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Assessment of liver fibrosis by non-invasive means is clinically important. Studies in chronic hepatitis delta (CHD) are scarce. We evaluated the performance of eight serum fibrosis markers [fibrosis-4 score (FIB-4), aspartate aminotransferase (AST) to alanine aminotransferase (ALT) ratio (AAR), age-platelet index (API), AST-to platelet-ratio-index (APRI), Goteborg University Cirrhosis Index (GUCI), Lok index, cirrhosis discriminant score (CDS) and Hui score] in CHD and chronic hepatitis B (CHB). Liver stiffness was assessed by transient elastography (TE) in CHD. The ability of fibrosis markers to detect significant fibrosis and cirrhosis were evaluated in 202 CHB and 108 CHD patients using published and new cut-offs through receiver operating characteristics (ROC) analysis. The latter was also applied to obtain cut-offs for TE. APRI, Fib-4, API and Hui score were assessed for significant fibrosis, and APRI, GUCI, Lok index, CDS and AAR for cirrhosis determination. Fibrosis markers displayed weak performance in CHB for significant fibrosis with area under ROC (AUROC) curves between 0.62 and 0.71. They did slightly better for CHD. TE displayed an AUROC of 0.92 and performed better than serum fibrosis markers (p < 0.05 for fibrosis markers). For cirrhosis determination, CDS and Lok Index displayed an AUROC of 088 and 0.89 in CHB and GUCI, Lok index and APRI displayed AUROCs around 0.90 in CHD. TE displayed the best AUROC (0.95). Hence TE is superior to serum fibrosis markers for diagnosing significant liver fibrosis and cirrhosis. GUCI, Lok index and APRI displayed a reasonable performance in CHD, which needs further confirmation.
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Hepatitis B Crónica , Hepatitis D Crónica , Hepatitis D , Humanos , Recuento de Plaquetas , Cirrosis Hepática/diagnóstico , Fibrosis , Pruebas de Función Hepática , Curva ROC , Hepatitis Crónica , Alanina Transaminasa , Biomarcadores , Aspartato Aminotransferasas , Hepatitis B Crónica/complicacionesRESUMEN
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.
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Infecciones por VIH , Hepatitis D Crónica , Alanina Transaminasa , Quimioterapia Combinada , Infecciones por VIH/tratamiento farmacológico , Hepatitis D Crónica/tratamiento farmacológico , Humanos , Interferón-alfa/uso terapéutico , Piperidinas , Piridinas , ARN , RitonavirRESUMEN
Background and Aim: This study was designed to predict the fibrosis stage with a clinical scoring system that may reduce the need for liver biopsy. Materials and Methods: The study cohort included the treatment of 430 chronic hepatitis B (CHB) and 170 chronic hepatitis C (CHC) of naive patients. The patients were divided into two groups as mild to moderate and severe fibrosis. After an index obtained in the study cohort, the index was tested in a validation cohort and compared with the FIB-4 Index. Results: The AUC of CHC index was found of 0.89 the sensitivity of 0.91 the specificity of 0.74, the positive predictive value (PPV) of 0.54 and the negative predictive value (NPV) of 0.96. The FIB-4 Index was applied to the CHC study cohort and the ATA Index Hepatitis C was found to be superior in terms of AUC (0.89-0.82), sensitivity (0.91-0.76) and NPV (0.96-0.86). The AUC of CHB Index was determined of 0.92, the sensitivity of 0.90, the specificity of 0.84, the PPV of 0.53 and the NPV of 0.98. Compared to the FIB-4 Index in CHB study cohort, the ATA Index Hepatitis B was predominant in terms of AUC (0.92-0.88), sensitivity (0.90-0.75), NPV (0.98-0.94) and PPV (0.53-0.49). Conclusion: ATA Indexes can predict the non-existence of severe fibrosis with an accuracy similar to FIB-4 Index and may reduce the need for liver biopsy.
