Satoshi Yoshimi

Long term persistence of NS5A inhibitor-resistant hepatitis C virus in patients who failed daclatasvir and asunaprevir therapy.

Although interferon‐free antiviral treatment is expected to improve treatment of hepatitis C, it is unclear to what extent pre‐existing drug‐resistant amino acid substitutions influence response to therapy. The impact of pre‐existing drug‐resistant substitutions on virological response to daclatasvir and asunaprevir combination therapy was studied in genotype 1b hepatitis C virus (HCV)‐infected patients. Thirty‐one patients were treated with daclatasvir and asunaprevir for 24 weeks. Twenty‐six patients achieved sustained virological response (SVR), three patients experienced viral breakthrough, and two patients relapsed. Direct sequencing analysis of HCV showed the existence of daclatasvir‐resistant NS5A‐L31M or ‐Y93H/F variants in nine out of 30 patients (30%) prior to treatment, while asunaprevir‐resistant NS3‐D168 mutations were not detected in any patient. All 21 patients with wild‐type NS5A‐L31 and ‐Y93 achieved SVR, whereas only four out of nine patients (44%) with L31M or Y93F/H substitutions achieved SVR (P = 0.001). Ultra‐deep sequencing analysis showed that treatment failure was associated with the emergence of both NS5A‐L31/Y93 and NS3‐D168 variants. NS5A‐L31/Y93 variants remained at high frequency through post‐treatment weeks 103 through 170, while NS3‐D168 variants were replaced by wild‐type in all patients. In conclusion, pre‐existence of NS5A inhibitor‐resistant substitutions compromised the response to daclatasvir and asunaprevir combination therapy, and treatment failure was associated with the emergence of both NS5A‐L31/Y93 and NS3‐D168 variants. While asunaprevir‐resistant variants that emerged during therapy returned to wild‐type, daclatasvir‐resistant variants tended to persist in the absence of the drug. J. Med. Virol. 87:1913–1920, 2015.

A novel TK-NOG based humanized mouse model for the study of HBV and HCV infections.

The immunodeficient mice transplanted with human hepatocytes are available for the study of the human hepatitis viruses. Recently, human hepatocytes were also successfully transplanted in herpes simplex virus type-1 thymidine kinase (TK)-NOG mice. In this study, we attempted to infect hepatitis virus in humanized TK-NOG mice and urokinase-type plasminogen activator-severe combined immunodeficiency (uPA-SCID) mice. TK-NOG mice were injected intraperitoneally with 6 mg/kg of ganciclovir (GCV), and transplanted with human hepatocytes. Humanized TK-NOG mice and uPA/SCID mice were injected with hepatitis B virus (HBV)- or hepatitis C virus (HCV)-positive human serum samples. Human hepatocyte repopulation index (RI) estimated from human serum albumin levels in TK-NOG mice correlated well with pre-transplantation serum ALT levels induced by ganciclovir treatment. All humanized TK-NOG and uPA-SCID mice injected with HBV infected serum developed viremia irrespective of lower replacement index. In contrast, establishment of HCV viremia was significantly more frequent in TK-NOG mice with low human hepatocyte RI (<70%) than uPA-SCID mice with similar RI. Frequency of mice spontaneously in early stage of viral infection experiment (8weeks after injection) was similar in both TK-NOG mice and uPA-SCID mice. Effects of drug treatment with entecavir or interferon were similar in both mouse models. TK-NOG mice thus useful for study of hepatitis virus virology and evaluation of anti-viral drugs.

Emergence of resistant variants detected by ultra-deep sequencing after asunaprevir and daclatasvir combination therapy in patients infected with hepatitis C virus genotype 1

Daclatasvir (DCV) and asunaprevir (ASV) are NS5A and NS3 protease‐targeted antivirals respectively, currently under development for the treatment of chronic hepatitis C virus (HCV) infection. We analysed the relationship between pre‐existing drug‐resistant variants and clinical outcome of the combination treatment with DCV and ASV. Ten patients with HCV genotype 1b were orally treated with a combination of ASV and DCV for 24 weeks. The frequencies of amino acid (aa) variants at NS3 aa positions 155, 156 and 168 and at NS5A aa31 and 93 before and after treatment were analysed by ultra‐deep sequencing. We established a minimum variant frequency threshold of 0.3% based on plasmid sequencing. Sustained virological response (SVR) was achieved in 8 out of 10 patients (80%), and relapse of HCV RNA after cessation of the treatment and viral breakthrough occurred in the other two patients. Pre‐existing DCV‐resistant variants (L31V/M and/or Y93H; 0.9–99.4%) were detected in three out of eight patients who achieved SVR. Pre‐existing DCV‐resistant variants were detected in a relapsed patient (L31M, Y93H) and in a patient with viral breakthrough (Y93H); however, no ASV‐resistant variants were detected. In these patients, HCV RNA rebounded with ASV‐ and DCV‐ double resistant variants (NS3 D168A/V plus NS5A L31M and Y93H). While pre‐existing DCV‐resistant variants might contribute to viral breakthrough in DCV and ASV combination therapy, the effectiveness of prediction of the outcome of therapy based on ultra‐deep sequence analysis of pre‐existing resistant variants appears limited.

