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Coulibaly TA, Koné A, Sissoko H, Goita A, Cissoko Y, Maiga M, Maiga AI, Kampo MI, Diakité M, Doumbia S, McFall S, Fofana DB. Quantification of Hepatitis B Virus DNA: Validation of a qPCR Approach to detect Pregnant Women at High Risk to Transmit the Virus in Mali. REVUE MALIENNE D'INFECTIOLOGIE ET DE MICROBIOLOGIE 2024; 19:50-55. [PMID: 39555246 PMCID: PMC11567684] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Subscribe] [Scholar Register] [Indexed: 11/19/2024]
Abstract
Introduction Mother-to-child transmission (MTCT) of hepatitis B virus (HBV) is one of the main causes of chronic hepatitis B in endemic regions such as West Africa. Its prevention constitutes an essential element to eliminate HBV. Without intervention, rates of vertical transmission of HBV vary depending on the level of viral replication. The management of this infection is a major concern, particularly the availability of the viral load at an affordable cost in countries with limited resources such as Mali. This study aimed to develop and validate a method for detecting and quantifying HBV DNA using qPCR in pregnant women, a population at risk of transmitting the virus to newborns. Methods We enrolled 74 pregnant women with positive AgHBs in this study. Their viral loads were previously determined at the Reference Centre Lab. We designed specific probes and primers for the HBV PreC gene, for detection and quantification by qPCR. We adapted this new qPCR for quantifying HBV DNA on different real-time PCR machines. Results Nine out of nine (9/9), 100% samples with a viral load (VL) between 10000-100000 IU/mL and 4/4,100% with a VL > 100,000 IU/mL were detected and quantified. Of the fifty-five (55) samples with a CV of 12-10000 IU/mL, 38/55, 69% samples with a CV > 1000 IU/mL were detected and 17/55, 30% samples between 12-1000 were not detected. No negative samples (6/6, 100%) were detected by our new qPCR. This in-house real-time PCR showed sensitivity and specificity of 75% and 100% respectively. Conclusion This work allowed the local development of a sensitive and efficient qPCR protocol for the detection of samples with CVs elevated (>1000 UI/mL). It will detect pregnant women who need to receive antiviral treatment in order to reduce the risk of HBV transmission. This tool could be extended to other high-risk populations such as immunocompromised people.
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Affiliation(s)
| | - A Koné
- University Clinical Research Center (UCRC)
- Université des sciences, des Techniques et des Technologies de Bamako (USTTB)
| | - H Sissoko
- Centre de santé de référence de la Commune III de Bamako (Csref C3)
| | - A Goita
- Centre de santé de référence de la Commune III de Bamako (Csref C3)
| | - Y Cissoko
- Université des sciences, des Techniques et des Technologies de Bamako (USTTB)
| | - M Maiga
- Institute for Global Health, Northwestern University, Chicago, IL 60208, USA
| | - A I Maiga
- University Clinical Research Center (UCRC)
| | - M I Kampo
- Université des sciences, des Techniques et des Technologies de Bamako (USTTB)
| | - M Diakité
- University Clinical Research Center (UCRC)
| | - S Doumbia
- University Clinical Research Center (UCRC)
| | - S McFall
- Institute for Global Health, Northwestern University, Chicago, IL 60208, USA
| | - D B Fofana
- University Clinical Research Center (UCRC)
- Université des sciences, des Techniques et des Technologies de Bamako (USTTB)
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Duarte G, Pezzuto P, Barros TD, Mosimann G, Martinez-Espinosa FE. Brazilian Protocol for Sexually Transmitted Infections 2020: viral hepatitis. Rev Soc Bras Med Trop 2021; 54:e2020834. [PMID: 33729415 PMCID: PMC8210490 DOI: 10.1590/0037-8682-834-2020] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2021] [Accepted: 03/10/2021] [Indexed: 12/09/2022] Open
Abstract
This article discusses viral hepatitis, a theme addressed by the Clinical Protocol and Therapeutic Guidelines to Comprehensive Care for People with Sexually Transmitted Infections and, more precisely, by the Clinical Protocols and Therapeutic Guidelines for Hepatitis B and Hepatitis C and Coinfections, published by the Brazilian Ministry of Health. Besides the broad spectrum of health impairment, hepatitis A, B, and C viruses also present different transmission forms, whether parenteral, sexual, vertical, or fecal-oral. Among the strategies suggested for the control of viral hepatitis, in addition to behavioral measures, are expanded diagnosis, early vaccination against hepatitis A and hepatitis B viruses, and access to available therapeutic resources. Considering vertical transmission of the hepatitis B and hepatitis C viruses, screening for pregnant women with chronic hepatitis B and C is an essential perinatal health strategy, indicating with precision those who can benefit from the prophylactic interventions. Viral hepatitis A, B, and C are responsible for more than 1.34 million deaths worldwide every year, from which 66% are the result of hepatitis B, 30% of hepatitis C, and 4% of hepatitis A.
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Affiliation(s)
- Geraldo Duarte
- Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto, SP, Brasil
| | - Paula Pezzuto
- Ministério da Saúde, Secretaria de Vigilância em Saúde, Brasília, DF, Brasil
| | - Tiago Dahrug Barros
- Ministério da Saúde, Secretaria de Vigilância em Saúde, Brasília, DF, Brasil
| | - Gláucio Mosimann
- Ministério da Saúde, Secretaria de Vigilância em Saúde, Brasília, DF, Brasil
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Duarte G, Pezzuto P, Barros TD, Mosimann Junior G, Martínez-Espinosa FE. [Brazilian Protocol for Sexually Transmitted Infections 2020: viral hepatitis]. ACTA ACUST UNITED AC 2021; 30:e2020834. [PMID: 33729415 DOI: 10.1590/s1679-4974202100016.esp1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2020] [Accepted: 10/03/2020] [Indexed: 01/01/2023]
Abstract
This article discusses viral hepatitis, a theme addressed by the Clinical Protocol and Therapeutic Guidelines to Comprehensive Care for People with Sexually Transmitted Infections and, more precisely, by the Clinical Protocols and Therapeutic Guidelines for Hepatitis B and Hepatitis C and Coinfections, published by the Brazilian Ministry of Health. Besides the broad spectrum of health impairment, hepatitis A, B and C viruses also present different forms of transmission, whether parenteral, sexual, vertical or oral. Among the strategies suggested for the control of viral hepatitis, in addition to behavioral measures, are expanded diagnosis, early vaccination against hepatitis A and hepatitis B viruses, and access to available therapeutic resources. Considering vertical transmission of the hepatitis B and hepatitis C viruses, screening for pregnant women with chronic hepatitis B and C is an important perinatal health strategy, indicating with precision those who can benefit from the prophylactic interventions.
