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High Prevalence of Preexisting HBV Polymerase Mutations in Pregnant Women Does Not Limit the Antiviral Therapy Efficacy. ACTA ACUST UNITED AC 2021; 2021:6653546. [PMID: 33986897 PMCID: PMC8079218 DOI: 10.1155/2021/6653546] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2020] [Revised: 02/05/2021] [Accepted: 03/15/2021] [Indexed: 11/18/2022]
Abstract
Background HBV-resistant mutants in treatment-naïve patients may lead to antiviral treatment failure. It is not clear if HBV mutants are present in pregnant women and about the influence of the preexisting mutants on the short-term antiviral therapy during pregnancy. Method We enrolled 73 pregnant women with high HBV DNA load and telbivudine (TBV) treatment during pregnancy in this retrospective study. The UDPS was used to detect the HBV mutations before and after the TBV treatment. Results Before TBV treatment, the complexity of HBV quasispecies of all subjects was 0.40 ± 0.09; 41.1% (30/73) and 53.4% (39/73) subjects had rtM204I/V and rtN236 T/A detected, respectively; and 9.6% (7/73) patients had more than 20% frequency mutation of rtM204I/V, which was also similar with high frequency of rtN236 T/A mutation (41.1% vs. 53.4%, P=0.136; frequencies >20%: 9.6% vs. 5.5%, P=0.347). After TBV treatment, 71.2% (52/73) subjects had HBV DNA load ≥ 103 IU/mL at delivery. Among them, 75.0% of patients with rtM204I positive had HBV DNA load ≥103 IU/mL at delivery, which was comparable with the subjects without rtM204I (75.0% vs. 70.8%, P=0.710). No changes were found in the frequencies and the complexity of HBV quasispecies of rtM204I mutation after the TVB treatment. Conclusion The prevalence of preexisting drug-resistant mutations among pregnant women was high using UPDS. However, the preexisting HBV mutation had limited influence on the efficacy of short-term TBV treatment, and TBV treatment during late pregnancy seemed not to increase the risk of emerging HBV-resistant mutants.
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Şahin E. Evaluation of antiviral resistant hepatitis B virus subpopulations in patients with chronic hepatitis B by using terminal restriction fragment length polymorphism. Virusdisease 2015; 26:267-75. [PMID: 26645037 PMCID: PMC4663704 DOI: 10.1007/s13337-015-0282-8] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2015] [Accepted: 10/05/2015] [Indexed: 11/25/2022] Open
Abstract
Antiviral therapies with nucleotide analogues (NA) is crucial in the treatment of chronic hepatitis B as it substantially protects patients from the complications of the disease . However in most of the available NA therapies, resistance emerges in the patients' HBV populations. Therefore, detection of antiviral resistance as early as possible by means of genotypically monitoring the patients' HBV pool during NA therapy is critical to manage treatment regime. In this research study we have investigated the sensitivity and specificity of the terminal restriction fragment length polymorphism (T-RFLP) method in detecting HBV subpopulations carrying antiviral resistance mutations. For this aim, differentiation of mutant strains from wild type strains was demonstrated by PCR-RFLP method. With using recombinant plasmids containing mutant and wild type HBV genomes, we constructed artificial HBV genome populations in order to determine the sensitivity of PCR-T-RFLP method in detecting antiviral resistant minor HBV populations. Finally by comparing with the DNA sequencing method, we demonstrated the specificity of T-RFLP method in genotyping HBV populations. As a result we showed that T-RFLP is able to detect HBV subpopulations representing as low as 1 % of the whole viral population. Additionally T-RFLP showed 100 % concordance with the DNA sequencing method in genotyping HBV populations. As a conclusion, considering the other genotyping methods used in evaluating HBV populations, T-RFLP showed high sensitivity and specificity profiles in detecting antiviral resistant HBV subpopulations. Therefore T-RFLP method can be easily employed in genotypic evaluation of patients' HBV populations during the course of antiviral treatment.