Rapid, Sensitive, and Accurate Evaluation of Drug Resistant Mutant (NS5A-Y93H) Strain Frequency in Genotype 1b HCV by Invader Assay.

Daclatasvir and asunaprevir dual oral therapy is expected to achieve high sustained virological response (SVR) rates in patients with HCV genotype 1b infection. However, presence of the NS5A-Y93H substitution at baseline has been shown to be an independent predictor of treatment failure for this regimen. By using the Invader assay, we developed a system to rapidly and accurately detect the presence of mutant strains and evaluate the proportion of patients harboring a pre-treatment Y93H mutation. This assay system, consisting of nested PCR followed by Invader reaction with well-designed primers and probes, attained a high overall assay success rate of 98.9% among a total of 702 Japanese HCV genotype 1b patients. Even in serum samples with low HCV titers, more than half of the samples could be successfully assayed. Our assay system showed a better lower detection limit of Y93H proportion than using direct sequencing, and Y93H frequencies obtained by this method correlated well with those of deep-sequencing analysis (r = 0.85, P <0.001). The proportion of the patients with the mutant strain estimated by this assay was 23.6% (164/694). Interestingly, patients with the Y93H mutant strain showed significantly lower ALT levels (p=8.8 x 10-4), higher serum HCV RNA levels (p=4.3 x 10-7), and lower HCC risk (p=6.9 x 10-3) than those with the wild type strain. Because the method is both sensitive and rapid, the NS5A-Y93H mutant strain detection system established in this study may provide important pre-treatment information valuable not only for treatment decisions but also for prediction of disease progression in HCV genotype 1b patients.

Elevated expression of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 in primary sclerosing cholangitis : Implications for cholangiocarcinogenesis

Cholangiocarcinoma (CCA) occurs frequently in primary sclerosing cholangitis (PSC). Cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) induced by inflammation are believed to mediate prostaglandin E2 (PGE2) production thereby promoting carcinogenesis. Their expression in PSC-associated CCA tissues and non-neoplastic bile duct epithelial cells (BDECs) in PSC was investigated. COX-2 and mPGES-1 levels in 15 PSC patients (7 with CCA) were scored using immunohistochemical staining. The results were compared with those obtained in CCA tissues and non-neoplastic BDECs (controls) of 15 sporadic CCA patients. Non-neoplastic BDECs from large and small bile ducts were investigated separately. The mRNA expression levels of COX-2 and mPGES-1 in CCA tissues were analyzed by quantitative polymerase chain reaction. Ki-67 immunostaining was performed to evaluate cell proliferation. COX-2 was strongly expressed in PSC-associated CCA tissues and non-neoplastic BDECs in PSC. This expression was significantly upregulated in both compared with sporadic CCA tissues and non-neoplastic BDECs in sporadic CCA (both P<0.01). mPGES-1 was expressed at moderate to strong levels in PSC. Compared with controls, the expression was significantly higher in non-neoplastic small BDECs (P<0.01). COX-2 mRNA levels were significantly higher in PSC-associated tissues than in sporadic CCA tissues (P<0.01). Conversely, no differences were observed in mPGES-1 mRNA levels. Ki-67 labeling indices were higher in PSC-associated CCA tissues and non-neoplastic BDECs in PSC than in controls. In conclusion, COX-2 and mPGES-1 were highly expressed in PSC-associated CCA tissues and non-neoplastic BDECs in PSC, suggesting the involvement of COX-2 and mPGES-1 in cholangiocarcinogenesis.

A comparative study of 4 Fr versus 6 Fr nasobiliary drainage catheters: a randomized, controlled trial

Despite the benefits of endoscopic nasobiliary drainage (NBD) in endoscopic retrograde cholangiopancreatography (ERCP), post‐ERCP pancreatitis (PEP) and nose/throat discomfort can result. We aimed to determine whether the use of a smaller catheter alleviates these complications.

Elimination of HCV via a non-ISG-mediated mechanism by vaniprevir and BMS-788329 combination therapy in human hepatocyte chimeric mice.

