Recent evidence of underestimated circulation of hepatitis C virus intergenotypic recombinant strain RF2k/1b in the Rhône-Alpes region, France, January to August 2014: implications for antiviral treatment.

Since the beginning of 2014, hepatitis C virus (HCV) recombinant forms RF2k/1b have been detected in the Rhône-Alpes French region in 10 patients originating from the Caucasus area. Circulation of this particular HCV strain is very likely to be underestimated. It is also prone to be misgenotyped when using genotyping methods based on the 5' region of the viral genome, which may lead to suboptimal treatment.

First next-generation sequencing full-genome characterization of a hepatitis C virus genotype 7 divergent subtype.

We report the near-full-length genome sequence of a hepatitis C virus (HCV) isolate from a man originating from Democratic Republic of Congo, the genotype of which could not be determined by the routinely used sequencing technique. The near-complete genome sequence of this variant BAK1 was obtained by the association of two next-generation sequencing technologies. Evolutionary analysis indicates that this isolate, BAK1, could be the first reported strain belonging to a new HCV-7b subtype. This new subtype has been incorrectly identified as genotype 2 by the Versant HCV Genotype 2.0 assay (LiPA). The requirement of three independent isolates has been filled, and a new subtype can be assigned. More examples of HCV-7 are required to better understand its origin, its pathogenicity and its relationship with genotype 2.

Amplification and pyrosequencing of near-full-length hepatitis C virus for typing and monitoring antiviral resistant strains.

Directly acting antiviral drugs have contributed considerable progress to hepatitis C virus (HCV) treatment, but they show variable activity depending on virus genotypes and subtypes. Therefore, accurate genotyping including recombinant form detection is still of major importance, as is the detection of resistance-associated mutations in case of therapeutic failure. To meet these goals, an approach to amplify the HCV near-complete genome with a single long-range PCR and sequence it with Roche GS Junior was developed. After optimization, the overall amplification success rate was 73% for usual genotypes (i.e. HCV 1a, 1b, 3a and 4a, 16/22) and 45% for recombinant forms RF_2k/1b (5/11). After pyrosequencing and subsequent de novo assembly, a near-full-length genomic consensus sequence was obtained for 19 of 21 samples. The genotype and subtype were confirmed by phylogenetic analysis for every sample, including the suspected recombinant forms. Resistance-associated mutations were detected in seven of 13 samples at baseline, in the NS3 (n = 3) or NS5A (n = 4) region. Of these samples, the treatment of one patient included daclatasvir, and that patient experienced a relapse. Virus sequences from pre- and posttreatment samples of four patients who experienced relapse after sofosbuvir-based therapy were compared: the selected variants seem too far from the NS5B catalytic site to be held responsible. Although tested on a limited set of samples and with technical improvements still necessary, this assay has proven to be successful for both genotyping and resistance-associated variant detection on several HCV types.

Dynamics of viral quasispecies during interferon therapy in non responder chronic hepatitis C patients.

BACKGROUND: the reference method to study the HCV complexity was cloning and sequence analysis of a sufficient number of clones. The evolution of the viral complexity in chronic non responder patients during treatment with standard doses of interferon was not very well investigate because this method was expensive and labour intensive when large series of patients were concerned. Meanwhile, with the alternative Single-Strand Conformation Polymorphism (SSCP) method, a rough estimation of the quasispecies present in a given sample could be obtained. OBJECTIVES: the aim of the study was to analyse the evolution of HCV heterogeneity, investigated by SSCP analysis targeted to the HVR-1, in 30 nonresponders chronic hepatitis C patients treated by Interferon-alpha 3MUI. RESULTS: genotype 1 was the main HCV type found in this population (77% of non responder patients). Before treatment, the SSCP assay revealed a high complexity pattern: the median of SSCP band number was 9. During IFN-alpha treatment, SSCP band number didn't change. However a significant decrease of the viral load was observed (P<0.01). Patients with variations in their SSCP patterns after therapy significantly decreased HCV RNA levels (P<0.002). In one third of patients the SSCP profile didn't change at all. CONCLUSIONS: we observed that viral heterogeneity didn't change in non responder chronic hepatitis C patients during IFN-alpha treatment. Nevertheless patients with a low number of pre-treatment quasispecies exhibited an improvement of the response (P<0.02). These phenomena were probably due to a selection of resistant variants present prior onset of therapy.

Quantitative detection of hepatitis B virus DNA in serum using chemiluminescence: comparison with radioactive solution hybridization assay.