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Affiliation(s)
- Geraldo Duarte
- Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto, SP, Brasil
| | - Paula Pezzuto
- Ministério da Saúde, Secretaria de Vigilância em Saúde, Brasília, DF, Brasil
| | - Tiago Dahrug Barros
- Ministério da Saúde, Secretaria de Vigilância em Saúde, Brasília, DF, Brasil
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Mulrooney-Cousins P, Michalak T. Molecular Testing in Hepatitis Virus Related Disease. DIAGNOSTIC MOLECULAR PATHOLOGY 2017:63-73. [DOI: 10.1016/b978-0-12-800886-7.00006-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
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Motahar M, Arabzadeh SA, Mollaei H, Iranmanesh Z, Nikpour N, Soleimani F. Evaluation of HBV resistance to tenofovir in patients with chronic hepatitis B using ZNA probe assay in Kerman, southeast of Iran. ASIAN PACIFIC JOURNAL OF TROPICAL DISEASE 2016. [DOI: 10.1016/s2222-1808(16)61079-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
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Morgnanesi D, Heinrichs EJ, Mele AR, Wilkinson S, Zhou S, Kulp JL. A computational chemistry perspective on the current status and future direction of hepatitis B antiviral drug discovery. Antiviral Res 2015; 123:204-15. [PMID: 26477294 DOI: 10.1016/j.antiviral.2015.10.014] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2015] [Revised: 10/02/2015] [Accepted: 10/11/2015] [Indexed: 12/11/2022]
Abstract
Computational chemical biology, applied to research on hepatitis B virus (HBV), has two major branches: bioinformatics (statistical models) and first-principle methods (molecular physics). While bioinformatics focuses on statistical tools and biological databases, molecular physics uses mathematics and chemical theory to study the interactions of biomolecules. Three computational techniques most commonly used in HBV research are homology modeling, molecular docking, and molecular dynamics. Homology modeling is a computational simulation to predict protein structure and has been used to construct conformers of the viral polymerase (reverse transcriptase domain and RNase H domain) and the HBV X protein. Molecular docking is used to predict the most likely orientation of a ligand when it is bound to a protein, as well as determining an energy score of the docked conformation. Molecular dynamics is a simulation that analyzes biomolecule motions and determines conformation and stability patterns. All of these modeling techniques have aided in the understanding of resistance mutations on HBV non-nucleos(t)ide reverse-transcriptase inhibitor binding. Finally, bioinformatics can be used to study the DNA and RNA protein sequences of viruses to both analyze drug resistance and to genotype the viral genomes. Overall, with these techniques, and others, computational chemical biology is becoming more and more necessary in hepatitis B research. This article forms part of a symposium in Antiviral Research on "An unfinished story: from the discovery of the Australia antigen to the development of new curative therapies for hepatitis B."
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Affiliation(s)
- Dante Morgnanesi
- Department of Chemistry, Baruch S. Blumberg Institute, Doylestown, PA 18902, USA
| | - Eric J Heinrichs
- Department of Chemistry, Baruch S. Blumberg Institute, Doylestown, PA 18902, USA
| | - Anthony R Mele
- Department of Chemistry, Baruch S. Blumberg Institute, Doylestown, PA 18902, USA
| | - Sean Wilkinson
- Department of Chemistry, Baruch S. Blumberg Institute, Doylestown, PA 18902, USA
| | - Suzanne Zhou
- Department of Chemistry, Baruch S. Blumberg Institute, Doylestown, PA 18902, USA
| | - John L Kulp
- Department of Chemistry, Baruch S. Blumberg Institute, Doylestown, PA 18902, USA.
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Zhang BG, Tanaka G, Aihara K, Honda M, Kaneko S, Chen L. Dynamics of an HBV Model with Drug Resistance Under Intermittent Antiviral Therapy. INTERNATIONAL JOURNAL OF BIFURCATION AND CHAOS 2015; 25:1540011. [DOI: 10.1142/s0218127415400118] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
Abstract
This paper studies the dynamics of the hepatitis B virus (HBV) model and the therapy regimens of HBV disease. First, we propose a new mathematical model of HBV with drug resistance, and then analyze its qualitative and dynamical properties. Combining the clinical data and theoretical analysis, we demonstrate that our model is biologically plausible and also computationally viable. Second, we demonstrate that the intermittent antiviral therapy regimen is one of the possible strategies to treat this kind of complex disease. There are two main advantages of this regimen, i.e. it not only may delay the development of drug resistance, but also may reduce the duration of on-treatment time compared with the long-term continuous medication. Moreover, such an intermittent antiviral therapy can reduce the adverse side effects. Our theoretical model and computational results provide qualitative insight into the progression of HBV, and also a possible new therapy for HBV disease.
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Affiliation(s)
- Ben-Gong Zhang
- School of Mathematics and Computer Science, Wuhan Textile University, Wuhan 430073, P. R. China
| | - Gouhei Tanaka
- Institute of Industrial Science, The University of Tokyo, 153-8505, Japan
| | - Kazuyuki Aihara
- Institute of Industrial Science, The University of Tokyo, 153-8505, Japan
| | - Masao Honda
- Department of Gastroenterology, Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa 920-8641, Japan
| | - Shuichi Kaneko
- Department of Gastroenterology, Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa 920-8641, Japan
| | - Luonan Chen
- Key Laboratory of Systems Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, P. R. China
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Makhlouf NA, Morsy KH, Othman EER, Eldin EN. Ante-natal screening of pregnant women for hepatitis B virus infection in Upper Egypt. EGYPTIAN LIVER JOURNAL 2014; 4:57-62. [DOI: 10.1097/01.elx.0000445723.55972.3a] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022] Open
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Zhang M, Ge G, Yang Y, Cai X, Fu Q, Cai J, Huang Z. Decreased antigenicity profiles of immune-escaped and drug-resistant hepatitis B surface antigen (HBsAg) double mutants. Virol J 2013; 10:292. [PMID: 24053482 PMCID: PMC3856468 DOI: 10.1186/1743-422x-10-292] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2013] [Accepted: 09/17/2013] [Indexed: 12/13/2022] Open
Abstract
BACKGROUND Selective pressure from either the immune response or the use of nucleoside analogs in antiviral therapy could be driving the emergence of HBV mutants. Because of the overlap of the open reading frame (ORF) S for the HBsAg and ORF P for viral polymerase, rtM204I and rtM204V mutations in the polymerase would produce sI195M and sW196S in the HBsAg. The combined effects of immune-escaped mutations (sT118M, sG145K, sG145R) and drug-resistant mutations (rtM204I, rtM204V) on the antigenicity profiles of HBsAg has not been widely explored. METHODS To determine the combined effects of immune-escaped and drug-resistant mutants on the antigenicity profiles of HBsAg, recombinant plasmids encoding HBsAg double mutants were constructed using site-directed mutagenesis. The supernatant from each plasmid transfection was analyzed for HBsAg in the western-blotting and five of the most commonly used commercial ELISA kits in China. RESULTS Western-blotting assay showed the successful expression of each HBsAg mutant. All five ELISA kits manifested similar avidity, which were demonstrated by the slope of the curves, for the sT118M mutant, and sT118M-rtM204I (sT118M-sI195M) and sT118M-rtM204V (sT118M-sW196S) double mutants, suggesting that drug-resistant YMDD mutants caused negligible losses in the antigenicity of immune-escaped sT118M HBsAg. In contrast, the presence of the rtM204I (sI195M) mutation, but not rtM204V (sW196S) in combination with the sG145K mutation significantly reduced the avidity of sG145K HBsAg. The rtM204I (sI195M) mutation also decreased the antigenicity profiles for sG145R HBsAg. CONCLUSIONS Drug-resistant mutations rtM204I (sI195M) and rtM204V (sW196S) caused significant reduction in antigenicity for the immune-escaped HBsAg mutants sG145K and sG145R, which may hamper HBV diagnosis and disease control from HBV blood-transfusion transmissions in China. The development of ELISA kits with a greater sensitivity for drug-resistant and immune-escaped HBsAg warrants further consideration.
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Affiliation(s)
- Mingshun Zhang
- Department of Infectious Disease, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
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Joshi H, Lewis K, Singharoy A, Ortoleva PJ. Epitope engineering and molecular metrics of immunogenicity: a computational approach to VLP-based vaccine design. Vaccine 2013; 31:4841-7. [PMID: 23933338 DOI: 10.1016/j.vaccine.2013.07.075] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2013] [Revised: 07/25/2013] [Accepted: 07/30/2013] [Indexed: 01/05/2023]
Abstract
Developing antiviral vaccines is increasingly challenging due to associated time and cost of production as well as emerging drug-resistant strains. A computer-aided vaccine design strategy is presented that could greatly accelerate the discovery process and yield vaccines with high immunogenicity and thermal stability. Our strategy is based on foreign viral epitopes engineered onto well-established virus-like particles (VLPs) and demonstrates that such constructs present similar affinity for antibodies as does a native virus. This binding affinity serves as one molecular metric of immunogenicity. As a demonstration, we engineered a preS1 epitope of hepatitis B virus (HBV) onto the EF loop of human papillomavirus VLP (HPV-VLP). HBV-associated HzKR127 antibody displayed binding affinity for this structure at distances and strengths similar to those for the complex of the antibody with the full HBV (PDBID: 2EH8). This antibody binding affinity assessment, along with other molecular immunogenicity metrics, could be a key component of a computer-aided vaccine design strategy.
