M. Bourlière

Impact of various handling and storage conditions on quantitative detection of hepatitis C virus RNA

BACKGROUND/AIMSnBoth HCV RNA viral load and HCV genotype have been described as important predicting factors determining the response to interferon in chronic hepatitis C. To investigate whether processing and storage conditions might influence the stability and could alter the concentration of the HCV RNA in serum, quantification of HCV RNA was performed by branched DNA assay.nnnMETHODSnWe studied serum samples obtained from seven patients with histologically proven chronic hepatitis C. These were subjected to the following physical conditions: (1) immediate quantification, (2) storage at room temperature for 5 days, (3) storage at 4 degrees C for 5 days, (4) storage at -20 degrees C for 5 days, (5) storage at -80 degrees C for 5 days, (6) five freeze-thaw cycles, (7) blood unspun for 4 h at room temperature then centrifuged and stored at -80 degrees C for 5 days, (8) storage at 4 degrees C for 6 months, (9) storage at -20 degrees C for 6 months, (10) storage at -80 degrees C for 6 months.nnnRESULTSnA loss of 100% HCV RNA titers was observed after storage at RT for 5 days and then storage at 4 degrees C for 6 months. A surprising decrease of HCV RNA titer (15.6%) was observed in sera stored for 5 days at -20 degrees C. Five freeze-thaw cycles resulted in a 16% decrease of the HCV RNA level. When centrifugation was performed after a 4 h delay at room temperature, a significant loss of HCV RNA titers of 29.5% was observed. Long-term stability (6 months) was observed at -80 degrees C with a slight loss of about of 10% HCV RNA titers, but a significant decrease in HCV RNA of 23% was observed at -20 degrees C. The reproducibility of the bDNA assay on five patient samples was performed eight times in duplicate and showed an average coefficient of variation of 9.1%.nnnCONCLUSIONSnThese data confirm the importance of storage and handling in measuring the amount of HCV RNA in clinical samples.

Epidemiological changes in hepatitis C virus genotypes in France: evidence in intravenous drug users

Hepatitis C virus (HCV) genotypes are distributed differently depending on geography and route of infection. We characterized the distribution of genotypes in a large cohort of patients with chronic hepatitis C in the South‐east of France and evaluated the relative prevalence according to time of acquisition. One thousand, one hundred‐and‐eighty‐three patients who were anti‐HCV‐positive were studied. HCV genotype distribution has changed significantly from the 1960s to 2000. The prevalence of genotype 1b decreased from 47% before 1978 to 18.8% in the 1990s while the prevalence of genotype 1a and 3a increased during the same period from 18% and 15.3% to 28.8% and 26.3%, respectively. The logistic regression model showed that genotype 1a was significantly more common in patients infected through intravenous drug injection odds ratio ((OR): 2.08, Pu2003<u20030.01) and after 1990 (OR: 1.98, Pu2003<u20030.05). Genotype 1b was significantly less frequent in patients infected through intravenous drug injection (OR: 0.17, Pu2003<u20030.001) and has decreased since 1978 (OR: 0.27, Pu2003<u20030.001). Genotype 3a was independently associated with intravenous drug injection (OR: 6.1, Pu2003<u20030.001) and tattooing (OR: 8.01, Pu2003<u20030.001) and was more frequent in the 1979–90 period (OR: 2.05 and 1.74, Pu2003<u20030.001 and Pu2003<u20030.05). Our results show a modification of HCV genotypes distribution over the last four decades due to an increase of intravenous drug use (IVDU) contamination and an evolution of HCV genotypes distribution only in IVDU population characterized by a decrease of genotype 1b, an increase of genotype 3a from 1970 to 1990 and a higher increase of genotype 1a which is currently the predominant genotype in our population.

Interferon γ receptor 2 gene variants are associated with liver fibrosis in patients with chronic hepatitis C infection

