The activity of the French National Reference Center for Viral Hepatitis B, C, and delta (NRC) is divided into three complementary branches that extensively cover the virology of hepatitis, from technological investigation to clinical and translational research.

• Technological expertise: Development, standardization, and evaluation of intrinsic performance, test indications, and new virology technologies for diagnostic purposes and therapeutic follow-up of chronic viral hepatitis.
• Molecular epidemiology: Molecular epidemiology and surveillance of viral hepatitis B, C, and delta.
• Clinical and Translational research: Study of natural genetic variability associated with antiviral chemotherapy of hepatitis B and C viruses, with a particular focus on the mechanisms underlying therapeutic efficacy and treatment failure and the role of antiviral resistance in treatment failure. The laboratory is also interested in the development of new broad-spectrum antiviral therapeutic approaches and the mechanisms of hepatic lesions associated, or not, with HCV infection, using cellular and transgenic mouse models.

Expertise

During the last decade, the NRC has carried out several expertise activities.

Expert activity in the field of diagnostic and typing tools

Several evaluations of virological diagnostic tests for HCV and HBV were conducted in partnership with the manufacturing companies which developed them. These evaluations concerned molecular biology techniques adapted to detect and/or quantify HCV RNA and HBV RNA by real-time PCR or TMA, as well as viral genomic typing techniques by reverse hybridization or sequencing to identify the viral genotype and subtype or resistance mutations after nucleotide sequence analysis (by direct or high-throughput sequencing).

Recent studies carried out within the scope of the missions of the NRC have included:

Performance of rapid diagnostic tests for the detection of HCV antibodies from capillary whole blood or crevicular fluid

A total of 531 individuals were included from two centers (September 2012 to November 2013) [Department of Hepatology and Gastroenterology, Henri Mondor Hospital; Department of Hepatology and Gastroenterology, Intercommunal Hospital Center of Creteil (CHIC)] over a 14-month period. Eighteen patients were excluded (16 HBV-positive patients, 2 HIV-positive patients). Four TROD tests were performed on each patient: one rapid test from crevicular fluid [OraQuick^® HCV Rapid Antibody Test (OraSure Technologies, Inc,)] and three tests using capillary whole blood from finger sticks [(OraQuick^® HCV Rapid Antibody Test, TOYO^® anti-HCV (TÜRKLAB TIBBI MALZEMELER SAN. TIC. A.S., Turkey) and Labmen^® HCV test (TÜRKLAB TIBBI MALZEMELER SAN. TIC. A.S., Turkey)].

The OraQuick and Toyo tests demonstrated satisfactory analytical performance in terms of specificity and sensitivity, with the OraQuick being superior. These tests are an interesting alternative for the detection of HCV antibodies in an at-risk population for HCV infection. Nevertheless, this study showed the importance of evaluating these tests before their use in clinical practice (Chevaliez et al., Clin Microbiol Infect 2016; 22(5):459.e1-6).

Evaluation of blotting paper (dried blood spot, DBS) for screening, diagnosis, and monitoring of HCV infection

More than 500 serum and blood samples were collected from three groups of patients (170 HCV-negative patients, 26 patients cured of HCV, and 315 patients with a chronic HCV infection). The detection of HCV antibodies from whole blood deposited on DBS was reliable after redefining a new cut off value (ratio signal/cutoff). This new cut off is linked to a sensitivity of 99.1% and a specificity of 98.2%. HCV RNA was detected in most patients with viral replication, but the values for viral load were significantly lower than those observed from serum (on average 1.6 to 4.17 log UI/mL less depending on the molecular test used), suggesting that only the presence or absence of HCV RNA or variations between two values should be taken into consideration. The detection and quantification of the capsid antigen from whole blood on DBS lacked sensitivity, as only 64.1% of the infected samples were positive. Determining the genotype was correct in most cases.

In conclusion, this study showed that blood collected on blotting paper is reliable for the diagnosis and monitoring of HCV infection. DBS could be useful for improving access to health care for remote populations, for whom traditional health structures are inaccessible (Soulier et al., J Infect Dis 2016;213(7):1087-95).

Clinical interest in the quantification of the capsid antigen in patients infected with chronic hepatitis C

The capsid antigen has been proposed as an indirect marker of viral replication. The detection and quantification of the capsid antigen can be used, in theory, instead of molecular biology techniques (viral genome detection, DVG) for the diagnosis and follow-up of chronic hepatitis C. In fact, the capsid antigen of HCV can be easily detected and quantified by serology, e.g., CMIA (chemiluminescent microparticle immunoassay) using the Architect immunoassay analyzer (Abbott). The objective of this study was to evaluate the clinical performance of the Abbott kit (HCV Ag Architect) for the detection and quantification of the capsid antigen in patients with chronic hepatitis C infected with a genotype 1 to 6 virus. The specificity of the HCV Ag Architect kit was 100% (IC95%:97.8%-100%). AgC titers were not influenced by HCV genotype. The detection limit of 3 fmol/L was equivalent to approximately 1,000 IU/mL HCV RNA, regardless of the RealTime PCR kit used (Abbott RealTime HCV and Cobas AmpliPrep/Cobas Taqman HCV v2.0). Antigen capsid titer positively correlated with the HCV viral load (r = 0.89 and r = 0.88 for the Abbott RealTime HCV and CAP/CTM 2.0 RealTime PCR kits, respectively, p < 0.001).

Overall, the HCV Ag Architect kit was specific and easy to use. This test could be used for the screening, diagnosis, and monitoring of an infection, particularly in the context of antiviral therapy development without interferon (Chevaliez et al., J Clin Virol. 2014;61(1):145-8)

Molecular Epidemiology

The Hospital Laboratory of Virology, in collaboration with the INSERM research group, has developed all the tools necessary for molecular typing and analysis of HBV and HCV genomes (PCR, cloning, conventional and high throughput sequencing, genetic and phylogenetic analyses) for molecular epidemiology studies, particularly in the context of grouped cases or an epidemic.

Studies or surveys in which the NRC participated from 2012 to 2016 that have contributed to epidemiological surveillance are presented in the table below:

Contribution to Alerts

Investigation of isolated and grouped cases of HBV and HCV transmission was carried out at the NRC, particularly in the context of nosocomial transmission of these viruses.

Clinical and translational research

The laboratory carries out numerous research activities. In clinical research, the laboratory has developed and is currently involved in clinical protocols to test the efficacy and tolerance of new molecules for the treatment of Hepatitis C in collaboration with the Hepatology service of the Henri Mondor Hospital. These molecules include protease inhibitors, nucleoside or nucleotide analogs, non-nucleoside inhibitors of HCV polymerase, second generation NS5A protein inhibitors used in combination, etc.

Therapeutic failure and HBV and HCV resistance to molecules constitute one of the main subjects of the laboratory and INSERM team associated with it, both its clinical and fundamental aspects (molecular mechanisms of antiviral efficacy and failure).  Several investigative studies have been carried out on this subject. Specifically, a phenotypic platform for characterizing HBV and HCV resistance to antivirals has been developed and implemented (see chapter Resistance Observatory).