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Affiliation(s)
- Harshad Joshi
- Center for Cell and Virus Theory, Department of Chemistry, Indiana University, Bloomington, IN 47405, USA
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Role of genotype G hepatitis B virus mixed infection on the progression of hepatic fibrosis in HIV positive patients over 5 years of follow-up. J Clin Virol 2013; 58:408-14. [PMID: 23958588 DOI: 10.1016/j.jcv.2013.07.018] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2013] [Revised: 07/08/2013] [Accepted: 07/26/2013] [Indexed: 12/19/2022]
Abstract
BACKGROUND Due to common routes of transmission, HIV and HBV are frequently found as concomitant infections. The dynamic of liver disease in co-infected patients is important to understand for appropriate clinical management. Conflicting data surround the role played by genotype-G HBV (HBV-G) during the course of HIV co-infection. OBJECTIVES This study aims to assess, using non-invasive methods, liver disease progression in HIV-HBV genotype-G co-infected patients. STUDY DESIGN Co-infected patients with residual HBV replication (n=125) were screened for HBV-G infection by specific real-time PCR. The impact of HBV-G on liver fibrosis progression, as assessed by a non invasive biomarker (Fibrotest), was evaluated first, by a cross sectional analysis comparing fibrosis between HBV-G (n=23) and non-G (n=55) infected patients and second, by a longitudinal study performed over a 5 year period. RESULTS Selected patients were mostly male (90%), with homogenous characteristics between the HBV-G and non-G infected groups, in terms of age, known duration of HIV disease, immune and virological status and duration of HIV/HBV treatment. HBV-G infected patients were exclusively from Western Europe with homosexual intercourses (83%) as principal risk of transmission. Cross sectional analysis revealed comparable liver disease severity distribution between HBV-G and non-G infected patients. Co-infection with other hepatitis viruses and low CD4-nadir, but not HBV-G co-infection, were associated with a 5-year risk of fibrosis progression. CONCLUSIONS This study suggests that HBV-G infection is not significantly associated with a more severe liver disease and does not have a deleterious impact on fibrosis progression in efficiently treated HIV-HBV co-infected patients.
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Fathi MS, EL-Folly RF, Hassan RA, El-Arab ME. Genotypic and phenotypic patterns of antimicrobial susceptibility of Helicobacter pylori strains among Egyptian patients. EGYPTIAN JOURNAL OF MEDICAL HUMAN GENETICS 2013. [DOI: 10.1016/j.ejmhg.2013.03.004] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
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Baxa DM, Thekdi AD, Golembieski A, Krishnan PV, Sharif O, Kizy A, Shetron-Rama L, Jovanovich J, Chappell BJ, Snow-Lampart A, Borroto-Esoda K, Gordon SC. Evaluation of anti-HBV drug resistant mutations among patients with acute symptomatic hepatitis B in the United States. J Hepatol 2013; 58:212-6. [PMID: 23022497 DOI: 10.1016/j.jhep.2012.09.014] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/08/2011] [Revised: 08/25/2012] [Accepted: 09/19/2012] [Indexed: 12/29/2022]
Abstract
BACKGROUND & AIMS Reported HBV drug resistance mutations among previously untreated patients with chronic hepatitis B are variable. Whether resistant HBV strains are transmitted in the acute setting is uncertain. We sought to document the presence of antiviral resistance (AVR) mutations in patients with acute HBV (AHB) infection. METHODS AHB infection was defined by HBsAg/IgM anti-HBc positivity, ALT>10X ULN and compatible clinical history. The TRUGENE HBV kit was used to perform genotyping and direct sequencing of the viral polymerase. INNO-LiPA HBV DRv2 and DRv3 were used to detect AVR mutations. Clonal sequencing was conducted on selected specimens. RESULTS Twenty-three patients were evaluated (mean age, 43 years; 54% male; 39% African American, 39% Caucasian, 13% Hispanic and 4% Asian). The mean peak ALT was 1554.2IU/L and mean peak total serum bilirubin was 12 mg/dl. The HBV DNA median viral load (N = 15) was 5.14 log(10)IU/ml. Nineteen patients were genotype A, and 1 each were genotype C, D, E and G. HBV drug resistance mutations were not detected by direct sequencing or INNO-LiPA. Clonal sequencing was conducted on 192 clones isolated from three patients and showed rtA181T, rtM250V and rtS202G mutations at an overall frequency of 1.54%, 1.39%, and 1.67% respectively. CONCLUSIONS We detected adefovir/lamivudine and entecavir relevant mutations in a minor population (<2%) of viral clones by clonal sequencing only. The clinical significance of these mutations is uncertain and may represent small populations of quasi-species vs. transmission of drug resistant strains.
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Affiliation(s)
- Dwayne M Baxa
- Infectious Disease, Henry Ford Hospital, Detroit, MI, USA.
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Teriaky A, Al-Judaibi B. Correlation between HBsAg quantitation and HBV DNA in HBeAg-negative HBV/D patients. Saudi J Gastroenterol 2013; 19:243-4. [PMID: 24195976 PMCID: PMC3958970 DOI: 10.4103/1319-3767.121030] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Affiliation(s)
- Anouar Teriaky
- Department of Medicine, Division of Gastroenterology and Hepatology, Western University and London Health Sciences Centre, London, Ontario, Canada
| | - Bandar Al-Judaibi
- Department of Medicine, Division of Gastroenterology and Hepatology, Western University and London Health Sciences Centre, London, Ontario, Canada,Department of Medicine, King Khalid University Hospital, King Saud University, Riyadh, Saudi Arabia. E-mail:
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Michailidis E, Kirby KA, Hachiya A, Yoo W, Hong SP, Kim SO, Folk WR, Sarafianos SG. Antiviral therapies: focus on hepatitis B reverse transcriptase. Int J Biochem Cell Biol 2012; 44:1060-71. [PMID: 22531713 DOI: 10.1016/j.biocel.2012.04.006] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2012] [Revised: 04/04/2012] [Accepted: 04/05/2012] [Indexed: 12/20/2022]
Abstract
Hepatitis B virus (HBV) is the etiologic agent of mankind's most serious liver disease. While the availability of a vaccine has reduced the number of new HBV infections, the vaccine does not benefit the approximately 350 million people already chronically infected by the virus. Most of the drugs approved by the FDA for the treatment of hepatitis B target the reverse transcriptase (RT or P gene product) and are nucleoside RT inhibitors (NRTIs) that suppress viral replication. However, prolonged monotherapies directed against a single target result in the emergence of viral resistance. HBV genotypic differences affect NRTI resistance, and because the reading frames of the S (surface antigen) and P genes partially overlap, genomic differences that affect the surface of the virus may also alter the viral polymerase sequence, function and drug susceptibility. The scope of this review is to assess the effects of HBV genotypic variation on the development of drug resistance to NRTIs. Some RT residues that vary among different genotypes are in the vicinity of residues that mutate and give rise to NRTI resistance. Interactions between these amino acids can help explain the effect of HBV genotype on the development of NRTI resistance during antiviral therapies, and might help in the design of improved therapeutic strategies.
