Velia Chiara Di Maio

Multiclass HCV resistance to direct-acting antiviral failure in real-life patients advocates for tailored second-line therapies

Despite the excellent efficacy of direct‐acting antivirals (DAA) reported in clinical trials, virological failures can occur, often associated with the development of resistance‐associated substitutions (RASs). This study aimed to characterize the presence of clinically relevant RASs to all classes in real‐life DAA failures.

A boceprevir failure in a patient infected with HCV genotype 1g: importance and limitations of virus genotyping prior to HCV protease-inhibitor-based therapy.

A patient classified as HCV-1a-positive by both LiPA Siemens 2.0 and Abbott RealTime HCV Genotype II was instead found to be infected with HCV-1g, as determined by phylogenetic analysis of NS3 sequences. HCV-1g NS3 sequences available to date naturally harbour the resistance substitution T54S, plus P131S and L135F changes, located in the highly conserved NS3 positions within the boceprevir-binding site, as determined by structural modelling. HCV-1g NS3 sequences show some similarities to HCV-4 and are poorly responsive to interferon/ribavirin and to boceprevir/telaprevir; this patient was also a null-responder to boceprevir treatment. Baseline genotypic resistance testing may provide crucial information for the management of first-generation protease-inhibitor-based regimens, including both HCV genotype/subtype and natural resistance.

Anti-HBV treatment induces novel reverse transcriptase mutations with reflective effect on HBV S antigen

INTRODUCTION The identification of novel reverse-transcriptase (RT) drug-resistance mutations is critical in predicting the probability of success to anti-HBV treatment. Furthermore, due to HBV-RT/HBsAg gene-overlap, they can have an impact on HBsAg-detection and quantification. METHODS 356 full-length HBV-RT sequences from 197 drug-naive patients and 159 patients experiencing virological-breakthrough to nucleoside/nucleotide-analogs (NUCs) were analyzed. Mutants and wild-type HBs-antigens were expressed in HuH7-hepatocytes and quantified in cell-supernatants and cell-lysates by Architect HBsAg-assay. RESULTS Ten novel RT-mutations (rtN53T-rtS78T-rtS85F-rtS135T-rtA181I-rtA200V-rtK212Q-rtL229V/F-rtM309K) correlated with specific NUC-treatments and classical drug-resistance mutations on divergent evolutionary pathways. Some of them reduced RT-binding affinity for anti-HBV drugs and altered S-antigen structure. Indeed, rtS78T (prevalence: 1.1% in drug-naïve and 12.2% in adefovir-failing patients) decreased the RT-affinity for adefovir more than the classical adefovir-resistance mutations rtA181 T/V (WT:-9.63 kcal/mol, rtA181T:-9.30 kcal/mol, rtA181V:-7.96 kcal/mol, rtS78T:-7.37 kcal/mol). Moreover, rtS78T introduced a stop-codon at HBsAg-position 69, and completely abrogated HBsAg-quantification in both supernatants and cell-lysates, indicating an impaired HBsAg-secretion/production. Furthermore, the HBsAg-mutation sP217L, silent in RT, significantly correlated with M204V/I-related virological-breakthrough and increased HBsAg-quantification in cell-lysate. CONCLUSIONS Mutations beyond those classically known can affect drug-binding affinity of mutated HBV-RT, and may have potential effects on HBsAg. Their cumulative effect on resistance and HBV-pathogenicity indicates the importance of preventing therapeutic failures.

Hepatitis C virus gene sequencing as a tool for precise genotyping in the era of new direct antiviral agents

MF, et al. Increased production of sonic hedgehog by ballooned hepatocytes. J Pathol 2011;224:401-410. 3) Machado MV, Michelotti GA, Pereira de Almeida T, Kruger L, Swiderska-Syn M, et al. Reduced lipoapoptosis, hedgehog pathway activation, and fibrosis in caspase-2 deficient mice with nonalcoholic steatohepatitis. Gut 2015;64:1148-1157. 4) Guy CD, Suzuki A, Zdanowicz M, Abdelmalek MF, Burchette J, Unalp A, Diehl AM; NASH CRN. Hedgehog pathway activation parallels histologic severity of injury and fibrosis in human nonalcoholic fatty liver disease. HEPATOLOGY 2012;55:1711-1721. 5) Michelotti GA, Xie G, Swiderska M, Choi SS, Karaca G, Kruger L, et al. Smoothened is a master regulator of adult liver repair. J Clin Invest 2013;123:2380-2394. 6) Guy CD, Suzuki A, Abdelmalek MF, Burchette JL, Diehl AM; for the NASH CRN. Treatment response in the PIVENS trial is associated with decreased hedgehog pathway activity. HEPATOLOGY 2015;61:98-107.

