Pre-existing, treatment-specific resistance-associated substitutions in hepatitis C virus genotype 1 and 3 and viral RNA titers during treatment with direct-acting antivirals.

The introduction of direct-acting antiviral (DAA) treatment of hepatitis C virus (HCV) infected patients has greatly increased treatment success rates. However, viral response kinetics to DAA treatment may depend on pre-existing resistance-associated substitutions (RASs) in HCV. The aim of this study was to describe how pre-existing RASs affect DAA treatment-induced reduction in HCV RNA titers in HCV genotypes 1- and 3-infected individuals. Patients with HCV genotype 1 infection (N = 31) treated with either sofosbuvir/ledipasvir/ribavirin or paritaprevir/ombitasvir/ritonavir/dasabuvir/ribavirin and HCV genotype 3-infected patients (N = 16) treated with either sofosbuvir/daclatasvir/ribavirin or sofosbuvir/ribavirin were analyzed. HCV RNA levels were determined at baseline and frequently during treatment, and RAS profiles were obtained by deep sequencing at baseline. In total, 33/47 (70.2%) of the patients had baseline RASs. However, treatment-specific RASs were detected at baseline only in 12.9% and 18.8% of HCV genotypes 1- and 3-infected patients, respectively. In genotype 1-infected individuals, reduction in HCV RNA titer during the first week of treatment was not affected by evidence of either treatment-specific RASs or cirrhosis or treatment regimen. In genotype 3-infected individuals receiving sofosbuvir/daclatasvir/ribavirin, the presence of daclatasvir-specific NS5A RASs at baseline correlated with a reduced decline of HCV RNA in the first treatment week. For both genotypes 1- and 3-infected individuals, cirrhosis but not treatment-specific RAS were associated with the time of clearance of HCV RNA. It is, however, important to note that this study involves DAA regimens that were used only during the original introduction of interferon-free DAA-based treatments.

Rapid in vivo development of resistance to daptomycin in vancomycin-resistant Enterococcus faecium due to genomic alterations.

Daptomycin is a cyclic lipopeptide used in the treatment of vancomycin-resistant Enterococcus faecium (VREfm). However, the development of daptomycin-resistant VREfm challenges the treatment of nosocomial VREfm infections. Resistance mechanisms of daptomycin are not fully understood. Here, we analyzed the genomic changes leading to a daptomycin-susceptible VREfm isolate becoming resistant after 50 days of daptomycin and linezolid combination therapy. A total of seven isogenic VREfm isolates from the same patient (daptomycin-susceptible and daptomycin-resistant) were analyzed using Illumina whole genome sequencing, and two isolates were further characterized with Nanopore sequencing. One nonsynonymous SNP in the rpoC gene previously shown to harbor mutations in daptomycin-resistant VREfm was identified in the daptomycin-resistant isolates. Whole genome comparative analysis identified the loss of a 46.5 kb fragment, duplication of a 29.7 kb fragment, and integration of two plasmids upon acquisition of daptomycin resistance. Transmission electron microscopy showed similar alterations in cell morphology and cell wall structure as have previously been described in daptomycin-resistant E. faecalis.

Characterization of a Novel Hepatitis C Virus Genotype 1 Subtype from a Patient Failing 4 Weeks of Glecaprevir-Pibrentasvir Treatment.

Limited information is available in relation to surveillance, genotyping, genome sequences, and treatment outcomes for rare hepatitis C virus variants. Here, we have characterized a novel subtype of major hepatitis C virus genotype 1, which was deep sequenced before and after treatment failure with 4 weeks of glecaprevir and pibrentasvir.

Inferior cure rate in pilot study of 4-week glecaprevir/pibrentasvir treatment with or without ribavirin of chronic hepatitis C.

