Skin advanced glycation end-products as indicators of the metabolic profile in diabetes mellitus: correlations with glycemic control, liver phenotypes and metabolic biomarkers.

INTRODUCTION: The production of advanced glycation end-products (AGEs) is a key pathomechanism related to the complications of diabetes mellitus. The measurement of HbA1c as one of the AGEs is widely used in the clinic, but also other proteins undergo glycation in the course of diabetes. Here, we measure skin AGEs (SAGEs) in patients with diabetes type 1 (DM1) and type 2 (DM2) and correlate them with metabolic markers as well as non-invasively measured liver fibrosis and steatosis. PATIENTS AND METHODS: In this cross-sectional study, a total of 64 patients with either DM1 or DM2 and 28 healthy controls were recruited. SAGEs were measured using autofluorescence (AGE Reader). Liver fibrosis and steatosis were quantified using transient elastography, which determines liver stiffness measurement (LSM) and controlled attenuation parameter (CAP). FGF19, FGF21 and GDF-15 were measured in blood samples using ELISA. RESULTS: SAGEs were elevated in both groups of patients with diabetes as compared to healthy controls (both p < 0.001) and were higher in patients with DM2 in comparison to DM1 (p = 0.006). SAGEs correlated positively with HbA1c (r = 0.404, p < 0.001), CAP (r = 0.260, p = 0.016) and LSM (r = 0.356, p < 0.001), and negatively with insulin growth factor binding protein 3 (p < 0.001). We also detected a positive correlation between GDF15 and SAGEs (r = 0.469, p < 0.001). CONCLUSIONS: SAGEs are significantly elevated in patients with both DM types 1 and 2 and correlate with metabolic markers, including HbA1c and GDF15. They might also help to detect patients with advanced liver injury in the setting of diabetes.

A phase 2 dose-finding study of lonafarnib and ritonavir with or without interferon alpha for chronic delta hepatitis.

BACKGROUND AND AIMS: Proof-of-concept studies demonstrated lonafarnib (LNF), a first-in-class oral prenylation inhibitor, efficacy in patients infected with HDV. The lonafarnib with ritonavir for HDV-2 (LOWR-2) study's aim was to identify optimal combination regimens of LNF + ritonavir (RTV) ± pegylated interferon alpha (PEG-IFNα) with efficacy and tolerability for longer-term dosing. Here we report the safety and efficacy at end of treatment for up to 24 weeks. APPROACH AND RESULTS: Fifty-five patients with chronic HDV were consecutively enrolled in an open-label, single-center, phase 2 dose-finding study. There were three main treatment groups: high-dose LNF (LNF ≥ 75 mg by mouth [po] twice daily [bid] + RTV) (n = 19, 12 weeks); all-oral low-dose LNF (LNF 25 or 50 mg po bid + RTV) (n = 24, 24 weeks), and combination low-dose LNF with PEG-IFNα (LNF 25 or 50 mg po bid + RTV + PEG-IFNα) (n = 12, 24 weeks). The primary endpoint, ≥2 log10 decline or < lower limit of quantification of HDV-RNA from baseline at end of treatment, was reached in 46% (6 of 13) and 89% (8 of 9) of patients receiving the all-oral regimen of LNF 50 mg bid + RTV, and combination regimens of LNF (25 or 50 mg bid) + RTV + PEG-IFNα, respectively. In addition, multiple patients experienced well-tolerated transient posttreatment alanine aminotransferase increases, resulting in HDV-RNA negativity and alanine aminotransferase normalization. The proportions of grade 2 and 3 gastrointestinal adverse events in the high-dose versus low-dose groups were 49% (37 of 76) and only 22% (18 of 81), respectively. CONCLUSIONS: LNF, boosted with low-dose RTV, is a promising all-oral therapy, and maximal efficacy is achieved with PEG-IFNα addition. The identified optimal regimens support a phase 3 study of LNF for the treatment of HDV.

Fibroblast Growth Factor 21 Response in a Preclinical Alcohol Model of Acute-on-Chronic Liver Injury.