Hepatitis C virus drug resistance associated substitutions and their clinical relevance: Update 2018

Nowadays, due to the development of potent Direct-Acting Antiviral Agents (DAAs) that specifically target NS3, NS5A and NS5B viral proteins, several new and highly efficacious options to treat chronic Hepatitis C virus (HCV) infection are available. The natural presence of resistance associated substitutions (RASs), as well as their rapid emergence during incomplete drug-pressure, are intrinsic characteristics of HCV that greatly affect treatment outcome and the chances to achieve a virolgical cure. To date, a high number of RASs in NS3, NS5A, and NS5B have been associated in vivo and/or in vitro with reduced susceptibility to DAAs, but no comprehensive RASs list is available. This review thus provides an updated, systematic overview of the role of RASs to currently approved DAAs or in phase II/III of clinical development against HCV-infection, discriminating their impact in different HCV-genotypes and DAAs, providing assistance for a fruitful use of HCV resistance testing in clinical practice.

Consequences of inaccurate hepatitis C virus genotyping on the costs of prescription of direct antiviral agents in an Italian district

Available commercial assays may yield inaccurate hepatitis C virus (HCV) genotype assignment in up to 10% of cases. We investigated the cost-effectiveness of re-evaluating HCV genotype by population sequencing, prior to choosing a direct acting antiviral (DAA) regimen. Between March and September 2015, HCV sequence analysis was performed in order to confirm commercial LiPA-HCV genotype (Versant® HCV Genotype 2.0) in patients eligible for treatment with DAAs. Out of 134 consecutive patients enrolled, sequencing yielded 21 (15.7%) cases of discordant results. For three cases of wrong genotype assignment, the putative reduction in efficacy was gauged between 15% and 40%. Among the eight cases for whom G1b was assigned by commercial assays instead of G1a, potentially suboptimal treatments would have been prescribed. Finally, for five patients with G1 and indeterminate subtype, the choice of regimens would have targeted the worst option, with a remarkable increase in costs, as in the case of the four mixed HCV infections for whom pan-genotypic regimens would have been mandatory. Precise assignment of HCV genotype and subtype by sequencing may, therefore, be more beneficial than expected, until more potent pan-genotypic regimens are available for all patients.

Implications of hepatitis C virus subtype 1a migration patterns for virus genetic sequencing policies in Italy

BackgroundIn-depth phylogeographic analysis can reveal migration patterns relevant for public health planning. Here, as a model, we focused on the provenance, in the current Italian HCV subtype 1a epidemic, of the NS3 resistance-associated variant (RAV) Q80K, known to interfere with the action of NS3/4A protease inhibitor simeprevir. HCV1a migration patterns were analysed using Bayesian phylodynamic tools, capitalising on newly generated and publicly available time and geo-referenced NS3 encoding virus genetic sequence data.ResultsOur results showed that both immigration and local circulation fuel the current Italian HCV1a epidemic. The United States and European continental lineages dominate import into Italy, with the latter taking the lead from the 1970s onwards. Since similar migration patterns were found for Q80K and other lineages, no clear differentiation of the risk for failing simeprevir can be made between patients based on their migration and travel history. Importantly, since HCV only occasionally recombines, these results are readily transferable to the genetic sequencing policy concerning NS5A RAVs.ConclusionsThe patient migration and travel history cannot be used to target only part of the HCV1a infected population for drug resistance testing before start of antiviral therapy. Consequently, it may be cost-effective to expand genotyping efforts to all HCV1a infected patients eligible for simeprevir-based therapies.

Hepatitis C virus RNA levels at week-2 of telaprevir/boceprevir administration are predictive of virological outcome