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BACKGROUND/AIMS: This study gives a clue about genotypes, subgenotypes and subtypes of HBV, HCV and HDV viruses in general population of Afghanistan. MATERIALS AND METHODS: A total of 234 HBsAg, 44 anti-HCV and 5 Anti-Delta positive patients belong to 25-70 age group were obtained through a rapid screening test among 5898 residents of Afghanistan. After quantifying viral load, genotyping of 61 HBV, 29 HCV and 1 HDV samples were accomplished by sequencing of a segment of the HBV Pre S, HCV NS5B, and HDV Delta antigen regions respectively. Clinically important variants of the HBV polymerase gene, the "a" determinant of HBsAg, HCV NS5B and NS3 regions were assessed. RESULTS: All HBV isolates were dispersed throughout the genotype D branch and ayw2 was the only subtypes found. The anti-HDV prevalence among HBsAg positive individuals was 2.2% and the single HDV sample, belonged to HDV genotype I. Analysis of HCV isolates revealed subtype HCV-1b in 75.86%, HCV-3a in 20.69% and HCV-3b in 3.44% patients. The observed mutant variants in the MHR of HBsAg were Y100 15%, Q101 5%, G102 15%, T115 45%, P120 5%, T131 5%. Likewise, S213T 10%, Q215P 5% and N248H 100% mutations were detected in the HBV polymerase region. C316N mutation was prevalent in 72.7% of HCV 1b participants. CONCLUSION: Genotypic variation in Afghan patients is in line with the ones existing in neighboring countries and regions. HBV genotypes D1, subtype ayw2, HDV RNA type I, and HCV RNA genotype 1b are likely to be dominant in Afghan patients.
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Hepacivirus/genética , Virus de la Hepatitis B/genética , Hepatitis B/epidemiología , Hepatitis C/epidemiología , Hepatitis D/epidemiología , Virus de la Hepatitis Delta/genética , Adulto , Afganistán/epidemiología , Femenino , Genotipo , Hepatitis B/virología , Hepatitis C/virología , Hepatitis D/virología , Humanos , Masculino , Persona de Mediana Edad , Epidemiología Molecular , ARN Viral/genética , Carga ViralRESUMEN
Background and Aim: The present study aims to investigate the role of genetic variability of the PNPLA3 gene (adiponectin) in predisposition to non-alcoholic fatty liver disease (NAFLD) by comparing NAFLD patients to healthy controls and to investigate any impact of the PNPLA3 genetic variability on the natural course of the disease. Materials and Methods: This cohort consisted of 174 patients with biopsy-proven NAFLD and 151 healthy controls. DNA was extracted from peripheral blood and the rs738409 C>G single nucleotide polymorphism was assessed using PCR-DNA sequencing. Results: The frequency distribution of the GG genotype was significantly higher in NAFLD patients than in controls (p=0.01). In patients with NAFLD, the GG genotype was associated with lower platelet counts (p=0.001), the presence of steatohepatitis (p=0.04) and hepatic fibrosis (p=0.016). After adjustment for age, gender, obesity, and diabetes mellitus, the GG genotype was an independent predictor of significant hepatic fibrosis (adjusted odds ratio =3.031 p=0.012). From the baseline to sequential liver biopsies, the progression of NAS in NAFLD patients was slightly higher in the GG genotype than that of CC and GG genotypes (p=0.18). Conclusion: The PNPLA3 GG genotype is a predisposing factor for the development of hepatic steatosis in NAFLD patients and related to a more severe liver disease.
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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.
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Técnicas de Genotipaje/normas , Garantía de la Calidad de Atención de Salud/normas , Control de Calidad , ARN Viral/sangre , Carga Viral/normas , Hepacivirus/genética , Hepatitis C Crónica/sangre , Hepatitis C Crónica/virología , Hepatitis D Crónica/sangre , Hepatitis D Crónica/virología , Virus de la Hepatitis Delta/genética , Humanos , Evaluación de Programas y Proyectos de Salud , Turquía , Carga Viral/métodosRESUMEN
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.