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Affiliation(s)
- Eleftherios Michailidis
- Christopher S. Bond Life Sciences Center, Department of Molecular Microbiology & Immunology, University of Missouri, Columbia, MO 65211, USA
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Sayed SK, Kobeisy MA. The relationship between core promoter mutation of hepatitis B virus, viral load and hepatitis B e antigen status in chronic hepatitis B patients. Cell Immunol 2012; 276:35-41. [PMID: 22551558 DOI: 10.1016/j.cellimm.2012.03.003] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2012] [Revised: 03/17/2012] [Accepted: 03/20/2012] [Indexed: 02/06/2023]
Abstract
The aim of this study is to detect the possible association of hepatitis B virus (HBV) core mutation, hepatitis B e antigen (HBeAg) status and the viral load in chronic hepatitis B (CHB) patients. Sixty-six patients with CHB were enrolled. Hepatitis markers and hepatitis C virus antibody (HCV-Ab) were tested using micro particle enzyme immunoassay kits. Viral load was measured by real-time polymerase chain reaction (PCR) and the mutation was analyzed by nested PCR followed by restriction fragment length polymorphism. Most of CHB patients were HBeAg (-ve). The HBeAg status did not have an influence on the presence or absence of T1762/A1764 mutation. HBV-DNA serum level was not significantly different in patients with core mutation and patients without core mutation in HBeAg (-ve) group, while in HBeAg (+ve) group HBV-DNA serum level was significantly higher in patients with core mutation. This study reports the predominance of HBeAg (-ve) and HBV core promoter mutation.
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Affiliation(s)
- Sohair K Sayed
- Department of Clinical Pathology, Assiut University School of Medicine, Egypt.
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Seto WK, Lai CL, Fung J, Wong DKH, Yuen JCH, Hung IFN, Yuen MF. Significance of HBV DNA levels at 12 weeks of telbivudine treatment and the 3 years treatment outcome. J Hepatol 2011; 55:522-528. [PMID: 21147187 DOI: 10.1016/j.jhep.2010.11.018] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/12/2010] [Revised: 11/25/2010] [Accepted: 11/30/2010] [Indexed: 02/07/2023]
Abstract
BACKGROUND & AIMS The significance of early HBV DNA suppression during telbivudine treatment in predicting long-term outcomes needs further investigation. METHODS We determined the cumulative rates of HBeAg seroconversion, ALT normalization, HBV DNA suppression (<12IU/ml) and telbivudine resistant mutations (using the highly sensitive line probe assay) for 117 treatment-native chronic hepatitis B (CHB) patients (61.5% HBeAg-positive) on telbivudine for 3years. The significance of serum HBV DNA at week 12 and 24 was compared. RESULTS The median age and duration of follow-up were 39years and 24.2months, respectively. 117, 105, 69, and 43 patients had been followed up for at least 6months and 1, 2, and 3years, respectively. The cumulative rates of HBeAg seroconversion, ALT normalization, HBV DNA undetectability were 46.8%, 80.5%, and 51.2%, respectively, at 3years. There was an incremental increase in virologic breakthroughs to 39.5% by year 3. The cumulative rate of telbivudine resistant mutations was 4.8%, 17.6%, and 34.0% for year 1, 2, and 3, respectively. Week 12 HBV DNA of <200IU/ml was predictive of a higher chance of HBV DNA undetectability (p=0.022) and lower chance of resistance (p=0.001) by year 3. Undetectable HBV DNA at week 24 was predictive of viral suppression at year 2 (p<0.001) but not at year 3 (p=0.241). CONCLUSIONS Continuous telbivudine resulted in improved biochemical and virologic outcomes, although there was an incremental increase in cumulative rate of resistance up to year 3. Week 12 HBV DNA of <200IU/ml was predictive of favorable long-term outcomes.
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Affiliation(s)
- Wai-Kay Seto
- Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong
| | - Ching-Lung Lai
- Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong
| | - James Fung
- Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong
| | - Danny Ka-Ho Wong
- Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong
| | - John Chi-Hang Yuen
- Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong
| | - Ivan Fan-Ngai Hung
- Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong
| | - Man-Fung Yuen
- Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong.
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18
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Abstract
Chronic hepatitis B (CHB) is a major public health problem affecting up to 400 million people globally. Complications of CHB including liver failure and hepatocellular carcinoma result in 1.2 million deaths per year, making CHB the 10th leading cause of mortality worldwide. The natural history of CHB is variable and complex. The past decade witnessed important developments for the therapy of hepatitis B and marked the new era of oral therapy. The ultimate goal of CHB therapy is to arrest the progression of liver injury and to prevent the development of liver failure and hepatocellular carcinoma. Currently, six agents are approved for the treatment of CHB. Each of these agents, given as monotherapy, has been shown to produce virological, biochemical, and histological benefits for both HBeAg positive and negative CHB. There are, however, limitations in spite of their efficacy. The significant side-effect profile of interferon, for example, limits its long-term use. The approved oral agents are tolerable with prolonged use but drug resistance could limit long-term monotherapy. To date, combination therapy with nucleoside analogue and pegylated interferon or two nucleos(t)ide analogues given for one year does not show superiority in durability of response compared to monotherapy. Ongoing research effort is critical to identify the ideal hepatitis B therapy that is safe, effective, and produces durable response with a finite course of therapy. It is equally important to conduct a well designed, prospective natural history study to identify predictors of disease progression. This will accurately guide treatment strategy for this important disease.
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Affiliation(s)
- Daryl T-Y Lau
- Associate Professor of Medicine, Harvard Medical School (HMS), Director of Translational Liver Research, Beth Israel Deaconess Medical Center, HMS Liver Center, Division of Gastroenterology, Department of Medicine, 110 Francis Street, Suite 4A, Boston, MA 02215.
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Seto WK, Yuen MF, Fung J, Lai CL. Tenofovir disoproxil fumarate for the treatment of chronic hepatitis B monoinfection. Hepatol Int 2011; 7:327-34. [PMID: 21688182 DOI: 10.1007/s12072-011-9282-y] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/15/2011] [Accepted: 05/30/2011] [Indexed: 02/06/2023]
Abstract
INTRODUCTION Resistance in nucleoside/nucleotide analog (NA) therapy has always been a challenge in the management of chronic hepatitis B (CHB). CLINICAL STUDIES Initially developed for the treatment of HIV infection, early in vitro and clinical observational studies had shown tenofovir disoproxil fumarate (TDF) to be also active against CHB. Recent data from various multicenter phase 3 and 4 clinical trials have confirmed TDF being able to achieve a high viral suppression in both NA-naive and -experienced CHB patients. There are also emerging data on the efficacy of TDF in decompensated CHB. Although there are in vitro studies identifying certain mutation loci associated with a reduced susceptibility to TDF, there have so far been no reports of virologic resistance to TDF in clinical studies. TDF has a favorable safety profile, although more long-term data would be needed. CONCLUSIONS TDF has the makings of an "ideal" first-line drug for the treatment of CHB.
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Affiliation(s)
- Wai-Kay Seto
- Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, Hong Kong
| | - Man-Fung Yuen
- Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, Hong Kong
| | - James Fung
- Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, Hong Kong
| | - Ching-Lung Lai
- Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, Hong Kong.
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20
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Abstract
Hepatitis B is a DNA virus affecting hundreds of millions of individuals worldwide. As the clinical sequelae of cirrhosis and hepatocellular cancer are increasingly recognized to be related to viral levels, the impetus increases to offer treatment to those previously not treated. With the development of more robust antivirals with reasonable safety profiles, long-term treatment is becoming more common. The oral nucleos(t)ide analogs have become the preferred first-line therapies for most genotypes of hepatitis B. Five are now available, all with different potencies and resistance profiles. Long-term data spanning several years are now available for most compounds in this arena. This article focuses on the common natural variants and those secondary to nucleos(t)ide therapy, as well as diagnostic methods to detect resistance.
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Affiliation(s)
- Fred Poordad
- David Geffen School of Medicine at UCLA, Hepatology and Liver Transplantation, Cedars-Sinai Medical Center, Suite 1060, Los Angeles, CA 90048, USA.