BACKGROUND & AIMS: Shortening the treatment duration for chronic hepatitis C may increase feasibility and reduce the cost of cure. The aims of this study were to compare 4 weeks of glecaprevir/pibrentasvir (GLE/PIB) treatment with and without ribavirin for patients with chronic hepatitis C and favourable baseline characteristics and to monitor the development of resistance-associated substitutions (RAS) and re-treatment outcomes if treatment failed. METHODS: We performed an open-label single-centre randomized controlled trial, in which patients with chronic hepatitis C were randomized 1:1 to GLE/PIB ± ribavirin, stratified by genotype 3. The main inclusion criteria were treatment-naive patients, aged 18-49 with all genotypes accepted, and absence of liver fibrosis, determined by liver stiffness measurement less than 8 kPa. Viral genome sequences were determined by deep sequencing at baseline and at the time of relapse. RESULTS: A total of 32 patients started treatment. Sustained virological response at week 12 (SVR12) was 59% (10/17) for GLE/PIB without ribavirin and 73% (11/15) for GLE/PIB with ribavirin. Drug target-specific NS5A RAS were detected at baseline for 45% (5/11) of patients with treatment failure and for 14% (3/21) of patients who achieved SVR12. Ten failure patients were retreated 12 weeks with sofosbuvir-based regimens; all have been cured. CONCLUSIONS: In this pilot study of 4-week treatment with GLE/PIB with and without ribavirin, we found that baseline RAS were more frequent in patients with virological failure. Development of RAS did occur after short treatment but did not result in retreatment failure with a different regimen. EudraCT no: 2017-005179-21.

Overcoming Culture Restriction for SARS-CoV-2 in Human Cells Facilitates the Screening of Compounds Inhibiting Viral Replication.

Efforts to mitigate the coronavirus disease 2019 (COVID-19) pandemic include the screening of existing antiviral molecules that could be repurposed to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Although SARS-CoV-2 replicates and propagates efficiently in African green monkey kidney (Vero) cells, antivirals such as nucleos(t)ide analogs (NUCs) often show decreased activity in these cells due to inefficient metabolization. SARS-CoV-2 exhibits low viability in human cells in culture. Here, serial passages of a SARS-CoV-2 isolate (original-SARS2) in the human hepatoma cell clone Huh7.5 led to the selection of a variant (adapted-SARS2) with significantly improved infectivity in human liver (Huh7 and Huh7.5) and lung cancer (unmodified Calu-1 and A549) cells. The adapted virus exhibited mutations in the spike protein, including a 9-amino-acid deletion and 3 amino acid changes (E484D, P812R, and Q954H). E484D also emerged in Vero E6-cultured viruses that became viable in A549 cells. Original and adapted viruses were susceptible to scavenger receptor class B type 1 (SR-B1) receptor blocking, and adapted-SARS2 exhibited significantly less dependence on ACE2. Both variants were similarly neutralized by COVID-19 convalescent-phase plasma, but adapted-SARS2 exhibited increased susceptibility to exogenous type I interferon. Remdesivir inhibited original- and adapted-SARS2 similarly, demonstrating the utility of the system for the screening of NUCs. Among the tested NUCs, only remdesivir, molnupiravir, and, to a limited extent, galidesivir showed antiviral effects across human cell lines, whereas sofosbuvir, ribavirin, and favipiravir had no apparent activity. Analogously to the emergence of spike mutations in vivo, the spike protein is under intense adaptive selection pressure in cell culture. Our results indicate that the emergence of spike mutations will most likely not affect the activity of remdesivir.

Global evolutionary analysis of chronic hepatitis C patients revealed significant effect of baseline viral resistance, including novel non-target sites, for DAA-based treatment and retreatment outcome.

Direct-acting antivirals (DAAs) have proven highly effective against chronic hepatitis C virus (HCV) infection. However, some patients experience treatment failure, associated with resistance-associated substitutions (RASs). Our aim was to investigate the complete viral coding sequence in hepatitis C patients treated with DAAs to identify RASs and the effects of treatment on the viral population. We selected 22 HCV patients with sustained virologic response (SVR) to match 21 treatment-failure patients in relation to HCV genotype, DAA regimen, liver cirrhosis and previous treatment experience. Viral-titre data were compared between the two patient groups, and HCV full-length open reading frame deep-sequencing was performed. The proportion of HCV NS5A-RASs at baseline was higher in treatment-failure (82%) than matched SVR patients (25%) (p = .0063). Also, treatment failure was associated with slower declines in viraemia titres. Viral population diversity did not differ at baseline between SVR and treatment-failure patients, but failure was associated with decreased diversity probably caused by selection for RAS. The NS5B-substitution 150V was associated with sofosbuvir treatment failure in genotype 3a. Further, mutations identified in NS2, NS3-helicase and NS5A-domain-III were associated with DAA treatment failure in genotype 1a patients. Six retreated HCV patients (35%) experienced 2nd treatment failure; RASs were present in 67% compared to 11% with SVR. In conclusion, baseline RASs to NS5A inhibitors, but not virus population diversity, and lower viral titre decline predicted HCV treatment failure. Mutations outside of the DAA targets can be associated with DAA treatment failure. Successful DAA retreatment in patients with treatment failure was hampered by previously selected RASs.

