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Background & Aims: Thrombopoietin receptor agonists are a new class of compounds licenced for the treatment of immune thrombocytopenic purpura. They are currently being studied for patients with thrombopenia in advanced liver disease or under therapy for hepatitis C. There are indications that the risk for development of portal vein thrombosis in patients with advanced liver cirrhosis might be increased under therapy with thrombopoietin receptor agonists. We report a case of a patient with Child class B liver cirrhosis with concurrent immune thrombocytopenic purpura that developed portal vein thrombosis under therapy with the thrombopoietin receptor agonist romiplostim.
Methods: A 50-year-old woman with hepatitis C virus associated immune thrombocytopenic purpura and Child class B liver cirrhosis presented in our emergency with rapidly evolving hydropic decompensation and general malaise. For immune thrombocytopenic purpura, the patient was started on the thrombopoietin receptor agonist romiplostim nine months ago.
Results: During hospitalization, the platelet count was measured above 330,000/μl and partial portal vein thrombosis was diagnosed by imaging studies. The thrombotic event was assumed to be associated with the romiplostim treatment for immune thrombocytopenic purpura via excessive elevation of platelet count. After anticoagulation with heparin and cessation of romiplostim treatment, complete recanalisation of the portal vein was achieved.
Conclusions: We conclude that romiplostim should be used with precaution in patients with hepatitis C-associated immune thrombocytopenic purpura and advanced liver cirrhosis as the risk for thrombotic complications may increase significantly.
Background The inhibitor telaprevir (VX-950) of the hepatitis C virus (HCV) protease NS3-4A has been tested in a recent phase 1b clinical trial in patients infected with HCV genotype 1. This trial revealed residue mutations that confer varying degrees of drug resistance. In particular, two protease positions with the mutations V36A/G/L/M and T54A/S were associated with low to medium levels of drug resistance during viral breakthrough, together with only an intermediate reduction of viral replication fitness. These mutations are located in the protein interior and far away from the ligand binding pocket. Results Based on the available experimental structures of NS3-4A, we analyze the binding mode of different ligands. We also investigate the binding mode of VX-950 by protein-ligand docking. A network of non-covalent interactions between amino acids of the protease structure and the interacting ligands is analyzed to discover possible mechanisms of drug resistance. We describe the potential impact of V36 and T54 mutants on the side chain and backbone conformations and on the non-covalent residue interactions. We propose possible explanations for their effects on the antiviral efficacy of drugs and viral fitness. Molecular dynamics simulations of T54A/S mutants and rotamer analysis of V36A/G/L/M side chains support our interpretations. Experimental data using an HCV V36G replicon assay corroborate our findings. Conclusion T54 mutants are expected to interfere with the catalytic triad and with the ligand binding site of the protease. Thus, the T54 mutants are assumed to affect the viral replication efficacy to a larger degree than V36 mutants. Mutations at V36 and/or T54 result in impaired interaction of the protease residues with the VX-950 cyclopropyl group, which explains the development of viral breakthrough variants.
Einleitung: Am 16.12.06 wurde im Eurotransplant-Gebiet der MELD-Score (MELD) als Allokationsbasis zur Lebertransplantation (OLT) eingeführt. Ziel ist eine Reduktion der Sterblichkeit auf der Warteliste. Material und Methoden: 100 Patienten wurden in die prospektive Analyse der MELD-Allokation vom 16.12.06 bis 15.09.07 einbezogen. Ergebnisse: Aktuell warten 68 Pat., 28 Pat. wurden transplantiert, 4 Pat. sind auf der Warteliste (WL) verstorben (4%). Der mittlere MELD auf der WL beträgt 17,2 +/- 5,2 (7-28). Bei 12 Pat. liegt eine Standard-exception (SE) (n=10 HCC, n=2 metabolische Erkrankung) mit einem Match-MELD von 25,6 +/-2,06 vor (24-28). Die Todesursachen der vier auf der WL verstorbenen Pat. waren eine akute Varizenblutung (MELD 9), zwei kardiale Versagen (MELD 13, 18) und eine MRSA-Sepsis (MELD 29, NT-Status). Die 28 transplantierten Pat. hatte zum Zeitpunkt der Transplantation einen mittleren MELD von 27,66 +/- 5,1 Punkten (21 bis 40). 20 Pat. wurden aufgrund des Labor-MELD (28,4 +/- 5,3, 24-40) transplantiert, wobei 7 Pat. einen MELD über 30 aufwiesen. Die Wartezeit lag bei 11,55 +/- 5,3 Tagen. 8 Pat. erhielten bei SE bei HCC (MELD 24 +/- 0, 24) ein Organ nach einer Wartezeit von 320 +/- 9,7 Tagen. Aktuell leben 23 der 28 transplantierten Pat. Bei zwei verstorbenen Pat. war die Todesursache ein kardiales Versagen, bei zwei Patienten eine primäre Non-Funktion sowie ein septisches Multiorganversagen. Schlussfolgerung: Während der ersten Monate der MELD Allokation lag die Letalität auf der WL in unserem Zentrum bei 4%. Patienten mit einem mittleren MELD über 27 erhielten Organangebote und konnten nach kurzer Wartezeit transplantiert werden.