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21
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Zaky S, Farghaly AM, Rashed HA, Hassan H, Faouzy E, Makhlouf N, Hussein MRA. Clinicopathologic features and genotyping of patients with chronic HBV infection in the Upper Egypt. Cell Immunol 2010; 265:97-104. [PMID: 20719306 DOI: 10.1016/j.cellimm.2010.07.008] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2010] [Revised: 07/17/2010] [Accepted: 07/24/2010] [Indexed: 02/06/2023]
Abstract
The aim of this study was to determine the clinicopathologic features and Hepatitis B virus genotypes in HBV-infected patients in the Upper Egypt. Eighty-three HBsAg-positive patients (28 carriers, 14 with chronic hepatitis, 32 with liver cirrhosis and 9 with hepatocellular carcinoma) were enrolled. Blood was collected and serum samples obtained were screened for Hepatitis markers genotyping was conducted for 6 HBV genotypes (A through F) using a method for genotyping HBV by primer specific polymerase chain reaction. Genotype D was the only genotype detected in different clinical forms of chronic HBV infection (carriers, chronic hepatitis, cirrhosis and hepatocellular carcinoma) and, in all patients who had elevated or normal alanine aminotransferase levels and in all ages. HBeAg was absent in 78 patients suggesting the presence of pre-core or core mutations. Positive correlation was found among serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), histological activity index and grade of hepatitis. This study provides the first indication about the clinicopathologic features of HBV-infected patients in the Upper Egypt. It also reports the predominance of genotype D in this region.
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Affiliation(s)
- Saad Zaky
- Department of Tropical Medicine and Gastroenterology, Assiut University School of Medicine, Egypt
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22
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Validation of the INNO-LiPA HBV DR assay (version 2) in monitoring hepatitis B virus-infected patients receiving nucleoside analog treatment. Antimicrob Agents Chemother 2010; 54:1283-9. [PMID: 20065049 DOI: 10.1128/aac.00970-09] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Hepatitis B virus (HBV) antiviral drug resistance mutations prevent successful outcome of treatment and lead to worsening of liver disease. Detection of its emergence permits opportune treatment with alternative drugs. Unfortunately, the use of newly approved antivirals, including adefovir dipivoxil, emtricitabine, and telbivudine, is also associated with the development of drug resistance, albeit to a lesser extent than the use of lamivudine. The objectives of this work were to assess the performance characteristics (sensitivity and accuracy) of an updated drug resistance test, the INNO-LiPA HBV DR v2, which includes detection of mutations associated with lamivudine, adefovir, emtricitabine, and telbivudine resistance, and to compare the results with consensus sequencing of serum samples from patients treated with HBV antivirals. Diagnostic sensitivity, defined as detection of a positive amplification line on the line probe assay (LiPA) strip, was 94.8% (95% confidence interval [CI], 89.7 to 97.9) after initial testing, increasing to 96.3% (95% CI, 91.6 to 98.8) after repeat test 1 and to 100% (95% CI, 97.3 to 100.0) after repeat test 2. In diagnostic accuracy determinations, full concordance was observed between sequencing and LiPA for 77.0% of the codons tested (620/805 codons [95% CI, 74.0 to 79.9]), whereas LiPA and sequencing were partially concordant 22% of the time (177/805 codons). In 167 out of 177 cases, LiPA detected a wild-type/mutant mixture whereas sequencing detected only one of the two results. Performance testing of the new LiPA test, the INNO-LiPA HBV DR v2, showed convincing diagnostic sensitivity and accuracy. The ability of the test to detect mixed infections and minority viral populations associated with resistance to the current generation of antivirals, including adefovir, emtricitabine, and telbivudine, makes it a useful tool for HBV therapy monitoring.
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23
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Sayan M, Akhan SC, Bozdayi M. Genotype A2/adw2 Strain of Hepatitis B Virus in Turkey. HEPATITIS MONTHLY 2010; 10:302-5. [PMID: 22312398 PMCID: PMC3271325] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/12/2010] [Revised: 02/15/2010] [Accepted: 08/27/2010] [Indexed: 12/11/2022]
Abstract
BACKGROUND AND AIMS Previous studies have demonstrated the dominance of genotype D subtype ayw in patients with hepatitis B virus infection in Turkey. The aim of the present study is to report, for the first time, genotype A2 subtype adw2 of hepatitis B virus in a patient who is an inactive hepatitis B carrier in Turkey. MATERIALS AND METHODS Hepatitis B virus DNA isolated from the serum sample was amplified by polymerase chain reaction. The polymerase gene segment of the hepatitis B virus was directly sequenced. A distance matrix/UPGMA comparison was used for phylogenetic analysis, and the genotype of the virus was identified accordingly. The subgenotype and subtype of hepatitis B virus were also detected. RESULTS The genotyping of the patient revealed that the isolated hepatitis B virus was genotype A2/adw2. DISCUSSION The subtype is inconsistent with the previous data from Turkey; specifically, the identification of the A2/adw2 subtype of the hepatitis B virus in an inactive carrier is the first such case in Turkey. This finding suggests that the transmission of another genotype besides genotype D subtype ayw of the hepatitis B virus is possible in Turkey.
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Affiliation(s)
- Murat Sayan
- Clinical Laboratory, PCR Unit, University of Kocaeli, Faculty of Medicine, Kocaeli, Turkey,Corresponding author at: Murat Sayan, University of Kocaeli, Faculty of Medicine, Clinical Laboratory, PCR Unit, Umuttepe Campus, İzmit, Kocaeli, Turkey. Tel.: +262-3038571, Fax: +262-3038085, E-mail:
| | - Sila Cetin Akhan
- Department of Clinical Bacteriology and Infectious Diseases, University of Kocaeli, Faculty of Medicine, Kocaeli, Turkey
| | - Mitha Bozdayi
- Institute of Hepatology, University of Ankara, Ankara, Turkey,Gastroenterology Department, Faculty of Medicine, University of Ankara, Ankara, Turkey
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24
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Sayan M. Molecular diagnosis of entecavir resistance. HEPATITIS MONTHLY 2010; 10:42-7. [PMID: 22308125 PMCID: PMC3270344] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/21/2009] [Revised: 01/11/2010] [Accepted: 01/14/2010] [Indexed: 12/11/2022]
Abstract
Entecavir (ETV) is a potent nucleoside analogue against hepatitis B virus (HBV). Because of development of ETV resistance requires at least three amino acid substitutions in HBV polymerase (pol) gene, emergence of ETV resistance is rare (~1%) in nucleoside-naive patients after up to 5 years of treatment. However, it has been suggested that lamivudine (LAM) therapy can preselect for HBV variants associated with resistance to ETV treatment. ETV resistance increased to 51% of patients after 5 years of ETV treatment in LAM refractory patients. The diagnosis of ETV resistance in chronic hepatitis B patients, mainly based on four types of molecular assays: direct sequencing, line probe assay, clonal analysis,and restriction fragment length polymorphism (RFLP) analysis. The applications of other assays are currently more specialized,and their use is more limited. The utility of these assays and their performance characteristics are reviewed below.Briefly, the monitoring of drug-resistant variants is important in the elucidation of the prevalence and mechanisms of resistance development and for the more effective management of treatment options.