Background Only a minority of patients with chronic hepatitis C virus (HCV) infection develops severe liver fibrosis, a process that may be controlled by human genetic factors. Objective To investigate the role of 384 single nucleotide polymorphisms (SNPs) located in 36 candidate genes related to the fibrogenesis/fibrolysis process. Methods Patients with chronic HCV infection were gathered from two French cohorts (prospectively and retrospectively). The overall sample consisted of 393 HCV-infected subjects without known risk factors for fibrosis progression, including 134 patients with severe liver fibrosis and 259 without severe fibrosis. Results Only two SNPs in strong linkage disequilibrium (LD) in the interferon γ receptor 2 gene (IFNGR2) were significantly associated with liver fibrosis in both the prospective and the retrospective samples. The strongest association (p=8×10−5) was observed with the G/A SNP rs9976971 with an OR of severe fibrosis for AA versus AG or GG subjects at 2.95 (95% CI 1.70 to 5.11). This effect was higher (p=9×10−7) when taking into account the time of follow-up, and the hazard ratio of progression towards severe fibrosis for AA patients was 2.62 (1.76 to 3.91). Refined sequencing and analysis of the IFNGR2 region identified two additional variants in strong LD with rs9976971. No haplotypes derived from this cluster of four variants provided stronger evidence for association than rs9976971 alone. Conclusions This identification of a cluster of four IFNGR2 variants strongly associated with fibrosis progression in chronic HCV infection underlines the role of IFNγ in the development of liver fibrosis that may pave the way for new treatments.

Myasthenia gravis and hepatitis C virus infection

SUMMARY. Chronic hepatitis C virus (HCV) infections are often associated with extrahepatic immunological manifestations, including various autoimmune disorders. The aims of this study were to determine the prevalence of HCV markers in patients with myasthenia gravis (MG) and to determine any relationship with HCV infection. Eighty‐three patients with MG, 40 men aged 20–93 years and 43 women aged 13–87 years (mean age 54 years) were studied. The MG patients were positive for antibody to acetylcholine receptor, in addition, their sera was analysed for antibody to HCV (HCVAb) and HCV RNA. HCVAb was detected in two of the 83 patients (2.4%). Four patients were repeatedly HCV RNA positive. They were infected by HCV genotype 1 (one patient), HCV genotype 2a (two patients) and an undetermined HCV genotype in one patient. They received plasmapheresis or intravenous immunoglobulin treatment. Among the four patients, one was infected after the onset of MG without receiving a blood transfusion or using intravenous drugs. The other three had chronic hepatitis C which was discovered at the same time as MG and only one patient had been exposed to blood products. The prevalence of HCV markers in patients with MG (4.8%) was higher than that reported for the general French population, about 1%. This prevalence is similar to that occurring in patients exposed to plasmapheresis or intravenous immunoglobulin. In conclusion, HCV appears to play little, if any, role in causing MG. The higher prevalence of infection among MG patients may be related to transmission in the course of therapy.

Combining non-invasive methods for assessment of liver fibrosis.

Non-invasive markers of liver fibrosis, including biochemical scores using simple biochemical parameters and transient elastography (TE), have been developed over the past decade to either replace or reduce the need for liver biopsy (LB) in the assessment of liver fibrosis. Although their diagnostic accuracy in liver fibrosis is promising, around 20% of patients are likely to be misclassified if these tests or LB are used alone. However, using a combination of several biochemical scores (Fibropaca algorithm, Leroy algorithm) or one biochemical score with TE (Bordeaux algorithm) will increase diagnostic accuracy for fibrosis and reduce the need for LB. Stepwise combination algorithms of non-invasive scores (SAFE biopsy) also improve the diagnostic performance in chronic hepatitis C (CHC) compared with the use of a single non-invasive score. Other sequential stepwise algorithms have been developed in CHC with similar performance results. Comparisons of different combinations of non-invasive methods indicate that the SAFE biopsy, Fibropaca algorithm and Bordeaux algorithm are excellent and comparable in the non-invasive diagnosis of liver fibrosis in HCV patients, and will markedly reduce the need for LB. They may also be useful in clinical practice and particularly for large-scale screening of HCV patients.

Malades atteints d'hépatite chronique C non répondeurs : définitions de la non-réponse et stratégies thérapeutiques

Resume Environ la moitie des patients atteints d’hepatite chronique C ne repondent pas au traitement actuel par bitherapie associant l’interferon pegyle et la ribavirine. On doit distinguer les patients « faux » non repondeurs qui n’ont pas eu un traitement optimal des « vrais » non-repondeurs qui ont eu un traitement optimal. Chez les patients « faux » non repondeurs, la correction des facteurs de non-reponse lies au patient (consommation d’alcool, surcharge ponderale…) ou l’amelioration de la tolerance du traitement (traitement antidepresseur, erythropoietine…) peut permettre un retraitement optimal avec des chances d’eradication virale. Au contraire, chez les patients « vrais » non repondeurs, la probabilite d’une eradication virale avec un retraitement est faible et on peut envisager, en cas de maladie severe (stade de fibrose F3 ou F4), un traitement d’entretien. L’objectif du traitement d’entretien est de diminuer l’activite de l’hepatite chronique et de stabiliser la fibrose et ainsi de diminuer le risque de complications et de carcinome hepatocellulaire. Les essais en cours permettront de definir les modalites optimales du traitement d’entretien. Les nouvelles molecules antivirales, essentiellement les antiproteases et antipolymerases, seront sans doute utilisees en tritherapie avec l’interferon et la ribavirine. Les molecules, actuellement en phase 1 et 2, qui feront la preuve de leur efficacite et de leur bonne tolerance ne devraient pas etre disponibles avant plusieurs annees.