The Q80K polymorphism in the NS3-4A protease of the hepatitis C virus is associated with treatment failure of direct-acting antiviral agents. This polymorphism is highly prevalent in genotype 1a infections and stably transmitted between hosts. Here, we investigated the underlying molecular mechanisms of evolutionarily conserved coevolving amino acids in NS3-Q80K and revealed potential implications of epistatic interactions in immune escape and variants persistence. Using purified protein, we characterized the impact of epistatic amino acid substitutions on the physicochemical properties and peptide cleavage kinetics of the NS3-Q80K protease. We found that Q80K destabilized the protease protein fold (p < 0.0001). Although NS3-Q80K showed reduced peptide substrate turnover (p < 0.0002), replicative fitness in an H77S.3 cell culture model of infection was not significantly inferior to the WT virus. Epistatic substitutions at residues 91 and 174 in NS3-Q80K stabilized the protein fold (p < 0.0001) and leveraged the WT protease stability. However, changes in protease stability inversely correlated with enzymatic activity. In infectious cell culture, these secondary substitutions were not associated with a gain of replicative fitness in NS3-Q80K variants. Using molecular dynamics, we observed that the total number of residue contacts in NS3-Q80K mutants correlated with protein folding stability. Changes in the number of contacts reflected the compensatory effect on protein folding instability by epistatic substitutions. In summary, epistatic substitutions in NS3-Q80K contribute to viral fitness by mechanisms not directly related to RNA replication. By compensating for protein-folding instability, epistatic interactions likely protect NS3-Q80K variants from immune cell recognition.
Different highly effective interferon-free treatment options for chronic hepatitis C virus (HCV) infection are currently available. Pre-existence of resistance associated variants (RAVs) to direct antiviral agents (DAAs) reduces sustained virologic response (SVR) rates by 3–53% in hepatitis C virus (HCV) genotype 1 infected patients depending on different predictors and the DAA regimen used. Frequencies of single and combined resistance to NS3, NS5A and NS5B inhibitors and consequences for the applicability of different treatment regimens are unknown. Parallel population based sequencing of HCV NS3, NS5A and NS5B genes in 312 treatment-naïve Caucasian HCV genotype 1 infected patients showed the presence of major resistant variants in 20.5% (NS3), 11.9% (NS5A), and 22.1% (NS5B) with important differences for HCV subtypes. In NS3, Q80K was observed in 34.7% and 2.1% of subtype 1a and 1b patients, respectively while other RAVs to second generation protease inhibitors were detected rarely (1.4%). Within NS5A RAVs were observed in 7.1% of subtype 1a and 17.6% in subtype 1b infected patients. RAVs to non-nucleoside NS5B inhibitors were observed in 3.5% and 44.4% of subtype 1a and 1b patients, respectively. Considering all three DAA targets all subtype 1a and 98.6% of subtype 1b infected patients were wildtype for at least one interferon free DAA regimen currently available. In conclusion, baseline resistance testing allows the selection of at least one RAVs-free treatment option for nearly all patients enabling a potentially cost- and efficacy-optimized treatment of chronic hepatitis C.