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Affiliation(s)
- Murat Sayan
- Faculty of Medicine, University of Kocaeli, Kocaeli, Turkey,Corresponding author at: Murat Sayan, Clinical Laboratory, Faculty of Medicine, Umuttepe Kampus,University of Kocaeli, 41340, İzmit, Kocaeli, Turkey. Tel.: +90 262 303 8571, Fax: +90 262 303 8085, E-mail:
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Ahmed CS, Wang ZH, Bin Z, Chen JJ, Kamal M, Hou JL. Hepatitis B virus genotypes, subgenotypes, precore, and basal core promoter mutations in the two largest provinces of Pakistan. J Gastroenterol Hepatol 2009; 24:569-573. [PMID: 19368634 DOI: 10.1111/j.1440-1746.2008.05642.x] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
BACKGROUND AND AIM Hepatitis B virus (HBV) genotyping has been done in most countries, but unfortunately, in Pakistan, HBV genotypic distribution is still unclear. The aim of the present study was to determine the prevalent genotype and subgenotype in the two most populated provinces in Pakistan: Punjab and Sind. METHODS In total, 236 HBV DNA-positive samples were selected for genotyping by polymerase chain reaction-restriction fragment length polymorphism (RFLP). The RFLP results were further confirmed with whole genome and partial genome sequencing. RESULTS Genotype D was detected as the most prevalent (93.22%) genotype in all eight cities of both provinces; genotype C was present in 5.93% and genotype A was present in 0.85% of the samples. The D1 subtype was present in 84%, and D2 was present in 8% of 25 whole genome-sequenced samples. The C2 subtype was detected in 58.33% of S gene-sequenced samples, while D1 was detected in the remaining 41.67% of 24 samples sequenced for the S gene. Subtype D1 is the most dominant in D, while C2 is dominant in genotype C. Eight- and 15-bp deletion mutations were also detected in genotype D samples. Other precore and basal core promoter (BCP) mutations included T1915 (100%), A1679 (86.96%), T1762 (39.13%), and A1764 (30.43%), which were also detected in the genotype D samples. CONCLUSION Genotype D subtype D1 is the most prevalent HBV strain in Pakistan with 8-bp deletion mutants the most common in HBV carriers.
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Affiliation(s)
- Choudhary Shoaib Ahmed
- Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
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26
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Use of massively parallel ultradeep pyrosequencing to characterize the genetic diversity of hepatitis B virus in drug-resistant and drug-naive patients and to detect minor variants in reverse transcriptase and hepatitis B S antigen. J Virol 2008; 83:1718-26. [PMID: 19073746 DOI: 10.1128/jvi.02011-08] [Citation(s) in RCA: 126] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Direct population sequencing and reverse hybridization (line probe assay [LiPA])-based methods are the most common methods for detecting hepatitis B virus (HBV) drug resistance mutations, although only mutations present in viral quasispecies with a prevalence of > or =20% can be detected by sequencing, and only known mutations are detected by LiPA. Massively parallel ultradeep pyrosequencing (UDPS; GS FLX platform) was used to analyze HBV quasispecies in reverse transcriptase (RT) and hepatitis B S antigen (HBsAg) from five drug-naive patients and eight drug-resistant patients. Eight primer pairs were used to obtain partially overlapping amplicons, covering the RT gene from codons 1 to 288 and the complete overlapping HBsAg sequence. A 1% mutation frequency was selected as the cutoff based on an error rate estimated on plasmid DNA. This technology enabled simultaneous analysis of between 2,852 and 18,016 clonally amplified fragments from each patient. The results indicate that UDPS has a relative sensitivity much higher than both direct sequencing and LiPA. In addition, the UDPS results are quantitative, allowing establishment of the relative frequency of both known mutations and novel substitutions. Some of the detected RT substitutions led to changes also in HBsAg. On the whole, genotype D presented a higher heterogeneity than genotype A. Considering the high quantity of information that can be provided by a single test from one patient, the short turnaround time, the information on substitution frequency, and the detection of rare variants, there are strong advantages conferred by UDPS, and the new method could play a relevant role in the clinical management of HBV infection and therapy.
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27
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Kim JK, Lee HJ, Lee YJ, Chun JY, Lee IK, Lim YS, Suh DJ, Ko SY, Kim MH, Oh HB. Direct detection of lamivudine-resistant hepatitis B virus mutants by a multiplex PCR using dual-priming oligonucleotide primers. J Virol Methods 2008; 149:76-84. [PMID: 18291537 DOI: 10.1016/j.jviromet.2008.01.003] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2007] [Revised: 01/04/2008] [Accepted: 01/10/2008] [Indexed: 01/18/2023]
Abstract
Mutations in the YMDD motif of the hepatitis B virus (HBV) polymerase gene increase lamivudine resistance of HBV, highlighting the clinical importance of accurate and sensitive detection of HBV mutants. Using dual-priming oligonucleotide primer technology, an assay that can detect mutations at codons 180 (L528M) and 204 (YVDD, YIDD, and YSDD) by a single-step multiplex PCR was developed. This Seeplex Lami-DR assay was sufficiently sensitive to detect 10(3)HBV/ml and was able to detect minor mutants comprising as little as 2% of the viral population. Mutants were detected in 57 of 65 serum samples (88%) from patients with chronic hepatitis B who had been treated with lamivudine (median, 32 months; range, 1-83 months). The agreement with direct sequencing was only 38.5% (25/65). Discrepancies between these methods resulted from detection of additional mutants by the Seeplex Lami-DR assay, as confirmed by a novel verification analysis. This assay is not only highly accurate and sensitive, but is also simple and cost-effective, requiring no expensive probes, laborious sequencing procedures, or digestion with restriction enzymes. Accordingly, the Seeplex HBV Lami-DR assay should be considered as a first-line, cost-effective tool for detecting viral mutations in patients with chronic hepatitis B receiving lamivudine therapy.
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Affiliation(s)
- Jong-Kee Kim
- Seegene Institute of Life Science, 65-5 Bangyi-Dong, Songpa-Gu, Seoul, Republic of Korea
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Wang XL, Xie SG, Zhang L, Yang WX, Wang X, Jin HZ. Comparison of ligase detection reaction and real-time PCR for detection of low abundant YMDD mutants in patients with chronic hepatitis B. World J Gastroenterol 2008; 14:120-4. [PMID: 18176973 PMCID: PMC2673375 DOI: 10.3748/wjg.14.120] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To compare the ligase detection reaction (LDR) and real-time PCR for detection of low abundant YMDD mutants in patients with chronic hepatitis B infection.
METHODS: Mixtures of plasmids and serum samples from 52 chronic hepatitis B patients with low abundant lamivudine-resistant mutations were tested with LDR and real-time PCR. Time required and reagent cost for both assays were evaluated.
RESULTS: Real-time PCR detected 100, 50, 10, 1 and 0.1% of YIDD plasmid, whereas LDR detected 100, 50, 10, 1, 0.1, and 0.01% of YIDD plasmid, in mixtures with YMDD plasmid of 106 copies/mL. Among the 52 clinical serum samples, completely concordant results were obtained for all samples by both assays, and 39 YIDD, 9 YVDD, and 4 YIDD/YVDD were detected. Cost and time required for LDR and real-time PCR are 60/80 CNY (8/10.7 US dollars) and 4.5/2.5 h, respectively.
CONCLUSION: LDR and real-time PCR are both sensitive and inexpensive methods for monitoring low abundant YMDD mutants during lamivudine therapy in patients with chronic hepatitis B. LDR is more sensitive and less expensive, while real-time PCR is more rapid.
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29
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Shih YH, Yeh SH, Chen PJ, Chou WP, Wang HY, Liu CJ, Lu SF, Chen DS. Hepatitis B virus quantification and detection of YMDD mutants in a single reaction by real-time PCR and annealing curve analysis. Antivir Ther 2008; 13:469-480. [PMID: 18672526 DOI: 10.1177/135965350801300403] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
Abstract
BACKGROUND Long-term antiviral therapy, although effective for treatment of hepatitis B, might select the emergence of drug-resistant hepatitis B virus (HBV) mutants. Detection of HBV mutants and determination of viral titre are two crucial parameters for monitoring treatment response and occurrence of mutants. In this study, we take lamivudine resistance as an example to develop a method that can determine both parameters in a single-tube PCR reaction. METHODS The method contained two consecutive steps: in the first step, real-time PCR was used for quantification; in the second step, a novel annealing curve analysis was used for detecting YMDD mutants. For accurate quantification, PCR primers and hybridization probes were chosen from highly conserved regions to ensure the equivalent amplification of all HBV genotypes. Within the sensor probe, there were signature nucleotide polymorphisms that could effectively differentiate YMDD mutants from wild type by distinct melting temperatures (Tm) values. The clinical applicability of the assay was tested in serial samples from 90 patients receiving lamivudine treatment. RESULTS This assay could readily differentiate YMDD, YIDD and YVDD mutants by their distinct Tm values. The quantification results showed great consistency in a linear range from 10(3) to 10(11) copies/ml. Moreover, this assay could detect YMDD mutants accounting for < or = 10% of the total viral population. Its clinical feasibility has been verified in primary specimens. CONCLUSIONS The newly designed YMDD detection method is simple, sensitive, cost-effective, time-saving and provides a useful tool for follow-up of patients treated with lamivudine or other antiviral drugs.
