Yves Benhamou

Overview of the diagnostic value of biochemical markers of liver fibrosis (FibroTest, HCV FibroSure) and necrosis (ActiTest) in patients with chronic hepatitis C

SummaryBackgroundRecent studies strongly suggest that due to the limitations and risks of biopsy, as well as the improvement of the diagnostic accuracy of biochemical markers, liver biopsy should no longer be considered mandatory in patients with chronic hepatitis C. In 2001, FibroTest ActiTest (FT-AT), a panel of biochemical markers, was found to have high diagnostic value for fibrosis (FT range 0.00–1.00) and necroinflammatory histological activity (AT range 0.00–1.00). The aim was to summarize the diagnostic value of these tests from the scientific literature; to respond to frequently asked questions by performing original new analyses (including the range of diagnostic values, a comparison with other markers, the impact of genotype and viral load, and the diagnostic value in intermediate levels of injury); and to develop a system of conversion between the biochemical and biopsy estimates of liver injury.ResultsA total of 16 publications were identified. An integrated database was constructed using 1,570 individual data, to which applied analytical recommendations. The control group consisted of 300 prospectively studied blood donors. For the diagnosis of significant fibrosis by the METAVIR scoring system, the areas under the receiver operating characteristics curves (AUROC) ranged from 0.73 to 0.87. For the diagnosis of significant histological activity, the AUROCs ranged from 0.75 to 0.86. At a cut off of 0.31, the FT negative predictive value for excluding significant fibrosis (prevalence 0.31) was 91%. At a cut off of 0.36, the ActiTest negative predictive value for excluding significant necrosis (prevalence 0.41) was 85%. In three studies there was a direct comparison in the same patients of FT versus other biochemical markers, including hyaluronic acid, the Forns index, and the APRI index. All the comparisons favored FT (P < 0.05). There were no differences between the AUROCs of FT-AT according to genotype or viral load. The AUROCs of FT-AT for consecutive stages of fibrosis and grades of necrosis were the same for both moderate and extreme stages and grades. A conversion table was constructed between the continuous FT-AT values (0.00 to 1.00) and the expected semi-quantitative fibrosis stages (F0 to F4) and necrosis grades (A0 to A3).ConclusionsBased on these results, the use of the biochemical markers of liver fibrosis (FibroTest) and necrosis (ActiTest) can be recommended as an alternative to liver biopsy for the assessment of liver injury in patients with chronic hepatitis C. In clinical practice, liver biopsy should be recommended only as a second line test, i.e., in case of high risk of error of biochemical tests.

Prediction of liver histological lesions with biochemical markers in patients with chronic hepatitis B

BACKGROUND AIMS Liver biopsy is the gold standard for assessing hepatitis B virus (HBV)-related histology. The aim was to determine the diagnostic utility of noninvasive serum markers in patients with chronic hepatitis B. METHODS The aminotransferases and indices including alpha(2)-macroglobulin, apolipoprotein A1, haptoglobin, gamma-glutamyl-transpeptidase (GGT), and total bilirubin (Fibrotest), and ALT (Actitest) were compared with liver histology. The primary outcomes were A2-A3 activity and F2-F4 fibrosis (METAVIR). RESULTS Two hundred and nine patients were included. Forty-one patients (20%) had A2-A3 activity and 61 (29%) had F2-F4 fibrosis. AST and GGT (P<0.001) were independently associated with A2-A3 activity. AST, ALT, and Actitest accurately predicted activity ((areas under receiver operating characteristic (ROC) curves (AUROC), 0.81-0.82+/-0.04)); an AST or ALT< or =30IU/l excluded significant activity with 96% certainty. Fibrotest accurately predicted F2-F4 fibrosis (AUROC, 0.78+/-0.04). Fibrotest scores (range, 0-1.0) < or =0.20 and >0.80 had negative and positive predictive values of 92%, respectively. Restricting biopsy to patients with intermediate scores (>0.20 and < or =0.80) may prevent liver biopsies in 46% of patients while maintaining 92% accuracy. CONCLUSIONS The aminotransferases and an index including five biochemical markers are accurate noninvasive markers of HBV-related activity and fibrosis, respectively.