BACKGROUND AND AIMS: Fibroblast growth factor (FGF) 21 has recently been shown to play a potential role in bile acid metabolism. We aimed to investigate the FGF21 response in an ethanol-induced acute-on-chronic liver injury (ACLI) model in Abcb4-/- mice with deficiency of the hepatobiliary phospholipid transporter. METHODS: Total RNA was extracted from wild-type (WT, C57BL/6J) and Abcb4-/- (KO) mice, which were either fed a control diet (WT-Cont and KO-Cont groups; n = 28/group) or ethanol diet, followed by an acute ethanol binge (WT-EtOH and KO-EtOH groups; n = 28/group). A total of 58 human subjects were recruited into the study, including patients with alcohol-associated liver disease (AALD; n = 31) and healthy controls (n = 27). The hepatic and ileal expressions of genes involved in bile acid metabolism, plasma FGF levels, and bile acid and its precursors 7α- and 27-hydroxycholesterol (7α- and 27-OHC) concentrations were determined. Primary mouse hepatocytes were isolated for cell culture experiments. RESULTS: Alcohol feeding significantly induced plasma FGF21 and decreased hepatic Cyp7a1 levels. Hepatic expression levels of Fibroblast growth factor receptor 1 (Fgfr1), Fgfr4, Farnesoid X-activated receptor (Fxr), and Small heterodimer partner (Shp) and plasma FGF15/FGF19 levels did not differ with alcohol challenge. Exogenous FGF21 treatment suppressed Cyp7a1 in a dose-dependent manner in vitro. AALD patients showed markedly higher FGF21 and lower 7α-OHC plasma levels while FGF19 did not differ. CONCLUSIONS: The simultaneous upregulation of FGF21 and downregulation of Cyp7a1 expressions upon chronic plus binge alcohol feeding together with the invariant plasma FGF15 and hepatic Shp and Fxr levels suggest the presence of a direct regulatory mechanism of FGF21 on bile acid homeostasis through inhibition of CYP7A1 by an FGF15-independent pathway in this ACLI model. Lay Summary: Alcohol challenge results in the upregulation of FGF21 and repression of Cyp7a1 expressions while circulating FGF15 and hepatic Shp and Fxr levels remain constant both in healthy and pre-injured livers, suggesting the presence of an alternative FGF15-independent regulatory mechanism of FGF21 on bile acid homeostasis through the inhibition of Cyp7a1.

Optimizing lonafarnib treatment for the management of chronic delta hepatitis: The LOWR HDV-1 study.

In a proof-of-concept (POC) study, the oral prenylation inhibitor, lonafarnib (LNF), decreased hepatitis D virus (HDV) RNA during 4 weeks of treatment. Here, we explored optimal LNF regimens. Fifteen patients (five groups; 3 per group) completed dosing as follows: (1) LNF 200 mg twice-daily (BID; 12 weeks); (2) LNF 300 mg BID (12 weeks); (3) LNF 100 mg thrice-daily (5 weeks); (4) LNF 100 mg BID + pegylated interferon alfa (PEG-IFNα) 180 µg once-weekly (QW; 8 weeks); and (5) LNF 100 mg BID + ritonavir (RTV) 100 mg once-daily (QD; 8 weeks). Tolerability and efficacy were assessed. Higher LNF monotherapy doses had greater decreases in HDV viral load than achieved in the original POC study. However, this was associated with increased gastrointestinal adverse events. Addition of RTV 100 mg QD to a LNF 100 mg BID regimen yielded better antiviral responses than LNF 300 mg BID monotherapy and with less side effects. A similar improvement was observed with LNF 100 mg BID + PEG-IFNα 180 µg QW. Two of 6 patients who received 12 weeks of LNF experienced transient posttreatment alanine aminotransferase (ALT) increases resulting in HDV-RNA negativity and ALT normalization. CONCLUSION: The cytochrome P450 3A4 inhibitor, RTV, allows a lower LNF dose to be used while achieving higher levels of postabsorption LNF, yielding better antiviral responses and tolerability. In addition, combining LNF with PEG-IFNα achieved more substantial and rapid HDV-RNA reduction, compared to historical responses with PEG-IFNα alone. Twelve weeks of LNF can result in posttreatment HDV-RNA negativity in some patients, which we speculate results from restoring favorable immune responses. These results support further development of LNF with RTV boosting and exploration of the combination of LNF with PEG-IFN. (Hepatology 2018;67:1224-1236).

HEV seroprevalence in blood donors in Turkey by two commercial total anti-HEV Ab ELISA kits.

