The Impact of Direct-Acting Antiviral Agents on Cytomegalovirus Reactivation in Chronic Hepatitis C Infection.

OBJECTIVE: The co-infection of HCV/CMV may accelerate the progression of liver diseases and worsen responsiveness to IFN treatment. The Direct-acting antiviral agents (DAAs), currently approved therapy for HCV, may cause a transient change in immune status, favoring the reactivation of other viruses. The current study aims to evaluate the impact of DAAs treatment on the reactivation of latent CMV in HCV patients. METHODS: The serological IgG, IgM Abs against CMV were detected by ELISA on192 HCV patients. The seronegative CMV IgM patients received (sofosbuvir/daclatasvir) regimen, then the CMV reactivation was examined by measuring the CMV IgM by ELISA and CMV DNA by real-time PCR. RESULTS: The serological data revealed that all patients were positive for CMV IgG (100%) while (64%) patients were positive for CMV IgM. The seronegative CMV IgM (36%) received the DAAs protocol. The sustained virological response was monitored by measuring the HCV RNA viremia in the patient sera. The serological data revealed that 28.6% of patients had a reactivation of CMV, while 18.5% of patients had detectable CMV DNA viremia. Moreover, there was a significant improvement in liver function as well as a decrease in FIB-4 and APRI scores at EOT. SVR was reached 97.4% among the total studied patients (N= 192). CONCLUSION: CMV co-infection has no impact on the response rate to DAAs. However, the CMV reactivation might have occurred after the complete eradication of HCV by DAAs.

Efficacy and Safety of Ombitasvir plus Paritaprevir, Ritonavir and Ribavirin in Non-cirrhotic Treatment-naïve and Treatment-experienced Egyptians with Chronic HCV Genotype-4 Infection.

Hepatitis C virus genotype 4 (HCV-GT4) is a risk factor for cirrhosis, hepatocellular carcinoma and liver failure. A combination of three new direct-acting antivirals ombitasvir, paritaprevir, and ritonavir has been recommended for treatment of HCV-GT4 infection. The current study was aimed to assess the efficacy and safety of this combination plus ribavirin in non-cirrhotic, treatment-naïve and -experienced Egyptians with HCV-GT4 infection in a real-world setting. A total of 255 Egyptians with HCV-GT4 infection were enrolled, including 82 treatment-experienced and 173 treatment-naïve patients. All of them completed 12-week treatment protocol of ombitasvir, paritaprevir and ritonavir as an oral dose combination with ribavirin. Virological response (VR) was measured, as well as the biochemical parameters related to treatment efficacy and adverse events at baseline and after treatment, at 4 (VR4) and 12 (VR12) weeks post-treatment. The results showed that the VR4 rates were 98.8% in both groups, and VR12 rates were 97.7% and 96.3% in treatment-naïve and -experienced patients, respectively, with no significant differences found between the groups concerning VR4 (P=0.9) and VR12 (P=0.3). The most common adverse events were headache and fatigue, which were significantly more common (P=0.001 and 0.003, respectively) in treatment-experienced than in treatment-naïve group. The quadruple regimen was well-tolerated, and the reported adverse events were generally mild to moderate. This real-world setting study confirms that the combination of ombitasvir, paritaprevir, ritonavir, and ribavirin is highly effective in the treatment of HCV- GT4 infection with a good safety and tolerability profile.

Possible Association of Elevated Plasma Levels of Growth Arrest-Specific Protein 6 and the Soluble Form of Tyrosine Kinase Receptor Axl with Low Hepatitis C Viral Load in Patients with Type 2 Diabetes Mellitus.

This study aimed to investigate the plasma levels of Gas6 and soluble Axl (sAxl) in patients with chronic hepatitis C virus (HCV) infection with and without type 2 diabetes mellitus (T2DM). The study involved four groups; 50 patients with chronic HCV, 50 patients with T2DM, 50 patients with chronic HCV and T2DM, and 31 age- and sex-matched healthy controls. T2DM was diagnosed according to American Diabetes Association criteria, HCV antibodies were detected by enzyme-linked immunosorbent assays (ELISA) and confirmed by real-time-polymerase chain reaction. Plasma Gas6 and sAxl levels were assayed in all groups by ELISA. Significant low levels of GAS 6 in HCV/T2DM group versus HCV group were detected (7.92 ± 5.18 vs. 16.09 ± 7.36, respectively, p = 0.000), but higher than T2DM and control groups (p ≥ 0.05), although nonsignificant. HCV load was higher in the HCV group than the HCV/T2DM group (1,888,300 ± 5,595,070 vs. 1,417,900 ± 4,066,460 copies/mL, respectively, p = 0.632). Among HCV group, significant positive correlations were detected between Gas6 and sAxl levels with HCV viral load (r = 0.48, p = 0.000 and r = 0.43, p = 0.002, respectively), while among HCV/T2DM group, significant negative correlations were detected (r = -0.29, p = 0.04 and r = -0.34, p = 0.014, respectively). Significant negative correlations were detected between Gas6/sAxl levels and glycated hemoglobin (r = -0.36, p = 0.01 and r = -0.4, p = 0.003, respectively) in T2DM despite the positive correlations detected in HCV/T2DM (r = 0.27, p = 0.053 and r = 0.55, p = 0.000, respectively). In conclusion, Gas6/Axl system in combined HCV/T2DM diseases may affect the pathogenesis and can alter the biomarkers and complications of both diseases in a manner that differs from a solitary disease.