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Affiliation(s)
- Yi-Hsien Shih
- NTU Center for Genomic Medicine, National Taiwan University College of Medicine, Taipei, Taiwan
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30
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França PHC, Coelho HSM, Brandão CE, Segadas JA, Quintaes RF, Carrilho FJ, Ono-Nita S, Mattos AA, Tovo C, Gouvea VS, Sablon E, Vanderborght BOM. The emergence of YMDD mutants precedes biochemical flare by 19 weeks in lamivudine-treated chronic hepatitis B patients: an opportunity for therapy reevaluation. Braz J Med Biol Res 2007; 40:1605-1614. [PMID: 17713642 DOI: 10.1590/s0100-879x2006005000169] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2007] [Accepted: 06/04/2007] [Indexed: 01/04/2023] Open
Abstract
Given the loss of therapeutic efficacy associated with the development of resistance to lamivudine (LMV) and the availability of new alternative treatments for chronic hepatitis B patients, early detection of viral genotypic resistance could allow the clinician to consider therapy modification before viral breakthrough and biochemical relapse occur. To this end, 28 LMV-treated patients (44 +/- 12 years; 24 men), on their first therapy schedule, were monitored monthly at four Brazilian centers for the emergence of drug resistance using the reverse hybridization-based INNO-LiPA HBV DR assay and occasionally sequencing (two cases). Positive viral responses (HBV DNA clearance) after 6, 12, and 18 months of therapy were achieved by 57, 68, and 53% of patients, while biochemical responses (serum alanine aminotransferase normalization) were observed in 82, 82, and 53% of cases. All viral breakthrough cases (N = 8) were related to the emergence of YMDD variants observed in 7, 21, and 35% of patients at 6, 12, and 18 months, respectively. The emergence of these variants was not associated with viral genotype, HBeAg expression status, or pretreatment serum alanine aminotransferase levels. The detection of resistance-associated mutations was observed before the corresponding biochemical flare (41 +/- 14 and 60 +/- 15 weeks) in the same individuals. Then, if highly sensitive LMV drug resistance testing is carried out at frequent and regular intervals, the relatively long period (19 +/- 2 weeks) between the emergence of viral resistance and the onset of biochemical relapse can provide clinicians with ample time to re-evaluate drug therapy.
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Affiliation(s)
- P H C França
- Departamento de Virologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.
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Cui J, Kang X, Dai Z, Huang C, Zhou H, Guo K, Li Y, Zhang Y, Sun R, Chen J, Li Y, Tang Z, Uemura T, Liu Y. Prediction of chronic hepatitis B, liver cirrhosis and hepatocellular carcinoma by SELDI-based serum decision tree classification. J Cancer Res Clin Oncol 2007; 133:825-834. [PMID: 17516088 DOI: 10.1007/s00432-007-0224-y] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2007] [Accepted: 04/12/2007] [Indexed: 01/17/2023]
Abstract
PURPOSE To screen potential serological biomarkers and develop decision tree classifications of chronic hepatitis B, liver cirrhosis (LC) and hepatocellular carcinoma (HCC), respectively, with high prediction score for improving diagnosis of liver diseases. METHODS The total serum samples were randomly divided into three training sets (41 HBV and 35 health; 36 LC and 35 health; 39 HCC and 35 health) and three testing groups (34 HBV and 38 health; 18 LC and 52 health; 42 HCC and 47 health). Selected WCX2 protein chip capture followed by SELDI-TOF-MS analysis was applied to generate the serum protein profiles. Subsequently serum protein spectra were normalized and aligned by Ciphergen SELDI Software 3.1.1 with Biomarker Wizard including baseline subtraction, mass accuracy calibration, automatic peak detection. Once the intensities of selected significant peaks from the training data set were transferred to further BPS analysis, an optimized classification tree with sequence-decision was established to divide training data set into disease group and control group successfully. A double blind test was employed to determine the clinical sensitivity and clinical specificity of three models. RESULTS After comparative analysis of SELDI based serum protein profile between the cases of disease and healthy, a HCC decision tree classification with sensitivity of 94.872% and specificity of 94.286%; a LC decision tree classification with sensitivity of 91.667% and specificity of 94.286% and a HBV decision tree classification with sensitivity of 95.122% and specificity of 94.286% were produced by BPS respectively. When three decision tree models were challenged by the double-blind test samples, clinical sensitivity and clinical specificity of these models were predicted in diagnosis of three liver diseases (HCC: 90.48 and 89.36%; cirrhosis: 100 and 86.5%; HBV: 85.29 and 84.21%). CONCLUSION SELDI-based decision tree classifications showed great advantages over conventional serological biomarkers in the diagnosis of chronic hepatitis B, LC as well as HCC.
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Affiliation(s)
- Jiefeng Cui
- Liver Cancer Institute, Zhongshan Hospital, Fudan University, 136 Yi Xue Yuan Road, Shanghai, 200032, China
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Khokhar U, Stevens D, Shipton LK, Lau DTY. Review. Gastroenterol Hepatol (N Y) 2007; 3:727-730. [PMID: 21960886 PMCID: PMC3104264] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/31/2023]
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Yang ZJ, Tu MZ, Liu J, Wang XL, Jin HZ. Comparison of amplicon-sequencing, pyrosequencing and real-time PCR for detection of YMDD mutants in patients with chronic hepatitis B. World J Gastroenterol 2006; 12:7192-6. [PMID: 17131486 PMCID: PMC4087785 DOI: 10.3748/wjg.v12.i44.7192] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To compare the sequencing of PCR products, pyrosequencing, and real-time PCR for detection of Tyrosine-methionine-aspartate-aspartate (YMDD) mutants in patients with chronic hepatitis B.
METHODS: Mixtures of plasmids and serum samples from 69 chronic hepatitis B patients treated with lamivudine were tested for YMDD mutations by sequencing of PCR products, pyrosequencing, and real-time PCR, respectively. Time required and reagent costs of the three assays were evaluated.
RESULTS: Real-time PCR detected 100%, 50%, 10%, 1% and 0.1% of YVDD plasmid in mixtures with 106 copies/mL of YMDD plasmid, whereas sequencing and pyrosequencing only detected 100% and 50% of YVDD plasmid in aliquots of the corresponding mixtures. Completely concordant results were obtained from 60 (87%) out of the 69 clinical serum samples by the three assays. Mutants were detected by real-time PCR in less than 20% of the total virus population, but no mutant was detected by sequencing and pyrosequencing. In addition, real-time PCR required less time and was more cost-effective than the other two assays. However, throughput of pyrosequencing was the highest.
CONCLUSION: Among the three assays compared, real-time PCR is the most sensitive, cost-effective, and time saving for monitoring YMDD mutants in patients with chronic hepatitis B on lamivudine therapy.
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Affiliation(s)
- Zhi-Jun Yang
- Management School, Shanghai Jiaotong University, No. 1289 Yishan Road, Shanghai 200233, China.
