M. Biermer

Viscoelasticity-based Staging of Hepatic Fibrosis with Multifrequency MR Elastography

PURPOSE To analyze the dynamics of the shear modulus of the liver to assess the optimal driving frequency and to determine the diagnostic accuracy of generalized frequency-independent elasticity cutoff values for staging hepatic fibrosis. MATERIALS AND METHODS This institutional review board-approved prospective study included 16 healthy volunteers and 72 patients with biopsy-proved liver fibrosis. After obtaining written informed consent, imaging was performed at 1.5-T by using a motion-sensitized echo-planar imaging sequence. Wave excitation was performed by an actuator introducing a superposition of four frequencies (25.0, 37.5, 50.0, 62.5 Hz) of shear waves. The elasticity µ value and the structure geometry parameter α were calculated by using the two-parameter springpot model. The performance of magnetic resonance (MR) elastography in staging liver fibrosis was assessed with area under the receiver operating characteristic curve (AUROC) analysis and Spearman correlation analysis. RESULTS Elasticity increased with stage of fibrosis, with mean values as follows: for volunteers, 2.25 kPa ± 0.43 (standard deviation); stage F1, 2.61 kPa ± 0.43; stage F2, 3.00 kPa ± 0.63; stage F3, 3.86 kPa ± 0.61; and stage F4, 5.86 kPa ± 1.22. Frequency-independent cutoff values derived for fibrosis and AUROC values, respectively, were as follows: stage F1 or higher, 2.84 kPa and 0.9128; stage F2 or higher, 3.18 kPa and 0.9244; stage F3 or higher, 3.32 kPa and 0.9744; and equivalent to stage F4, 4.21 kPa and 0.9931. The geometry of the tissue (α value) did not correlate with fibrosis. Frequencies of 50.0 Hz and 62.5 Hz displayed the highest diagnostic accuracy. CONCLUSION The diagnostic performance of multifrequency MR elastography in determining the degree of hepatic fibrosis increases with stage of fibrosis. Metrics obtained at the higher frequencies provide better diagnostic performance compared with the lower frequencies. Results of the AUROC analysis demonstrate the high accuracy of frequency-independent cutoff values for staging higher grades of hepatic fibrosis.

Successful prevention of hepatitis C virus (HCV) liver graft reinfection by silibinin mono-therapy

To the Editor:Therapeutic measurements to control hepatitis C reinfection afterorthotopic liver transplantation (OLT) still pose many problemsand remain unsatisfactory [1]. While hepatitis C viremiadecreases strongly during the anhepatic phase of OLT, initiationof viral replication does occur within hours or days [2,3]. At thisearly stage after OLT, i.e. once chronic HCV reinfection is estab-lished, the virologic response is often markedly reduced.Although specifically targeted antiviral therapeutic regimes forhepatitis C (STAT-C) are currently extensively studied, none ofthem have been tried in the post-OLT period treatment.However, as recently described by Ferenci et al. [4], high doseintravenous silibinin (Legalon SIL ) can express potent antiviralactivity. This effect was shown to be driven by direct inhibitionof the viral RNA polymerase NS5B [5,6].In this letter, we can report the first successful prevention ofHCV reinfection after OLT by the administration of silibinin(1400 mg/d). This drug was applied immediately after OLT bydaily infusions for 14 days.At the time of OLT, the 57-year-old male patient exhibited aMELD Score of 23, Child-Pugh stage C liver cirrhosis, and a25 mm hepatocellular carcinoma in the left lobe. HCV infection(genotype 3a) was first diagnosed in 1997 and three interferon-based treatment regimens – at last PegInterferon alpha and Riba-virin were given for 48 weeks 6 years before OLT – failed toinduce a sustained virologic response (SVR). The anhepatic phaseduring OLT surgical procedures lasted 61 min and postoperativecare transpired without complication after a short phase of renalinsufficiency and haemodialysis treatment on day 1 and 2.Immunosuppressive therapy included methyl-prednisolone,mycophenolatmofetil, and belatacept. Aminotransferase levelsreached normal values within 12 postoperative days. Bilirubinlevels rose to a maximum of 9.5 mg/dl 7 days after OLT andshowed a protracted mild elevation until 4 weeks later. Thepatient was discharged from the hospital 21 days after OLT.The levels of HCV RNA 3 months prior to OLT were rather low(17.800 IU/ml), and further declined significantly during theanhepatic phase. Silibinin infusions were started 8 h after OLT.At this time HCV RNA levels measured 182 IU/ml, and droppedagain to 127 IU/ml after 48 h (RNA levels measured directly afterhaemodialysis are not available). Already from day 3 onwards,HCV RNA levels were below <15 IU/ml and became undetectableat day 9. During follow-up HCV RNA remained negative whenexamined at day 14, 21, 66, 84, and 168 (Fig. 1).This is the first report of the successful suppression of earlyHCV reinfection after OLT with a 14 day course of silibininmono-therapy. This new treatment regimen thus induced anSVR, as after 6 months of follow-up, RNA for HCV was undetect-able. Accordingly, liver histology 6 months after OLT did notshow any cellular inflammation. Low pre-transplant HCV RNAlevels may be taken as a favourable prognostic factor for theunexpected therapeutic efficiency of silibinin. Applying silibininduring the anhepatic phase or even in the days before transplantmay expand the number of patients benefiting from thisapproach. This report may stimulate further trials and the con-cept of interferon-free HCV clearance induced by directantivirals.Conflicts of interestThe Authors have declared that they received funding from thedrug companies involved in order to carry out their research inthis manuscript.References