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Donantes de Sangre , Ensayo de Inmunoadsorción Enzimática , Anticuerpos Antihepatitis/sangre , Virus de la Hepatitis E/inmunología , Hepatitis E/epidemiología , Adolescente , Adulto , Anciano , Femenino , Genotipo , Hepatitis E/sangre , Hepatitis E/inmunología , Virus de la Hepatitis E/genética , Humanos , Inmunoglobulina G/sangre , Masculino , Persona de Mediana Edad , ARN Viral/genética , Juego de Reactivos para Diagnóstico , Estudios Seroepidemiológicos , Turquía/epidemiología , Adulto JovenRESUMEN
Although a few studies have been done on transmissible blood-borne viral infections in high-risk groups, little attention has been given to assessing the infection status of the general population in Afghanistan. To investigate the epidemiological status in the general population, we tested the serological markers of hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis delta virus (HDV), human immunodeficiency virus 1 (HIV-1) and human T-cell leukemia virus (HTLV) infections. In total, 492 samples were selected randomly from Nangarhar, Herat, Mazar-e Sharif, Kandahar, and Kabul from subjects between 25 and 70 years old. The samples were tested for the presence of HBsAg, anti-HBs, anti-HBc, anti-HDV, anti-HCV, anti-HIV-1 and anti-HTLV I/II antibodies using chemiluminescent immunoassays on Abbott Architect automated platforms. In addition, 220 HBsAg-positive samples identified among 5897 samples from the general population of the same regions of Afghanistan were included in the study and tested for both HBsAg and anti-HDV to investigate HDV prevalence in the country. Viral loads of HBV, HCV and HDV were determined in all seropositive samples using Ampliprep/Cobas TaqMan HBV, HCV, Test Roche (CA, USA), and an in-house method, respectively. Out of 492 samples, 31 (6.3%), 136 (27.6%) and 149 (30.3%) were found to be positive for HBsAg, anti-HBs and anti-HBc, respectively. Anti-HDV positivity was detected in five (2.1%) out of 234 HBsAg-positive samples (including 14 of the randomly selected samples that were not among the 220 previously identified as HBsAg positive). Only eight out of 492 (1.6%) subjects were positive for anti-HCV antibodies. Seven out of 489 (1.4%) were positive for anti-HIV-1 antibodies, and three out of 466 cases (0.6%) were positive for anti-HTLV I/II antibodies. These results suggest that Afghanistan is an intermediate endemic region for HBV, HDV and HCV infection. The prevalence of HIV-1 seems to be significantly higher than the global prevalence and that of the eastern Mediterranean region. In addition, the HTLV I/II screening results suggest that these viruses should be monitored in Afghanistan to confirm the trend observed in the current study.
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Virosis/epidemiología , Adulto , Afganistán/epidemiología , ADN Viral/genética , Femenino , Infecciones por VIH/epidemiología , Infecciones por VIH/inmunología , VIH-1/inmunología , Hepacivirus/inmunología , Anticuerpos Antihepatitis/inmunología , Hepatitis B/epidemiología , Hepatitis B/inmunología , Anticuerpos contra la Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis C/epidemiología , Hepatitis C/inmunología , Anticuerpos contra la Hepatitis C/inmunología , Hepatitis D/epidemiología , Hepatitis D/inmunología , Virus de la Hepatitis Delta/inmunología , Humanos , Masculino , Persona de Mediana Edad , Carga Viral/métodos , Virosis/inmunologíaRESUMEN
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.
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Técnicas de Laboratorio Clínico , VIH-1 , Hepatitis B , Hepatitis C , Control de Calidad , Técnicas de Laboratorio Clínico/normas , ADN Viral/genética , Hepacivirus/genética , Hepatitis B/diagnóstico , Virus de la Hepatitis B/genética , Hepatitis C/diagnóstico , Humanos , ARN Viral/genética , TurquíaRESUMEN
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.
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Antivirales/administración & dosificación , Antivirales/uso terapéutico , Hepatitis D Crónica/tratamiento farmacológico , Interferones/administración & dosificación , Interferones/uso terapéutico , Adulto , Biomarcadores , Esquema de Medicación , Femenino , Antígenos de Superficie de la Hepatitis B/sangre , Humanos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Recuento de Plaquetas , Estudios RetrospectivosRESUMEN
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).
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Antivirales/administración & dosificación , Inhibidores Enzimáticos/administración & dosificación , Hepatitis D Crónica/tratamiento farmacológico , Virus de la Hepatitis Delta/efectos de los fármacos , Piperidinas/administración & dosificación , Piridinas/administración & dosificación , Adolescente , Adulto , Anciano , Antivirales/efectos adversos , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Inhibidores Enzimáticos/efectos adversos , Femenino , Estudios de Seguimiento , Virus de la Hepatitis Delta/genética , Humanos , Interferón-alfa/administración & dosificación , Interferón-alfa/efectos adversos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Piperidinas/efectos adversos , Piridinas/efectos adversos , ARN Viral , Ritonavir/administración & dosificación , Ritonavir/efectos adversos , Resultado del Tratamiento , Adulto JovenRESUMEN
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.
