Françoise Lunel

A novel panel of blood markers to assess the degree of liver fibrosis

The objective was to develop new blood tests to characterize different fibrosis parameters in viral and alcoholic chronic liver diseases. Measurements included 51 blood markers and Fibrotest, Fibrospect, ELFG, APRI, and Forns scores. The clinically significant fibrosis was evaluated via Metavir staging (F2‐F4), and image analysis was used to determine the area of fibrosis. In an exploratory step in 383 patients with viral hepatitis, the area under the receiving operator characteristic (AUROC) curve for stages F2‐F4 in a test termed the “Fibrometer” test combining platelets, prothrombin index, aspartate aminotransferase, α2‐macroglobulin (A2M), hyaluronate, urea, and age was 0.883 compared with 0.808 for the Fibrotest (P = .01), 0.820 for the Forns test (P = .005), and 0.794 for the APRI test (P < 10−4). The Fibrometer AUROC curve was 0.892 in the validating step in 120 patients. The AUROC curve for stages F2‐F4 in a test combining prothrombin index, A2M, hyaluronate, and age was 0.962 in 95 patients with alcoholic liver diseases. The area of fibrosis was estimated in viral hepatitis by testing for hyaluronate, γ‐glutamyltransferase, bilirubin, platelets, and apolipoprotein A1 (aR2 = 0.645), and in alcoholic liver diseases by testing for hyaluronate, prothrombin index, A2M, and platelets (aR2 = 0.836). In conclusion, the pathological staging and area of liver fibrosis can be estimated using different combinations of blood markers in viral and alcoholic liver diseases. Whereas the Fibrometer has a high diagnostic accuracy for clinically significant fibrosis, blood tests for the area of liver fibrosis provide a quantitative estimation of the amount of fibrosis, which is especially useful in cirrhosis. (HEPATOLOGY 2005.)

Cryoglobulinemia in chronic liver diseases: Role of hepatitis C virus and liver damage

BACKGROUND/AIMS Mixed cryoglobulinemia is frequently associated with liver diseases. The respective role of hepatitis C virus (HCV) and liver damage in the pathogenesis of cryoglobulinemia is investigated in this study. METHODS The prevalence of cryoglobulinemia in 226 consecutive patients with chronic liver diseases (hepatitis C, 127; hepatitis B, 40; other diseases, 59) was studied, and the epidemiological, biological, histological, and virological features in these three groups were analyzed. Anti-HCV antibodies, HCV proteins, and HCV RNA were searched in the cryoprecipitates. RESULTS The prevalence of mixed cryoglobulinemia was high (41.5%) in patients with liver diseases and higher in patients with hepatitis C (54.3%) than in patients with hepatitis B (15%) or other causes of liver disease (32%). Patients with cryoglobulinemia had cirrhosis more frequently and had a longer history of hepatitis. In patients with hepatitis C, HCV RNA sequences and HCV proteins were detected in the cryoprecipitate. Cryoglobulins became undetectable in 21 of 43 patients treated with interferon. CONCLUSIONS These findings suggest that HCV is a major cause of cryoglobulinemia. Besides viral infection itself, multiple factors appear to be responsible for the production of cryoglobulins, including cirrhosis and duration of liver disease.

Comparison of blood tests for liver fibrosis specific or not to NAFLD

BACKGROUND/AIMS To compare blood tests of liver fibrosis specific for NAFLD: the FibroMeter NAFLD and the NAFLD fibrosis score (NFSA) with a non-specific test, APRI. METHODS Two hundred and thirty-five NAFLD patients with liver Metavir staging and blood markers from two independent centres were randomly assigned to a test (n=121) or a validation population (n=114). RESULTS The highest accuracy--91%--for significant fibrosis was obtained with the FibroMeter whose (i) AUROC (0.943) was significantly higher than those of NFSA (0.884, p=0.008) and APRI (0.866, p<10(-3); p=0.309 vs NFSA) in the whole population, and (ii) misclassification rate (9%) was significantly lower than those of NFSA (14%, p=0.04) and APRI (16%, p=0.002) and did not vary according to centre (14 vs 7%, p=0.07), unlike those of NFSA (25 vs 9%, p=0.001) and APRI (29 vs 11%, p<10(-3)). By using thresholds of 90% predictive values, liver biopsy could have been avoided in most patients: FibroMeter: 97.4% vs NFSA: 86.8% (p<10(-3)) and APRI: 80.0% (p<10(-3)). A new classification provided three reliable diagnosis intervals: F0/1, F0/1/2, F2/3/4 with 91.4% accuracy for FibroMeter, avoiding biopsy in all patients. CONCLUSIONS FibroMeter NAFLD had high performance and provided reliable diagnosis for significant fibrosis, significantly outperforming NFSA and APRI.

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

Sequence analysis of the NS5A protein of European hepatitis C virus 1b isolates and relation to interferon sensitivity.

