Switching from combination therapy with entecavir hydrate plus tenofovir alafenamide fumarate to tenofovir alafenamide fumarate monotherapy in patients with chronic hepatitis B based on nucleotide sequences of hepatitis B virus pregenome RNA.

AIM: Patients with chronic hepatitis B virus (HBV) infection experiencing viral breakthrough (BTH) or partial response (PR) during lamivudine (LAM) or entecavir hydrate (ETV) administration often took ETV plus tenofovir alafenamide fumarate (TAF) due to the emergence of a drug-resistance mutation. However, in patients lacking drug-resistance mutation against TAF, sufficient antiviral effects may be achievable with TAF monotherapy. We assessed the drug-resistance profile through nucleotide sequences of HBV pregenome RNA, and subsequently changed to TAF monotherapy from ETV plus TAF. METHODS: This prospective study included 25 patients with serum HBV-DNA below 20 IU/mL under ETV plus TAF administration. Pregenome RNA nucleotide sequences of HBV in the sera were analyzed using direct sequencing and deep sequencing. ETV was discontinued in patients without rtA194T and rtS106C + rtH126Y + rtD134E + rtL269I quadruple mutations in direct sequencing. RESULTS: LAM-PR, LAM-BTH, ETV-PR, and ETV-BTH were observed in 1, 16, 7, and 1 patient(s), respectively. Pregenome RNA nucleotide sequences were analyzable in 20 patients. Among the 12 patients classified as LAM-BTH, six patients showed rtL180M + rtM204V/I in direct sequencing, and one patient showed minor clones containing rtL180M + rtM204V + A194T in deep sequencing at a frequency of 0.3%. In the six patients classified as ETV-PR, one patient harbored rtM204I. No clones showing rtS106C + rtH126Y + rtD134E + rtL269I quadruple mutation were detected in deep sequencing. Subsequently, ETV was discontinued, and serum HBV-DNA remained undetectable up to 48 weeks in all patients. CONCLUSION: Patients receiving ETV plus TAF due to partial response or BTH during initial LAM or ETV administration were able to safely transition to TAF monotherapy based on nucleotide sequences of HBV pregenome RNA in the sera.

Significance of portal venous blood flow as a factor to determine liver function in patients with decompensated cirrhosis due to hepatitis C virus infection following achievement of sustained viral response by sofosbuvir plus velpatasvir.

AIM: To determine the outcomes concerning portal venous blood flow and portosystemic shunts in patients with decompensated cirrhosis due to hepatitis C virus (HCV) infection who achieved sustained viral response (SVR) following antiviral therapy. METHODS: Portal hypertension-related events and liver function were evaluated in 24 patients achieving SVR following sofosbuvir plus velpatasvir therapy. RESULTS: Serum albumin level (median; g/dL) increased from 2.9 at baseline to 3.5 at 12 weeks after the end of treatment (EOT) (p = 0.005), while liver volumes (cm3 ) decreased from 1260 to 1150 (p = 0.0002). Portal hypertension-related events developed in 10 patients (41.7%), and the cumulative occurrence rates after the EOT were 29.2%, 33.3%, and 46.1% at 24, 48, and 96 weeks, respectively. Multivariate logistic regression analysis revealed that the maximal diameter of the shunts (p = 0.0235) was associated with the development of the events, with a cut-off value of 8.3 mm (p = 0.0105). Meanwhile, multiple linear regression analysis revealed that portal venous blood flow, liver volume, serum albumin, and bilirubin levels at baseline were associated with serum albumin levels at 12 weeks after EOT (p = 0.0019, p = 0.0154, p = 0.0010, and p = 0.0350, respectively). CONCLUSION: In patients with decompensated cirrhosis due to HCV infection, the baseline portal venous blood flow and liver volume and function were predictive of liver function following SVR, while the maximal diameter of portosystemic shunts predicted the occurrence of portal hypertension-related events.

Evaluation of an immunochromatography-based rapid antigen test, Inspecter Kowa® SARS-CoV-2, using saliva specimens for the detection of SARS-CoV-2.

