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Comparison of multiplex restriction fragment mass polymorphism and sequencing analyses for detecting entecavir resistance in chronic hepatitis B.

Authors
 Kyung Ho Han  ;  Sun Pyo Hong  ;  Seo Hee Choi  ;  Soo-Kyung Shin  ;  Sung Won Cho  ;  Sang Hoon Ahn  ;  Ji-Sook Hahn  ;  Soo-Ok Kim 
Citation
 ANTIVIRAL THERAPY, Vol.16(1) : 77-87, 2011 
Journal Title
ANTIVIRAL THERAPY
ISSN
 1359-6535 
Issue Date
2011
MeSH
Adenine/administration & dosage ; Adenine/analogs & derivatives ; Adenine/therapeutic use ; Antiviral Agents/administration & dosage ; Antiviral Agents/therapeutic use ; Drug Resistance, Viral/genetics* ; Genotype ; Guanine/administration & dosage ; Guanine/analogs & derivatives ; Guanine/therapeutic use ; Hepatitis B virus/drug effects ; Hepatitis B virus/genetics ; Hepatitis B, Chronic/diagnosis* ; Hepatitis B, Chronic/drug therapy ; Hepatitis B, Chronic/genetics* ; Hepatitis B, Chronic/virology ; Humans ; Lamivudine/administration & dosage ; Lamivudine/therapeutic use ; Mass Spectrometry/methods ; Mutation ; Organophosphonates/administration & dosage ; Organophosphonates/therapeutic use ; Polymerase Chain Reaction/methods* ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length* ; Sequence Analysis, DNA/methods ; Viral Load
Abstract
BACKGROUND: Drug resistance is a major limitation to the long-term efficacy of controlling chronic hepatitis B (CHB). There is a growing need to analyse multiple mutations associated with drug resistance because sequential or combinational use of antivirals is increasingly being used in treatment. In this study, we introduced a multiplex restriction fragment mass polymorphism (RFMP) assay for detecting mutations conferring entecavir and lamivudine resistance, and compared its performance with direct or clonal sequencing assays.

METHODS: Multiplex PCR was performed with mixed primers designed to interrogate rt184, rt202, rt204 and rt250. The PCR products were digested with restriction enzymes and the resulting fragments were analysed by mass spectrometry. A total of 251 serum samples, taken serially from 45 patients who received entecavir treatment after confirmed diagnosis of lamivudine resistance and inadequate adefovir dipivoxil response, were analysed by the multiplex RFMP assay.

RESULTS: The multiplex RFMP assay correctly identified known viral sequences with sufficient analytical sensitivity to detect as few as 100 IU/ml of HBV and with superior ability to determine haplotypes composed of neighbouring variations. Complex mutational patterns and relative abundances determined by multiplex RFMP assay were in good concordance with results obtained by direct or clonal sequencing analyses. Defined mixtures were successfully and consistently identified at 2% relative concentration of mutant versus wild-type virus by the assay.

CONCLUSIONS: The multiplex RFMP assay is an accurate and sensitive means to detect entecavir and lamivudine resistance mutations, simultaneously. The method is expected to enable early and efficient diagnosis of multiple drug resistance mutations for optimal management of CHB.
Full Text
http://www.intmedpress.com/journals/avt/article.cfm?id=1702&pid=88&sType=AVT
DOI
10.3851/IMP1702
Appears in Collections:
1. College of Medicine (의과대학) > Dept. of Internal Medicine (내과학교실) > 1. Journal Papers
Yonsei Authors
Ahn, Sang Hoon(안상훈) ORCID logo https://orcid.org/0000-0002-3629-4624
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/95226
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