Christophe Rodriguez

New Virologic Tools for Management of Chronic Hepatitis B and C

Molecular biology techniques are routinely used to diagnose and monitor treatment of patients with chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. These tools can detect and quantify viral genomes and analyze their sequence to determine their genotype or subtype and to identify nucleotide or amino acid substitutions associated with resistance to antiviral drugs. They include real-time target amplification methods, which have been standardized and are widely used in clinical practice to diagnose and monitor HBV and HCV infections, and next-generation sequencing techniques, which are still restricted to research laboratories. In addition, new enzyme immunoassays can quantify hepatitis B surface and hepatitis C core antigens, and point-of-care tests and alternatives to biologic tests that require whole-blood samples obtained by venipuncture have been developed. We review these new virologic methods and their clinical and research applications to HBV and HCV infections.

Characterization of the dynamics of hepatitis B virus resistance to adefovir by ultra‐deep pyrosequencing

Hepatitis B virus (HBV) resistance to nucleoside/nucleotide analogs is frequent. Ultra‐deep pyrosequencing (UDPS) is a powerful new tool that can detect minor viral variants and characterize complex quasispecies mixtures. We used UDPS to analyze the dynamics of adefovir‐resistant HBV variants in patients with chronic HBV infection in whom adefovir resistance occurred during treatment. Amino acid substitutions known to confer resistance to adefovir were detected at baseline in most patients. The dynamics of adefovir‐resistant variants were complex and differed among patients as a result of evolving differences in variant fitness. UDPS analysis revealed successive waves of selection of HBV populations with single and multiple amino acid substitutions. Adefovir‐resistant variants were partially inhibited by lamivudine, but remained fit in its presence. Conclusion: Substitutions conferring HBV resistance to nucleoside/nucleotide analogs exist before treatment, and that the dynamics of adefovir‐resistant populations are much more complex and heterogeneous than previously thought and involve thus far unknown amino acid substitutions. The UDPS‐based approach described here is likely to have important implications for the assessment of antiviral drug resistance in research and clinical practice. (Hepatology 2013;53:890–901)

Complex dynamics of hepatitis B virus resistance to adefovir

In patients with hepatitis B e antigen‐negative chronic hepatitis B, adefovir dipivoxil administration selects variants bearing reverse transcriptase rtN236T and/or rtA181V/T substitutions in 29% of cases after 5 years. The aim of this study was to characterize the dynamics of adefovir‐resistant variant populations during adefovir monotherapy in order to better understand the molecular mechanisms underlying hepatitis B virus resistance to this class of nucleotide analogues. Patients included in a 240‐week clinical trial of adefovir monotherapy who developed adefovir resistance‐associated substitutions were studied. The dynamics of hepatitis B virus populations were analyzed over time, after generating nearly 4,000 full‐length reverse transcriptase sequences, and compared with the replication kinetics of the virus during therapy. Whatever the viral kinetics pattern, adefovir resistance was characterized by exclusive detection of a dominant wild‐type, adefovir‐sensitive variant population at baseline and late and gradual selection by adefovir of several coexisting resistant viral populations, defined by the presence of amino acid substitutions at position rt236, position rt181, or both. The gain in fitness of one or the other of these resistant populations during adefovir administration was never associated with the selection of additional amino acid substitutions in the reverse transcriptase. Conclusion: Our results suggest that adefovir administration selects poorly fit preexisting or emerging viral populations with low‐level adefovir resistance, which subsequently compete to fill the replication space. Viral kinetics depends on the initial virological response to adefovir. Lamivudine add‐on restores some antiviral efficacy, but adefovir‐resistant variants remain predominant. Whether these adefovir resistance–associated substitutions may confer cross‐resistance to tenofovir in vivo will need to be determined. (HEPATOLOGY 2009;49:50‐59.)

Highly frequent HIV-1 minority resistant variants at baseline of the ANRS 139 TRIO trial had a limited impact on virological response

OBJECTIVES To assess the prevalence of minority resistant variants (MRVs) at baseline and their impact on the virological response. The ANRS 139 TRIO trial evaluated the combination of raltegravir, etravirine and darunavir, plus an optimized background therapy, in 87% of cases. Patients were highly experienced and harboured multiresistant viruses, but were naive to the three drugs, and showed a high level of virological suppression. METHODS Ultra-deep sequencing of reverse transcriptase, protease and integrase regions was performed at the trial baseline, and sequences were interpreted according to the ANRS algorithm. MRVs were assessed using MiSeq and 454 technologies (limit of detection 1%). RESULTS At baseline, minority variants with at least one NRTI, one NNRTI, one PI, one major PI or an integrase inhibitor resistance-associated mutation were present in 46%, 45%, 68%, 24% and 13% of patients, respectively. When minority variants are taken into account, the prevalence of resistance to etravirine, darunavir and raltegravir at baseline was 29%, 40% and 9%, respectively. No difference was observed in the prevalence of MRVs between patients with virological failure and those with virological success, except a trend for patients exhibiting baseline etravirine MRVs (50% versus 26%, P = 0.09). CONCLUSIONS We have shown a high level of MRVs at baseline in highly pre-treated patients harbouring multiresistant viruses. However, these MRVs were not associated with an increased risk of virological failure, except for a trend for etravirine MRVs.

