Phylogenetic and Phylodynamic Analysis of Delta Strains Circulating in Italy.

The hepatitis delta virus (HDV) exhibits high genetic and evolutionary variability and is classified into eight genotypes (HDV-1 to -8). HDV-1 is the most widespread genotype worldwide and includes several subtypes. It predominates mainly in Europe, the Middle East, North America, and Northern Africa, and is associated with both severe and mild forms of liver disease. In this study, we performed phylogenetic and phylodynamic analyses of HDV strains circulating in Regione Lazio, Italy, to understand when these strains were introduced into the Lazio region and to define their genetic variability in Italy. Fifty HDV RNA positive patient samples were amplified using a nested RT-PCR approach targeting the HDV R0 region and sequenced. A phylogenetic tree of patient-derived sequences and reference sequences representing HDV-1 to -8 was constructed using the GTRGAMMA model in RAxML v8. The results indicated that HDV-1 was the predominant genotype with HDV-1d being the most frequently inferred subtype. HDV-1 sequences clustering with subtypes 1b and 1e were also identified. A phylodynamic analysis of HDV-1 sequences employing a Bayesian birth-death model inferred a clock rate of 3.04 × 10-4 substitutions per site per million years, with a 95% Highest Posterior Density (HPD) interval of 3.45 × 10-5 to 5.72 × 10-4. A Bayesian birth-death analysis with tree calibration based on a sample dating approach indicated multiple original sources of infection (from the late 1950s to late 1980s). Overall, these results suggest that HDV sequences from the native Italian and non-Italian patients analyzed in this study represent multiple lineages introduced across a wide period. A common ancestral origin should be excluded.

Phylogenetic and Phylodynamic Analyses of HCV Strains Circulating among Patients Using Injectable Drugs in Central Italy.

Approximately 71 million people worldwide are infected with the hepatitis C virus (HCV). Injectable drug use represents the most common route of transmission in Europe and other developed countries. We studied the molecular characteristics of the HCV infection among mono-infected people who used drugs (PWUD) in Italy. Among 208 PWUD with anti-HCV antibodies, 101 (48.6%) were HCV RNA-positive, the majority (47%) were infected with the HCV genotype (Gt)1a, followed by Gt3a (34.9%), Gt4 (9.1%), Gt1b (4.5%), and Gt2 (4.5%). Bayesian phylogenetic analyses of clustered HCV NS5B sequences from 66 HCV-positive PWUDs with available plasma samples indicated age and neighborhood proximity as the most common characteristics between closely related HCV strains. Population dynamics, as measured by a coalescent Bayesian skyline analysis, revealed an increase in HCV Gt1a infections from the mid-1980s to mid-1990s. While HCV Gt3a infections were first detected in the 1980s, patient numbers with this genotype subtype remained relatively constant. For both Gt1a and Gt3a, Birth-Death Bayesian Skyline analyses produced higher reproduction numbers post 2014. For earlier time intervals, slow growths were observed for both Gt1a and Gt3a with reproduction numbers (Re) of approximately 1. The evolutionary rates for Gt1a and Gt3a were estimated as 2.23 × 10-4 and 3.85 × 10-4, respectively.

Late Relapse and Reinfection in HCV Patients Treated with Direct-Acting Antiviral (DAA) Drugs.

The risk of hepatitis C virus (HCV) recurrence after direct-acting antiviral (DAA) treatment is <0.5%. However, the distinction between HCV RNA late relapse and reinfection still represents a challenge in virological diagnostics. The aim of this study was to employ next-generation sequencing (NGS) to investigate HCV RNA recurrence in patients achieving a sustained virologic response (SVR) at least six months post-treatment. NGS was performed on plasma samples from six HCV-positive patients (Pt1-6) treated with DAA. NGS of HCV NS5B was analyzed before treatment (T0), after HCV RNA rebound (T1), and, for Pt3, after a second rebound (T2). Reinfection was confirmed for Pt5, and for the first rebound observed in Pt3. Conversely, viral relapse was observed when comparing T0 and T1 for Pt6 and T1 and T2 for Pt3. Z-scores were calculated and used to predict whether HCV-positive patient samples at different time points belonged to the same quasispecies population. A low Z-score of <2.58 confirmed that viral quasispecies detected at T0 and T1 were closely related for both Pt1 and Pt2, while the Z-score for Pt4 was suggestive of possible reinfection. NGS data analyses indicate that the Z-score may be a useful parameter for distinguishing late relapse from reinfection.