Étude de l'association hépatite auto-immune et antiphospholipides

Introduction nOnly few series have reported the association of autoimmune hepatitis with antiphospholipid antibodies. The aim of our study is to investigate the frequency of these antibodies in a series of autoimmune hepatitis and to search for a correlation with clinical, biological or histological characteristics.

Analogs and fibrosis regression in hepatitis B

Nucleoside or nucleotide analogs (NUCs) are a major step forward in the treatment of hepatitis B virus (HBV) infection. Apart from their proven antiviral efficacy, these drugs have proved able to significantly improve liver fibrosis as short-term treatment. Using different definitions of fibrosis regression, after 1 year of treatment, improvement in liver fibrosis was observed among HBe antigen (HBeAg)-positive naive patients: 35-61% with lamivudine; 41% with adefovir; 68% with telbivudine; 39% with entecavir; and 74% with tenofovir. Among HBeAg-negative patients, after 1 year of treatment, improvement in liver fibrosis was seen in: 36-46% with lamivudine; 48% with adefovir; 56% with telbivudine; 36% with entecavir; and 71% with tenofovir. Long-term treatment is often required with NUCs; the response continue to improve over time, reaching up to 63% of patients with lamivudine and 71% of patients with adefovir, although the development of antiviral drug resistance can blunt any histological improvement. Also, there is now growing evidence that non-invasive methods of fibrosis diagnosis, such as surrogate markers, are likely to become as important as liver biopsy for taking the initial decision to biopsy and for treatment, and in the follow-up of treated chronic HBV patients.

Traitement de l’hépatite chronique B : nouvelles perspectives

New antiviral drugs, therapeutic vaccines and immunotherapies are interesting therapeutic option for treatment of chronic hepatitis B. Tenofovir disoproxil fumarate and clevudine are very effective in suppressing viral load with lack of resistance at short term. However antiviral drug rarely eliminate the virus. Immunotherapies by inducing HBV - specific T-cell responses may contribute to virus control. Adoptive T-cell therapy is an interesting approach to eliminate persistently infected cells. Therapeutic vaccines with either protein-based or DNA vaccines induce an HBV - specific T-cell responses leading to a more often transient decrease of viral load. Combination of antiviral drug and therapeutic vaccines may be an interesting therapeutic option. However additional larger scale and more systematic studies of the understanding of HBV specific T-cell response have to be performed in order to select which patient can benefit of such therapies.

Évaluation économique des stratégies de dépistage de l'hépatite chronique C

Resume Objectifs Estimer lefficacite, les couts et les benefices de plusieurs strategies de depistage de lhepatite chronique C en fonction de la prevalence de linfection. Methode Une analyse de decision a permis de comparer le cout et lefficacite de strategies de depistage avec la strategie statu quo qui consiste a ne pas depister. Le nombre de pathologies evitees a ete retenu comme critere defficacite. Les benefices retires dun depistage et dune prise en charge precoce ont ete evalues par les couts evites et pris en compte dans le calcul du cout net total. Deux hypotheses de prise en charge ont ete evaluees (mono- et bitherapie). Une modelisation de lhistoire naturelle de la maladie a permis dintegrer dans lanalyse les consequences a long terme (10 ans) dun programme de depistage. Resultats Loption statu quo est dominee quelle que soit la strategie de depistage consideree. La strategie consistant en un seul test ELISA, suivi dun ELISA de confirmation en cas de positivite est la plus cout-efficace. Depister et traiter demblee par interferon et ribavirine les sujets naifs depistes savere aujourdhui plus cout-efficace que le traitement par bitherapie des seuls rechuteurs. Conclusion Cette evaluation economique privilegie la strategie du depistage et du traitement des patients porteurs chroniques du virus C par rapport a une strategie de statu quo.