Background: Liver fibrosis in human immunodeficiency virus (HIV)-infected individuals is mostly attributable to co-infection with hepatitis B or C. The impact of other risk factors, including prolonged exposure to combined antiretroviral therapy (cART) is poorly understood. Our aim was to determine the prevalence of liver fibrosis and associated risk factors in HIV-infected individuals based on non-invasive fibrosis assessment using transient elastography (TE) and serum biomarkers (Fibrotest [FT]).
Methods: In 202 consecutive HIV-infected individuals (159 men; mean age 47 ± 9 years; 35 with hepatitis-C-virus [HCV] co-infection), TE and FT were performed. Repeat TE examinations were conducted 1 and 2 years after study inclusion.
Results: Significant liver fibrosis was present in 16% and 29% of patients, respectively, when assessed by TE (≥ 7.1 kPa) and FT (> 0.48). A combination of TE and FT predicted significant fibrosis in 8% of all patients (31% in HIV/HCV co-infected and 3% in HIV mono-infected individuals). Chronic ALT, AST and γ-GT elevation was present in 29%, 20% and 51% of all cART-exposed patients and in 19%, 8% and 45.5% of HIV mono-infected individuals. Overall, factors independently associated with significant fibrosis as assessed by TE (OR, 95% CI) were co-infection with HCV (7.29, 1.95-27.34), chronic AST (6.58, 1.30-33.25) and γ-GT (5.17, 1.56-17.08) elevation and time on dideoxynucleoside therapy (1.01, 1.00-1.02). In 68 HIV mono-infected individuals who had repeat TE examinations, TE values did not differ significantly during a median follow-up time of 24 months (median intra-patient changes at last TE examination relative to baseline: -0.2 kPa, p = 0.20).
Conclusions: Chronic elevation of liver enzymes was observed in up to 45.5% of HIV mono-infected patients on cART. However, only a small subset had significant fibrosis as predicted by TE and FT. There was no evidence for fibrosis progression during follow-up TE examinations.
Hepatitis C virus (HCV) substantially affects lipid metabolism, and remodeling of sphingolipids appears to be essential for HCV persistence in vitro. The aim of the current study is the evaluation of serum sphingolipid variations during acute HCV infection. We enrolled prospectively 60 consecutive patients with acute HCV infection, most of them already infected with human immunodeficiency virus (HIV), and serum was collected at the time of diagnosis and longitudinally over a six-month period until initiation of antiviral therapy or confirmed spontaneous clearance. Quantification of serum sphingolipids was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Spontaneous clearance was observed in 11 out of 60 patients (18.3%), a sustained viral response (SVR) in 43 out of 45 patients (95.5%) receiving an antiviral treatment after follow-up, whereas persistence of HCV occurred in six out of 60 patients (10%). C24-ceramide (C24-Cer)-levels increased at follow-up in patients with spontaneous HCV eradication (p < 0.01), as compared to baseline. Sphingosine and sphinganine values were significantly upregulated in patients unable to clear HCV over time compared to patients with spontaneous clearance of HCV infection on follow-up (p = 0.013 and 0.006, respectively). In summary, the persistence of HCV after acute infection induces a downregulation of C24Cer and a simultaneous elevation of serum sphingosine and sphinganine concentrations.