Rapid suppression of hepatitis C viremia induced by intravenous silibinin plus ribavirin.

ear Sir: In the November 2008 issue of GASTROENTEROLOGY, erenci et al1 presented their interesting, preliminary ndings on the efficacy of silibinin administered intraveously to patients with hepatitis C virus (HCV) with revious nonresponse to full-dose of pegylated interferonlfa plus ribavirin combination therapy. Their dose-findng study revealed that a 7-day course of 5, 10, 15, or 20 g/kg per day of silibinin monotherapy led to a viral oad decrease ranging from 0.55 to 3.02 log IU/mL. The ddition of pegylated interferon-alfa plus ribavirin to he ongoing silibinin therapy at day 8 resulted in a urther decline in the viremia levels (log drop 3.69 or 4.85 n the 15 or 20 mg/kg group), including 6 patients who chieved a complete virologic response at day 15 (HCV NA 15 IU/mL). A re-increase in HCV RNA levels, owever, was observed in some of these patients when ntravenous silibinin administration was stopped at this ime, despite the continuous pegylated interferon-alfa lus ribavirin treatment. Herein, we present a case report of a 65-year-old oman who had previously failed any antiviral treatment trategies. Two courses of pegylated interferon and ribairin had been applied unsuccessfully in 2002 and 2004. n February 2008, low-dose maintenance therapy with 50 g pegylated interferon-alfa-2b had to be terminated ecause of a solitary hepatocellular carcinoma (3 cm in iameter) that developed in compensated liver cirrhosis. ecause the patient refused liver transplantation, a local blative therapy was performed that resulted in complete blation of the tumor. Confronted with the need to radicate effectively the HCV infection to reduce the risk f tumor recurrence and disease progression, we adapted he silibinin protocol as described by Ferenci et al.1 We tarted first a monotherapy with ribavirin to evaluate the ighest tolerable dosage and also to reach a steady-state lasma ribavirin level before starting silibinin and pegyated interferon-alfa. During the 4-week ribavirin monoherapy (800 mg/d) a 0.6-log drop in viremia was oberved (from 1,610,000 to 363,330 IU/mL; Figure 1). fter silibinin was initiated intravenously at 20 mg/kg odyweight with daily infusions over 2 hours, a further arked reduction in HCV RNA levels was reached (day 8: ,105 IU/mL corresponding to an additional 2.4-log deline). Ribavirin was continued during silibinin adminisration. On day 8, we initiated weekly subcutaneous inm ections with 180 g pegylated interferon alfa-2a. At day 7, 3 days after stopping the 14-day silibinin course, ndetectable HCV RNA levels were obtained. No re-inrease of HCV RNA was found after stopping intraveous silibinin. It has to be pointed out that our treatment strategy as slightly different from Ferenci’s protocol in that we dministered silibinin in the context of a ribavirin loadng dosage protocol. It might, therefore, be speculated hat this approach augmented the efficacy of silibinin. It emains speculative whether a complete virologic reponse (HCV RNA undetectable) could have been inuced only by the combination of ribavirin plus silibinin, hen both substances were given for extended periods nd not only for 2 weeks. At least, a continuous reducion in viremia from 1,600,000 to 1000 IU/mL was aleady achieved with ribavirin and silibinin alone. After he application of pegylated interferon-alfa, there was no nflection point in the viral decay suggesting that the nduction of a complete virologic suppression may be ossible by the combined application of silibinin and ibavirin only. In conclusion, the application of high-dose, intraveous silibinin in an HCV-positive, cirrhotic patient reractory to 3 courses of interferon-based therapy led to a apid virologic response as documented by undetectable CV RNA levels. The significant antiviral effect may have een augmented by the concurrent use of ribavirin. This ase report could, therefore, favor further studies that ompare the antiviral efficacy of silibinin with and withut concurrent ribavirin use.