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Affiliation(s)
- Ergin Şahin
- />Department of Biology, Faculty of Science, Ankara University, Tandogan, 06100 Ankara, Turkey
- />Institute of Hepatology, Ankara University, Ankara, Turkey
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Tan YW, Ge GH, Zhao W, Gan JH, Zhao Y, Niu ZL, Zhang DJ, Chen L, Yu XJ, Yang LJ. YMDD motif mutations in chronic hepatitis B antiviral treatment naïve patients: a multi-center study. Braz J Infect Dis 2012. [PMID: 22729192 DOI: 10.1016/s1413-8670(12)70319-7] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
OBJECTIVE This study aimed to determine the natural prevalence of variants of tyrosine-methionine-aspartic acid-aspartic acid (YMDD) motif in patients with chronic hepatitis B (CHB), and to explore its relation with demographic and clinical features, hepatitis B virus (HBV) genotypes, and HBV DNA levels. METHODS A total of 1,042 antiviral treatment naïve CHB patients (including with lamivudine [LAM]) in the past year were recruited from outpatient and inpatient departments of six centers from December 2008 to June 2010. YMDD variants were analyzed using the HBV drug resistance line probe assay (Inno-Lipa HBV-DR). HBV genotypes were detected with polymerase chain reaction (PCR) microcosmic nucleic acid cross-ELISA, and HBV deoxyribonucleic acid (DNA) was quantitated with real-time PCR. All serum samples underwent tests for HBV, HCV, and HDV with ELISA. RESULTS YMDD variants were detected in 23.3% (243/1042) of CHB patients. YMDD mutation was accompanied by L180M mutation in 154 (76.9%) patients. Both wild-type HBV and YMDD variant HBV were present in 231 of 243 patients. Interestingly, 12 patients had only YIDD and/or YVDD variants without wild YMDD motif. In addition, 27.2% (98/359) of HbeAg-positive patients had YMDD mutations, which was higher than that in HbeAg-negative patients (21.2%, 145/683). The incidence of YMDD varied among patients with different HBV genotypes, but the difference was not significant. Moreover, the incidence of YMDD in patients with high HBV DNA level was significantly higher than that in those with low HBV DNA level. CONCLUSION Mutation of YMDD motif was detectable at a high rate in CHB patients in this study. The incidence of YMDD may be correlated with HBeAg and HBV DNA level.
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Affiliation(s)
- You-Wen Tan
- Department of Liver Diseases, No. 3 Hospital of Zhenjiang, Zhenjiang, Jiangsu, China.
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Tan Y, Ding K, Su J, Trinh X, Peng Z, Gong Y, Chen L, Cui Q, Lei N, Chen X, Yu R. The naturally occurring YMDD mutation among patients chronically infected HBV and untreated with lamivudine: a systematic review and meta-analysis. PLoS One 2012; 7:e32789. [PMID: 22479339 PMCID: PMC3314000 DOI: 10.1371/journal.pone.0032789] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2011] [Accepted: 01/31/2012] [Indexed: 01/30/2023] Open
Abstract
BACKGROUND Several recent reports have demonstrated that tyrosine (Y)-methionine (M)-aspartic acid (D)-aspartic acid (D) (YMDD) motif mutations can naturally occur in chronic HBV patients without antiviral treatment such as lamivudine therapy. This paper aims to assess the overall spontaneous incidence and related risk factors of YMDD-motif mutations among lamivudine-naïve chronic HBV carriers, so as to provide some clue for clinical treatment of hepatitis B. METHODOLOGY/PRINCIPAL FINDINGS Chinese and English literatures were searched for studies reporting natural YMDD mutations among untreated chronic HBV patients from 2001 to 2010. The incidence estimates were summarized and analyzed by meta-analyses. Forty-seven eligible articles from eight countries were selected in this review (13 in English and 34 in Chinese). The pooled incidence of YMDD-motif mutation among untreated chronic HBV patients from eight countries was 12.21% (95% CI: 9.69%-14.95%). China had an incidence of 13.38% (95% CI: 10.90%-16.07%) and seven other countries had an incidence of 9.90% (95% CI: 3.28%-19.55%), respectively. Lamivudine therapy would increase the risk of mutations 5.23 times higher than the untreated patients. A higher HBV DNA copy number was associated with increased incidence of natural YMDD mutation. No significant difference was found in YMDD mutation incidence between groups of different gender, age, HBeAg status, patients' ALT (alanine aminotransferase) level, and between the groups of HBV genotype B and C. CONCLUSIONS The YMDD-motif mutations can occur spontaneously with a relatively high incidence in CHB patients untreated with lamivudine. These mutations might be the consequence of accumulated base mismatch due to the nature of viral polymerase. More fundamental and clinical studies are needed to clarify the influence of YMDD mutations in hepatitis B progression and antiviral treatment.