Incidence and Predictors of Severe Liver Fibrosis in Human Immunodeficiency Virus—Infected Patients with Chronic Hepatitis C: A European Collaborative Study

A study was performed in 10 European health care centers in which 914 patients coinfected with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) who had elevated serum alanine aminotransferase (ALT) levels underwent liver biopsy during the period of 1992 through 2002. Overall, the METAVIR liver fibrosis stage was F0 in 10% of patients, F1 in 33%, F2 in 22%, F3 in 22%, and F4 in 13%. Predictors of severe liver fibrosis (METAVIR stage, F3 or F4) in multivariate analysis were age of >35 years (odds ratio [OR], 2.95; 95% confidence interval [CI], 2.08-4.18), alcohol consumption of >50 g/day (OR, 1.61; 95% CI, 1.1-2.35), and CD4+ T cell count of <500 cells/mm3 (OR, 1.43; 95% CI, 1.03-1.98). Forty-six percent of patients aged >40 years had severe liver fibrosis, compared with 15% of subjects aged <30 years. The use of antiretroviral therapy was not associated with the severity of liver fibrosis. In summary, severe liver fibrosis is frequently found in HCV-HIV-coinfected patients with elevated serum ALT levels, and its severity increases significantly with age. The rate of complications due to end-stage liver disease will inevitably increase in this population, for whom anti-HCV therapy should be considered a priority.

