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.
References
1.
MalikA.H., LeeW.M.Chronic hepatitis B virus infection: treatment strategies for the next millennium. Ann Intern Med2000; 132: 723–731.
2.
GanemD., PrinceA.M.Hepatitis B virus infection – natural history and clinical consequences. N Engl J Med2004; 350: 1118–1129.
3.
IloejeU.H., YangH., SuJ.Predicting cirrhosis risk based on the level of circulating hepatitis B viral load. Gastroenterology2006; 130: 678–686.
4.
ChenC.J., YangH.I., SuJ.Risk of hepatocellular carcinoma across a biological gradient of serum hepatitis B virus DNA level. JAMA2006; 295: 65–73.
5.
LeungN.W., LaiC.L., ChangT.T.Extended lamivudine treatment in patients with chronic hepatitis B enhances hepatitis B e antigen seroconversion rates: results after 3 years of therapy. Hepatology2001; 33: 1527–1532.
LaiC.L., ChienR.N., LeungN.W.A one-year trial of lamivudine for chronic hepatitis B. Asia Hepatitis Lamivudine Study Group. N Engl J Med1998; 339: 61–68.
8.
LokA.S.The maze of treatments for hepatitis B. N Engl J Med2005; 352: 2743–2746.
9.
LocarniniS.Molecular virology and the development of resistant mutants: implications for therapy. Semin Liver Dis2005; 25: 9–19.
10.
KeeffeE.B., DieterichD.T., HanS.H.A treatment algorithm for the management of chronic hepatitis B virus infection in the United States: 2008 update. Clin Gastroenterol Hepatol2008; 6: 1315–1341.
ZoulimF.Entecavir: a new treatment option for chronic hepatitis B. J Clin Virol2006; 36: 8–12.
13.
TenneyD.J., LevineS.M., RoseR.E.Clinical emergence of entecavir-resistant hepatitis B virus requires additional substitutions in virus already resistant to lamivudine. Antimicrob Agents Chemother2004; 48: 3498–3507.
14.
RaptiI., DimouE., MitsoulaP.Adding-on versus switching-to adefovir therapy in lamivudine-resistant HBeAg-negative chronic hepatitis B. Hepatology2007; 45: 307–313.
15.
MukaideM., TanakaY., Shin-IT.. The mechanism of entecavir resistance of hepatitis B virus with viral breakthrough as determined by long-term clinical assessment and molecular docking simulation. Antimicrob Agents Chemother2010; 54: 882–889.
16.
SheldonJ., RodesB., ZoulimF.Mutations affecting the replication capacity of the hepatitis B virus. J Viral Hepat2006; 13: 427–434.
17.
FungS.K., FontanaR.J.Management of drug-resistant chronic hepatitis B. Clin Liver Dis2006; 10: 275–302.
18.
de Vries-SluijsT.E., van der EijkA.A., HansenB.E.Wild type and YMDD variant of hepatitis B virus: no difference in viral kineitcs on lamivudine/tenofovir therapy in HIV–HBV co-infected patients. J Clin Virol2006; 36: 60–63.
19.
PasS.D., de ManR.A., FriesE.The dynamics of mutations in the YMDD motif of the hepatitis B virus polymerase gene during and after lamivudine treatment as determined by reverse hybridization. J Clin Virol2002; 25: 63–71.
20.
PaikY.H., HanK.H., HongS.P.The clinical impact of early detection of the YMDD mutant on the outcomes of long-term lamivudine therapy in patients with chronic hepatitis B. Antivir Ther2006; 11: 447–455.
21.
HongS.P., KimN.K., HwangS.G.Detection of hepatitis B virus YMDD variants using mass spectrometric analysis of oligonucleotide fragments. J Hepatol2004; 40: 837–844.
22.
KimH.S., HanK.H., AhnS.H.Evaluation of methods for monitoring drug resistance in chronic hepatitis B patients during lamivudine therapy based on mass spectrometry and reverse hybridization. Antivir Ther2005; 10: 441–449.
23.
HongS.P., ShinS.K., LeeE.H.High-resolution human papillomavirus genotyping by MALDI–TOF mass spectrometry. Nat Protoc2008; 3: 1476–1484.
24.
OhH.B., KimS.O., ChaC.H.Identification of hepatitis C virus genotype 6 in Korean patients by analysis of 5′ untranslated region using a matrix assisted laser desorption/ ionization time of flight-based assay, restriction fragment mass polymorphism. J Med Virol2008; 80: 1712–1719.
25.
LeeY.S., SuhD.J., LimY.S.Increased risk of adefovir resistance in patients with lamivudine-resistant chronic hepatitis B after 48 weeks of adefovir dipivoxil monotherapy. Hepatology2006; 43: 1385–1391.
26.
KimY.J., KimS.O., ChungH.J.Population genotyping of hepatitis C virus by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry analysis of short DNA fragments. Clin Chem2005; 51: 1123–1131.
27.
LeeC.H., KimS.O., ByunK.S.Predominance of hepatitis B virus YMDD mutants is prognostic of viral DNA breakthrough. Gastroenterology2006; 130: 1144–1152.
28.
YeonJ.E., YooW., HongS.P.Resistance to adefovir dipivoxil in lamivudine resistant chronic hepatitis B patients treated with adefovir dipivoxil. Gut2006; 55: 1488–1495.
29.
CheongJ.Y., ChoS.W., YooJ.H.Selection of precore mutants during lamivudine treatment in patients with chronic hepatitis B. Hepatogastroenterology2008; 55: 1029–1033.
30.
AberleS.W., KletzmayrJ., WatschingerB.Comparison of sequence analysis and the INNO-LiPA HBV DR line probe assay for detection of lamivudine-resistant hepatitis B virus strains in patients under various clinical conditions. J Clin Microbiol2001; 39: 1972–1974.
31.
OnoY., OndaH., SasadaR.The complete nucleotide sequences of the cloned hepatitis B virus DNA; subtype adr and adw. Nucleic Acids Res1983; 11: 1747–1757.