Clinical Relevance of Torque Teno Virus (TTV) in HIV/HCV Coinfected and HCV Monoinfected Patients Treated with Direct-Acting Antiviral Therapy.

Torque Teno virus (TTV) is a ubiquitous virus that causes chronic infection in humans with unknown clinical consequences. Here, we investigated the influence of TTV infection on HCV direct-acting antiviral (DAA) efficacy in HIV/HCV coinfected and HCV monoinfected patients as controls. Of 92 study patients, 79.3% were TTV DNA positive; untreated patients exhibited a significantly higher proportion of TTV DNA-positivity vs. sustained virological response (SVR) patients (100.0% vs. 65.2%, p < 0.001), while TTV positivity was not significant in DAA failure patients vs. SVR patients despite HIV/HCV coinfection. TTV DNA viral load was higher among HCV monoinfected patients vs. HIV/HCV coinfected, although marginally significant (p = 0.074) and no significant viral load difference was detected between DAA failures and SVR patients, while untreated vs. SVR patients had a significantly higher viral load (19,884, IQR 5977-333,534, vs. 469, IQR 10-4124, p = 0.004). Alpha-genogroup 3 TTV was the most prevalent genetic group, and no specific strain or genogroup was observed in relapser patients. Among HIV/HCV patients with HCV RNA detectable at end of treatment (EOT), TTV DNA was detected in 9/17 treatment responder patients and 3/5 relapser patients, thus, TTV infection does not appear to influence the control HCV viremia after EOT. Levels of IL-6 IL-4, and CD14 were not significantly different between TTV PCR-positive and -negative patients. These results suggest no association between TTV DNA positivity or viral load and HCV DAA failure whether patients were HIV/HCV coinfected or HCV monoinfected.

Anti-HEV IgG Avidity Testing: Utility for Diagnosing Acute and Resolved Genotype 3 Infections.

European Association of the Study of the Liver (EASL) guidelines specify HEV RNA, as well as anti-HEV IgG and IgM as positive markers for acute HEV infection. HEV RNA assay sensitivity limitations may lead to false negative test results in patients with low levels of viremia. Moreover, anti-HEV IgM positivity is not a reliable indicator for distinguishing between acute and resolved infections given the ability of this antibody to persist several months after a resolved infection. Our study aims were to assess HEV IgG avidity for diagnosing acute and resolved infections, regardless of the anti-HEV IgM serostatus, and examine assay reliability when evaluating different genotype 3 (GT3) HEV subtypes. Patient serum samples (n = 104) were tested for HEV IgG avidity by utilizing the DIA.PRO kit on a DSX automated instrument. Among patients identified with acute HEV infections, 32 were infected with GT3: GT3c (n = 5), GT3e (n = 8), 3f (n = 17) and GT3-unsubtyped (n = 2). Avidity sensitivity was 91.2% and specificity was 100%. For patients with long-lasting anti-HEV IgM persistence, an Avidity Index >70% was observed. Thus, the DIA.PRO avidity assay may be utilized to distinguish between recently acquired and resolved HEV GT3 infections. However, for equivocal results (Avidity Index > 40-70%), HEV RNA molecular testing will be required to confirm a recent infection.

Discovery of BMS-986144, a Third-Generation, Pan-Genotype NS3/4A Protease Inhibitor for the Treatment of Hepatitis C Virus Infection.