High-dose silibinin rescue treatment for HCV-infected patients showing suboptimal virologic response to standard combination therapy.

Summary.  Incomplete suppression of hepatitis C virus (HCV) replication with persistence of minimal viremia (partial virologic response) leading to treatment failure can be observed in a significant proportion of HCV type 1‐infected patients during antiviral therapy. Recently, high‐dose intravenous silibinin has demonstrated strong antiviral activity against HCV. We were therefore interested in whether patients with partial virologic response can be rescued by the on‐treatment addition of a short‐term course of high‐dose intravenous silibinin infusions. Twenty patients who failed to achieve a complete virologic response to different interferon‐based regimens qualified for the rescue strategy and received 1400 mg/day silibinin infusions on two consecutive days. Complete viral suppression (below the limit of detection <6 IU/mL, TMA assay) could be induced in 13 of 20 patients within the first week after the short‐term silibinin infusion, and all but one of them also remained HCV RNA negative during the subsequent follow‐up period on continued peginterferon plus ribavirin treatment. In the remaining seven patients, no complete suppression could be achieved although four showed a significant HCV RNA reduction in response to silibinin. Silibinin infusions were generally well tolerated, and activation of abdominal peristalsis with nausea, diarrhoea and vomiting were the most prominent side effects. Of the twelve patients who exhibited a durable response to peginterferon and ribavirin treatment, three achieved an SVR, two achieved a week 12 SVR and four suffered a viral relapse. Three patients could not complete the assigned antiviral treatment with peginterferon alpha and ribavirin for nonvirological reasons. Short‐term administration of high‐dose intravenous silibinin might be an interesting approach to rescue patients with ongoing minimal residual viremia while on interferon‐based therapy. These preliminary findings may stimulate further studies to evaluate more refined therapeutic strategies.

A Preliminary Study on the Relationship between Platelet Serotonin Transporter Functionality, Depression, and Fatigue in Patients with Untreated Chronic Hepatitis C

Objective and Methods. Although the interaction between fatigue and depression in patients with chronic hepatitis C infection (HCV) has been recognized, the biological correlates of this observation have yet to be reported. We addressed this issue by examining serotonin transporter- (SERT-) driven [14C]-serotonin uptake rate (SUR) and serotonin content in platelets of 65 untreated HCV patients and 65 healthy control subjects (HCS). All patients completed report questionnaires for fatigue, depression, and general psychopathology. Structured interviews were conducted by a board-certified psychiatrist. Results. Whereas 36 of the patients experienced fatigue of moderate-to-severe intensity, only 16 reported symptoms of depression (BDI score > 10). Mean SUR in patients with depressive symptoms was significantly higher relative to the HCS, corresponding to a large Cohens effect size of d = 1.45 (95% CI = 0.66—1.83). Patients who rated their fatigue to have a marked impact on mood and activity displayed a moderate relationship between the BDI score and SUR (n = 18, r = 0.563, P = 0.015), which becomes stronger after controlling for age, gender, and thrombocytopenia (r part = 0.710, P = 0.003). In the univariate analysis, high fatigue interference score, thrombocytopenia, and high SUR were all significant predictors of depression. Conclusions. High SERT activity could be implicated in the expression of depressive symptoms especially in a subgroup of HCV patients who are feeling fatigue as markedly distressing.