BACKGROUND: In the context of the coronavirus disease 2019 (COVID-19) pandemic, a rapid and reliable point-of-care test is an essential tool for controlling the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In particular, an immunochromatography test (ICT) that uses saliva specimens for rapid antigen detection not only reduces the risk of secondary infections but also reduces the burden on medical personnel. METHODS: The newly developed salivary antigen test kit "Inspecter Kowa® SARS-CoV-2" is an ICT to which saliva specimens can be directly applied. We evaluated its usefulness in comparison with reverse transcription quantitative PCR (RT-qPCR) and the Espline® SARS-CoV-2 Kit for the detection of SARS-CoV-2 using nasopharyngeal swab specimens. In this study, 140 patients with suspected symptomatic COVID-19 who visited our hospital were enrolled, and nasopharyngeal swab and saliva specimens were collected after they consented to participate in the study. RESULTS: Inspector Kowa SARS-CoV-2 was positive in 45 of 61 (73.8%) saliva that were positive by RT-qPCR and the Espline® SARS-CoV-2 Kit was also positive in 56 of 60 (93.3%) Np swabs that were positive by RT-qPCR. Good antigen detection was achieved by ICT with saliva and nasopharyngeal swab specimens when viral load was ≥105 copies/mL, whereas detection sensitivity was low when viral load was <105 copies/mL, especially in saliva specimens. CONCLUSION: This ICT for the detection of SARS-CoV-2 salivary antigen is an attractive tool that does not require specialized equipment and allows patients to perform the entire process from sample collection to self-diagnose and to reduce the burden on medical care during a pandemic.

Significance of furosemide in patients with cirrhosis treated with or without zinc acetate hydrate supplementation.

BACKGROUND: The Japanese guidelines for the treatment of cirrhosis suggest zinc supplementation to prevent hepatic encephalopathy in patients with cirrhosis and zinc deficiency, although the factors that are associated with therapeutic efficacy remain unknown. METHOD: A total of 159 patients with chronic liver diseases but without previous zinc supplementation were analyzed. Factors associated with serum zinc levels as well as the therapeutic efficacy of zinc supplementation were evaluated. RESULT: Serum zinc levels decreased with the progression of liver diseases. A multiple linear regression analysis revealed that the serum levels of albumin and cholinesterase and the daily furosemide dose were independently associated with the serum zinc levels. The optimal furosemide cut-off dosage for patients with zinc deficiency (<60 µg/dl) was 5 mg/day. Among 34 patients receiving zinc acetate hydrate, overt hepatic encephalopathy occurred in 12 patients (35.4%). A multivariate analysis identified a minimal serum zinc level of 50 µg/dl after more than 12 weeks of zinc supplementation as a factor associated with overt encephalopathy development, while furosemide use was not associated. The Child-Pugh score at baseline was the only factor associated with the maintenance of sufficient serum zinc levels. CONCLUSION: Although the furosemide dose was negatively correlated with the serum zinc level in patients with chronic liver diseases, furosemide use was not associated with the occurrence of overt encephalopathy in those receiving zinc supplementation. Serum zinc levels of ≥50 µg/dl were required to prevent overt encephalopathy development during zinc supplementation in both patients with and those without furosemide administration.

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.

Furosemide as a factor to deteriorate therapeutic efficacy of rifaximin in patients with decompensated cirrhosis.

