Detection of Hepatitis B Virus Genotypic Resistance Mutations by Coamplification at Lower Denaturation Temperature-PCR Coupled with Sanger Sequencing

Detection of anti-hepatitis B virus (HBV) drug resistance mutations is critical for therapeutic decisions for chronic hepatitis B virus infection. We describe a real-time PCR-based assay using multicolor melting curve analysis (MMCA) that could accurately detect 24 HBV nucleotide mutations at 10 amino acid positions in the reverse transcriptase region of the HBV polymerase gene. The two-reaction assay had a limit of detection of 5 copies per reaction and could detect a minor mutant population (5% of the total population) with the reverse transcriptase M204V amino acid mutation in the presence of the major wild-type population when the overall concentration was 10 4 copies/μl. The assay could be finished within 3 h, and the cost of materials for each sample was less than $10. Clinical validation studies using three groups of samples from both nucleos(t)ide analog-treated and -untreated patients showed that the results for 99.3% (840/846) of the samples and 99.9% (8,454/8,460) of the amino acids were concordant with those of Sanger sequencing of the PCR amplicon from the HBV reverse transcriptase region (PCR Sanger sequencing). HBV DNA in six samples with mixed infections consisting of minor mutant subpopulations was undetected by the PCR Sanger sequencing method but was detected by MMCA, and the results were confirmed by coamplification at a lower denaturation temperature-PCR Sanger sequencing. Among the treated patients, 48.6% (103/212) harbored viruses that displayed lamivudine monoresistance, adefovir monoresistance, entecavir resistance, or lamivudine and adefovir resistance. Among the untreated patients, the Chinese group had more mutation-containing samples than did the Pakistani group (3.3% versus 0.56%). Because of its accuracy, rapidness, wide-range coverage, and cost-effectiveness, the real-time PCR assay could be a robust tool for the detection if anti-HBV drug resistance mutations in resource-limited countries.

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Application of Coamplification at Lower Denaturation Temperature-PCR Sequencing for Early Detection of Antiviral Drug Resistance Mutations of Hepatitis B Virus

Journal of Clinical Microbiology ◽

10.1128/jcm.00343-14 ◽

2014 ◽

Vol 52 (9) ◽

pp. 3209-3215 ◽

Cited By ~ 5

Author(s):

Danny Ka-Ho Wong ◽

Ottilia Tsoi ◽

Fung-Yu Huang ◽

Wai-Kay Seto ◽

James Fung ◽

...

Keyword(s):

Drug Resistance ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Early Detection ◽

Antiviral Drug ◽

Resistance Mutations ◽

Denaturation Temperature ◽

Drug Resistance Mutations ◽

B Virus ◽

Antiviral Drug Resistance

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Low-Level Persistence of Drug Resistance Mutations in Hepatitis B Virus-Infected Subjects with a Past History of Lamivudine Treatment

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01601-12 ◽

2012 ◽

Vol 57 (1) ◽

pp. 343-349 ◽

Cited By ~ 18

Author(s):

Severine Margeridon-Thermet ◽

Evguenia S. Svarovskaia ◽

Farbod Babrzadeh ◽

Ross Martin ◽

Tommy F. Liu ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Sanger Sequencing ◽

Past History ◽

Resistance Mutation ◽

Resistance Mutations ◽

Direct Pcr ◽

B Virus ◽

Sanger Sequence ◽

Minority Variants

ABSTRACTWe sought to determine the prevalence of hepatitis B virus (HBV) lamivudine (LAM)-resistant minority variants in subjects who once received LAM but had discontinued it prior to virus sampling. We performed direct PCR Sanger sequencing and ultradeep pyrosequencing (UDPS) of HBV reverse transcriptase (RT) of plasma viruses from 45 LAM-naive subjects and 46 LAM-experienced subjects who had discontinued LAM a median of 24 months earlier. UDPS was performed to a depth of ∼3,000 reads per nucleotide. Minority variants were defined as differences from the Sanger sequence present in ≥0.5% of UDPS reads in a sample. Sanger sequencing identified ≥1 LAM resistance mutations (rtL80I/V, rtM204I, and rtA181T) in samples from 5 (11%) of 46 LAM-experienced and none of 45 LAM-naive subjects (0%;P= 0.06). UDPS detected ≥1 LAM resistance mutations (rtL80I/V, rtV173L, rtL180M, rtA181T, and rtM204I/V) in 10 (22%) of the 46 LAM-experienced subjects, including 5 in whom LAM resistance mutations were not identified by Sanger sequencing. Overall, LAM resistance mutations were more likely to be present in LAM-experienced (10/46, 22%) than LAM-naive subjects (0/45, 0%;P= 0.001). The median time since LAM discontinuation was 12.8 months in the 10 subjects with a LAM resistance mutation compared to 30.5 months in the 36 LAM-experienced subjects without a LAM resistance mutation (P< 0.001). The likelihood of detecting a LAM resistance mutation was significantly increased using UDPS compared to Sanger sequencing and was inversely associated with the time since LAM discontinuation.