Previous hepatitis E virus (HEV) seroprevalence studies in Turkey have shown high variabilities, leading to conflicting results. We aimed to re-evaluate HEV seroprevalence among blood donors in Turkey using the Wantai (Beijing, China) and the Dia.Pro (Milan, Italy) total anti-HEV antibody (Ab) enzyme-linked immunosorbent assay (ELISA) kits and compare their performances and to investigate the presence of HEV RNA in blood donors. Serum total anti-HEV antibodies were determined in a total of 2011 volunteer blood donor samples collected from different regions of Turkey (807 from Ankara, 243 from Kayseri, 284 from Izmir, 200 from Malatya, 200 from Kahramanmaras, and 277 from Van). HEV RNA was evaluated by a real-time polymerase chain reaction in a total of 272 anti-HEV seropositive samples. The country-wide HEV seroprevalence was calculated as 11.5% (Dia.Pro) and 12.2% (Wantai) with seropositivity rates of 12.0%-12.5% in Ankara, 7.4%-8.2% in Kayseri, 14.5%-15.5% in Malatya, 8.1%-8.8% in Izmir, 15.0%-16.0% in Kahramanmaras, and 12.6%-13.4% in Van by Dia.Pro and Wantai kits, respectively. The lowest detectable Ab concentrations were 0.16 and 0.14 units/mL WHO, for the Dia.Pro and the Wantai assays, respectively, showing no significant difference between assays. HEV RNA was not detected in any of the anti-HEV seropositive samples. Compared with previous studies, HEV was shown to have a higher overall seroprevalence in Turkey. Despite its limitation, the current study represents the most comprehensive HEV seroprevalence study in Turkey performed with two different commercial ELISA assays with high sensitivities so far. Further investigation is required to determine HEV genotypes in Turkey.

Interferon Treatment Duration in Patients With Chronic Delta Hepatitis and its Effect on the Natural Course of the Disease.

Background: Interferon is the only treatment option in chronic delta hepatitis (CDH). A CDH database (333 patients, 161 with interferon treatment history) was analyzed for effects of treatment duration on virologic response and clinical outcomes. Methods: Ninety-nine CDH patients who received at least 6 months of interferon were selected. Maintained virologic response (MVR) was defined as hepatitis D virus RNA negative for 2 years after treatment discontinuation. Cumulative median interferon treatment duration was 24 months (range 6-126 months), with a median of 2 courses (range 1-8). Post-treatment median follow-up was 55 months (24-225 months). Results: Thirty-five patients achieved MVR. Cumulative probability of MVR increased with treatment duration and reached 50% at 5 years. Patients with MVR were less likely to die from liver disease or develop complications compared to patients without MVR (P = .032, P = .006, respectively). Cirrhosis at baseline and no response to therapy (odds ratio 16.1 and 5.23, respectively) predicted an adverse endpoint. Hepatitis B surface antigen clearance occurred in 37% of patients with MVR. Conclusion: Viral response to interferon increases with treatment duration and favorably affects the natural course of disease. Interferon treatment duration has to be individualized with careful post-treatment assessment.

[Evaluation of 2015-2016 MOTAKK HBV DNA and HCV RNA external quality assessment national program results]. / MOTAKK HBV DNA ve HCV RNA dis kalite kontrol ulusal programi 2015-2016 sonuçlarinin degerlendirilmesi.