AIM: To optimize the therapeutic strategy for cirrhotic patients manifesting hepatic encephalopathy, factors affecting the outcome of patients receiving rifaximin were evaluated. METHODS: The subjects were 95 patients receiving rifaximin. Serum ammonia levels were measured serially during rifaximin treatment. Factors associated with long-term outcomes and cumulative survival rates were evaluated. RESULTS: Serum ammonia levels were decreased at 4 weeks after rifaximin treatment compared to the levels at baseline even in patients receiving rifaximin as an add-on therapy with lactitol hydrate (P < 0.001) and reduction values were negatively correlated with the maximal diameter of portosystemic shunts (r = -0.275, P = 0.009). Overt encephalopathy occurred in 37 patients (38.9%) during rifaximin treatment, and the hazard function analysis identified 90 days as a high-risk term for developing the first-time overt encephalopathy. Thus, the long-term outcome was judged as favorable in 77 patients (81.1%) in whom overt encephalopathy was absent for at least 90 days during rifaximin initiation. A multivariate analysis revealed that furosemide, especially at daily doses of ≥20 mg both at baseline and during rifaximin treatment, was a significant factor associated with unfavorable outcome (P = 0.009 and P = 0.022, respectively) as well as occurrence and recurrence of overt encephalopathy (P = 0.012). Moreover, furosemide treatment significantly deteriorated the cumulative survival rate of patients receiving rifaximin (P = 0.026). CONCLUSION: Furosemide contributed to the deteriorated outcome of patients receiving rifaximin. Consequently, rifaximin should be given before increasing the furosemide dose, and the furosemide dose should not be increased during rifaximin treatment.

Furosemide as a factor to deteriorate therapeutic efficacy of tolvaptan in patients with decompensated cirrhosis.

AIM: To optimize the therapeutic strategy for patients with decompensated cirrhosis manifesting hepatic ascites and/or edema, factors affecting the outcome of patients receiving tolvaptan were evaluated. METHODS: The subjects were 165 patients receiving tolvaptan including 116 patients (70%) also treated with furosemide. The therapeutic efficacy of tolvaptan was defined as "effective" when a body weight reduction of 1.5 kg or more was obtained within 1 week. The long-term outcome was defined as "favorable" when the ascites-related events-free duration was prolonged following tolvaptan treatment, compared with that before treatment, or ascites-related events were absent for at least 120 days during treatment based on the hazard function analysis. RESULTS: Tolvaptan was effective in 115 patients (70%). Among them, the long-term outcome was evaluated in 99 patients and was favorable in 70 patients (71%). A multivariate analysis revealed that the serum blood urea nitrogen levels at baseline (odds ratio 0.960 per +1 mg/dL, P = 0.021) and the type of tolvaptan initiation (planned vs. emergent; 3.695, P < 0.001) were associated with therapeutic efficacy, while the furosemide dose (0.280 per +20 mg/day, P = 0.014) and previous ascites-related events (0.074, P < 0.001) were associated with the long-term outcome. Receiver operating curve analyses identified the optimal cut-off values for the furosemide dose as 15 mg/day (P < 0.001). Furthermore, the cumulative survival rates in patients receiving furosemide at 15 mg/day or less were significantly higher than those in the remaining patients (P = 0.048). CONCLUSION: Furosemide given at baseline contributed to an unfavorable outcome in patients receiving tolvaptan; consequently, tolvaptan should be given before increasing the furosemide dose.

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.

Deteriorated outcome of recent patients with acute liver failure and late-onset hepatic failure caused by infection with hepatitis A virus: A subanalysis of patients seen between 1998 and 2015 and enrolled in nationwide surveys in Japan.

AIM: A nationwide survey of acute liver failure (ALF) and late-onset hepatic failure (LOHF) has revealed that the outcomes of recent patients whose diseases were caused by infection with hepatitis A virus (HAV) have worsened, compared with those of previously reported patients. The factors associated with this deterioration were evaluated. METHODS: A total of 83 patients with HAV infection seen between 1998 and 2015 were enrolled. All the patients had a prothrombin time-international normalized ratio of 1.5 or more and hepatic encephalopathy of grade 2 or more severe. The demographic and clinical features of 45 patients seen prior to 2003 (cohort 1) and 38 patients seen during 2004 and thereafter (cohort 2) were compared. RESULTS: Three and four patients in cohort 1 and cohort 2, respectively, received liver transplantations; the survival rates among the remaining patients were 56% for cohort 2 and 79% for cohort 1 (P < 0.05). The mean age (±standard deviation) of the patients was higher in cohort 2 than in cohort 1 (58 ± 11 vs. 48 ± 13 years; P < 0.01). The percentages of patients with underlying metabolic diseases were 22% in cohort 1 and 61% in cohort 2 (P < 0.01). Diabetic mellitus was more common among deceased patients than among rescued patients (29% vs. 8%; P < 0.05) among patients who did not receive liver transplantations, and a multivariate analysis revealed that patient age and disease type were significantly and independently associated with the outcome. CONCLUSION: The outcomes of recent patients with ALF or LOHF caused by HAV infection have recently worsened mainly because of an increase in underlying metabolic diseases as a consequence of aging.