Improved detection of resistance at failure to a tenofovir, emtricitabine and efavirenz regimen by ultradeep sequencing

OBJECTIVES Resistant minority variants present before ART can be a source of virological failure. This has been shown for NRTIs, NNRTIs and CCR5 inhibitors. However, very few data are available for the detection of such minority resistant variants that could be selected at virological failure and not detected using classical Sanger sequencing. METHODS We studied 26 patients treated with tenofovir, emtricitabine and efavirenz with their first virological failure (defined as two consecutive viral loads >50 copies/mL). We performed standard Sanger sequencing and ultradeep sequencing (UDS; Roche 454(®) Life Sciences) in plasma at failure. For UDS, mutations >1% were considered. We compared the presence of reverse transcriptase mutations between the two techniques, using the latest ANRS algorithm. RESULTS UDS detected more resistance mutations in 38.5% of cases (10/26 patients) and the genotypic sensitivity score (GSS) was reduced for 6 of them (23.1%). The GSS was impacted more often for NRTIs than for NNRTIs, for which most mutations were already detected by Sanger sequencing. Resistant minority variants were detected even in patients with low viral load at failure. CONCLUSIONS These results strongly argue for the use of next-generation sequencing in patients failing on an NRTI+NNRTI regimen, as UDS has the potential to modify the choice of the subsequent regimen.

Virological factors associated with outcome of dual maraviroc/raltegravir therapy (ANRS-157 trial)

OBJECTIVES ROCnRAL ANRS-157 was a single-arm study designed to evaluate a switch to a maraviroc (300 mg twice a day) plus raltegravir (400 mg twice a day) regimen in virologically suppressed HIV-1-infected patients (ClinicalTrials.gov: NCT01420523). The aim of this work was to investigate the factors associated with virological failure (VF) (5/44 patients) or virological rebound defined as one viral load (VL) >50 copies/mL or VL >1 copy/mL. METHODS At baseline (BL), ultradeep sequencing (UDS) of DNA gp120 V3 and integrase regions and quantification of HIV DNA were performed in PBMCs. Tropism, VL, BL ultrasensitive HIV RNA VL, BL HIV DNA VL, subtype, age, ethnicity, transmission group, AIDS status, nadir CD4 and BL CD4 cell count, time since HIV diagnosis, duration of ART and suppressed viraemia, VL zenith, CD4/CD8 ratio and BL CD8 cell count were investigated as potential factors associated with virological rebound. RESULTS The proportion of patients with VL <1 copy/mL did not evolve over time. Among the 44 included patients, 3 had minority X4-tropic viruses determined by UDS at BL and one of them presented VF. Minority resistant variants in the integrase gene were detected at BL at two positions (E138 and G140) for three patients who did not have VF. Among all studied factors, none was associated with virological rebound. CONCLUSIONS Maraviroc plus raltegravir failed to maintain virological suppression in virologically suppressed HIV-1-infected patients. However, neither minority viral variants nor ultrasensitive viraemia was found to be a predictive factor of VF or virological rebound in this context.

Ultradeep Pyrosequencing and Molecular Modeling Identify Key Structural Features of Hepatitis B Virus RNase H, a Putative Target for Antiviral Intervention

ABSTRACT Last-generation nucleoside/nucleotide analogues are potent against hepatitis B virus (HBV) and have a high barrier to resistance. However, delayed responses have been observed in patients previously exposed to other drugs of the same class, long-term resistance is possible, and cure of infection cannot be achieved with these therapies, emphasizing the need for alternative therapeutic approaches. The HBV RNase H represents an interesting target because its enzyme activity is essential to the HBV life cycle. The goal of our study was to characterize the structure of the HBV RNase H by computing a 3-dimensional molecular model derived from E. coli RNase H and analyzing 2,326 sequences of all HBV genotypes available in public databases and 958,000 sequences generated by means of ultradeep pyrosequencing of sequences from a homogenous population of 73 treatment-naive patients infected with HBV genotype D. Our data revealed that (i) the putative 4th catalytic residue displays unexpected variability that could be explained by the overlap of the HBx gene and has no apparent impact on HBV replicative capacity and that (ii) the C-helix-containing basic protrusion, which is required to guide the RNA/DNA heteroduplex into the catalytic site, is highly conserved and bears unique structural properties that can be used to target HBV-specific RNase H inhibitors without cross-species activity. The model shows substantial differences from other known RNases H and paves the way for functional and structural studies as a prerequisite to the development of new inhibitors of the HBV cell cycle specifically targeting RNase H activity.