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Affiliation(s)
- Youwen Tan
- Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China
- Department of Hepatosis, The Third People's Hospital of Zhenjiang City, Zhenjiang, China
| | - Keqin Ding
- Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China
| | - Jing Su
- Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China
| | - Xuan Trinh
- Department of Pharmacology, University of Gothenburg, Göteborg, Sweden
| | - Zhihang Peng
- Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China
| | - Yuhua Gong
- Department of Hepatosis, The Third People's Hospital of Zhenjiang City, Zhenjiang, China
| | - Li Chen
- Department of Hepatosis, The Third People's Hospital of Zhenjiang City, Zhenjiang, China
| | - Qian Cui
- Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China
| | - Na Lei
- Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China
| | - Xin Chen
- Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China
| | - Rongbin Yu
- Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China
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Mirandola S, Campagnolo D, Bortoletto G, Franceschini L, Marcolongo M, Alberti A. Large-scale survey of naturally occurring HBV polymerase mutations associated with anti-HBV drug resistance in untreated patients with chronic hepatitis B. J Viral Hepat 2011; 18:e212-6. [PMID: 21692935 DOI: 10.1111/j.1365-2893.2011.01435.x] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Drug resistance is a major limitation for the long-term efficacy of antiviral therapy with nucleos(t)ide analogues (NAs) in chronic hepatitis B (CHB). Antiviral resistance mutations may pre-exist in the overall viral population of untreated patients. We aimed to assess the prevalence of such hepatitis B virus (HBV) variants in a large cohort of NAs-naïve patients with CHB and to explore possible association with viral and host variables. Serum samples from 286 NAs-naïve consecutive patients with CHB were tested for serum HBV-DNA, and 255 of them having HBV-DNA > 1000 IU/mL were further analysed for drug resistance mutations by INNO-LiPA HBV DRv2/v3. NAs-naïve patients analysed were mainly men (73%), Caucasians (85%), hepatitis B e Antigen (HBeAg) negative (79%) and genotype D (69%), with a mean age of 43.2 ± 13.4 years. HBV mutations associated with antiviral drug resistance were detected in 13 (5%) patients: three patients infected with HBV genotype C had the rtM204V + rtL180M mutations associated with lamivudine (LMV) resistance. Four patients had the rtI233V mutation that may reduce sensitivity to adefovir, and three patients had the rtM250L/V mutation typical of entecavir resistance. LMV compensatory mutations rtL80V and rtV173L were seen in two and one patients, respectively. No relationship was seen between presence of resistant or compensatory mutations and HBV-DNA levels, HBeAg/anti-HBe status or previous IFN therapy. These results confirm that HBV mutations, which confer resistance against currently available anti-HBV NAs, may already exist in patients who have never received the drug.
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Affiliation(s)
- S Mirandola
- Venetian Institute of Molecular Medicine, Padova, Italy
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Rapid detection of the hepatitis B virus YMDD mutant using AllGlo™ probes. Clin Chim Acta 2011; 412:1018-21. [PMID: 21324308 DOI: 10.1016/j.cca.2011.02.012] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2010] [Revised: 01/27/2011] [Accepted: 02/08/2011] [Indexed: 12/16/2022]
Abstract
BACKGROUND The early detection of hepatitis B virus (HBV) mutants in clinical samples is important when monitoring chronic HBV patients with lamivudine-resistant mutations during lamivudine therapy. METHODS The AllGlo™ probes were designed to distinguish between wild-type (YMDD) and mutant (YVDD and YIDD) strains of HBV. The sensitivity and specificity of the assay were evaluated using a series of diluted mixtures of wild-type and mutant plasmids. This assay was compared with direct sequencing and the mutation-specific primer assay. RESULTS Each YMDD, YVDD, and YIDD probe only detected its corresponding plasmid. Moreover, the assay correctly identified negative samples from 40 non-HBV infected patients and 100 healthy controls. The detection limit of this assay was 50 copies/ml for YVDD and YIDD. The assay could detect the mutant strains when they were present at ≥10% within a mixed virus population. The assay was fully concordant with direct sequencing in 34 samples (56.7%) and partially concordant in 26 samples (43.3%), and detected more types of the HBV motif than direct sequencing. CONCLUSIONS AllGlo™ probe assay is a novel, sensitive and specific assay to detect lamivudine-related HBV mutants, therefore, may be useful for monitoring chronic HBV patients treated with lamivudine.
