Significance of switching of the nucleos(t)ide analog used to treat Japanese patients with chronic hepatitis B virus infection from entecavir to tenofovir alafenamide fumarate.

The significance of switching of the nucleos(t)ide analog used to treat patients with hepatitis B virus (HBV) from entecavir (ETV) to tenofovir alafenamide fumarate (TAF) is uncertain. The subjects of this study were 159 patients with HBV who received treatment with ETV followed by TAF. Among these patients, serial changes in the HBV marker levels were monitored in 92 patients in whom the serum HBsAg levels were ≥100 IU/mL during the 48-week period immediately before and after the switching. A questionnaire survey for medication compliance was performed in 127 patients. The serum HBsAg levels (log IU/mL) decreased by 0.041 during the ETV treatment period and by 0.068 during the TAF administration period. The degree of reduction was higher during the TAF administration period than during the ETV administration period in patients without cirrhosis (P = .030), patients with genotype B HBV (P = .014), and patients with undetectable serum HBcrAg (P = .038). Multivariate analysis revealed the HBV genotype (B vs C; odds ratio, 3.400; P = .025) and serum aspartate aminotransferase level (every 1+; 1.111; P = .015) at the time of switching as factors influencing the treatment efficacy. Thirty-six patients (28%) responded that the number of days that they forgot to take the drug decreased after the drug switching, and 77 patients (61%) reported feeling satisfied with the drug switching. Switching of the nucleos(t)ide analog used from ETV to TAF may be useful in the treatment of patients with HBV infection, as it is associated with both a decrease in the serum HBsAg level and improvement of the medication compliance.

Involvement of portosystemic shunts in impaired improvement of liver function after direct-acting antiviral therapies in cirrhotic patients with hepatitis C virus.

AIM: Factors responsible for impaired improvement of liver function despite sustained viral response after direct-acting antiviral agents therapies in cirrhotic patients with hepatitis C virus need to be elucidated. METHODS: Liver function and the extent of portosystemic shunting were evaluated for 79 patients with compensated cirrhosis, in whom sustained viral response had been achieved after direct-acting antiviral agents therapies for hepatitis C virus at least 3 years earlier. RESULTS: Portosystemic shunts were observed in 63 patients (80%). Improvement and worsening, as compared with the baseline, of esophageal/gastric varices after direct-acting antiviral agents therapies was seen in three patients (4%) and 10 patients (13%), respectively. Portal hypertension-related events, such as varices and ascites requiring treatment, were observed in six patients (8%), in whom three patients showing worsening of Child-Pugh scores were included. Multivariate analysis showed that maximal diameter of the shunts (P = 0.012) and serum Mac-2 binding protein glycosylation isomer levels at the end of treatment (P = 0.005) were associated with the development of portal hypertension-related events, with cut-off values of 5.25 mm (P = 0.001) and 6.84 cut-off index (P < 0.001), respectively. The increase of serum albumin levels at 3 years, as compared with the baseline, was smaller in 22 patients having shunts with maximal diameters of ≥5 mm than in the remaining 57 patients (P = 0.034), whereas no such difference was seen between the patients with and without elevation of serum Mac-2 binding protein glycosylation isomer level of ≥6.8 cut-off index. CONCLUSIONS: A large size of portosystemic shunts was found to be a crucial determinant of impaired improvement of liver function, as well as of the development of portal hypertension-related events, even after sustained viral response in patients with compensated cirrhosis.

A case of genotype-3b hepatitis C virus in which the whole genome was successfully analyzed using third-generation nanopore sequencing.

A 42-year-old Chinese man with chronic hepatitis C virus (HCV) infection visited our hospital for antiviral therapy. The subgenotype could not be determined using the HCV GENOTYPE Primer Kit (Institute of Immunology, Tokyo, Japan), which can identify genotype 3a HCV exclusively among genotype 3 HCV. Thus, the whole-genome sequence of HCV was analyzed using the MinION nanopore sequencer (Oxford Nanopore Technologies, Oxford, UK), a third-generation single-molecule sequencing platform. Consequently, a total of 9442 bases with a 73.6 mean depth, corresponding to the sequences between nt25 and PolyU/UC were determined (LC414155.2). The similarity analysis revealed that the obtained sequence was classified into genotype 3b HCV and showed nucleotide identities from 87.6% to 93.9% with those of 12 previously reported strains. Furthermore, possible resistance-associated substitutions in non-structural protein (NS)3, NS5A, and NS5B based on consensus sequences of 12 genotype 3b HCV strains, including NS5A-Y93H and NS5B-S282 T substitutions, were absent. In conclusion, the MinION nanopore sequencer is useful for analyzing the HCV genome, especially the genomes of genotype 3 HCV strains for which standardized real- time PCR methods for all subgenotypes have not been established.