A quantitative, non-radioactive hybrid capture HBV DNA assay (Digene Diagnostics), which uses an efficient solution hybridization procedure coupled to a sensitive chemiluminescent signal amplification system, was compared with the quantitative, radioactive solution hybridization assay (Genostics, Abbott Laboratories), in hepatitis B virus carriers, particularly in those undergoing antiviral therapy. The qualitative reproducibility of the chemiluminescent method, tested on 30 sera, was acceptable, with a reproducibility rate of 93.3%. A comparison of this hybrid capture HBV DNA assay with the radioactive test on 113 sera obtained from 48 patients (39 HBsAg-positive patients) gave a sensitivity of 87.2%, a specificity of 100% and an agreement between the two tests of 89.4% (101 sera including 82 HBV DNA positive and 19 negative samples). Changes in HBV DNA levels measured by the two assays showed a good correlation with each other during interferon therapy. However, the hybrid capture values were higher than the radioactive assay values, with the ratio of the two values being variable in the same patient during the course of treatment. The Genostics assay therefore seems to be a more accurate procedure for evaluating changes in viral replication, particularly at high HBV DNA levels. However, the hybrid capture method is faster and has the advantage of being a non-radioactive procedure. This chemiluminescent assay is easy to perform as a routine diagnostic procedure and may be a useful alternative to the radioactive solution hybridization method.

c-Ha-ras polymorphism in patients with hepatocellular carcinoma. Comparison of healthy subjects and alcoholic patients with cirrhosis.

It has been observed that certain rare alleles of the c-Ha-ras gene occur more frequently in patients with certain malignant tumors than in healthy individuals, suggesting that these alleles may serve as markers for particular types of cancer. In this study, we compared the restriction fragment length polymorphism at the c-Ha-ras gene locus in 40 patients with cirrhosis and hepatocellular carcinoma with that in 39 patients with cirrhosis and in 42 normal subjects, all of Caucasian origin. Southern blotting of leukocyte DNA from the above patients, after digestion with either BamH1 or AvaII, revealed the presence of allele fragments of different sizes, corresponding to 4 common alleles and 3 rare alleles. The occurrence of the 3 rare alleles was not significantly different in the 3 populations studied. On the other hand, 2 of the common alleles, a3 (P < 0.01) and a4 (P < 0.03), were found at a significantly higher frequency in patients with cancer than in the 2 other groups. These results suggest that, in hepatocellular carcinoma, there is no increase in the frequency of occurrence of the rare alleles of the c-Ha-ras, but that the distribution of the common alleles may be modified.

IRES complexity before IFN-alpha treatment and evolution of the viral load at the early stage of treatment in peripheral blood mononuclear cells from chronic hepatitis C patients.

At the early stage of treatment, IFN alpha-2a induces inhibition of HCV replication. The viral load reflects mainly the degradation rate of the viruses. However, differences in the behavior of the viral population depend on changes, which occurred in the HCV-IRES genome. In this study, cloning and sequencing strategies permitted the generation of a large number of IRES sequences from the PBMCs of 18 patients (5 women, 13 men) with chronic hepatitis C. The HCV IRES appeared to be highly conserved structurally. However, some variability was found between the different isolates obtained: 467 substitutions with a median of 7 variants/patients. No relationship was observed between pre-treatment IRES complexity and the viral load at the beginning. However, on review of the evolution of viral load in the PBMCs during the first 3 days of IFN alpha-2a treatment, patients could be classified into two groups: Group 1, in which the viral population continued to replicate and Group 2, in which the viral load decreased significantly (P = 0.01727). Positioning of the mutations on the predicted IRES secondary structure showed that the distribution of the mutations and their apparition frequency were different between the two groups. At the early stage of treatment, IFN alpha-2a was efficient in reducing the viral replication in a significant number of patients; mechanisms of response might affect the virus directly. However, pre-treatment genomic variations observed in the 5'NCR of HCV were not a parameter of a later response to antiviral therapy in chronic hepatitis C patients. (244)

Interest of the detection of hepatitis C virus RNA in patients with alcoholic liver disease. Comparison with the HBV status.

HCV-RNA detection was investigated in 66 chronic alcoholic patients divided into 3 groups according to the severity of liver injury: group 1 included 22 chronic alcoholics without cirrhosis, group 2, 20 patients with alcoholic cirrhosis and group 3, 24 patients with alcoholic cirrhosis and hepatocellular carcinoma. The 'nested' polymerase chain reaction (PCR) technique amplifying the 5' non-coding region was used to detect HCV-RNA. For comparison, ELISA1, ELISA2 and RIBA2 tests (Ortho Diagnostics System) were also used to detect anti-HCV antibodies. Finally HBV markers (HBsAg, anti-HBc and anti-HBs antibodies) were detected in all patients as well as HBV-DNA by PCR. In group 1, only 1 patient (4.5%) showed an HCV-RNA-positive PCR, while 3 patients (13.6%) were found to have anti-HCV antibodies detected by RIBA2. In group 2, 3 patients (15%) showed positive PCRs, whereas 4 patients (20%) had anti-HCV antibodies. Finally, in group 3, the PCR was positive in 3 patients (12.5%), while 9 (37.5%) had anti-HCV antibodies. All patients with positive PCRs showed positive anti-HCV antibodies detected by second-generation assays. On the other hand, these patients often had past HBV infection markers but rarely had HBV-DNA detected by PCR. These results suggest that in chronic alcoholic patients, regardless of the severity of liver injury, HCV replication is rarely observed by PCR. Indeed, replication is only observed when anti-HCV antibody detection is positive in second-generation assays, particularly with strong reactivity against C33-C and C22-3 antigens. The relatively high prevalence of anti-HCV antibodies in this population compared to the usual rates could be explained by the age, geographic and perhaps even socioeconomic origin of the patients.