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Lupo J, Larrat S, Hilleret MN, Germi R, Boyer V, Nicod S, Barguès G, Leroy V, Seigneurin JM, Zarski JP, Morand P. Assessment of selective real-time PCR for quantitation of lamivudine and adefovir hepatitis B virus-resistant strains and comparison with direct sequencing and line probe assays. J Virol Methods 2008; 156:52-8. [PMID: 19041345 DOI: 10.1016/j.jviromet.2008.10.028] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2008] [Revised: 10/06/2008] [Accepted: 10/13/2008] [Indexed: 02/06/2023]
Abstract
A selective real-time PCR (sPCR) assay has been developed to detect the rtM204V/I and rtN236T mutations of hepatitis B virus (HBV) associated with resistance to lamivudine and adefovir. Using mixtures of mutant and wild-type plasmids, this sPCR was able to detect 0.1% of mutated strain in a total plasmid population of 10(5) copies and was more sensitive in detecting resistant strains than the line probe INNO-LiPA-DR-v2 assay and a direct sequencing assay. The comparison of these methods on 20 clinical specimens from treated patients confirmed the plasmid results: the three methods were concordant for the detection of the mutant strains in 72% of the cases and the discrepant results were caused mainly by the sequencing assay's lack of sensitivity. The line probe assay was more sensitive for detecting mutations than sPCR when the viral load was less than 10(4) copies/ml; conversely, the sPCR provided a more sensitive detection when the viral load was greater than 10(4) copies/ml. Although difficult to perform in clinical practice, sPCR appears to be a reliable technique for detecting and quantifying quasi-species resistant to lamivudine (LAM) and adefovir (ADV) and can be useful to gain a better understanding of the natural history of antiviral resistance during the treatment of chronic hepatitis B (CHB).
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Affiliation(s)
- Julien Lupo
- Unit of Virus Host Cell Interactions, CNRS-EMBL-UJF-UMR 5233, University Hospital and University of Joseph Fourier, Grenoble, France
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Hua R, Tanaka Y, Fukai K, Tada M, Seto M, Asaoka Y, Ohta M, Goto T, Kanai F, Kato N, Yoshida H, Kawabe T, Yokosuka O, Omata M. Rapid detection of the hepatitis B virus YMDD mutant using TaqMan-minor groove binder probes. Clin Chim Acta 2008; 395:151-154. [PMID: 18602379 DOI: 10.1016/j.cca.2008.06.013] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2008] [Revised: 05/29/2008] [Accepted: 06/11/2008] [Indexed: 10/21/2022]
Abstract
BACKGROUND TaqMan-minor groove binder (MGB) probes were used in a real-time PCR-based assay for the rapid and accurate detection of hepatitis B virus (HBV) YMDD mutants. METHODS TaqMan-MGB probes were designed to distinguish between wild-type (YMDD) and mutant (YVDD and YIDD) strains of HBV. The detection limit and sensitivity of the assay were determined using a dilution series of a mixture of wild-type and mutant plasmids. Serum samples collected from four patients with chronic mutant HBV infections during lamivudine therapy were analyzed using this method. RESULTS The detection limit for YVDD and YIDD was 10 and 50 copies, respectively, whereas the sensitivity was 10% within a mixed virus population. In the clinical samples, mutant strains of HBV could be detected at levels <2.6 log copies/ml of HBV DNA. While 15 of the 21 samples tested by this method were positive for the YMDD mutant, direct sequencing and a reverse hybridization line probe assay (INNO-LiPA HBV DR v2) detected the mutant strain in only 11 and 9 samples, respectively. Moreover, the data for 6 samples analyzed by TA cloning were fully consistent with our TaqMan PCR results. CONCLUSIONS We successfully established a sensitive and accurate assay for the YMDD mutant of HBV. This method may be useful for monitoring patients treated with lamivudine.
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Affiliation(s)
- Rui Hua
- Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655, Japan
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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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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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