Background: FibroTest (FT) is the most frequently used serum fibrosis marker and consists of an algorithm of five fibrosis markers (alfa2-macroglobulin, apolipoproteinA1, haptoglobin, GGT, bilirubin). The Enhanced Liver Fibrosis (ELF) test consists of an algorithm of three fibrosis markers (hyaluronic acid, amino-terminal propeptide-of-type-III-collagen, tissue-inhibitor of matrix-metaloproteinase-1). While a systematic review has shown comparable results for both individual markers, there has been no direct comparison of both markers. Methods: In the present study, the ELF-test was analyzed retrospectively in patients with chronic liver disease, who received a liver biopsy, transient elastography (TE) and the FibroTest using histology as the reference method. Histology was classified according to METAVIR and the Ludwig's classification (F0-F4) for patients with chronic hepatitis C and B virus (HCV, HBV) infection and primary biliary cirrhosis (PBC), respectively. Results: Seventy-four patients were analysed: 36 with HCV, 10 with HBV, and 28 with PBC. The accuracy (AUROC) for the diagnosis of significant fibrosis (F[greater than or equal to]2) for ELF and FibroTest was 0.78 (95%CI:0.67-0.89) and 0.69 (95%-CI:0.57-0.82), respectively (difference not statistically significant, n.s.). The AUROC for the diagnosis of liver cirrhosis was 0.92 (95%CI:0.83-1,00), and 0.91 (95%CI:0.83-0.99), respectively (n.s.). For 66 patients with reliable TE measurements the AUROC for the diagnosis of significant fibrosis (cirrhosis) for TE, ELF and FT were 0.80 (0.94), 0.76 (0.92), and 0.67 (0.91), respectively (n.s.). Conclusion: FibroTest and ELF can be performed with comparable diagnostic accuracy for the non-invasive staging of liver fibrosis. Serum tests are informative in a higher proportion of patients than transient elastography.
Variants resistant to compounds specifically targeting HCV are observed in clinical trials. A multi-variant viral dynamic model was developed to quantify the evolution and in vivo fitness of variants in subjects dosed with monotherapy of an HCV protease inhibitor, telaprevir. Variant fitness was estimated using a model in which variants were selected by competition for shared limited replication space. Fitness was represented in the absence of telaprevir by different variant production rate constants and in the presence of telaprevir by additional antiviral blockage by telaprevir. Model parameters, including rate constants for viral production, clearance, and effective telaprevir concentration, were estimated from 1) plasma HCV RNA levels of subjects before, during, and after dosing, 2) post-dosing prevalence of plasma variants from subjects, and 3) sensitivity of variants to telaprevir in the HCV replicon. The model provided a good fit to plasma HCV RNA levels observed both during and after telaprevir dosing, as well as to variant prevalence observed after telaprevir dosing. After an initial sharp decline in HCV RNA levels during dosing with telaprevir, HCV RNA levels increased in some subjects. The model predicted this increase to be caused by pre-existing variants with sufficient fitness to expand once available replication space increased due to rapid clearance of wild-type (WT) virus. The average replicative fitness estimates in the absence of telaprevir ranged from 1% to 68% of WT fitness. Compared to the relative fitness method, the in vivo estimates from the viral dynamic model corresponded more closely to in vitro replicon data, as well as to qualitative behaviors observed in both on-dosing and long-term post-dosing clinical data. The modeling fitness estimates were robust in sensitivity analyses in which the restoration dynamics of replication space and assumptions of HCV mutation rates were varied.
Background & Aims: HBV genotype G (HBV/G) is mainly found in co-infections with other HBV genotypes and was identified as an independent risk factor for liver fibrosis. This study aimed to analyse the prevalence of HBV/G co-infections in healthy European HBV carriers and to characterize the crosstalk of HBV/G with other genotypes.
Methods: A total of 560 European HBV carriers were tested via HBV/G-specific PCR for HBV/G co-infections. Quasispecies distribution was analysed via deep sequencing, and the clinical phenotype was characterized regarding qHBsAg-/HBV-DNA levels and frequent mutations. Replicative capacity and expression of HBsAg/core was studied in hepatoma cells co-expressing HBV/G with either HBV/A, HBV/D or HBV/E using bicistronic vectors.
Results: Although no HBV/G co-infection was found by routine genotyping PCR, HBV/G was detected by specific PCR in 4%-8% of patients infected with either HBV/A or HBV/E but only infrequently in other genotypes. In contrast to HBV/E, HBV/G was found as the quasispecies major variant in co-infections with HBV/A. No differences in the clinical phenotype were observed for HBV/G co-infections. In vitro RNA and DNA levels were comparable among all genotypes, but expression and release of HBsAg was reduced in co-expression of HBV/G with HBV/E. In co-expression with HBV/A and HBV/E expression of HBV/G-specific core was enhanced while core expression from the corresponding genotype was markedly diminished.
Conclusions: HBV/G co-infections are common in European inactive carriers with HBV/A and HBV/E infection, but sufficient detection depends strongly on the assay. HBV/G regulated core expression might play a critical role for survival of HBV/G in co-infections.