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Hosseini SY, Sabahi F, Amini-Bavil-Olyaee S, Alavian SM, Merat S. A novel accurate ACRS-PCR method with a digestion internal control for identification of wild type and YMDD mutants of hepatitis B virus strains. J Virol Methods 2006; 137:298-303. [PMID: 16962669 DOI: 10.1016/j.jviromet.2006.07.008] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2006] [Revised: 06/29/2006] [Accepted: 07/04/2006] [Indexed: 12/11/2022]
Abstract
As a consequence of the point mutation in the YMDD motif of the hepatitis B virus (HBV) polymerase gene, lamivudine-resistant mutants have been reported in chronic hepatitis B patients who underwent lamivudine therapy. The objective of the study was to develop a novel accurate artificially created restriction site (ACRS) method with a digestion internal control for identification of YMDD, YIDD and YVDD HBV strains. Three conserved, specific and diagnostic primers introducing NdeI, SspI and AleI cleavage sites were designed in order to identify YMDD, YIDD and YVDD strains, respectively; while, their reverse primers also modified with the above recognition sites in order to enzyme correctness monitoring and false outcome avoiding. Thirty-two chronic hepatitis B patients who had taken lamivudine for 1-3 years and checked by the Inno-LiPA HBV DR kit, were evaluated by the ACRS method and then compared to sequencing data. The results of the ACRS method revealed the YMDD mutant strain in 20 patients, YMDD plus YIDD pattern in 1 patient, YMDD plus YVDD in 4 patients, the YIDD in 4 patients and mixed infection with each three strains in 1 patient. The sequencing and Inno-LiPA results were in agreement with the ACRS results. The novel ACRS method is a reliable, rapid and a cost-effective technique for determination of HBV strains with the wild type and YMDD mutant patterns.
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Affiliation(s)
- Seyed Younes Hosseini
- Virology Department, Faculty of Medical Sciences, Tarbiat Modarres University, Tehran, Iran
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Chieochansin T, Chutinimitkul S, Payungporn S, Theamboonlers A, Tangkijvanich P, Komolmit P, Poovorawan Y. Rapid detection of lamivudine-resistant hepatitis B virus mutations by PCR-based methods. TOHOKU J EXP MED 2006; 210:67-78. [PMID: 16960347 DOI: 10.1620/tjem.210.67] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
Hepatitis B virus (HBV) is one of the major causes of liver disease worldwide, and chronic HBV infection may progress to cirrhosis and hepatocellular carcinoma. Mutations at the active site of DNA polymerase of HBV, tyrosine-methionine-aspartate-aspartate (YMDD) motif, render infected patients resistant to antiviral drug (Lamivudine) therapy. Hence, sensitive and specific methods aimed at detecting the mutants are essential. The purpose of this study was to develop methods for detecting the mutations at YMDD by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and real-time PCR using locked nucleic acid (LNA)-mediated TaqMan probes. The results obtained by these methods were compared with those examined by conventional direct sequencing on serum samples of 77 patients treated with lamivudine. Our results show that both PCR-RFLP and real-time PCR could detect wild type, YMDD, and its mutants, tyrosine-isoleucine-aspartate-aspartate and tyrosine-valine-aspartate-aspartate. In addition, the mixtures of the wild-type virus and its mutants in the serum sample were detected. Importantly, real-time PCR is less time-consuming, and more sensitive for the detection of mixed populations than PCR-RFLP. The real-time PCR with LNA-mediated TaqMan probes is a sensitive, specific and rapid detection method for mutations at the YMDD motif, which will be essential for monitoring patients undergoing lamivudine antiviral therapy.
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Affiliation(s)
- Thaweesak Chieochansin
- Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
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Shi M, Yang ZJ, Wang RS, Zhang H, Zhu YF, Xu YP, Lin QY, Jin LJ. Rapid quantitation of lamivudine-resistant mutants in lamivudine treated and untreated patients with chronic hepatitis B virus infection. Clin Chim Acta 2006; 373:172-5. [PMID: 16814763 DOI: 10.1016/j.cca.2006.05.023] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2006] [Revised: 05/10/2006] [Accepted: 05/10/2006] [Indexed: 01/26/2023]
Abstract
BACKGROUND Long-term lamivudine treatment induces emergence of lamivudine-resistant hepatitis B virus (HBV) in a significant number of patients with chronic HBV infection. Rapid and quantitative methods to determine the percentage of lamivudine-resistant mutants in total HBV are important during lamivudine therapy. METHODS We established a quantitative real-time PCR method with selective primers and TaqMan probe to detect the percentage of lamivudine-resistant mutants in total HBV without the need of external DNA standards. This percentage was calculated as the PCR efficiency raised to the differences between threshold cycle number (DeltaCt) of mutant and control reactions. Clones of the HBV polymerase gene containing the different YMDD variants were diluted in series and tested. Serum samples from 145 lamivudine-treated and 98 untreated patients with chronic hepatitis B virus infection were analyzed using this method and compared with DNA sequencing. RESULTS As little as 0.1% mutant plasmids in 10(6)-10(9) copies/ml of wild-type plasmids were detected. Among the 145 patients treated with lamivudine, 42 of them had mutants with percentages of 5-100%. In six discordant results between real-time PCR and DNA sequencing, real-time PCR detected mutants with percentages of 5-20%, which were concordant with subclone sequencing. Five of 98 lamividine-untreated patients had mutants of 10-20% in wild-type virus populations. Compared to DNA sequencing, real-time PCR was fast and cost-effective. CONCLUSION This real-time PCR is a rapid, sensitive and cost-effective method for relative quantitation of YMDD mutants of HBV.
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Affiliation(s)
- Ming Shi
- Department of Biotechnology, Da Lian University of Technology, Da Lian 116023, Liaoning Province, China
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Affiliation(s)
- Tim Shaw
- Victorian Infectious Diseases Reference Laboratory, Locked Bag 815, Carlton South, Vic. 3053, Australia.
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Tanaka J, Valdez-Salazar HA, Juárez-Barreto V, Dehesa-Violante M, Torres J, Muñoz-Hernández O, Alvarez-Muñoz MT. Hepatitis B epidemiology in Latin America. Vaccine 2000; 4:6. [PMID: 17217533 PMCID: PMC1781063 DOI: 10.1186/1743-422x-4-6] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2006] [Accepted: 01/11/2007] [Indexed: 12/12/2022]
Abstract
Background Hepatitis B virus (HBV) is a small DNA-containing virus with 4 genes, C, S, X and P. The S gene codes for the surface antigen (HBsAg), which contains the "a" determinant, the main region for induction of a protective humoral immune response. To compare the genotype and sequence of the "a" determinant between strains isolated from asymptomatic and symptomatic Mexican HBV carriers. Results 21 asymptomatic (blood donors) and 12 symptomatic (with clinical signs and with >1 year lamivudine treatment) HBV carriers were studied; all patients were positive for the HBsAg in serum. Viral load, genotypes, and subtypes were determined in plasma. A fragment of the S gene including the "a" determinant was PCR amplified and sequenced to determine genotype, subtype and to identify mutations. Mean viral load was 0.7965 × 104 copies/ml in asymptomatic carriers and 2.73 × 106 copies/ml in symptomatic patients. Genotypes H, C, and F were identified in asymptomatic individuals; whereas H was dominant in symptomatic patients. A fragment of 279 bp containing the "a" determinant was amplified from all 33 carriers and sequences aligned with S gene sequences in the GenBank. Mutations identified were Y100N, T126I, Q129H and N146K in the asymptomatic group, and F93I and A128V in the symptomatic group. Conclusion Differences in genotype and in mutations in the "a" determinant were found between strains from asymptomatic and symptomatic HBV Mexican carriers.
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Affiliation(s)
- J Tanaka
- Biological Division, SmithKline Beecham Biologicals, Av. Insurgentes sur 1605-Piso 20, Col. San Jose Insurgentes, Delegacion Benito Juarez, Mexico City, Mexico
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