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Performance of LigAmp Assay for Sensitive Detection of Drug-Resistant Hepatitis B Virus Minor Variants in Comparison with Standard Nucleotide Sequencing

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Abstract

Background and Objectives

A virus population often exists as a complex mixture of genetic populations. Antiviral-resistant mutants could be circulating as minority variants in the mixed virus population that are not detected by standard sequencing methods. The role of minor drug-resistant variants and clinical outcome is slowly evolving and there is a need to employ sensitive methods for detection of minority variants that emerge as dominant species and subsequently affect the antiviral efficacy. This study was intended to develop a technique called the ligation amplification assay (LigAmp) to identify minor drug-resistant variants of hepatitis B virus (HBV).

Methods

A LigAmp HBV assay was developed and clinical samples were tested from chronic hepatitis B subjects on antiviral treatment. Nucleotide sequencing of HBV reverse transcriptase (rt) region was performed and the results were compared with LigAmp assay. The performance of LigAmp assay was validated by clonal sequencing. Virological response was measured using HBV DNA levels and the results were correlated with antiviral-resistant mutations detected by sequencing and LigAmp assays.

Results

A total of 80 reactions of LigAmp assay were performed for rtM204V and rtM204I (ATT) mutant detection. Samples were obtained from 40 chronic hepatitis B subjects. Among these subjects, rtM204V and rtM204I (ATT) mutations were identified by standard sequencing in 10 (25 %) and 12 (30 %) subjects, respectively. LigAmp detected both rtM204V and rtM204I (ATT) mutations in 13 (32.5 %) subjects, rtM204I mutation in 12 (30 %) subjects and rtM204V mutation in 1 (2.5 %) subject, respectively. LigAmp detected primary resistant mutants in 69.4 % of lamivudine non-responders while sequencing detected resistant mutations in only 55.6 % subjects (p < 0.001).

Conclusions

This data shows significantly higher sensitivity of LigAmp for detection of minority rtM204V and rtM204I (ATT) mutations over standard sequencing. Therefore, LigAmp has potential clinical utility for appropriate monitoring and tailoring of HBV therapy.

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Acknowledgments

The study was funded by a Fluid research grant (IRB Min. No. 7189) and Clinical Virology Department Research funds, Christian Medical College, Vellore 632 004, India.

AMI conceived and designed the study, performed the experiments, analyzed the data, and wrote the manuscript. JS performed the validation of LigAmp results by clonal sequencing. CEE helped in recruitment of patients for the study and critically reviewed the manuscript. RK and PA supervised the study and critically reviewed the manuscript.

Conflicts of interest

The authors have no conflicts of interest that are directly relevant to the content of this article.

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Authors and Affiliations

  1. Departments of Clinical Virology, Christian Medical College, Vellore, 632004, Tamil Nadu, India

    Ashrafali Mohamed Ismail, Jaiprasath Sachithanandham, Rajesh Kannangai & Priya Abraham

  2. Department of Gastrointestinal Sciences, Christian Medical College, Vellore, 632004, Tamil Nadu, India

    Chundmannil Eapen Eapen

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  1. Ashrafali Mohamed Ismail
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  2. Jaiprasath Sachithanandham
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Correspondence to Priya Abraham.

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Ismail, A.M., Sachithanandham, J., Eapen, C.E. et al. Performance of LigAmp Assay for Sensitive Detection of Drug-Resistant Hepatitis B Virus Minor Variants in Comparison with Standard Nucleotide Sequencing. Mol Diagn Ther 18, 655–663 (2014). https://doi.org/10.1007/s40291-014-0119-y

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  • DOI: https://doi.org/10.1007/s40291-014-0119-y

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