MOTAKK, as a national external quality control program has been launched to evaluate the molecular detection of viral infections including HBV DNA and HCV RNA in molecular microbiology diagnostic laboratories in Turkey. This program is prepared in compliance with ISO 17043:2010 (Conformity assessment general requirements for proficiency testing) standards, and aims to take the place of external quality control programs from abroad, contributing to standardization and accuracy of molecular diagnostic tests in our country. The aim of this study was to evaluate 2015 and 2016 results of the MOTAKK External Quality Control Program for HBV DNA and HCV RNA viral load . The calls were announced on the web page of MOTAKK (www.motakk.org). The quality control samples were sent to participating laboratories in 2015 and 2016. Main stocks were prepared from patients with chronic hepatitis B and C who had viral load detection with reference methods according to WHO reference materials for viral load studies to improve quality control sera. From these main stocks, samples with different viral loads were prepared from dilutions of plasma with HBV, HCV, HAV, HIV, Parvovirus B19 and CMV negative serologic markers. Quality control samples were sent to the participating laboratories along with the negative samples in the cold chain. The laboratories accomplished the related tests within 2-3 weeks and entered their results on the MOTAKK web page. These results were analysed according to ISO 13528 (Statistical methods for use in proficiency testing by interlaboratory comparison) and scoring reports were created by a software developed by MOTAKK and sent to participating labs. Each laboratory evaluated their own results in comparison with the other laboratory results, reassessed the tests via observing the distance from the mean result and the reference values. The number of laboratories participating in the HBV DNA and HCV RNA external quality control program was 70-73 in 2015-2016. Participants were able to comply with the program tools, registering, entering results and receiving the results reports without problem. In HBV panel, 72.6-89.1% and 84.7-90.3% of the participant laboratories were in 1 standard deviation (SD) in 2015-2016, respectively. In HCV panel, 70.8-89.1% and 84.7-90.3% of the participant laboratories were in 1 SD in 2015-2016, respectively. A national external quality control program for HBV DNA and HCV RNA in Turkey has been prepared for the first time with this project and implemented successfully. All the data provided in the MOTAKK external quality control program final report, compensate all the data provided by the quality control program final reports from abroad; additionally, the report allows comparison of used technologies and commercial products.

Interleukin-28 gene polymorphisms may contribute to HBsAg persistence and the development of HBeAg-negative chronic hepatitis B.

BACKGROUND & AIMS: Aim of this study was to investigate whether a potential association exists between several single nucleotide polymorphisms (SNPs) of the IL-28B gene (rs12979860, rs1188122, rs8099917, rs8105790, rs12980275) and HBsAg persistence. Further, a potential effect on the development of HBeAg-negative CHB vs. inactive HBsAg carrier state was assessed in a genotype D HBV cohort. A cohort of chronic HDV patients was also used to see if they behave differently compared to chronic HBV patients. METHODS: This study was conducted in three main patient cohorts: Group 1 consisted of 482 patients with HBsAg persistence. Of them 143 were inactive carriers, 94 had HBeAg-positive chronic hepatitis B (CHB) and 245 had anti-HBe-positive CHB. Group 2 represents spontaneously recovered HBV patients; they were anti-HBs and anti-HBc positive. Group 3 consisted of 176 chronic hepatitis delta (CHD) patients with antidelta and HDV-RNA positivity. DNA sequencing was performed for genotyping. RESULTS: When patients with HBsAg persistence were compared with spontaneously recovered patients, a significant difference was observed for rs8105790 (P < 0.0001), rs12980275 (P < 0.02). Patients who had the CC/TC genotype for rs8105790 (P < 0.0001) and AA genotype for 1188122 (P < 0.02) were more likely to be inactive HBsAg carriers, when inactive HBsAg carriers were compared with HBeAg-negative CHB patients. Comparison of CHD patients vs. recovered HBV patients was parallel to that of HBV persistence vs. recovered HBV with similar significant differences in same SNPs. CONCLUSION: These results suggest that IL-28B polymorphisms may contribute to HBsAg persistence and the development of the inactive HBsAg carrier state.

[Production of a recombinant CagA protein for the detection of Helicobacter pylori CagA antibodies]. / Helicobacter pylori CagA antikorlarinin saptanmasi için rekombinant bir CagA proteininin üretilmesi.