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Antivirales/uso terapéutico , Hepatitis B Crónica/tratamiento farmacológico , Lamivudine/uso terapéutico , Adulto , Anciano , Antivirales/efectos adversos , Biomarcadores , ADN Viral , Quimioterapia Combinada , Femenino , Estudios de Seguimiento , Antígenos de Superficie de la Hepatitis B/sangre , Antígenos e de la Hepatitis B/sangre , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/sangre , Hepatitis B Crónica/diagnóstico , Hepatitis B Crónica/mortalidad , Humanos , Estimación de Kaplan-Meier , Lamivudine/efectos adversos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Factores de Tiempo , Resultado del Tratamiento , Carga ViralRESUMEN
BACKGROUND/AIMS: Hepatitis C virus (HCV) genotyping has a considerable effect on therapy. The aim was to determine the change in prevalence of HCV genotypes in Turkey during the last decade and to compare the performance of DNA sequencing of different targets in the HCV genome (NS5B, E1, and 5'UTR). MATERIALS AND METHODS: Five hundred HCV RNA-positive patients (226 males, 274 females) were included in the study. The NS5B, E1, and 5'UTR regions of the HCV genome were amplified by polymerase chain reaction (PCR) in patients where possible. Amplified PCR products were sequenced directly, and phylogenetic analysis was performed. A commonly used database, namely www.hcv.lanl.gov, was also used to determine the genotypes. RESULTS: Phylogenetic analysis of the NS5B, E1, and 5'UTR regions showed that 1b was the most frequent genotype, with percentages of 92.5%, 93.5%, and 87.7%, respectively. Genotype 1a was the second most prevalent genotype, with ratios of 6.7%, 5.6%, and 6.6%, whereas genotype 2a was detected in proportions of 0.4%, 0.2%, and 0.8%, respectively. Genotype 5 or 6 was not detected among patients. The phylogenetic analysis showed discordant results with 18 patients' genotypes for different targets. The phylogenetic analysis showed similar results with the hcv.lanl.gov database for the E1 and NS5B sequences. CONCLUSION: There has been no change in genotyping profiles of Turkey during the last decade, representing 1b as the most prevalent subtype, followed by 1a. Phylogenetic analysis of HCV indicated high performance compared with the hcv.lanl.gov database when sequences of E1 and NS5B regions were analyzed.
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Regiones no Traducidas 5'/genética , Genotipo , Hepacivirus/genética , Hepatitis C/virología , Proteínas del Envoltorio Viral/genética , Proteínas no Estructurales Virales/genética , Anciano , Femenino , Técnicas de Genotipaje , Humanos , Masculino , Persona de Mediana Edad , Filogeografía , Análisis de Secuencia de ADN/métodos , Factores de Tiempo , TurquíaRESUMEN
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.
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Anticuerpos Antibacterianos/aislamiento & purificación , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Helicobacter pylori/inmunología , Factores de Virulencia/inmunología , Secuencia de Aminoácidos , Anticuerpos Antibacterianos/genética , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopsia , Western Blotting , Clonación Molecular , ADN Bacteriano/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Mucosa Gástrica/microbiología , Regulación Bacteriana de la Expresión Génica , Helicobacter pylori/genética , Helicobacter pylori/patogenicidad , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Internal controls (ICs), are the main components of any real-time PCR based amplification methods, which are co-purified and co-amplified with the actual target. The existence of free circulating nucleic acids in plasma and serum (CNAPS) has been known for many years. The aim of this study was to verify whether CNAPS can be used as ICs in real-time PCR based detection and quantification of DNA or RNA targets in plasma and serum samples. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene, was chosen at random as CNAPS to serve as an intrinsic internal control in two different real-time PCR based quantification models in plasma and serum. Viral loads of hepatitis B virus (HBV) DNA and hepatitis delta virus (HDV) RNA were quantified as actual targets in parallel to GAPDH as IC in a total of 519 serum or plasma samples including 21 healthy controls, 202 positive chronic hepatitis delta patients, 37 chronic hepatitis C patients, 168 chronic hepatitis B patients, 52 patients with hepatocellular carcinoma, and 39 patients with non-alcoholic steatohepatitis/non-alcoholic fatty liver disease. GAPDH levels did not show significant variance in different patient groups and yielded positive signals in all 519 patients with persistent cycle threshold (CT) values 27.85±1.57 (mean±standard deviation (SD)). Reproducibility of the GAPDH amplification in HDV RNA and HBV DNA quantifications was shown with a SD value of CT ranging from 0.42 to 2.14 (mean SD; 1.18) and 0.24 to 1.75 (mean SD; 1.03), respectively. In conclusion, the freely circulating nucleic acids can clearly be used as internal controls for real-time PCR based detection and quantification of any RNA and mainly DNA targets (pathogens) in serum or plasma and this simply excludes the compulsory external addition of any IC molecules into the reaction.