Effects of Lamivudine on Replication of Hepatitis B Virus in HIV-Infected Men

Chronic infection with hepatitis B virus (HBV) affects about 5% of the worlds population [1]. Of persons infected with the human immunodeficiency virus (HIV) as many as 10% are carriers of hepatitis B surface antigen (HBsAg) [2]. The high prevalence of HBV infection in HIV-infected patients occurs partly because the two viruses share routes of transmission. Carriers of HBsAg are usually asymptomatic, but they may develop cirrhosis, liver failure, and hepatocellular carcinoma. The 5-year survival rate of patients with established cirrhosis associated with chronic active hepatitis does not exceed 55% [3]. A high level of HBV replication or the presence of hepatitis B e antigen (HBeAg) predicts poor survival [4] and is common in persons infected with both HIV and HBV. In these patients, chronic HBV infection is more frequent than in the general population [5, 6] and has a peculiar course. Histologic and biological activities are lower and serum concentrations of HBV DNA are higher than those seen in HIV-negative patients [2, 7]. Interferon- is a cytokine that has antiviral and immunomodulatory properties [8] and is often proposed for the treatment of chronic HBV infection. In HIV-negative patients, the rate of response to interferon- (loss of serum HBV DNA and HBeAg) is 20% greater than the spontaneous seroconversion rate of 12% after 6 to 12 months of follow-up [9]. However, HIV infection is known to diminish the response to interferon-; these decreased response rates range from 0% [10] to 8% [11]. Therapies for HIV infection are already associated with increased survival [12]. Thus, the frequency of chronic HBV infection, which leads to cirrhosis and its complications, may increase in the population of HIV-infected patients who are coinfected with HBV. Therapies that are more effective against HBV than is interferon- alone are still needed in both HIV-positive and HIV-negative patients. Lamivudine, the enantiomer of 2-deoxy-3-thiacytidine, an inhibitor of HIV type 1 and HIV type 2 reverse transcriptase [13], has been shown to have antiretroviral activity in HIV-infected patients [14]. Lamivudine is also a potent selective inhibitor of HBV replication because this replication depends on reverse transcription of an intermediate RNA to a minus-stranded DNA, which then serves as a template for the synthesis of plus-stranded DNA [15]. Experimental studies have shown that lamivudine inhibits HBV DNA replication in transfected cell line 2.2.15 and in HBV-infected chimpanzees [15, 16]. Results of a phase II clinical trial [17] showed that lamivudine inhibited HBV replication in patients with chronic HBV infection. However, no information is available about the activity of lamivudine against HBV in HIV-infected patients. An open-label program of lamivudine therapy (NUCB 3004) has been in progress in France since December 1993. This program is for patients with progressive HIV disease who are refractory to or unable to tolerate therapies other than lamivudine. We evaluated the efficacy of lamivudine against HBV replication in 40 consecutive patients who were infected with both HIV and HBV and who entered this program. We report our results after 1 year of follow-up. Methods Patients From April to September 1994, 228 HIV-infected patients who were followed at the infectious diseases department at Groupe Hospitalier Pitie-Salpetriere and were eligible for the open-label lamivudine trial were prospectively tested for serologic markers of HBV infection (HBsAg, antibodies to HBsAg, HBeAg, and antibodies to HBeAg), IgG and IgM antibodies to hepatitis delta virus, and antibodies to hepatitis C virus (HCV). Forty of these patients were HBsAg carriers; these patients were consecutively included in our study and were followed for 1 year. Patients were seen at baseline and every 2 months. At each visit, physical examination was done and all clinical events were recorded. Blood samples were obtained for complete blood cell counts, measurement of CD4 lymphocyte counts, serum biochemistry tests, tests for serologic markers of HBV, and measurement of serum HBV DNA concentrations. Laboratory Assays Commercially available enzyme immunoassays were used to measure serologic markers of HBV (Abbott Diagnostics, Chicago, Illinois), IgG and IgM antibodies to hepatitis delta virus (Pasteur, Marne la Coquette, France), and antibodies to HCV (Ortho HCV ELISA, Ortho Diagnostic Systems, Inc., Raritan, New Jersey; RIBA HCV, Chiron Corp., Emeryville, California). Serum HBV DNA was assessed quantitatively and qualitatively. Serum concentrations of HBV DNA were measured by molecular hybridization through hybrid capture (Murex Diagnostics, Dartford, United Kingdom). Hybrid capture has a cut-off point of 5 pg/mL and allows titration to 2000 pg/mL. If the titer is greater than 2000 pg/mL, the test result is positive but exact titration cannot be done. We qualitatively assessed HBV DNA by using PCR at baseline and at months 2, 6, and 12 of lamivudine therapy only if serum concentrations of HBV DNA were less than 5 pg/mL as determined by molecular hybridization. We extracted DNA by heating 100 L of serum for 45 minutes. The two sets of primer used for the amplification were from the core region (HBV Core; Sorin-Biomedica, Saluggia, Italy). Polymerase chain reaction was done with the extracted DNA, and 35 cycles were run in a programmable thermoblock (Gen Amp PCR system 9600; Perkin Elmer Cetus Corp., Norwalk, Connecticut). To avoid contamination, each step of the PCR assay was done in a separate room and each series was done with at least three negative controls. We identified DNA by using DNA enzyme immunoassay (HBV Core Gene, ETI-K DEIA; Sorin-Biomedica). Single-stranded DNAs were hybridized with an oligonucleotide probe specific for the amplified region and were assayed by a monoclonal antibody that reacted only with double-stranded DNA. This assay can detect seven equivalent genomes per sample [18]. Antiviral Therapies In the French open-label treatment program, lamivudine was given to patients at a dosage of 300 mg twice daily from December 1993 to May 1995. Then, in May 1995, a protocol amendment recommended reducing the dose by 50% (to 150 mg twice daily). All study patients followed this protocol amendment. Thus, during the 12-month study period, 14 patients received 300 mg of lamivudine twice daily for 1 year and 26 patients received 300 mg of lamivudine twice daily for 9.5 1.2 months followed by 150 mg of lamivudine twice daily for 2.4 1.3 months. Seven patients with high HBV replication at baseline and 3 patients with low HBV replication at baseline received lamivudine in combination with zidovudine, 250 mg/d. The remaining 30 patients received lamivudine alone. Two patients with cytomegalovirus retinitis and no evidence of HBV replication (positivity for antibodies to HBeAg and an HBV DNA concentration < 5 pg/mL) started receiving lamivudine simultaneously with treatment for cytomegalovirus infection (1 patient received foscarnet and 1 received ganciclovir). No patients received interferon- during the course of the study. Other Concurrent Therapies At baseline, 38 patients (29 patients with high HBV replication at baseline and 9 patients with low HBV replication at baseline) were receiving sulfamethoxazole (800 mg/d) and trimethoprim (160 mg/d) as prophylaxis for Pneumocystis carinii pneumonia. One patient with high HBV replication and 1 patient with low HBV replication received sulfadiazine (2 g/d) and pyrimethamine (25 mg/d) as prophylaxis for recurrence of cerebral toxoplasmosis. Two patients with high replication and 1 patient with low replication were receiving bleomycin (5 mg/d for 3 days twice monthly) for cutaneous Kaposi sarcoma. Statistical Analysis Results are expressed as the mean SD. Data were compared using the Mann-Whitney test; a P value less than 0.05 was considered to be significant. Results The clinical and biological characteristics of the 40 study patients are shown in Table 1. No statistically significant differences in age, ratio of men to women, Centers for Disease Control and Prevention clinical stage of HIV infection [19], known duration of HIV infection, or CD4 lymphocyte counts were seen between the high-replication group and the low-replication group (data not shown). Table 1. Primary Clinical Findings at Baseline in Patients Infected with Both HIV and Hepatitis B Virus* Thirty-four patients had progressive HIV disease (a progressive decrease in CD4 lymphocyte count) despite receiving therapy with zidovudine and didanosine; 6 patients had progressive HIV disease despite receiving therapy with zidovudine and did not tolerate didanosine. All patients carried HBsAg for at least 1 year. In 27 patients, HBsAg and antibodies to HIV were found simultaneously in serum 5.9 2.8 years (range, 1 to 10 years) before lamivudine therapy was started. No patients had a history of initial acute hepatitis or decompensated liver disease or were infected with the hepatitis delta virus. One patient had detectable antibodies to HCV in serum. At baseline, two groups of patients were retrospectively identified according to serologic markers of HBV and serum concentrations of HBV DNA as measured by molecular hybridization. The first group comprised patients with high HBV replication (patients who were positive for HBeAg and had serum concentrations of HBV DNA > 5 pg/mL as determined by molecular hybridization) (n = 30). Liver biopsy was done in 6 of these patients 27 9 months (range, 12 to 36 months) before lamivudine therapy was started. These 6 patients had histologic findings consistent with chronic HBV infection, had a mean Knodell score [20] of 6 2.7 (range, 4 to 10), and did not lose serum HBV DNA while receiving interferon- therapy (5 million U/m2 body surface area three times a week for 6 months). Measurements of serum HBV DNA were available for 17 patients from 12 and 6 months before lamivudine therapy was started and for 4 patient