At present, Helicobacter pylori infections affect approximately 50% of the world population. It is known that H.pylori is related with several gastric diseases including chronic atrophic gastritis, peptic and gastric ulcers as well as gastric carcinomas. CagA (Cytotoxin-associated gene A) protein which is one of the most important virulence factors of H.pylori, is thought to be responsible for the development of gastric cancer. CagA is a 128 kDa hydrophilic protein which binds to the epitelial stomach cells and is known to be phosphorylated on its EPIYA regions. The EPIYA regions are highly variable and carry a higher risk of developing gastric cancer than CagA negative strains. The aim of this study was to construct a prokaryotic expression system expressing a recombinant CagA protein, which can be used for the detection of anti-CagA antibodies. For the isolation of H.pylori genomic DNA, a total of 112 gastric biopsy samples obtained from patients who were previously found positive for rapid urease (CLO) test, were used. H.pylori DNAs were amplified from 57 of those samples by polymerase chain reaction (PCR) and of them 35 were found positive in terms of cagA gene. Different EPIYA motifs were detected in 25 out of 35 cagA positive samples, and one of those samples that contained the highest number of EPIYA motif, was chosen for the cloning procedure. Molecular cloning and expression of the recombinant fragment were performed with Champion Pet151/D expression vector (Invitrogen, USA), the expression of which was induced by the addition of IPTG (Isopropyl-beta-D-thiogalactopyranoside) into the E.coli culture medium. Expression was observed with anti-histidin HRP (Horse Radish Peroxidase) antibodies by SDS-PAGE and Western Blot (WB) analysis. In our study, two clones possessing different fragments from the same H.pylori strain with three different EPIYA motifs were succesfully expressed. Since CagA antigen plays a signicant role in the pathogenesis of H.pylori infections, the detection of anti-CagA antibodies done by non-invasive commercial ELISA or WB methods, is important in diagnosis. Recombinant CagA protein produced in this study could easily be used in the ELISA tests to be developed for screening anti-CagA antibodies in the patients' sera. However, since an ELISA method using this antigen has not been developed yet, its diagnostic performance could not be evaluated in this study. In conclusion, the construction of such a system for recombinant CagA antigen expression may be a pilot study for the development of our own ELISA tests in Turkey, and also will help the clinicians for the prediction of disease outcome and decision of the appropriate antimicrobial treatment by the help of anti-CagA antibody detection.

Intra-pituitary follicle-stimulating hormone signaling regulates hepatic lipid metabolism in mice.

Inter-organ communication is a major hallmark of health and is often orchestrated by hormones released by the anterior pituitary gland. Pituitary gonadotropes secrete follicle-stimulating hormone (FSH) and luteinizing hormone (LH) to regulate gonadal function and control fertility. Whether FSH and LH also act on organs other than the gonads is debated. Here, we find that gonadotrope depletion in adult female mice triggers profound hypogonadism, obesity, glucose intolerance, fatty liver, and bone loss. The absence of sex steroids precipitates these phenotypes, with the notable exception of fatty liver, which results from ovary-independent actions of FSH. We uncover paracrine FSH action on pituitary corticotropes as a mechanism to restrain the production of corticosterone and prevent hepatic steatosis. Our data demonstrate that functional communication of two distinct hormone-secreting cell populations in the pituitary regulates hepatic lipid metabolism.

Entecavir treatment of chronic hepatitis D.

BACKGROUND: Hepatitis D virus (HDV) requires hepatitis B surface antigen (HBsAg) to propagate infection and cause disease. Entecavir is a nucleoside analog with potent antiviral efficacy, and in the woodchuck animal model it also decreased hepatitis B virus (HBV) cccDNA and woodchuck surface antigen. The aim of this study was to investigate the efficacy of entecavir in chronic hepatitis D (CHD). METHODS: This single-center study was conducted in patients with compensated liver disease. All patients had to have detectable hepatitis HDV RNA and elevated levels of alanine aminotransferase (ALT). Entecavir was given at a dosage of 1 mg/d for 1 year. The primary end point was achievement of undetectable HDV RNA at the end of treatment. RESULTS: Thirteen consecutive patients were assessed. All patients had detectable HDV RNA, and 8 had detectable HBV DNA at baseline. At the end of treatment, HBV DNA became undetectable in all patients (P = .001). No significant decline in HDV RNA, ALT, or quantitative HBsAg levels was observed. The primary end point of undetectable HDV RNA at the end of treatment was achieved in 3 patients who had significantly lower baseline HDV RNA levels than nonresponders (2.99 log(10) copies/mL ± .70 vs 4.68 ± .97; P = .0185). In all 3 patients, ALT levels were also normal at the end of treatment. CONCLUSIONS: One year of entecavir treatment is ineffective in CHD. Any generalized beneficial effect of nucleoside/nucleotide analog treatment may necessitate prolonged treatment. Patients with CHD with HBV dominance, which is likely to occur in the later phases of CHD, may be a reasonable patient cohort in which to target nucleoside/nucleotide analog therapy.

Complete genome sequences and phylogenetic analysis of hepatitis delta viruses isolated from nine Turkish patients.