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Hepatitis B virus quasispecies susceptibility to entecavir confirms the relationship between genotypic resistance and patient virologic response

Journal of Hepatology ◽

10.1016/j.jhep.2007.12.024 ◽

2008 ◽

Vol 48 (6) ◽

pp. 895-902 ◽

Cited By ~ 48

Author(s):

Carl J. Baldick ◽

Betsy J. Eggers ◽

Jie Fang ◽

Steven M. Levine ◽

Kevin A. Pokornowski ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Virologic Response ◽

Genotypic Resistance ◽

B Virus ◽

The Relationship

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Characterization of antiviral resistance mutations among the Eastern Indian Hepatitis B virus infected population

Virology Journal ◽

10.1186/1743-422x-10-56 ◽

2013 ◽

Vol 10 (1) ◽

pp. 56 ◽

Cited By ~ 10

Author(s):

Rajesh Panigrahi ◽

Avik Biswas ◽

Binay Krishna De ◽

Sekhar Chakrabarti ◽

Runu Chakravarty

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Antiviral Resistance ◽

Resistance Mutations ◽

Infected Population ◽

B Virus

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Frequency of Hepatitis B Virus Resistance Mutations in Women Using Tenofovir Gel as Pre-Exposure Prophylaxis

Viruses ◽

10.3390/v11060569 ◽

2019 ◽

Vol 11 (6) ◽

pp. 569

Author(s):

Cheryl Baxter ◽

Sinaye Ngcapu ◽

Jason T Blackard ◽

Eleanor A Powell ◽

Patricia K Penton ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Systemic Exposure ◽

Resistance Mutations ◽

Dosing Regimen ◽

Hbv Genotype ◽

B Virus ◽

Tenofovir Gel ◽

Exposure Prophylaxis ◽

The Impact

Intermittent use of a single antiretroviral agent in the presence of a replicating virus could potentially increase the development of antiviral resistance. The pericoital, before-and-after sex, dosing regimen used in the Centre for the AIDS Programme of Research in South Africa (CAPRISA) 004 tenofovir gel trial meant that women who were infected with hepatitis B virus (HBV) were exposed intermittently to tenofovir during their participation. The impact of this dosing regimen on HBV resistance was assessed by amplification of the HBV polymerase region from 37 stored plasma samples of women who were HBV surface antigen positive. All samples belonged to HBV genotype A. None of the known tenofovir resistance mutations (M240V/I, L180M, A194T, V214A, N238T) were identified in any individuals. While it is reassuring that no resistance mutations were found among women using topical tenofovir, the rapidly expanding access to oral tenofovir-containing HIV pre-exposure prophylaxis (PrEP), with higher systemic exposure to the drug, makes monitoring for potential HBV drug resistance important.

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Coevolution analysis of amino-acids reveals diversified drug-resistance solutions in viral sequences: a case study of hepatitis B virus

Virus Evolution ◽

10.1093/ve/veaa006 ◽

2020 ◽

Vol 6 (1) ◽

Cited By ~ 1

Author(s):

Elin Teppa ◽

Francesca Nadalin ◽

Christophe Combet ◽

Diego Javier Zea ◽

Laurent David ◽

...

Keyword(s):

Drug Resistance ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Resistance Mutations ◽

Drug Resistance Mutations ◽

Coevolution Analysis ◽

B Virus ◽

Compensatory Mutations ◽

Viral Sequences

Abstract The study of mutational landscapes of viral proteins is fundamental for the understanding of the mechanisms of cross-resistance to drugs and the design of effective therapeutic strategies based on several drugs. Antiviral therapy with nucleos(t)ide analogues targeting the hepatitis B virus (HBV) polymerase protein (Pol) can inhibit disease progression by suppression of HBV replication and makes it an important case study. In HBV, treatment may fail due to the emergence of drug-resistant mutants. Primary and compensatory mutations have been associated with lamivudine resistance, whereas more complex mutational patterns are responsible for resistance to other HBV antiviral drugs. So far, all known drug-resistance mutations are located in one of the four Pol domains, called reverse transcriptase. We demonstrate that sequence covariation identifies drug-resistance mutations in viral sequences. A new algorithmic strategy, BIS2TreeAnalyzer, is designed to apply the coevolution analysis method BIS2, successfully used in the past on small sets of conserved sequences, to large sets of evolutionary related sequences. When applied to HBV, BIS2TreeAnalyzer highlights diversified viral solutions by discovering thirty-seven positions coevolving with residues known to be associated with drug resistance and located on the four Pol domains. These results suggest a sequential mechanism of emergence for some mutational patterns. They reveal complex combinations of positions involved in HBV drug resistance and contribute with new information to the landscape of HBV evolutionary solutions. The computational approach is general and can be applied to other viral sequences when compensatory mutations are presumed.

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Hepatitis B virus genotype C encoding resistance mutations that emerge during adefovir dipivoxil therapy: in vitro replication phenotype

Hepatology International ◽

10.1007/s12072-012-9411-2 ◽

2012 ◽

Vol 7 (2) ◽

pp. 443-450 ◽

Cited By ~ 5

Author(s):

Wenpeng Li ◽

Nadia Warner ◽

Vitina Sozzi ◽

Lilly Yuen ◽

Danni Colledge ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Adefovir Dipivoxil ◽

Resistance Mutations ◽

Virus Genotype ◽

In Vitro Replication ◽

B Virus ◽

Genotype C

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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

Journal of Virology ◽

10.1128/jvi.02011-08 ◽

2008 ◽

Vol 83 (4) ◽

pp. 1718-1726 ◽

Cited By ~ 124

Author(s):

Mariacarmela Solmone ◽

Donatella Vincenti ◽

Mattia Carlo Felice Prosperi ◽

Alessandro Bruselles ◽

Giuseppe Ippolito ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Reverse Transcriptase ◽

Direct Sequencing ◽

Relative Sensitivity ◽

Massively Parallel ◽

Drug Resistant ◽

Resistance Mutations ◽

B Virus ◽

Drug Naïve

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 ≥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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