Japanese studies have defined the discrete 2209-2248 amino acid region of the non-structural 5A protein (NS5A(2209-2248)) of hepatitis C virus genotype 1b (HCV 1b) isolates as the interferon sensitivity determining region (ISDR). European studies did not confirm these results since most of the ISDR sequences harboured an intermediate profile. Recently, a direct interaction between the NS5A protein, involving the ISDR, and the interferon-induced protein kinase (PKR) has been reported and presented as a possible explanation of HCV interferon resistance. In the present study, the entire NS5A amino acid sequence from 11 resistant and eight sensitive strains from European HCV 1b isolates was inferred from direct sequencing. The previously described important amino acid stretches and positions in NS5A were compared between the resistant and sensitive groups. Although some variations were observed, no clear differences could be directly correlated with the interferon sensitivity. However, sensitive strains were different, owing to more amino acid changes when compared to a consensus sequence from all strains. The carboxy-terminal region and especially the previously reported NS5A/V3 region showed most of the variations. Moreover, the conformational analysis of NS5A by secondary structure prediction allowed the differentiation of most sensitive strains from resistant ones. It was concluded that other regions different from ISDR were involved in resistance to interferon maybe via the interaction between NS5A and PKR.

Mutations of hepatitis C virus 1b NS5A 2209–2248 amino acid sequence do not predict the response to recombinant interferon-alfa therapy in French patients

BACKGROUND/AIMS Studies of HCV quasispecies during interferon treatment have shown the selection of resistant clones. Enomoto et al. have defined the interferon sensitivity-determining region in an amino acid stretch of the HCV-1b NS5A region. Patients with a mutant strain before treatment were complete responders, whereas those with wild-type HCV-J strain were resistant to interferon. The same region was studied in HCV isolates of French patients. METHODS Forty-three HCV-1b chronically infected patients, consisting of 26 non-responders and 17 complete responders to interferon-alfa treatment (3 MUI tiw for 6 months), were included retrospectively. We directly sequenced the NS5A(2209-2248) HCV region of these patients before treatment. The viral load could be obtained from six complete responders and 15 non-responders. RESULTS We detected wild-type and intermediate strains, but only two mutant strains were present. One of them was found in a non-responder. In three complete responders, we found a wild-type strain. The distribution of the various strains was rather different from that found in Japan. Before treatment, the viral load was lower in complete responders (p=0.01). CONCLUSIONS Only two mutant strains were detected in our study. This could partially explain the low response rate to interferon treatment of French HCV-1b-infected patients, although the dose regimen was lower than in Japanese studies. Also, wild-type strains were found in some complete responders, and no correlation was determined between the mutation number in the NS5A(2209-2248) region and response to alfa interferon therapy. This may be related to epidemiological differences between HCV-1b strains present in France and those in Japan. Searching for the mutant NS5A pattern before treatment does not appear to be useful in French patients as it is too uncommon.

Liver-Stage Development of Plasmodium falciparum in a Humanized Mouse Model

BACKGROUND The liver stage of the human malaria parasite Plasmodium falciparum is the least known, yet it holds the greatest promise for the induction of sterile immunity and the development of novel drugs. Progress has been severely limited by the lack of adequate in vitro and in vivo models. METHODS Recently, it was found that immunodeficient mice transgenic for the urokinase plasminogen activator allow survival of differentiated human hepatocytes. We confirm this finding but show that hepatocyte survival is short lived unless nonadaptive defenses are simultaneously depleted. RESULTS By controlling macrophages and NK cells, we readily effected the long-term secretion of human serum albumin and human alpha-1 antitrypsin in mouse serum (at 3 months, the proportion of repopulated mice increased from 0% to 60% and from 22% to 80%, respectively; P<.0001). P. falciparum sporozoites delivered intravenously into mice readily infected transplanted human hepatocytes and developed into liver schizonts. Their size was twice as large as what was seen in vitro and was comparable to that found in humans and chimpanzees. CONCLUSION These results emphasize the importance of nonadaptive defenses against xenotransplantation and lead to development of small laboratory models that, because they can harbor human hepatocytes, provide novel opportunities to study intrahepatic pathogens, such as those causing malaria and hepatitis.

Comparison of Hepatitis C Virus NS5b and 5′ Noncoding Gene Sequencing Methods in a Multicenter Study

ABSTRACT A national evaluation study was performed in 11 specialized laboratories with the objective of assessing their capacities to genotype hepatitis C virus (HCV) and define the applicability of a given genotyping method. The panel consisted of 14 samples positive for HCV RNA of different genotypes (including 3 samples with two different artificially mixed genotypes) and 1 HCV-negative sample. Seventeen sets of data were gathered from the 11 participating laboratories. The sensitivities ranged from 64.3 to 100% and from 42.7 to 85.7% for the methods that used sequencing of the NS5b region and the 5′ noncoding (5′ NC) region, respectively. When the data for the artificially mixed samples were excluded, NS5b genotyping gave correct results for 80% of the samples, 1.7% of the samples were misclassified, and 18.3% of the samples had false-negative results. By 5′ NC-region genotyping methods, 58.3% of the results were correct, 29.7% were incomplete, 8.3% were misclassifications, 1.2% were false positive, and 2.4% were false negative. Only two procedures based on NS5b sequencing correctly identified one of the three samples with mixtures of genotypes; the other methods identified the genotype corresponding to the strain with the highest viral load in the sample. Our results suggest that HCV 5′ NC-region genotyping methods give sufficient information for clinical purposes, in which the determination of the subtype is not essential, and that NS5b genotyping methods are more reliable for subtype determination, which is required in epidemiological studies.