Hepatitis delta virus (HDV) is a subviral agent of hepatitis B virus (HBV), and its life cycle is dependent on HBV. It is commonly accepted that HDV has eight distinct genotypes. In this study, the complete nucleotide sequences of HDV genomes isolated from nine Turkish patients were obtained by RT-PCR using two pairs of primers that cover the entire HDV genome. PCR products were sequenced directly. The results showed that these 9 isolates were approximately 1680 base pairs in length and clustered in the genotype HDV-1 branch when phylogenetic analysis was done with the sequences together with the complete sequences of HDV genomes representing each genotype retrieved from GenBank. Analysis of a portion of the large hepatitis D antigen (L-HDAg) gene showed that sequence similarity among these Turkish isolates is between 87.4 and 97.1%, and the Turkish isolates have the most sequence similarity to HDV-1 (90.5%), while they have the least sequence similarity to HDV-3 (64.1%). Full-genome analysis indicates that the sequence similarity is between 80.7 and 95.4%, and the highest sequence similarity is 84.8% (between the Turkish isolates and HDV-1). The lowest sequence similarity is 56.4% (between the Turkish isolates and HDV-3). In conclusion, phylogenetic analysis shows that the Turkish HDV isolates belong to HDV-1.

Vitamin D in Preclinical Models of Fatty Liver Disease.

Simple steatosis in non-alcoholic fatty liver disease (NAFLD) progresses to non-alcoholic steatohepatitis (NASH) when excessive fat accumulation is accompanied by ballooning, inflammation, and progressive hepatocellular injury. Due to the increasing global incidence of NAFLD/NASH and the lack of effective drugs, current treatment options are currently dominated by lifestyle interventions, including dietary and physical activity modifications. In this regard, vitamin D has received widespread attention in recent years. In line with its pleiotropic physiological effects, preclinical animal models and patient cohorts have demonstrated anti-inflammatory, anti-fibrotic and anti-proliferative effects of vitamin D on NAFLD and NASH. Several animal models have confirmed the association of vitamin D deficiency and NALFD/NASH severity in humans and revealed potential benefits of dietary vitamin D supplementation. These preclinical models also provide critical guidance to define the roles and therapeutic potential of vitamin D as well as its downstream functional mechanisms in the pathogenesis of fatty liver disease. This review summarizes vitamin D research in currently available animal models of fatty liver disease.

Effect of alcohol on the interleukin 6-mediated inflammatory response in a new mouse model of acute-on-chronic liver injury.

BACKGROUND AND AIMS: ACLF is usually associated with a precipitant in the setting of a chronically damaged liver. We aim to combine a mouse model with a pre-injured liver (Abcb4/Mdr2-/-) with a recently standardized ethanol feeding model to dissect alcohol-related inflammatory responses in this model. METHOD: Ten (n = 64) and 15 (n = 64) week old wild-type (WT) C57BL/6 J and Abcb4-/- knock-out (KO) mice were either fed control (WT/Cont and KO/Cont groups) or liquid ethanol diet (5% v/v) followed by an ethanol binge (4 mg/kg) (WT/EtOH and KO/EtOH groups). Hepatic mRNA levels of IL6, IFN-G, IL-1B, TGFB1, TNF-A, CCL2, HGF, CRP, RANTES, PNPLA3 and COL3A1 were evaluated using the 2-ΔΔCt method. IL6 and HGF plasma levels were quantified by ELISA. RESULTS: Older mice in KO/EtOH group displayed higher IL6 expressions compared to KO/Cont, WT/EtOH and WT/Cont groups of the same age, whereas HGF did not differ. Significant over-expression of CCL2 also corresponded to the same group. Males in KO/EtOH group exhibited higher IL6 expression than females. Lipid droplets were observed in about 80% of mice challenged with ethanol. There was a profound downregulation in PNPLA3 and RANTES levels after ethanol exposure. Mean size of the LDs was inversely correlated with hepatic PNPLA3 levels. CONCLUSION: We propose a novel promising approach to model alcohol-related ACLI. Acute inflammatory IL6-driven response might help transition from a stable chronic state to a progressive liver damage in Abcb4-/- mice. Repression of PNPLA3 resulted in a notable expansion in size of lipid droplets, indicating lipid remodeling in this model.