Asunto(s)
Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Ácidos Nucleicos/análisis , Plasma/química , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Estándares de Referencia , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis B Crónica/diagnóstico , Hepatitis B Crónica/virología , Hepatitis D/diagnóstico , Hepatitis D/virología , Virus de la Hepatitis Delta/aislamiento & purificación , Humanos , Masculino , Reproducibilidad de los Resultados , Carga ViralRESUMEN
BACKGROUND: Hepatitis delta virus (HDV) RNA viral load measurement is critical in diagnosis and monitoring the response to antiviral treatment. OBJECTIVES: Our aim is to design a real time PCR method for accurate quantitation of HDV RNA in clinical specimens using an armored RNA as external standard, and an intrinsic internal control. STUDY DESIGN: A plasmid bearing delta antigen region of genotype I HDV genome was used to develop an armored RNA. Serial dilutions of the armored HDV RNA standard with 10(12)copy/mL were used as standards for quantitation. A primer-probe set derived from HDAg region was used in one step EZ RT PCR kit chemistry which uses rTth enzyme allowing reverse transcription and polymerization in the same tube. The kit also uses the advantage of uracil-N-glycosylase (UNG) enzyme treatment to prevent PCR contamination. RESULTS: The established assay has a dynamic range of 10(2)-10(11)copy/mL with a PCR efficiency of 96.9%. Detection limit was 858±32copy/mL with 95% confidence interval. Intra- and inter-assay variabilities were low for high, medium and low levels of viremia. Incorporation of freely circulating GAPDH in serum into the assay as an intrinsic internal control prevented false negative results and failures in PCR amplifications due to inhibitors, inefficient extraction procedures or enzymatic reactions. CONCLUSION: In conclusion, this study defines a novel assay for sensitive and reliable quantification of HDV RNA using an armored HDV RNA as a standard and GAPDH in plasma or serum as an intrinsic internal control in a single tube.
Asunto(s)
Virus de la Hepatitis Delta/aislamiento & purificación , ARN Viral/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Carga Viral/métodos , Femenino , Virus de la Hepatitis Delta/genética , Humanos , Masculino , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Carga Viral/normasRESUMEN
BACKGROUND: Tenofovir (TDF) has similar antiviral efficacy in both treatment-naive and lamivudine-resistant chronic hepatitis B (CHB) patients. Data on TDF use in patients with adefovir (ADV) resistance is inconsistent. The aim of our study was to assess antiviral efficacy of TDF against nucleoside analogue-naive (NN) and ADV-resistant (ADV-R) CHB and suboptimal responders to ADV (ADV-S). METHODS: A database of 135 CHB patients treated with TDF was analysed. A total of 37 patients with incomplete data were excluded and analysis was performed in 98 (44 NN, 30 ADV-R and 24 ADV-S). Patients with primary ADV-R mutations had either A181T/V or N236T mutations or both. HBV DNA was measured at 3-month intervals until month 24. Primary outcome measures were comparison of the decline of HBV DNA between the three treatment groups. RESULTS: NN patients had higher baseline HBV DNA compared with ADV-R and ADV-S patients (6.08 log10 IU/ml versus 5.53 and 4.88, respectively; P=0.002). By exponential regression analysis, HBV DNA decline kinetics differed between the three groups. HBV DNA decline was faster in NN patients compared to ADV-R and ADV-S CHB patients (P=0.002 and P=0.004, respectively). Undetectable HBV DNA was achieved in 77.2%, 60% and 75% of NN, ADV-R and ADV-S CHB patients, respectively, at month 12 (P= not significant). CONCLUSIONS: HBV DNA decline is slower in ADV-experienced patients compared with treatment-naive patients. The clinical significance of this slow response may be important in patients with critical liver reserve and high viral load. Optimal combination treatment (TDF+ entecavir) could be considered in these patients.