Chronic hepatitis caused by hepatitis C virus showing a discrepancy between serogroup and genotype because of intergenotypic 2b/1b recombination: A pitfall in antiviral therapy with direct-acting antivirals.

A 40-year-old male patient with virologic relapse after daclatasvir plus asunaprevir therapy for a serogroup 1 hepatitis C virus (HCV) infection visited our hospital for retreatment. Virologic examinations revealed that a genotype 2b HCV strain carrying both NS3-S122N / D168A and NA5A-R30Q / L31M / Q54H / Y93H mutations had relapsed. The patient received sofosbuvir plus ribavirin therapy, but virologic relapse occurred once again. Sequencing of the HCV genome clarified an intergenotypic recombination of 2b and 1b with an estimated crossover point between nucleotides 3114 and 3115, corresponding to the N-terminal end of the NS3 region (DDBJ/EMBL/GenBank databases accession no. LC273304). The NS5B-S282T mutation was not detected in the HCV strain, and resistance-association substitutions in the NS3 and NS5A regions were similar to those at baseline. Direct sequencing of the core and NS4A regions corresponding to the targeting sites of genotyping and serogrouping, respectively, is useful to determine the combination of direct-acting antivirals when a discrepancy is observed between the serogroup and genotype of HCV strains.

Retreatment with sofosbuvir/ledipasvir with or without lead-in interferon-ß injections in patients infected with genotype 1b hepatitis C virus after unsuccessful daclatasvir/asunaprevir therapy.

AIM: To improve the therapeutic efficacy of sofosbuvir/ledipasvir (SOF/LDV) for the retreatment of patients after daclatasvir/asunaprevir (DCV/ASV), a customized therapy with or without lead-in interferon (IFN)-ß injections was formulated according to the types of resistance-associated substitutions (RAS) in the non-structural protein (NS)5A region of genotype 1b hepatitis C virus (HCV). METHODS: Thirty-three patients failing prior DCV/ASV received SOF/LDV for 12 weeks. Patients with HCV carrying unfavorable NS5A-RAS and/or those previously treated with simeprevir were given lead-in IFN-ß injections twice a day for 2 weeks; sequential changes in the NS5A-RAS during the injection period were evaluated using deep sequencing. RESULTS: Lead-in injections were not undertaken in 27 patients; a sustained viral response (SVR) was achieved in 26 patients, while viral relapse occurred in 1 patient with HCV carrying NS5A-L28M/R30H/Y93H mutations. Among the 6 patients receiving lead-in injections, viral relapse occurred in 2 patients who had an unfavorable IFN-λ3-related gene single nucleotide polymorphism allele; both patients had been previously treated with simeprevir, and HCV carrying NS5A-L31V/Y93H mutations had emerged after DCV/ASV. Deep sequencing revealed no changes in the NS5A-RAS profiles during the lead-in injection period in either patient. In contrast, in a patient with a favorable allele who was infected with similar unfavorable HCV strains, NS5A-L31/Y93 wild-type strains appeared during the injection period, enabling an SVR. CONCLUSION: Using customized therapies based on the NS5A-RAS profiles, a high SVR rate was obtained after SOF/LDV in patients failing prior DCV/ASV. Lead-in IFN-ß injections did not improve the efficacy in patients with HCV carrying unfavorable NS5A-RAS except in those with a favorable IFN-λ3-related gene allele.

NS5A-P32 deletion as a factor involved in virologic failure in patients receiving glecaprevir and pibrentasvir.