Outcome and adverse events in patients with chronic hepatitis C treated with direct-acting antivirals: a clinical randomized study.

OBJECTIVE: New potent direct-acting antiviral (DAA) regimens against hepatitis C virus have been approved in recent years. However, information about the rate of adverse events (AEs) across different DAA regimens is limited. We aimed to evaluate differences in AEs and treatment efficacy in patients with chronic hepatitis C (CHC), genotype (GT) 1 or 3, randomized to two different treatment arms, correspondingly. PATIENTS AND METHODS: We randomly assigned 96 patients in a 1 : 1 ratio, to treatment for 12 weeks with either paritaprevir/ombitasvir/ritonavir/dasabuvir/ribavirin (RBV) or ledipasvir/sofosbuvir (SOF)/RBV if infected with GT1 (72 patients) or to daclatasvir/SOF/RBV for 12 weeks or SOF/RBV for 24 weeks, if infected with GT3 (24 patients). Data on AEs were collected throughout the entire study period. RESULTS: A total of 70 (97%) patients with CHC with GT1 and 20 (83%) patients with GT3 achieved cure. The GT3 treatment arm was prematurely terminated, owing to change in national treatment guidelines. Thus, only AEs for GT1 patients are described. AEs occurred in 70 (97%) GT1 patients, and most common AEs were anemia (n=56/78%), fatigue (n=53/74%), and headache (n=33/46%). No difference was observed in relation to treatment group (P=1.0), anemia (P=1.0), or liver cirrhosis (P=0.53). In seven (11%) patients, AEs assessed by the investigator to be possibly related to the DAA regimen were still present 12 weeks after treatment. CONCLUSIONS: We found no difference in AEs possibly related to the DAA regimen in patients with CHC, but surprisingly, AEs possibly related to the DAA regimen persisted in a significant number of patients after treatment. This finding can be of importance for clinicians in relation to patient information concerning AEs possibly related to DAA treatment.

Direct acting antiviral treatment of chronic hepatitis C in Denmark: factors associated with and barriers to treatment initiation.

OBJECTIVES: We describe factors associated with and barriers to initiation of Direct Acting Antiviral (DAA) treatment in patients with chronic hepatitis C, who fulfill national fibrosis treatment guidelines in Denmark. MATERIALS AND METHODS: In this nationwide cohort study, we included patients with chronic hepatitis C from The Danish Database for Hepatitis B and C (DANHEP) who fulfilled fibrosis treatment criteria. Factors associated with treatment initiation and treatment failure were determined by logistic regression analyses. Medical records were reviewed from patients who fulfilled fibrosis treatment criteria, but did not initiate DAA treatment to determine the cause. RESULTS: In 344 (49%) of 700 patients, who fulfilled treatment criteria, factors associated with DAA treatment initiation were transmission by other routes than injecting drug use odds ratio (OR) 2.13 (CI: 1.38-3.28), previous treatment failure OR 2.58 (CI: 1.84-3.61) and ALT above upper limit of normal OR 1.60 (CI: 1.18-2.17). The most frequent reasons for not starting treatment among 356 (51%) patients were non-adherence to medical appointments (n = 107/30%) and ongoing substance use (n = 61/17%). Treatment failure with viral relapse occurred in 19 (5.5%) patients, who were more likely to have failed previous treatment OR 4.53 (CI: 1.59-12.91). CONCLUSIONS: In this nationwide cohort study, we found non-adherence to medical appointments and active substance use to be major obstacles for DAA treatment initiation. Our findings highlight the need for interventions that can overcome these barriers and increase the number of patients who can initiate and benefit from curative DAA treatment.