The discovery of a pan-genotypic hepatitis C virus (HCV) NS3/4A protease inhibitor based on a P1-P3 macrocyclic tripeptide motif is described. The all-carbon tether linking the P1-P3 subsites of 21 is functionalized with alkyl substituents, which are shown to effectively modulate both potency and absorption, distribution, metabolism, and excretion (ADME) properties. The CF3Boc-group that caps the P3 amino moiety was discovered to be an essential contributor to metabolic stability, while positioning a methyl group at the C1 position of the P1' cyclopropyl ring enhanced plasma trough values following oral administration to rats. The C7-fluoro, C6-CD3O substitution pattern of the P2* isoquinoline heterocycle of 21 was essential to securing the targeted potency, pharmacokinetic (PK), and toxicological profiles. The C6-CD3O redirected metabolism away from a problematic pathway, thereby circumventing the time-dependent cytochrome P (CYP) 450 inhibition observed with the C6-CH3O prototype.

Clinical Characteristics of Acute Hepatitis E and Their Correlation with HEV Genotype 3 Subtypes in Italy.

Genotype 3 (GT3) is responsible for most European autochthonous hepatitis E virus (HEV) infections. This study analyzed circulating genotypes and GT3 subtypes in the Lazio region, Italy, between 2011 and 2019, as well as their pathogenic characteristics. Of the 64 evaluable HEV GT3 patient-derived sequences, identified subtypes included GT3f (n = 36), GT3e (n = 15), GT3c (n = 9), GT3a (n = 1) and three unsubtyped GT3 sequences. GT3c strains were similar to Dutch sequences (96.8-98.1% identity), GT3e strains showed high similarity (96.8%) with a United Kingdom sequence, while the most related sequences to GT3f Italian strains were isolated in France, Belgium and Japan. One sequence was closely related to another Italian strain isolated in raw sewage in 2016. The liver functioning test median values for 56 evaluable GT3 patients were: alanine aminotransferase (ALT), 461 (range 52-4835 U/L); aspartate aminotransferase (AST), 659 (range 64-6588 U/L); and total bilirubin, 3.49 (range 0.4-33 mg/dL). The median HEV RNA viral load for 26 evaluable GT3 patients was 42,240 IU/mL (range 5680-895,490 IU/mL). Of the 37 GT3 patients with available clinical information, no correlation was observed between HEV clinical manifestations and GT3 subtype. HEV symptoms were comparable among GT3c/e/f patients across most analyzed categories except for epigastric pain, which occurred more frequently in patients with HEV GT3e (75%) than in patients with GT3c (50%) or GT3f (19%) (p = 0.01). Additionally, patients with HEV GT3c exhibited significantly higher median international normalized ratio (INR) than patients with GT3e and GT3f (p = 0.033). The severity of GT3 acute hepatitis E was not linked to HEV RNA viral load or to the GT3 subtype.

Developments in the treatment of HCV genotype 3 infection.

Introduction: Unlike other hepatitis C virus (HCV) genotypes (GTs), patients infected with GT3 are associated with an increased risk of accelerated liver disease progression. Although early immuno-modulator therapies yielded moderate sustained virologic response (SVR) rates, treatment of GT3 patients has proven more challenging in the era of direct-acting antivirals (DAAs). Areas covered: The review provides an overview of the evolution of therapies against GT3 since the approval of the first immunomodulatory agent nearly 30 years ago. Expert opinion: A greater choice of treatment options is now available for HCV GT3-infected patients. In treatment-naïve patients with or without compensated cirrhosis, SVR rates are comparably high approaching 100% irrespective of treatment option. For treatment-experienced patients, choosing the right therapy is important, especially for those with advanced liver disease. For the few patients who fail with multiple persistent highly resistant DAA substitutions, retreatment options are limited. Additional real-world treatment comparisons are required to confirm differences in SVR in these more difficult-to-treat patients. This also includes patients infected with GT3 subtypes such as GT3b where multiple DAA-resistant substitutions occur naturally. In the absence of new drugs with non-overlapping drug-resistant profiles, an interferon-based therapy may still be beneficial in select patient populations with high-level multiple DAA-resistant substitutions.

Impact of Preexisting Hepatitis C Virus Genotype 6 NS3, NS5A, and NS5B Polymorphisms on the In Vitro Potency of Direct-Acting Antiviral Agents.

