clinical development. Monotherapy with protease inhibitors has shown high antiviral activity, but is associated with frequent selection of resistant HCV variants, often resulting in viral breakthrough. However, there is encouraging evidence from phase 2/3 trials indicating that the addition of a protease inhibitor (e.g. telaprevir and boceprevir) to pegylated interferon-α/ribavirin substantially improves sustained virological response rates in both treatment-naïve and treatment-experienced patients with HCV genotype 1. Nucleos(t)ide inhibitors of the HCV NS5B polymerase have shown variable antiviral activity against different HCV genotypes, but seem to have a higher genetic barrier to resistance than protease inhibitors. In addition, several allosteric binding sites have been identified for non-nucleoside inhibitors of the NS5B polymerase. However, the development of a substance with high antiviral activity and a high genetic barrier to resistance seems to be difficult. Among the different host cell-targeting compounds in early clinical development, cyclophilin inhibitors have shown the most promising results. Although advances have also been made in improving interferons, combinations of antiviral agents with different mechanisms of action may lead to the eventual possibility of interferon-free regimens.