Real Life Egyptian Experience of Daclatasvir Plus Sofosbuvir with Ribavirin in Naïve Difficult to Treat HCV Patients.

BACKGROUND: Chronic infection with Hepatitis C virus (HCV) is considered as a major cause for developing liver cirrhosis and hepatocellular carcinoma. A new era in HCV treatment is ongoing using Direct Acting Antiviral activity (DAA). The first approved DAA drug was Sofosbuvir which has a high tolerability and preferable pharmacokinetic profile. Another recently developed drug is Daclatasvir a first-in-class HCV NS5A replication complex inhibitor. Both drugs are administered orally once daily and have potent antiviral activity with wide genotypic coverage. METHODS: In the outpatient clinic, one hundred and fifty naïve difficult to treat chronic HCV patients were recruited from Tropical Medicine Department at Fayoum public hospital. A combination of Daclatasvir (60 mg) and Sofosbuvir (400 mg) (DCV/SOF) has been administered for those patients once daily with Ribavirin (1200 mg or 1000 mg based on patients' weight on two divided doses) over a period of 12 weeks. All patients have been followed up for clinical, laboratory assessment and HCV PCR to detect the efficacy and safety of the therapy. RESULTS: Sustained Virologic Response rate (SVR12) was achieved in the vast majority of patients (90.67%). Cirrhotic patients showed lower SVR compared to non-cirrhotic patients (88.89% vs 90.91%, respectively). Around half of the patients (49.33%) developed adverse events (AEs) during treatment. The most common AEs were headache, fatigue and abdominal pain. CONCLUSION: The available evidence seems to suggest that combination therapy of (DCV/SOF with RBV) in the treatment of chronic HCV genotype IV naïve difficult to treat patients either cirrhotic or non-cirrhotic is safe and effective. Monitoring for clinical and laboratory hepatic parameters was the basis for these findings.

High SVR rate following retreatment of non-sustained virological responders to sofosbuvir based anti-HCV therapies regardless of RAS testing: A real-life multicenter study.

Aim: Evaluation of the efficacy and safety of sofosbuvir/daclatasvir/ribavirin (SOF/DCV/RBV) in treating non-sustained virological responders (non-SVR12) to prior sofosbuvir-based therapy, in absence of RAS testing in mass treatment, and determination of the optimal timing to start re-treatment. Methods: Real-life prospective observational study included prior non-responders to 24-weeks SOF-RBV (n = 679, 67%) or 12-weeks SOF- RBV- PEG (n = 335, 33%). Patients were re-treated with daily SOF/DCV/RBV for 12 (n = 270) or 24 weeks (n = 744). The primary efficacy endpoint was SVR12. The primary safety endpoints were reported adverse events (AEs) from baseline to SVR12 time point. Results: We included 1,014 patients [age 52 ± 9 years, 58.48% men]. Cirrhosis was documented in 46.98% and 27.5% of SOF-RBV and SOF-RBV-PEG non-responders respectively. Overall, SVR12 was 90.6% [92.2% for 12 weeks therapy and 90.05% for 24 weeks therapy]. Mild AEs occurred in 5.13% (n=52) and 3.1% (n=32) discontinued treatment including eight on-treatment mortalities. Higher baseline FIB-4 and shorter interval before starting retreatment (<6 months) were independent predictors of non-SVR12 on multivariate regression analysis. Conclusion: SOF/DCV/RBV is an effective and safe treatment option for non-responders to prior sofosbuvir-based therapy. Six months interval before retreatment is optimal for achieving favorable SVR.

Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems.

CRISPR-Cas adaptive immune systems in prokaryotes boast a diversity of protein families and mechanisms of action, where most systems rely on protospacer-adjacent motifs (PAMs) for DNA target recognition. Here, we developed an in vivo, positive, and tunable screen termed PAM-SCANR (PAM screen achieved by NOT-gate repression) to elucidate functional PAMs as well as an interactive visualization scheme termed the PAM wheel to convey individual PAM sequences and their activities. PAM-SCANR and the PAM wheel identified known functional PAMs while revealing complex sequence-activity landscapes for the Bacillus halodurans I-C (Cascade), Escherichia coli I-E (Cascade), Streptococcus thermophilus II-A CRISPR1 (Cas9), and Francisella novicida V-A (Cpf1) systems. The PAM wheel was also readily applicable to existing high-throughput screens and garnered insights into SpyCas9 and SauCas9 PAM diversity. These tools offer powerful means of elucidating and visualizing functional PAMs toward accelerating our ability to understand and exploit the multitude of CRISPR-Cas systems in nature.