HCV genotype 6 (GT-6) is found predominantly in East and Southeast Asia. Clinical studies have focused on patients infected with hepatitis C virus (HCV) GT-6a, where high sustained virologic response (SVR) rates to direct-acting antivirals (DAAs) have been achieved. However, GT-6 is highly diverse, with 29 reported subtypes. We explored the diversity of GT-6 polymorphisms at residues associated with DAA resistance, their impact on DAA in vitro potency when evaluated in a GT-6a consensus replicon, and their association with specific GT-6 subtypes. GT-6 sequences from 25 patient-derived samples and 105 sequences from the U.S. HCV database were compared, and substitutions at resistance-associated residue positions were phenotyped against different DAAs. Preexisting resistance-associated substitutions (RASs) to NS3 protease (A156V and D168E) and NS5B nucleotide (L159F and S282C) inhibitors were rare (<4%). Preexisting RASs to NS5A inhibitors were common, especially at L28 (A/F/G/M/T/V) and R30 (E/N/S). In vitro susceptibilities of NS5A-L28A and -L28T were dramatically reduced against all tested NS5A drugs (90% effective concentration [EC90] range, 119 to 2,032 nM) compared with susceptibilities against a GT-6a consensus replicon (EC90 range, 0.1 to 19 nM). These L28 RASs preexisted in combination with R30S (EC90 [L28A-R30S] of ≥720 nM or EC90 [L28T-R30S] of ≥128 nM against tested DAAs) or as L28T-L31I (EC90 [tested DAAs] of >5,000 nM) and were detected in evaluated GT-6b and -6f sequences. NS5A-L28A-R30A, observed in GT-6r, did not replicate. In conclusion, HCV GT-6b, GT-6f, and GT-6r sequences harbored highly resistant RASs to all evaluated NS5A drugs. Therefore, monitoring SVR in patients infected with these GT-6 subtypes treated with NS5A drug-containing regimens is suggested to confirm any association between noted NS5A polymorphisms and treatment failure.

Daclatasvir and sofosbuvir with ribavirin for 24 weeks in chronic hepatitis C genotype-3-infected patients with cirrhosis: a Phase III study (ALLY-3C).

BACKGROUND: Optimal treatment for patients with HCV genotype-3 infection and liver cirrhosis remains a medical priority. Daclatasvir+sofosbuvir and ribavirin is a recommended option for such patients, but clinical trial data are lacking for treatment >16 weeks. METHODS: This was a single-arm, Phase III study of daclatasvir+sofosbuvir+ribavirin for 24 weeks in patients with compensated cirrhosis and HCV genotype-3 infection. The primary end point was sustained virological response at post-treatment week 12 (SVR12); the primary objective was to demonstrate statistical superiority to historical SVR12 data for 12 weeks' daclatasvir+sofosbuvir without ribavirin in genotype-3-infected patients with cirrhosis (95% CI lower bound >79.0%). RESULTS: A total of 78 patients were treated (54 treatment-naive, 24 treatment-experienced including 8 with prior sofosbuvir exposure). SVR12 was achieved by 87% (68/78; 95% CI 77.7, 93.7%) of patients in the primary analysis of central laboratory data. One additional patient achieved SVR12 by local testing resulting in an overall SVR12 rate of 88% (95% CI 79.2, 94.6%) and the lower bound of the 95% CI above the historical threshold. SVR12 rates were 93% (50/54) for treatment-naive and 79% (19/24) for treatment-experienced patients. Of the nine non-SVR12 patients, four were lost to follow-up, two relapsed (both sofosbuvir-experienced), two had end-of-treatment virological failure and one discontinued early. There were no unexpected safety signals; only one patient discontinued for an adverse event. CONCLUSIONS: Daclatasvir+sofosbuvir+ribavirin for 24 weeks was well tolerated and efficacious in HCV genotype-3-infected patients with compensated cirrhosis, with SVR12 outcomes comparable to previously reported outcomes in patients treated with this regimen for 12-16 weeks. ClinicalTrials.gov ID NCT02673489.