We previously reported that interferon (IFN)-free direct-acting antiviral combination treatment succeeded in eradicating genotype 1b hepatitis C virus (HCV) in human hepatocyte chimeric mice. In this study, we examined the effect of vaniprevir (MK7009, NS3/4A protease inhibitor) and BMS-788329 (NS5A inhibitor) combination treatment on HCV genotype 1b and the expression of IFN-stimulated genes (ISGs) using a subgenomic replicon system and the same animal model. Combination treatment with vaniprevir and BMS-788329 significantly reduced HCV replication compared to vaniprevir monotherapy in HCV replicon cells (Huh7/Rep-Feo cells). HCV genotype 1b-infected human hepatocyte chimeric mice were treated with vaniprevir alone or in combination with BMS-788329 for four weeks. Vaniprevir monotherapy reduced serum HCV RNA titers in mice, but viral breakthrough was observed in mice with high HCV titers. Ultra-deep sequence analysis revealed a predominant replacement by drug-resistant substitutions at 168 in HCV NS3 region in these mice. Conversely, in mice with low HCV titers, HCV was eradicated by vaniprevir monotherapy without viral breakthrough. In contrast to monotherapy, combination treatment with vaniprevir and BMS-788329 succeeded in completely eradicating HCV regardless of serum viral titer. IFN-alpha treatment significantly increased ISG expression; however, vaniprevir and BMS-788329 combination treatment caused no increase in ISG expression both in cultured cells and in mouse livers. Therefore, combination treatment with vaniprevir and BMS-788329 eliminated HCV via a non-ISG-mediated mechanism. This oral treatment might offer an alternative DAA combination therapy for patients with chronic hepatitis C.

Occupational Radiation Exposure during Endoscopic Retrograde Cholangiopancreatography and Usefulness of Radiation Protective Curtains

Objective. To evaluate the effectiveness of radiation protective curtains in reducing the occupational radiation exposure of medical personnel. Methods. We studied medical staff members who had assisted in 80 consecutive therapeutic endoscopic retrograde cholangiopancreatography (ERCP) procedures. Use of radiation protective curtains mounted to the X-ray tube was determined randomly for each procedure, and radiation doses were measured with electronic pocket dosimeters placed outside the protective apron. Results. When protective curtains were not used, the mean radiation doses to endoscopists, first assistants, second assistants, and nurses were 340.9, 27.5, 45.3, and 33.1 µSv, respectively; doses decreased to 42.6, 4.2, 13.1, and 10.6 µSv, respectively, when protective curtains were used (P < 0.01). When the patient had to be restrained during ERCP (n = 8), the radiation dose to second assistants without protective curtains increased by a factor of 9.95 (P < 0.01) relative to cases in which restraint was not required. Conclusions. During ERCP, not only endoscopists, but also assistants and nurses were exposed to high doses of radiation. Radiation exposure to staff members during ERCP was reduced with the use of protective curtains.

Timing of radiological improvement after steroid therapy in patients with autoimmune pancreatitis.

Abstract Objective. We retrospectively studied the timing of radiological improvement after steroid therapy in patients with autoimmune pancreatitis (AIP). Material and methods. Patients with AIP (n = 31) received steroids followed by diagnostic imaging within 1 month. Pancreatic swelling, pancreatic and bile duct features, and apparent diffusion coefficient (ADC) were compared before and after treatment. The period from treatment initiation to evaluation was divided into five phases: early phase (days 3–5), week 1 (days 6 and 7), week 2 (days 8–14), week 3 (days 15–21), and week 4 (days 22–28). Five gastroenterologists evaluated pancreatic swelling and duct features (good/intermediate/no response), and the “good response” rate (response rate) was calculated for each phase. In addition, pancreatic volumes were measured with a 3D workstation before and after treatment, and the percentage change in volume was calculated. ADC values were calculated in 14 patients. Results. The median ratio of pancreatic volume after relative to before treatment was 0.89, 0.79, 0.67, 0.59, and 0.47 for early phase, week 1, week 2, week 3, and week 4, respectively. The response rate of the pancreatic swelling was 37.5%, 57.1%, 83.3%, 100%, and 100%; response rate of the pancreatic duct was 0%, 20%, 75%, 75% and 100%; and response rate of the bile duct was 0%, 66.7%, 83.3%, 100%, and 80%. The ADC increased after treatment in all 14 patients, including the 7 patients evaluated in the early phase. Conclusions. Evaluation of pancreatic swelling and duct features is recommended in week 2 and thereafter. The ADC increased soon after treatment initiation, suggesting its usefulness for evaluating early treatment responses.