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.

A multicenter pilot survey to clarify the clinical features of patients with acute-on-chronic liver failure in Japan.

AIM: To establish diagnostic criteria for acute-on-chronic liver failure (ACLF) in Japan, a multicenter pilot survey was carried out to examine the usefulness of overseas criteria in patients with chronic liver diseases manifesting acute decompensation. METHODS: Patients fulfilling the Asian-Pacific Association for the Study of the Liver (APASL), European Association for the Study of the Liver (EASL), or Chinese Medical Association (CMA) criteria for decompensation were enrolled from eight institutions in Japan, and the clinical features were evaluated. RESULTS: Among 112 patients, 109 patients (97.3%) fulfilled the APASL criteria for decompensation; 7 patients were excluded because the decompensation had been provoked by gastrointestinal bleeding. Consequently, 102 patients (91.1%) were diagnosed as having ACLF according to the APASL definition. Among the patients who fulfilled the APASL criteria for decompensation, the etiologies of the underlying liver diseases were alcohol abuse in 59 cases (54.1%) and hepatitis B or hepatitis C virus infection in 24 (22.0%). The acute insults were alcohol abuse in 50 (45.9%), bacterial infection in 26 (23.9%), and exacerbation of underlying liver disease in 14 (12.8%). Fifty-four patients (49.5%) satisfied the CMA criteria, but the survival rates were similar between patients who did and those who did not meet the criteria. When 84 patients with underlying cirrhosis were classified according to the EASL-Chronic Liver Failure (Clif) Consortium criteria, the survival rates differed according to grade: 67.6% (23/34) for patients without ACLF, and 41.2% (14/34) and 18.8% (3/16) for those with grade 1/2 and grade 3 ACLF, respectively. CONCLUSION: The APASL definition was suitable for screening Japanese patients with ACLF, including those whose conditions were triggered by gastrointestinal bleeding, and the EASL-Clif Consortium criteria were useful for predicting outcome.

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.

"Reversi-type virologic failure" involved in the development of non-structural protein 5A resistance-associated variants (RAVs) in patients with genotype 1b hepatitis C carrying no signature RAVs at baseline.

AIMS: The therapeutic efficacy of daclatasvir/asunaprevir was inferior in patients with non-structural protein 5A (NS5A)-R30Q mutant hepatitis C virus strains at baseline, compared with those with wild-type strains, even though the half maximal effective concentration of NS5A inhibitors was lower in mutant strains than in wild-type strains. In these patients, R30Q and Y93H mutant strains, which are highly resistant to NS5A inhibitors, emerged at virologic failure. The mechanisms involved in such virologic failure were examined. METHODS: The NS5A resistance-associated variants were evaluated using direct sequencing in 88 patients with virologic failure after daclatasvir/asunaprevir therapy. In patients with R30Q and Y93H mutant strains at virologic failure, the original strains responsible for the multiple mutations were evaluated using baseline sera samples. RESULTS: L28 M and/or R30Q, L31 M, and Y93H mutant strains were found in 36, 46, and 65 patients, respectively, and R30Q and Y93H mutants were seen in 23 patients. R30Q mutant strains were detected in baseline sera samples available from eight of these patients; cycling-probe real-time polymerase chain reaction showed that the Y93H mutant strain to total strain ratio was less than 1% in four patients and ranged from 1% to 98% in four patients. A phylogenetic tree analysis undertaken after deep sequencing revealed that the R30Q and Y93H mutant strains originated from minor strains with both mutations at baseline, even in patients with a ratio of less than 1%. CONCLUSION: In patients with genotype 1b hepatitis C virus strains with R30Q mutation, minor strains with Y93H as well as R30Q mutations contributed to the development of virologic failure after treatment with NS5A inhibitors.