BACKGROUND: This study sought to clarify the factors involved in virologic failure in patients with HCV receiving retreatment with glecaprevir/pibrentasvir (GLE/PIB) in real-world practice. METHODS: Forty-two patients who had previously received direct-acting antivirals (DAAs) therapies consisting of 35, 3, 3, and 1 patient(s) with genotype (GT)-1b, GT-2a, GT-2b, and GT-3b HCV, respectively, received GLE/PIB for 12 weeks. Resistance-associated substitutions (RASs) at baseline were evaluated, and the dynamics of NS5A-RASs were assessed by deep sequencing in patients showing virologic failure. RESULTS: Baseline NS5A-RASs were found in all the patients with GT-1b HCV including 16 patients with NS3-RASs. In contrast, both NS5A-RASs and NS3-RASs were absent in 3 and 2 patients with GT-2a and GT-2b HCV, respectively. Virologic failure occurred in 3 patients with GT-1b HCV with NS5A-P32del, while a sustained virologic response (SVR) was achieved in the remaining 39 patients including those with GT-1b HCV carrying NS5A-L31V + Y93H and NS5A-A92K. Virologic failure even occurred in a patient in whom the NS5A-P32del HCV strains had become undetectable by direct sequencing, and the percentage of such strains relative to the total HCV strains was 10%, as determined by deep sequencing. In the other patient with GT-1b HCV with NS5A-P32del, NS3-A156A/V/S were found at 4 weeks after GLE/PIB therapy, but had disappeared at 11 weeks, as determined by direct sequencing. CONCLUSIONS: GLE/PIB was effective for patients with HCV who failed to achieve an SVR after prior DAA therapies except in those with GT-1b HCV carrying NS5A-P32del even when such strains became undetectable by direct sequencing.

Serum asunaprevir concentrations showing correlation with the extent of liver fibrosis as a factor inducing liver injuries in patients with genotype-1b hepatitis C virus receiving daclatasvir plus asunaprevir therapy.

AIMS: Liver injury can occur during antiviral therapies with direct-acting antivirals (DAAs), potentially necessitating discontinuation of the therapies, with consequent worsening of the sustained viral response (SVR) rates, in patients with hepatitis C virus (HCV). To clarify the mechanisms involved in serum transaminase level elevation, we performed a retrospective evaluation of the serum concentrations of daclatasvir and asunaprevir, both classified as DAAs, in patients receiving treatment with a combination of the two drugs. METHODS: Subjects were 278 Japanese patients with genotype-1b HCV who received daclatasvir plus asunaprevir therapy for more than 4 weeks. Serum concentrations of both the DAAs were measured at 4 weeks after the initiation of therapy. RESULT: Liver injuries including serum AST and/or ALT level elevation to 150 U/L or over were found in 34 patients (12.2%). Multivariate logistic regression analysis identified serum asunaprevir concentrations as being significantly associated with developing liver injury, with an odds ratio of 1.046 (95% confidence interval 1.011-1.082, p<0.05). Serum asunaprevir concentrations showed correlation with the extent of liver fibrosis, estimated by peripheral platelets counts and serum albumin levels and baseline and FIB4 index and serum Mac-2 binding protein glycosylation isomer (M2BPGi) levels at 4 weeks of the therapy; the concentrations were significantly higher among patients showing 3.0 or more of M2BPGi levels than among those with the levels less than 3.0; on the other hand, no such correlation/difference was found in serum daclatasvir concentrations. CONCLUSION: High serum concentrations of serum asunaprevir, which were associated with the extent of liver fibrosis, appear to provoke the occurrence of liver injury in patients with genotype-1b HCV receiving combined daclatasvir plus asunaprevir therapy.

Significance of NS5B Substitutions in Genotype 1b Hepatitis C Virus Evaluated by Bioinformatics Analysis.

To evaluate the effects of HCV NS5B amino acid substitutions on treatment outcome in Ledipasvir (LDV)/Sofosbuvir (SOF) for Japanese patients with genotype 1b HCV infection, NS5B sequences were examined in i) seven patients experiencing virologic failure after LDV/SOF in real-world practice, ii) 109 SOF-naïve patients, iii) 165 patients enrolled in Phase-3 LDV/SOF trial. A218S and C316N were detected in all patients with viral relapse; the percentages of these substitutions in SOF-naïve patients were 64.2% and 55.0%, respectively. Genotype 1b HCV strains with NS5B-C316N mutation were located in the leaves different from those in which HCV strains without such substitutions were present on the phylogenetic tree. Structural modeling revealed that amino acid 218 was located on the surface of the NTP tunnel. Free energy analysis based on molecular dynamics simulations demonstrated that the free energy required to pass through the tunnel was larger for triphosphate SOF than for UTP in NS5B polymerase carrying A218S, but not in wild-type. However, no susceptibility change was observed for these substitutions to SOF in replicon assay. Furthermore, the SVR rate was 100% in patients enrolled the Phase-3 trial. In conclusion, NS5B A218S and C316N were detected in all patients who relapsed following LDV/SOF in real-world practice. These substitutions did not impact the overall SVR rate after LDV/SOF, however, further studies are needed to elucidate the impact of these substitutions.