BACKGROUND Triple therapy with telaprevir/boceprevir + pegylated-interferon+ribavirin can achieve excellent antiviral efficacy, but it can be burdened with resistance development at failure. AIMS To evaluate kinetics of hepatitis C virus (HCV) RNA decay and early resistance development, in order to promptly identify patients at highest risk of failure to first generation protease inhibitors. METHODS HCV-RNA was prospectively quantified in 158 patients receiving pegylated-interferon+ribavirin+telaprevir (N = 114) or+boceprevir (N = 44), at early time-points and during per protocol follow-up. Drug resistance was contextually evaluated by population sequencing. RESULTS HCV-RNA at week-2 was significantly higher in patients experiencing virological failure to triple-therapy than in patients with sustained viral response (2.3 [1.9-2.8] versus 1.2 [0.3-1.7]log IU/mL, p < 0.001). A 100 IU/mL cut-off value for week-2 HCV-RNA had the highest sensitivity (86%) in predicting virological success. Indeed, 23/23 (100%) patients with undetectable HCV-RNA reached success, versus 26/34 (76.5%) patients with HCV-RNA<100 IU/mL, and only 11/31 (35.5%) with HCV-RNA > 100 IU/mL (p < 0.001). Furthermore, differently from failing patients, none of the patient with undetectable HCV-RNA at week-2 had baseline/early resistance. CONCLUSIONS With triple therapy based on first generation protease inhibitors, suboptimal HCV-RNA decay at week-2 combined with early detection of resistance can help identifying patients with higher risk of virological failure, thus requiring a closer monitoring during therapy.

Kinetics of hepatitis C virus RNA decay, quasispecies evolution and risk of virological failure during telaprevir-based triple therapy in clinical practice

BACKGROUND The used first generation protease inhibitors may be hampered by virological failure in partially interferon-sensitive patients. AIM To investigate early hepatitis C virus (HCV)-RNA decay and quasispecies modifications, and disclose viral dynamics underlying failure. METHODS Viraemia decay at early time-points during telaprevir treatment was modelled according to Neumann et al. (1998). NS3-sequences were obtained by population-sequencing and ultradeep-454-pyrosequencing. RESULTS 13 treatment-experienced (8 non-responders, 5 relapsers), and two cirrhotic naïve patients, received telaprevir+pegylated-interferon-α+ribavirin. Viraemia decay was biphasic. In all patients, first-phase was rapid and consistent, with a median [interquartile-range] viraemia decay of 2.8 [2.6-3.2]logIU/ml within 48h. Second-phase decay was slower, especially in failing patients: 3/3 showed <1logIU/ml decay between 48h and 2 weeks, and HCV-RNA >100IU/ml at week 2. Only one patient experiencing sustained viral response showed similar kinetics. By pyrosequencing, mutational freeze was observed in all 15 patients within the first 24h, but only in patients with sustained response afterwards. Indeed, 2/2 failing patients showed early resistance, as minor (V36A-T54A: prevalence <26% at 48h) or major (V36M/A-R155K: prevalence, 99.8% at week 2) variants. CONCLUSIONS Following telaprevir administration, first-phase HCV-RNA decay is consistent with mutational freeze and limited/no viral replication, while second-phase is significantly slower in failing patients (with appearance of resistance), suggesting the usefulness of early HCV-RNA monitoring.

Improvement of ALT decay kinetics by all-oral HCV treatment: Role of NS5A inhibitors and differences with IFN-based regimens

Background Intracellular HCV-RNA reduction is a proposed mechanism of action of direct-acting antivirals (DAAs), alternative to hepatocytes elimination by pegylated-interferon plus ribavirin (PR). We modeled ALT and HCV-RNA kinetics in cirrhotic patients treated with currently-used all-DAA combinations to evaluate their mode of action and cytotoxicity compared with telaprevir (TVR)+PR. Study design Mathematical modeling of ALT and HCV-RNA kinetics was performed in 111 HCV-1 cirrhotic patients, 81 treated with all-DAA regimens and 30 with TVR+PR. Kinetic-models and Cox-analysis were used to assess determinants of ALT-decay and normalization. Results HCV-RNA kinetics was biphasic, reflecting a mean effectiveness in blocking viral production >99.8%. The first-phase of viral-decline was faster in patients receiving NS5A-inhibitors compared to TVR+PR or sofosbuvir+simeprevir (p<0.001), reflecting higher efficacy in blocking assembly/secretion. The second-phase, noted δ and attributed to infected-cell loss, was faster in patients receiving TVR+PR or sofosbuvir+simeprevir compared to NS5A-inhibitors (0.27 vs 0.21 d-1, respectively, p = 0.0012). In contrast the rate of ALT-normalization, noted λ, was slower in patients receiving TVR+PR or sofosbuvir+simeprevir compared to NS5A-inhibitors (0.17 vs 0.27 d-1, respectively, p<0.001). There was no significant association between the second-phase of viral-decline and ALT normalization rate and, for a given level of viral reduction, ALT-normalization was more profound in patients receiving DAA, and NS5A in particular, than TVR+PR. Conclusions Our data support a process of HCV-clearance by all-DAA regimens potentiated by NS5A-inhibitor, and less relying upon hepatocyte death than IFN-containing regimens. This may underline a process of “cell-cure” by DAAs, leading to a fast improvement of liver homeostasis.