Ribavirin exposure after the first dose is predictive of sustained virological response in chronic hepatitis C

The impact of ribavirin exposure on sustained virological response (SVR) in patients with chronic hepatitis C is unknown. Preliminary studies showed marked inter‐individual variability of ribavirin concentrations despite dose adjustment for body weight (BW) and suggested there was a correlation between single time point concentrations and SVR. None of them evaluated the global exposure to ribavirin. This study was conducted to determine whether early ribavirin global exposure is related with SVR. An exploratory pharmacokinetic‐pharmacodynamic (PK‐PD) study was conducted in genotype 1 hepatitis C patients treated with peginterferon alfa‐2a and ribavirin (dose‐adjusted for BW) for 12 weeks, to which amantadine was added for the following 36 weeks. Full and abbreviated ribavirin area under the concentration time curves (AUC0–12h, AUC0–4h) were derived from plasma concentration profiles at day 0 (D0), week 12 (W12), W12 + 1 day, and W24. Virological follow‐up was performed at D0 (0, 12, and 24 hours), W2, W4, W6, and monthly until W72 (TaqMan polymerase chain reaction, cut‐off 15 international units/mL). Twenty‐eight patients were enrolled in the study and 24 completed it. Patients with a SVR had a significantly higher D0 AUC0–12h (3695 [1571–6916] versus 2937 [1266–4913] μg/hour/L, P = 0.03) and D0 AUC0–4h (2010 [615–3175] versus 1340 [622–2246] μg/hour/L, P = 0.03). Patients with D0 AUCs above the cut‐off values defined by receiver operating characteristic curves (3014 μg/hour/L and 1755 μg/hour/L for AUC0–12h and AUC0–4h, respectively) had a significantly better chance of achieving an SVR than patients with AUCs under the thresholds (odds ratio = 16.0, 95% confidence interval 1.54–166.6, P = 0.02 and odds ratio = 8.9, 95% confidence interval, 1.4–56.6; P = 0.02). Conclusion: Ribavirin exposure at D0 is significantly related to SVR. To our knowledge, this is the first study to give an early pharmacokinetic predictor of SVR. We propose a minimum AUC0–4h threshold of 1755 μg/hour/L at D0 as a target for ribavirin dose adjustment. (HEPATOLOGY 2008;47:1453–1461.)

Comparative evaluation of the total hepatitis C virus core antigen, branched-DNA, and amplicor monitor assays in determining viremia for patients with chronic hepatitis C during interferon plus ribavirin combination therapy.

ABSTRACT An assay prototype designed to detect and quantify total hepatitis C virus [HCV] core antigen (HCV core Ag) protein in serum and plasma in the presence or absence of anti-HCV antibodies has been recently developed by Ortho-Clinical Diagnostics. The aim of the study was to evaluate the sensitivity, specificity, and reproducibility of the Total HCV core Ag assay in comparison with two quantitative assays for HCV RNA: Quantiplex HCV RNA 2.0 (bDNA v2.0) or Versant HCV RNA 3.0 (bDNA v3.0) assays and the Cobas Amplicor HCV Monitor version 2.0 (HCM v2.0) test. We have studied samples of a well-characterized panel and samples from patients with chronic hepatitis C treated with interferon alone or with ribavirin. We have also compared the kinetics of HCV core Ag and HCV RNA in the follow-up of treated patients. The HCV core Ag assay exhibited linear behavior across samples from different genotypes. The coefficients of variation for intra- and interassay performance were 5.11 and 9.95%, respectively. The specificity of the assay tested in blood donors was 99.5%. Samples from HCV-infected patients showed that the correlation between the HCV core Ag and the two HCV RNA quantitative assays (bDNA and HCM v2.0) was 0.8 and 0.7, respectively. This correlation was maintained across different genotypes of HCV (r2 = 0.64 to 0.94). Baseline HCV core Ag values were significantly lower in sustained responders to interferon (IFN) than in other groups of patients (5.31 log10 [104 pg/ml] versus 5.99 log10 [104 pg/ml]; P < 0.001). In patients treated with IFN or combination therapy, we found an association between a decrease of more than 2 log IU/ml in viral load, undetectable HCV core Ag, and sustained response. Among sustained responders to IFN alone or combination therapy and among relapsers after IFN alone, 84 out of 101 (83.2%) had undetectable HCV core Ag, and 76 out of 96 (79.2%) had a viral load decrease of ≥2 log IU/ml, after 1 month of treatment. In conclusion, the Total HCV core Ag assay is a new useful test for the detection of HCV viremia and the monitoring of patients treated with IFN alone or in combination with ribavirin.