Development of rare resistance-associated variants that are extremely tolerant against NS5A inhibitors during daclatasvir/asunaprevir therapy by a two-hit mechanism.

AIM: The virologic characteristics of resistance-associated variants (RAVs) developing in patients receiving dual oral therapy with daclatasvir/asunaprevir, including those with previous triple therapy with simeprevir, were evaluated. METHODS: A total of 206 patients with genotype-1b HCV infection, including 5 patients with previous simeprevir therapy, were treated with daclatasvir/asunaprevir for 24 weeks. Resistance-associated variants in the NS5A regions at baseline and during/after therapy were evaluated using cycling-probe real-time polymerase chain reaction combined with direct sequencing. The dynamics of rare RAVs were also assessed using ultra-deep sequencing. RESULTS: A sustained virologic response (SVR12) was achieved in 180 patients (87%); the rates were 95% in patients without baseline NS5A-RAVs and 83%, 59%, and 77% in those with hepatitis C virus (HCV) strains carrying NS5A-L31M, NS5A-Y93H/C, and NS5A-R30Q/H/L mutations, respectively. A multivariate analysis revealed baseline NS5A-R30Q/H/L mutation and NS5A-Y93H mutations as significant factors associated with SVR12. Virologic failure developed in all 5 patients with previous simeprevir treatment, and rare RAVs (HCV strains with NS5A-R30H, NS5A-A92K, NS5A-P29del, and NS5A-P32del) developed at virologic failure. Ultra-deep sequencing revealed that HCV strains with NS5A-P29del or NS5A-P32del were absent at baseline and emerged within 4 weeks of dual oral therapy among the strains appearing after simeprevir administration. CONCLUSION: NS5A-R30Q/H/L and NS5A-Y93H mutations at baseline determined the therapeutic efficacy of dual oral therapy with daclatasvir/asunaprevir, but rare NS5A-RAVs developed frequently in patients with previous simeprevir treatment. Such RAVs may develop in a two-hit manner, with simeprevir altering the quasispecies of HCV strains in the NS5A regions, leading to the emergence of HCV strains with NS5A-P29del and NS5A-P32del during exposure to daclatasvir/asunaprevir.

Novel hepatitis B virus strain developing due to recombination between genotypes H and B strains isolated from a Japanese patient.

AIM: In Japan, genotypes B and C are the predominant genotypes isolated from patients with chronic hepatitis B, while genotype A predominates in patients with acute hepatitis B. Globalization, however, appears to have changed the distribution of the hepatitis B virus (HBV) genotypes. Thus, the viral characteristics of HBV genotypes other than genotypes A, B and C were examined. METHODS: Screening of genotypes was performed by enzyme immunoassay and/or polymerase chain reaction INVADER method in 222 patients with HBV. The full-length nucleotide sequences of unusual strains were compared to those in the database, followed by construction of a phylogenetic tree. RESULTS: Unusual HBV strains were isolated from two patients: a 27-year-old Japanese bisexual man with acute hepatitis B with HIV co-infection and a 52-year-old Japanese man with chronic hepatitis B. The former strain was classified as genotype H, showing an overall identity of 99.8% to the Thailand strain (EU498228), while the nucleotide sequence of the latter strain showed similarity to the genotype B strains isolated in Malaysia (JQ027316) and Indonesia (JQ429079) between DR2 and DR1 in the X region, with identities of 96.9%. However, this strain was classified as genotype H by full-length sequence analysis, and the sequence between nt2023 and nt2262 showed no similarity to that in any previously reported strains. CONCLUSION: HBV strains showing recombination between genotype B and H strains were found even in chronic hepatitis patients in Japan. Globalization may yield HBV strains of possible novel genotypes containing novel nucleotide sequences in the precore/core region.