Fine-scale structure analysis shows epidemic patterns of clonal complex 95, a cosmopolitan Escherichia coli lineage responsible for extraintestinal infection

Escherichia coli clonal complex 95 represents a cosmopolitan, genetically diverse lineage, and the extensive substructure observed in this lineage is epidemiologically and clinically relevant. The frequency with which CC95 strains are responsible for extraintestinal infection appears to have been stable over the past 15 years. However, the different subgroups identified within this lineage have an epidemic structure depending on the host, sample, continent, and time. Thus, the evolution and spread of strains belonging to CC95 are very different from those of another cosmopolitan human-associated clonal complex, CC131, which has increased significantly in frequency as a cause of extraintestinal infection over the past 15 years due to the evolution and spread of two very closely related, nearly monomorphic lineages. ABSTRACT The Escherichia coli lineage known as clonal complex 95 (CC95) is a cosmopolitan human-associated lineage responsible for a significant fraction of extraintestinal infections of humans. Whole-genome sequence data of 200 CC95 strains from various origins enabled determination of the CC95 pangenome. The pangenome analysis revealed that strains of the complex could be assigned to one of five subgroups that vary in their serotype, extraintestinal virulence, virulence gene content, and antibiotic resistance gene profile. A total of 511 CC95 strains isolated from humans living in France, Australia, and the United States were screened for their subgroup membership using a PCR-based method. The CC95 subgroups are nonrandomly distributed with respect to their geographic origin. The relative frequency of the subgroups was shown to change through time, although the nature of the changes varies with continent. Strains of the subgroups are also nonrandomly distributed with respect to source of isolation (blood, urine, or feces) and host sex. Collectively, the evidence indicates that although strains belonging to CC95 may be cosmopolitan, human movement patterns have been insufficient to homogenize the distribution of the CC95 subgroups. Rather, the manner in which CC95 strains evolve appears to vary both spatially and temporally. Although CC95 strains appeared globally as pandemic, fine-scale structure analysis shows epidemic patterns of the CC95 subgroups. Furthermore, the observation that the relative frequency of CC95 subgroups at a single locality has changed over time indicates that the relative fitness of the subgroups has changed. IMPORTANCE Escherichia coli clonal complex 95 represents a cosmopolitan, genetically diverse lineage, and the extensive substructure observed in this lineage is epidemiologically and clinically relevant. The frequency with which CC95 strains are responsible for extraintestinal infection appears to have been stable over the past 15 years. However, the different subgroups identified within this lineage have an epidemic structure depending on the host, sample, continent, and time. Thus, the evolution and spread of strains belonging to CC95 are very different from those of another cosmopolitan human-associated clonal complex, CC131, which has increased significantly in frequency as a cause of extraintestinal infection over the past 15 years due to the evolution and spread of two very closely related, nearly monomorphic lineages.

Severe Ketoalkalosis as Initial Presentation of Imported Human Rabies in France

ABSTRACT We report a patient with an unusual initial metabolic presentation of imported human rabies who became symptomatic within 2 weeks of returning from Mali to France. This is the single case of imported human rabies identified in France within the past 11 years and the first report of viral RNA in bronchial secretions.

A novel standardized deep sequencing-based assay for hepatitis C virus genotype determination

Hepatitis C virus (HCV) genotype and subtype (1a/1b) identification is needed to tailor anti-HCV therapy. Currently available methods accurately identify the genotype and differentiate subtypes 1a from 1b. However, these assays have not been designed to identify other HCV subtypes, nor to recognize mixed genotype/subtype infections, emphasizing the need for a high-resolution system based on phylogenetic analysis of reads obtained by deep sequencing of a relevant genome region. The aim of this study was to evaluate the performance of the Sentosa SQ HCV Genotyping Assay, a novel deep sequencing-based assay targeting the HCV nonstructural 5B (NS5B) region, in clinical samples from patients with an indication for anti-HCV therapy. A high concordance rate with Sanger sequencing of the NS5B region, the reference method, was found for genotype 1 to 6 determination, 1a/1b subtype identification, and genotype 4, 5 and 6 subtyping. Discrepancies were seen essentially for HCV genotype 2 subtyping. Overall, the performance of the deep sequencing-based assay in generating the genotypes/subtype information needed to tailor anti-HCV treatment was adequate in this study. Further improvements, such as a longer NS5B fragment analyzed and enriching the database of reference prototype strains used for subtype assignment would make it a method of choice for HCV genotyping and subtyping for future clinical practice and research.