Significance of variants associated with resistance to NS5A inhibitors in Japanese patients with genotype 1b hepatitis C virus infection as evaluated using cycling-probe real-time PCR combined with direct sequencing.

BACKGROUND: Dual oral therapy with daclatasvir plus asunaprevir yielded an SVR rate of 85% among patients with genotype 1b HCV. Treatment failure mainly occurred in patients with pre-existing HCV with NS5A-Y93H mutation. The significance of the mutation was evaluated. METHODS: The percent of serum NS5A-Y93H strains relative to the total strains was quantified using cycling-probe real-time PCR combined with direct sequencing in 444 patients with genotype 1b HCV, and the factors associated with mutation were analyzed. The mutation rates during interferon therapy were measured sequentially. RESULTS: NS5A-Y93H strains (1-100% of the total strains) were detected in 87 patients (19.6%). Mutant strains were detected more frequently among women than among men, in patients with a favorable allele in the IL28B-related gene SNP than among those with unfavorable alleles, and among patients without HCC and/or with serum AFP levels less than 6.0 ng/ml than among those with HCC and/or levels of 6.0 ng/ml or more. A multivariate analysis revealed that IL28B-related gene polymorphisms were significant factors associated with mutant strains. Although the frequency of patients with mutant strains was equivalent among patients depending on their previous interferon therapies, a sequential analysis during the interferon administrations revealed that the mutant strains disappeared earlier than the wild-type strains. CONCLUSIONS: NS5A-Y93H mutation was associated with sex, serum AFP levels, and IL28B-related gene polymorphisms in patients infected with genotype 1b HCV. The indications for NS5A inhibitor use should be determined based on these factors, since mutant strains seem to be sensitive to interferon.

Long-term efficacy of endoscopic coagulation for different types of gastric vascular ectasia.

AIM: To examine the long-term therapeutic efficacies of endoscopic cauterization for gastric vascular ectasia, according to the type of lesion. METHODS: Thirty-eight patients with hemorrhagic gastric vascular ectasia (VE) were treated by endoscopic cauterization: 13 by heater probe coagulationand 25 by argon plasma coagulation. Depending on the number of lesions, 14 and 24 patients were classified into localized VE (≤ 10; LVE) and extensive VE (> 10; EVE), respectively. The patients were followed-up by repeated endoscopic examinations after the therapy, and the incidences of VE recurrence and re-bleeding from the lesions were evaluated. RESULTS: Although the VE lesions disappeared initially in all the patients after the therapy, the recurrence of VE developed in 25 patients (66%) over a mid-term observation period of 32 mo, and re-bleeding occurred in 15 patients (39%). The recurrence of VE was found in all patients with EVE, with re-bleeding occurring in 14 patients (58%). In contrast, only 1 patient (7%) with LVE showed recurrence of the lesions and complicating hemorrhage. Both the cumulative recurrence-free rates and cumulative re-bleeding-free rates were significantly lower in the EVE group than in the LVE group (P < 0.001 and P < 0.001, respectively). Moreover, the cumulative re-bleeding-free rate in the EVE group was 47.6% at 1 year and 25.4% at 2 years in patients with chronic renal failure, which were significantly lower than the rates in the patients without chronic renal failure (83.3% and 74.1%, respectively) (P < 0.05). CONCLUSION: The recurrence of VE and re-bleeding from the lesions was more frequent in the patients with EVE, especially in those with complicating renal failure.

A case of liver cirrhosis due to hepatits C virus infection complicating giant anorectal varices treated with balloon-occluded retrograde transvenous obliteration.

A 73-year-old man with liver cirrhosis due to hepatitis C virus infection was admitted to our hospital because of massive bleeding from external varices. Colonoscopic examination revealed that giant anorectal varices had developed between the anus and rectal ampulla, and had ruptured at the perianal site. On three-dimensional computed tomography imaging, the feeding and drainage vessels of the varices were identified as the inferior mesenteric vein and right inferior hemorrhoidal vein, respectively. Endoscopic therapies were not employed for the bleeding varices, because the blood flow volume of the feeding vessel was extremely large. Balloon-occluded retrograde transvenous obliteration (B-RTO) was therefore carried out through the drainage vessels. The variceal blood flow disappeared after B-RTO therapy, and the varices decreased in size with thrombus formation verified by colonoscopy. Bleeding from the external varices also ceased. B-RTO therapy may be an effective approach for giant anorectal varices presenting as a complication in liver cirrhosis patients in whom the main drainage vessels can be determined.