Evaluation of the results of MOTAKK hepatitis C virus RNA genotyping and hepatitis delta virus external quality assessment programs during 2015-2016.

BACKGROUND/AIMS: To evaluate the HCV RNA genotyping and HDV RNA tests that are performed in molecular microbiology laboratories in Turkey as part of a national external quality assessment programme, MOTAKK (Moleküler Tanida Kalite Kontrol) (English translation: Quality control in molecular diagnostics). MATERIALS AND METHODS: Plasmas having different HCV RNA genotypes were used to prepare HCV genotype control sera. The HDV RNA main stock was prepared from patients with chronic delta hepatitis who had a significant amount of viral load detected, as per the WHO reference materials on viral load studies that were compiled for the purpose of developing HDV RNA control sera. Samples with different viral loads were prepared from this main stock by dilution. The prepared controls were delivered to the registered laboratories. The laboratories carried out the relevant tests and entered their results via the MOTAKK web page. External quality assessment (EQA) reports of the participants were uploaded to the website as well. RESULTS: In total, there were 23 participating laboratories, out of which 20 exclusively performed HCV genotyping, and 15 and 16 only performed HDV RNA in 2015 and 2016, respectively. The success rate of the results of the HCV genotype was 56-96% in 2015 and 30-95% in 2016. The tube with a 30% success rate had a recombinant type of HCV, therefore, it could not be detected in most of the laboratories. The HDV RNA results were evaluated qualitatively. Accordingly, HDV RNA detection rates of participant laboratories were 71-100% in 2015 and 50-100% in 2016. CONCLUSION: This study was the first national external quality control program in Turkey regarding HCV RNA genotyping and HDV RNA in the field of molecular microbiology, and it was implemented successfully.

Antimicrobial and antiviral screening of novel indole carboxamide and propanamide derivatives.

A few series of indole derivatives were screened for antimicrobial, antifungal and anti-HBV activities. The compounds were tested for their in vitro antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and for their antifungal activity against Candida albicans using a disc diffusion method, which measures the diameter of the inhibition zone around a paper disc soaked in a solution of the test compounds. The antimicrobial activity results showed that all compounds are as a active as the standard compound ampicillin against Staphylococcus aureus. It was also found that indole carboxamide derivatives, substituted at 3-position with several benzyl groups, showed better inhibition of Bacillus subtilis than their congeners substituted at 2-position. Activity patterns of the compounds against Escherichia coli and Staphylococcus aureus were found slightly different by the same method. In this case, there was no correlation between structure and activity of the compounds. The antifungal activity of carboxamide derivatives was found higher compared to that of the propanamide derivatives. The minimum inhibitory concentration (MIC) values of some indole derivatives were also determined by the tube dilution technique. The MIC values of the compounds were found nearly 20- to 100-fold smaller compared to the standard compounds ciprofloxacin and ampicillin (1.56-3.13 microg/ml and 1.56-12.5 microg/ml, respectively) against Staphylococcus aureus, Bacillus subtilis and Escherichia coli. The MIC values of the tested compounds showed that these are better inhibitors for Candida albicans. Indole derivatives were screened by the anti-HBV susceptibility test. No compound showed good inhibition against the HBV virus.

A novel mutation pattern emerging during lamivudine treatment shows cross-resistance to adefovir dipivoxil treatment.

AIMS: This study was conducted to clarify the resistance profile of a novel mutation pattern emerging during lamivudine (3TC) therapy and showing cross-resistance to adefovir dipivoxil (ADV) in a patient with chronic hepatitis B. METHODS AND RESULTS: Successful suppression of hepatitis B virus (HBV) replication by sequential therapy of 9 MU thrice weekly interferon (IFN) and 3TC was followed by genotypical resistance detected at month 28 of therapy (month 19 of lamivudine treatment). ADV was added to 3TC therapy on month 44 of antiviral treatment. Neither alanine aminotransferase normalization nor a stable decrease in HBV viral load was observed, although ADV was used for more than 40 months. The HBV pol region was amplified from serum samples obtained before and after ADV treatment. The complete genome was cloned into a TA vector. PCR products and 7-10 clones from each cloned vector were sequenced. A novel mutation, A181S, in the reverse transcriptase gene leading to a conversion of W172C in the overlapping surface antigen gene was detected along with a M2041 mutation. The complete genome comprising the A181S+M2041 pattern was cloned into an expression vector and its in vitro susceptibility to 3TC, ADV, tenofovir (PMPA), clevudine (L-FMAU) and emtricitabine (FTC) were determined in transiently transfected Huh7 cells. This mutation pattern displayed more than 1000-fold resistance to the nucleoside analogues 3TC and FTC and approximately sixfold resistance to L-FMAU, while it confers 28.23- and 5.57-fold resistance for the nucleotide analogues ADV and PMPA, respectively. CONCLUSION: A new mutation pattern, A181S+M2041, arising under lamivudine treatment confers cross-resistance to ADV both in vivo and in vitro.