Long-term evaluation of patients with sustained virologic remission by highly sensitive HCV RNA assays: no evidence for viral persistence.

The induction of a sustained virologic response (SVR) in hepatitis virus (HCV) infected patients, defined as undetectable serum HCV NA levels 6 months after stopping therapy, is believed to herald he determination of the chronic HCV infection.1,2 However, recent ndings suggest that residual HCV RNA can persist in PBMC, lymhatic or liver tissue3–5 indicating that minimal residual hepatitis viremia can escape standard HCV RNA assays.6 Now Fytili et al. eported in this Journal that, minimal residual HCV RNA can be etected in more than 20% of SVR patients when serum samples ere tested with a highly sensitive RealTime PCR assay (Abbott, 2000 RealTime PCR).7 To further substantiate these findings, we re-evaluated 160 erum samples of 145 SVR patients with two different highly senitive HCV RNA assays, the Abbott RealTime PCR (m2000rt) system nd the Siemens Versant TMA® assay. Two different RNA extraction ethods were applied for the RealTime PCR assay: the manual RNA xtraction method was used in 97 patients while the automated ersion was employed in 63 patients. Median time after EOT was .1 years (range: 0.5–12 y). By using the TMA assay, only one sample out of 160 was found to e HCV-RNA positive (0.6%). In contrast, in 10% (n = 16) of the SVR atients HCV RNA was detectable by the RealTime PCR. 14 out of hese 16 positive samples have been treated by the manual extracion method (14.5% of those analysed manually, 9% of all), whereas CV RNA could be detected only in 2 out of 63 patients (3.2%, 1.4% of ll) when the fully automated version of the assay was applied. All ut one of the HCV RNA positive samples had levels below the limit f quantification of the assay (i.e. <12 IU/mL) see Fig. 1. But most mportantly, in none of the patient the positive HCV RNA result ould be confirmed by the TMA assay or vice versa. These findings ould be also confirmed by testing a second serum sample from a ifferent time point in 8 out of the 16 HCV RNA positive patients. oth assays, the TMA and the fully automated RealTime PCR test ave negative results. Thus our study contradicts Fytili’s conclusion and rather indiates that positive HCV RNA signals detectable by RealTime PCR re due to either unspecific amplification or contamination. This nterpretation is strengthens by our finding that the ratio of HCV NA positive samples by real time PCR was significantly higher hen using the manual extraction (14.5%) as compared to the fully utomated system (3%). We argue that most probably false posi-

Improved pharmacodynamics and pharmacokinetics after i.v. application of peginterferon alfa-2a in hepatitis C null responders.

Mechanisms of non‐responsiveness to peginterferon alfa‐2a are not completely understood. Inadequate plasma levels may contribute to reduced response. The aim of this prospective, multicentre, crossover, Phase 1 study was to evaluate the pharmacokinetics and viral kinetics of intravenous vs. subcutaneous peginterferon alfa‐2a in patients with genotype 1 chronic hepatitis C infection who showed null response to previous peginterferon/ribavirin.