Guide RNA functional modules direct Cas9 activity and orthogonality.

The RNA-guided Cas9 endonuclease specifically targets and cleaves DNA in a sequence-dependent manner and has been widely used for programmable genome editing. Cas9 activity is dependent on interactions with guide RNAs, and evolutionarily divergent Cas9 nucleases have been shown to work orthogonally. However, the molecular basis of selective Cas9:guide-RNA interactions is poorly understood. Here, we identify and characterize six conserved modules within native crRNA:tracrRNA duplexes and single guide RNAs (sgRNAs) that direct Cas9 endonuclease activity. We show the bulge and nexus are necessary for DNA cleavage and demonstrate that the nexus and hairpins are instrumental in defining orthogonality between systems. In contrast, the crRNA:tracrRNA complementary region can be modified or partially removed. Collectively, our results establish guide RNA features that drive DNA targeting by Cas9 and open new design and engineering avenues for CRISPR technologies.

A CRISPR design for next-generation antimicrobials.

Two recent publications have demonstrated how delivering CRISPR nucleases provides a promising solution to the growing problem of bacterial antibiotic resistance.

Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems.

UNLABELLED: CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems in bacteria and archaea employ CRISPR RNAs to specifically recognize the complementary DNA of foreign invaders, leading to sequence-specific cleavage or degradation of the target DNA. Recent work has shown that the accidental or intentional targeting of the bacterial genome is cytotoxic and can lead to cell death. Here, we have demonstrated that genome targeting with CRISPR-Cas systems can be employed for the sequence-specific and titratable removal of individual bacterial strains and species. Using the type I-E CRISPR-Cas system in Escherichia coli as a model, we found that this effect could be elicited using native or imported systems and was similarly potent regardless of the genomic location, strand, or transcriptional activity of the target sequence. Furthermore, the specificity of targeting with CRISPR RNAs could readily distinguish between even highly similar strains in pure or mixed cultures. Finally, varying the collection of delivered CRISPR RNAs could quantitatively control the relative number of individual strains within a mixed culture. Critically, the observed selectivity and programmability of bacterial removal would be virtually impossible with traditional antibiotics, bacteriophages, selectable markers, or tailored growth conditions. Once delivery challenges are addressed, we envision that this approach could offer a novel means to quantitatively control the composition of environmental and industrial microbial consortia and may open new avenues for the development of "smart" antibiotics that circumvent multidrug resistance and differentiate between pathogenic and beneficial microorganisms. IMPORTANCE: Controlling the composition of microbial populations is a critical aspect in medicine, biotechnology, and environmental cycles. While different antimicrobial strategies, such as antibiotics, antimicrobial peptides, and lytic bacteriophages, offer partial solutions, what remains elusive is a generalized and programmable strategy that can distinguish between even closely related microorganisms and that allows for fine control over the composition of a microbial population. This study demonstrates that RNA-directed immune systems in bacteria and archaea called CRISPR-Cas systems can provide such a strategy. These systems can be employed to selectively and quantitatively remove individual bacterial strains based purely on sequence information, creating opportunities in the treatment of multidrug-resistant infections, the control of industrial fermentations, and the study of microbial consortia.

Clinical and laboratory evaluation of patients with end-stage liver cell failure injected with bone marrow-derived hepatocyte-like cells.

AIM: One of the defining features of the liver is the capacity to maintain a constant size despite injury. Extrahepatic stem cells especially bone marrow-derived stem cells are thought to undertake an important role in liver repopulation. This study was carried out to evaluate the outcome of autologous bone marrow-derived hepatocytes transplantation in patients with end-stage liver cell failure due to chronic hepatitis C. METHODS: Forty patients were included, divided into two groups. Group I: 20 patients receiving autologous bone marrow-derived mesenchymal stem cells stimulated to hepatic lineage. They were subdivided into two groups regarding the route of transplantation: intrasplenic (10) and intrahepatic (10). Group II: included 20 patients who received traditional supportive treatment. Patients were followed up using examination, laboratory investigations, abdominal ultrasonography, and evaluated by Child score, Model for End Stage Liver Disease score, fatigue scale, and performance status. RESULTS: The results showed significant improvement in group I regarding ascites, lower limb edema, and serum albumin, over the control group. Group I also showed statistically significant improvement in Child score, Model for End Stage Liver Disease score, fatigue scale, and performance status over the controls. No difference was observed between intrahepatic and intrasplenic groups. CONCLUSION: This study demonstrated the safety and short-term efficacy of autologous bone marrow-derived mesenchymal stem cell injection in liver cell failure. Further study is necessary to standardize the cell dose, determine the life span of the injected cells, and detect the appearance of long-term complications.