Discontinuation of lamivudine treatment in HBeAg-negative chronic hepatitis B: a pilot study with long-term follow-up.

BACKGROUND: Finite treatment of hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (CHB) with nucleoside/nucleotide analogues (NAs) is important in resource-limited countries. Outcome of treatment discontinuation in patients on long-term lamivudine (LVD) was assessed in a single centre observational pilot study in the current study. METHODS: Non-cirrhotic patients on LVD for at least 5 years with undetectable HBV DNA on at least two consecutive assessments were offered to stop treatment. Biochemical, serological and virological measures were determined at 3-6 month intervals after treatment discontinuation. Serum quantitative hepatitis B surface antigen (HBsAg) was determined at treatment discontinuation and 5-6 years thereafter. NA treatment was re-instituted in patients with confirmed viral rebound defined as HBV DNA >20,000 IU/ml. Relapser patients were no longer followed but were re-assessed 6 years after treatment cessation. RESULTS: LVD was discontinued in 23 patients. 8 patients relapsed within 1 year and NA treatment was restarted; 15 patients (65%) were non-relapsers. Thirteen of them were followed for at least 5 years. Two patients had undetectable HBV DNA throughout the follow-up period. In the rest, HBV DNA fluctuated at low levels. Two patients cleared HBsAg 24 and 36 months after stopping treatment. Quantitative HBsAg levels 5-7 years after treatment discontinuation were lower in non-relapser compared to relapser patients (1.21 IU/ml ±0.98 versus 2.71 ±0.76; P=0.002). Of 8 relapser patients 1 patient had HBsAg levels less than 100 IU/ml compared to 11 out of 13 non-relapser patients (P=0.0022). CONCLUSIONS: These data suggest that cessation of NA treatment is a viable option after a reasonable treatment duration in patients with HBeAg-negative CHB and that HBsAg clearance may become an achievable target in these patients.

Clinical and pathological characteristics of gastrointestinal stromal tumor (GIST) metastatic to bone.

Our aim in this study was to describe the clinical, morphological, and molecular profile of gastrointestinal stromal tumor (GIST) metastatic to bone. We analyzed the morphological, phenotypic, and molecular characteristics of seven cases, and in addition reviewed 17 cases from literature. Sequence analysis of KIT and PDGFRA genes was possible for six cases. For the GIST cases with bone metastasis, the most common primaries were small intestine (29%), stomach (25%), and rectum (21%). Sites of bone metastases were vertebrae (11), pelvis (8), femur (8), ribs (6), humerus (5), skull (3), scapula (1), and mandible (1). The size ranged from 1.5 to 13 cm (median, 3.8 cm). Bone metastases without involvement of any other organ were seen in 17% of the cases and were solitary in 14 (58%). Adjacent soft tissue involvement was present in nearly half of the patients. Bone metastasis was either manifest at the time of diagnosis (28%) or occurred after a mean period of 4.7 years (3 months-20 years). Morphologically, neoplastic cells were spindle in 67%, epithelioid in 13%, and mixed epithelioid and spindle in 20%. CD117, DOG1, and CD34 were positive in 88, 86, and 85% of the cases, respectively. KIT Exon 11 mutations were the most frequent gene alteration (78%), followed by KIT Exon 13 mutations. Of 17 of the cases with available follow-up information, 7 (41%) patients developed bone metastasis under imatinib therapy. Five patients (29%) died of disease within a mean of 17 months. Bone metastases from GIST are usually found in patients with advanced disease and typically present as lytic masses with occasional soft tissue involvement. We could not identify any KIT or PDGFRA alterations predisposing to bone metastasis.