Antiviral efficacy of NS3‐serine protease inhibitor BILN‐2061 in patients with chronic genotype 2 and 3 hepatitis C

BILN‐2061, a specific and potent peptidomimetic inhibitor of the HCV NS3 protease, has recently been shown to markedly lower serum hepatitis C virus (HCV)‐RNA levels in patients chronically infected with HCV genotype 1 in three 2‐day proof of principle studies. The aim of the current study was to assess the antiviral efficacy of BILN‐2061 in patients with genotypes 2 and 3 HCV infection. The antiviral efficacy, pharmacokinetics, and tolerability of 500 mg twice‐daily BILN‐2061 given as monotherapy for 2 days in 10 patients chronically infected with non–genotype 1 HCV (genotype 2: n = 3; genotype 3: n =7) and minimal liver fibrosis (Ishak score 0‐2) were assessed in a placebo‐controlled (placebo n = 2), double‐blind pilot study. HCV‐RNA levels decreased by ≥1 log10 copies/mL in 4 of 8 patients treated with BILN‐2061. One patient showed a weak response of <1 log10 copies/mL. Three of 8 treated patients showed no response. There was no correlation between baseline viral concentration or genotype and response. BILN‐2061 exhibited good systemic exposure after oral administration and was well tolerated. In conclusion, the antiviral efficacy of the HCV serine protease inhibitor BILN‐2061 is less pronounced and more variable in patients with HCV genotype 2 or 3 infection compared with previous results in patients with HCV genotype 1. A lower affinity of BILN‐2061 for the NS3 protease of genotypes 2 and 3 HCV is most likely a major contributor to these findings. (HEPATOLOGY 2005.)

Anti‐hepatitis B virus efficacy of tenofovir disoproxil fumarate in HIV‐infected patients

Tenofovir disoproxil fumarate (TDF) has shown in vitro activity against both HIV and hepatitis B virus (HBV). We retrospectively evaluated the efficacy of TDF (300 mg/d), administered as a part of anti‐retroviral therapy, in a large cohort of HIV/HBV‐coinfected patients. Sixty‐five HIV/HBV‐coinfected patients who received TDF for at least 6 months with serum HBV DNA levels above 2.3 log10 copies/mL at TDF initiation and who had stored serum samples before and during TDF therapy were included. Serum HBV DNA was measured on stored samples. The median follow‐up period was 12 (Q1‐Q3: 8‐17) months. Serum hepatitis B e antigen (HBeAg) was positive in 54 patients (83.1%). Fifty‐two patients (80.0%) were receiving lamivudine (LAM) (150 mg twice a day), and 68.8% had documented LAM resistance at baseline. Among HBeAg‐positive patients, the median reduction from baseline (8.17; Q1‐Q3 = 7.30‐8.30 log10 copies/mL) of serum HBV DNA was 4.56 log10 copies/mL (Q1‐Q3 = 3.33‐5.55) (P < .0001). In HBeAg‐negative patients, serum HBV DNA decline from baseline (4.83; Q1‐Q3 = 2.69‐6.40 log10 copies/mL) was 2.53 log10 copies/mL (Q1‐Q3 = 0.39‐4.10). At the end of the study, HBV DNA became undetectable in 29.6% and 81.6% of the HBeAg‐positive and HBeAg ‐negative patients, respectively. Serum HBeAg became negative in 4 patients, 2 of whom acquired serum hepatitis B e antibody. In conclusion, this retrospective analysis demonstrates the efficacy of TDF against wild‐type, presumed precore mutants and LAM‐resistant HBV when used as a part of anti‐retroviral therapy in HIV‐coinfected patients. (HEPATOLOGY 2006;43:548–555.)