Usefulness of atezolizumab plus bevacizumab as second-line therapy for patients with unresectable hepatocellular carcinoma.

AIM: To clarify the efficacy of atezolizumab (ATZ) plus bevacizumab (BEV) as the second-line therapy for patients with unresectable hepatocellular carcinoma (HCC). METHODS: The subjects were 82 patients with HCC receiving ATZ/BEV, including 33 patients with previous therapies with molecular-targeted agents (MTA). Therapeutic efficacy was evaluated using contrast-enhanced CT according to the mRECIST. RESULTS: The Child-Pugh scores were 5, 6,7 and 8 in 40, 35, 5 and 2 patients, respectively, and the extents of HCC progression were BCLC stage A, B and C in 3, 31 and 48 patients, respectively. Early therapeutic efficacy was evaluated in 67 patients, and percentages of patients achieving CR/PR/SD/PD until 12 weeks were 3.0%/29.9%/49.3%/17.9%, respectively, indicating ORR of 32.8% and DCR of 82.1%, The ORR was higher in MTA-naïve patients (40.5%) than in those after discontinuation of lenvatinib due to PD (7.7%, P = 0.0410), while the DCR was equivalent between both patients (83.3% vs 80.0%, P = 0.1184), and the multivariate analysis revealed previous MTA therapies with lenvatinib alone as a factor to deteriorate the ORR (HR of 4.846 (P = 0.0619)). The OS rates at 24 and 48 weeks were 86% and 72%, respectively, and the rates did not differ between MTA-naïve and MTA-experienced patients. Multivariate analyses revealed that achievement of CR, PR or SD and peripheral neutrophil/lymphocyte ratio were associated with a favorable outcome (HR of 0.124, P<0.0001 and 0.351, P = 0.0303). CONCLUSIONS: ATZ/BEV merits consideration even for MTA-experienced patients, since the OS was equivalent to those in MTA-naïve patients despite of an unfavorable early therapeutic efficacy.

Long-term effects of ipragliflozin and pioglitazone on metabolic dysfunction-associated steatotic liver disease in patients with type 2 diabetes: 5 year observational follow-up of a randomized, 24 week, active-controlled trial: Effect of ipragliflozin in MASLD.

AIMS/INTRODUCTION: We conducted a 5 year post-trial monitoring study of our previous randomized 24 week, open-label, active-controlled trial that showed beneficial effects of ipragliflozin on metabolic dysfunction-associated steatotic liver disease (MASLD), identical to those of pioglitazone. MATERIALS AND METHODS: In our previous trial, 66 patients with MASLD and type 2 diabetes were randomly assigned to receive either ipragliflozin (n = 32) or pioglitazone (n = 34). Upon its conclusion, 61 patients were monitored for 5 years for outcome measures of MASLD, glycemic, and metabolic parameters. Differences between the two groups were analyzed at baseline, 24 weeks, and 5 years; changes in outcome measures from baseline were also evaluated. RESULTS: At 5 years, the mean liver-to-spleen attenuation ratio increased by 0.20 (from 0.78 ± 0.24 to 0.98 ± 0.20) in the ipragliflozin group and by 0.26 (from 0.76 ± 0.26 to 1.02 ± 0.20) in the pioglitazone group (P = 0.363). Similarly, ipragliflozin and pioglitazone significantly improved serum aminotransferase, HbA1c, and fasting plasma glucose levels over 5 years. In the ipragliflozin group, significant reductions in body weight and visceral fat area observed at 24 weeks were sustained throughout the 5 years (-4.0%, P = 0.0075 and -7.6%, P = 0.045, respectively). Moreover, ipragliflozin significantly reduced the values of fibrosis markers (serum ferritin and FIB-4 index), was well tolerated, and had a higher continuation rate for 5 years compared with pioglitazone. CONCLUSIONS: Ipragliflozin and pioglitazone improved MASLD and glycemic parameters over 5 years. In the ipragliflozin group, significant reductions in body weight and visceral fat mass persisted for 5 years.