Asunto(s)
Adenina/análogos & derivados , Antivirales/uso terapéutico , Hepatitis B Crónica/tratamiento farmacológico , Organofosfonatos/uso terapéutico , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Adenina/uso terapéutico , Adulto , Anciano , ADN Viral , Farmacorresistencia Viral , Femenino , Hepatitis B Crónica/diagnóstico , Hepatitis B Crónica/virología , Humanos , Masculino , Persona de Mediana Edad , Retratamiento , Estudios Retrospectivos , Tenofovir , Factores de Tiempo , Resultado del Tratamiento , Carga Viral , Adulto JovenRESUMEN
BACKGROUND: Clonal analysis of quasispecies of resistant HBV genomes in patients with entecavir (ETV) resistance receiving lamivudine (3TC) plus adefovir (ADV) rescue therapy has never been performed. METHODS: A sample of 10 patients with ETV resistance who were switched to 3TC+ADV treatment were analysed for changes in viral quasispecies. Serum samples at baseline, and at months 3 and 6 of 3TC+ADV treatment could be clonally analysed in 7 of 10 patients; 3-82 clones per sample (total 1,068 clones, mean 63) were sequenced. RESULTS: 3TC+ADV therapy led to a modest decline in HBV DNA. Almost all clones had L180M and M204V 3TC resistance mutations before and during combination therapy. All clones had ≥1 of the S202G, T184F, T184A, T184L, T184I and M250V ETV resistance mutations. The percentages of detected clones bearing 3TC (rtL180M and rtM204V) and ETV mutations did not change with rescue 3TC+ADV therapy. In 7 of 8 patients with detectable HBV DNA (median 5.17 log(10) copies/ml) after a median 24 months of ADV therapy, HBV DNA became undetectable with 3TC plus tenofovir after 6 months of treatment. CONCLUSIONS: In patients with ETV resistance tenofovir is effective. Clonal analysis data indicate no selection of specific HBV mutants during rescue 3TC+ADV.
Asunto(s)
Adenina/análogos & derivados , Antivirales/farmacología , Farmacorresistencia Viral/genética , Guanina/análogos & derivados , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Hepatitis B Crónica/tratamiento farmacológico , Organofosfonatos/uso terapéutico , Adenina/farmacología , Adenina/uso terapéutico , Adulto , Antivirales/uso terapéutico , Farmacorresistencia Viral/efectos de los fármacos , Quimioterapia Combinada , Femenino , Genoma Viral/efectos de los fármacos , Genoma Viral/genética , Guanina/farmacología , Guanina/uso terapéutico , Virus de la Hepatitis B/clasificación , Hepatitis B Crónica/virología , Humanos , Lamivudine/uso terapéutico , Masculino , Persona de Mediana Edad , Mutación , Organofosfonatos/farmacología , Tenofovir , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND/AIMS: The safety and efficacy of interferons in advanced delta hepatitis have not been explored. The aim of this subanalysis of a multicenter clinical trial was to compare the efficacy and safety of 48 weeks of pegylated interferon alpha-2a (180 µg weekly) with or without adefovir (10 mg daily) in patients with chronic delta hepatitis-induced advanced liver disease and in those with non-advanced liver disease. MATERIALS AND METHODS: Thirty-one patients with advanced and 27 patients with non-advanced liver disease were assessed. Patients were considered to have advanced liver disease when biopsy disclosed a fibrosis score of ≥4 according to Ishak or when imaging studies were indicative of cirrhosis. Virologic response, defined as achievement of undetectable hepatitis D virus RNA, was assessed at the end of treatment and end of 24 weeks of treatment-free follow-up. RESULTS: Patients with advanced disease had lower hepatitis D virus RNA levels and platelet counts (p=0.014 and p=0.0015, respectively). End of treatment and end of follow-up virologic responses in patients with advanced vs. non-advanced liver disease were similar (29% vs. 19% and 32% vs 23%). Proportion of adverse events did not differ between groups except that thrombocytopenia was noted more often in the advanced liver disease group. Further, four cases of clinically important adverse events including two cases of hepatic decompensation and one case of tuberculosis reactivation occurred in the advanced liver disease group. CONCLUSIONS: Pegylated interferon is as effective in patients with advanced liver disease due to chronic delta hepatitis as in patients with non-advanced liver disease, but patients should be monitored closely for clinically important side effects.
Asunto(s)
Hepatitis D Crónica/tratamiento farmacológico , Interferón-alfa/administración & dosificación , Cirrosis Hepática/tratamiento farmacológico , Hígado/patología , Polietilenglicoles/administración & dosificación , Adolescente , Adulto , Anciano , Antivirales/administración & dosificación , Biopsia , Relación Dosis-Respuesta a Droga , Hepatitis D Crónica/complicaciones , Hepatitis D Crónica/virología , Virus de la Hepatitis Delta/genética , Humanos , Cirrosis Hepática/etiología , Cirrosis Hepática/patología , Persona de Mediana Edad , ARN Viral/genética , Proteínas Recombinantes/administración & dosificación , Resultado del Tratamiento , Adulto JovenRESUMEN
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.