819 IMPROVED PHARMACODYNAMICS AND PHARMACOKINETICS AFTER INTRAVENOUS APPLICATION OF PEG-INTERFERON alfa-2a 180 μg IN CHRONIC HEPATITIS C GENOTYPE 1 NULLRESPONDERS TO PEGINTERFERON/RIBAVIRIN. THE IVAN STUDY

819 IMPROVED PHARMACODYNAMICS AND PHARMACOKINETICS AFTER INTRAVENOUS APPLICATION OF PEG-INTERFERON alfa-2a 180mg IN CHRONIC HEPATITIS C GENOTYPE 1 NULLRESPONDERS TO PEGINTERFERON/RIBAVIRIN. THE IVAN STUDY B. Fueloep, M. Biermer, R. Heyne, H. Wedemeyer, M. Cornberg, K. Port, S. Zeuzem, K.-H. Peiffer, S. Koenig, A. Herber, A. Boehlig, J. Wiegand, P. Buggisch, S. Stoll, U. Alshuth, T. Berg, IVAN Study Group. Division of Gastroenterology and Hepatology, University Hospital Leipzig, Leipzig, Liver and Study Center Checkpoint, Center of Gastroenterology and Liver Center, Berlin, Dept. of Gastroenterology, Hepatology & Endocrinology, Hannover Medical School, Hannover, J.W. Goethe University Hospital, Frankfurt/M, ifi-Institute for Interdisciplinary Medicine, Hamburg, Virology, Roche Pharma AG, Grenzach-Wyhlen, Germany E-mail: balzs.fueloep@medizin.uni-leipzig.de

259 ANTIVIRAL EFFICACY OF RIBAVIRIN MONOTHERAPY FOLLOWED BY STANDARD COMBINATION TREATMENT IN CHRONIC HEPATITIS C

(IP-10 or CXCL10) level might be a predictive factor for sustained virological response (SVR) to pegylated IFN plus ribavirin (PegIFN/RBV) therapy in patients infected with hepatitis C virus (HCV) genotype 1. We aimed to evaluate association of pretreatment and during treatment serum IP-10 levels with virological response to PegIFN/RBV therapy in genotype 1-infected Japanese patients with chronic hepatitis C (CHC). Methods: A total of 110 patients treated with PegIFN/RBV therapy. Serum IP-10 levels were determined by enzyme linked immunosorbent assay before therapy, after 12 weeks of treatment, and endpoint of therapy. Results: 40 (36.4%) patients obtained SVR and 72 (63.6%) resulted in non-responder (NR). From univariate analysis, age (51.8 vs. 57.8: P = 0.004); IFN treatment (First/Re-treatment) (30/10 vs. 33/37: P = 0.005); Stage (F0–2 vs. F3–4) (29/11 vs. 34/36: P = 0.015); Total volume of PegIFN (3194.8 vs 2494.4: P = 0.005); Total volume of RBV (192.4 vs.161.3: P = 0.004); HCV-RNA levels (1615.7 vs. 2110.0: P = 0.046); AST level (60.1 vs.80.4: P = 0.005); PLT level (17.8 vs.16.5: P = 0.021); g-GTP level (44.2 vs.72.9: P < 0.001); AFP level (6.4 vs. 24.2: P < 0.001); treatment period (weeks) (46.4 vs. 40.1: P = 0.005); pretreatment IP-10 levels (448.9 vs. 590.1: P = 0.008); after 12 weeks of treatment IP-10 levels (243.1 vs. 328.4: P = 0.033); endpoint of therapy IP-10 levels (280.7 vs. 374.8: P = 0.047); Rapid virological response (RVR) (12/28 vs. 1/69: P < 0.001); Early virological response (EVR) (36/4 vs. 14/56: P < 0.001); wild type at amino acid 70 in the HCV core region (30/10 vs. 33/37: P = 0.005). By multivariate analysis, Age (odds ratio (OR) 9.19 (95% confidence interval (CI) 1.52–55.72; P = 0.016), treatment period (OR 153.54 (95%CI 3.44–6860.09; P = 0.009), early virological response (EVR) (OR 126.32 (95%CI 7.87-.2026.60; P < 0.001) and after 12 weeks of treatment (OR 16.14 (95%CI 1.29-.202.13; P = 0.031) were identified as predictive factor of SVR. Conclusions: Our data suggest that pretreatment and after 12 weeks of treatment serum IP-10 levels predict SVR to PegIFN/RBV therapy in CHC genotype 1 infected patients.