Care of HIV patients with chronic hepatitis B: Updated recommendations from the HIV-Hepatitis B Virus International Panel

Nearly 10% of the estimated 36 million people having HIV worldwide suffer from chronic hepatitis B virus (HBV) infection. The advent of new antiviral agents against HBV and the recent availability of improved molecular diagnostic tools have revolutioned the management of HIV/HBV coinfected patients. The present study represents an update of the current knowledge about HBV/HIV coinfection and an intent to provide practical advise about how to give the best care to HIV-infected persons with chronic hepatitis B.

Tenofovir disoproxil fumarate in patients with HIV and lamivudine-resistant hepatitis B virus.

To the Editor: Mutations in the YMDD (tyrosine, methionine, aspartate, aspartate) motif of the DNA polymerase resulting in phenotypic hepatitis B virus (HBV) resistance to lamivudine monotherapy ha...

Coexistence of Hepatitis B Surface Antigen (HBs Ag) and Anti-HBs Antibodies in Chronic Hepatitis B Virus Carriers: Influence of “a” Determinant Variants

ABSTRACT In chronic hepatitis B (CHB), the persistence of hepatitis B surface antigen (HBs Ag) is sometimes associated with antibodies (Ab) to HBs (anti-HBs). To assess the hypothesis of the selection of HBs Ag immune escape variants in CHB patients, the variability of the HBV S gene was determined for patients persistently carrying both HBs Ag and anti-HBs antibodies and patients solely positive for HBs Ag. We selected 14 patients who presented both markers (group I) in several consecutive samples and 12 patients positive for HBs Ag only (group II). The HBs Ag-encoding gene was amplified and cloned, and at least 15 clones per patient were sequenced and analyzed. The number of residue changes within the S protein was 2.7 times more frequent for group I than for group II patients and occurred mostly in the “a” determinant of the major hydrophilic region (MHR), with 9.52 versus 2.43 changes per 100 residues (P = 0.009), respectively. Ten patients (71%) from group I, but only three (25%) from group II, presented at least two residue changes in the MHR. The most frequent changes in group I patients were located at positions s145, s129, s126, s144, and s123, as described for immune escape variants. In CHB patients, the coexistence of HBs Ag and anti-HBs Ab is associated with an increase of “a” determinant variability, suggesting a selection of HBV immune escape mutants during chronic carriage. The consequences of this selection process with regard to vaccine efficacy, diagnosis, and clinical evolution remain partially unknown.

Potency, safety, and pharmacokinetics of the NS3/4A protease inhibitor BI201335 in patients with chronic HCV genotype-1 infection

BACKGROUND & AIMS BI201335 is a highly specific and potent HCV protease inhibitor. This multiple rising dose trial evaluated antiviral activity and safety in chronic HCV genotype-1 patients. METHODS Thirty-four treatment-naïve patients were randomized to monotherapy with placebo or BI201335 at 20-240 mg once-daily for 14 days, followed by combination with pegylated interferon alfa/ribavirin (PegIFN/RBV) through Day 28. Nineteen treatment-experienced patients received 48-240 mg BI201335 once-daily with PegIFN/RBV for 28 days. HCV-RNA was measured with Roche COBAS TaqMan. RESULTS In treatment-naïve patients, median maximal viral load (VL) reductions during 14-day monotherapy were -3.0, -3.6, -3.7, and -4.2 log(10) for the 20, 48, 120, and 240 mg groups. VL breakthroughs (≥1 log(10) from nadir) were seen in most patients on monotherapy and were caused by NS3/4A variants (R155K, D168V) conferring in vitro resistance to BI201335. Adding PegIFN/RBV at Days 15-28 led to continuous viral load reductions in most patients. In treatment-experienced patients, treatment with BI201335 and PegIFN/RBV achieved VL<25 IU/ml at Day 28 in 3/6, 4/7, and 5/6 patients in the 48, 120, and 240 mg dose groups. VL breakthroughs were observed during triple combination in only 3/19 patients. BI201335 was generally well tolerated. Mild rash or photosensitivity was detected in four patients. Mild unconjugated hyperbilirubinemia was the only dose-dependent laboratory abnormality of BI201335. BI201335 elimination half-life supports once-daily dosing. CONCLUSIONS BI201335 combined with PegIFN/RBV was well tolerated and induced strong antiviral responses. These results support further development of BI201335 in HCV genotype-1 patients.