Journal of Virological Methods 159 (2009) 265–270

Contents lists available at ScienceDirect

Journal of Virological Methods

journa l homepage: www.e lsev ier .com/ locate / jv i romet

Impact of hepatitis B virus genotypes and surface antigen variants on theperformance of HBV real time PCR quantification

Vincent Thibaulta,∗, Syria Lapercheb, Sepideh Akhavana, Annabelle Servant-Delmasb,Dalila Belkhiri c, Anne-Marie Roque-Afonsoc,d,e

a Laboratoire de Virologie, AP-HP Hôpital de la Pitié-Salpêtrière, Paris, Franceb Centre National de Référence pour les Hépatites B et C en Transfusion, Institut National de la Transfusion Sanguine, Paris, Francec AP-HP Hôpital Paul Brousse, Virologie, Villejuif, Franced Inserm, Unité 785, Villejuif, Francee Université Paris-Sud, UMR-S 785, Villejuif, France

Article history:Received 26 February 2009Received in revised form 10 April 2009Accepted 20 April 2009Available online 3 May 2009

Keywords:Hepatitis B virusGenotypes

a b s t r a c t

Quantitative PCR assays used to monitor hepatitis B virus (HBV) load differ in their ability to detectdifferent HBV variants. This study evaluated the performance of the Abbott RT PCR assay for quantitatingDNA from different HBV genotypes and from HBV variants bearing HBsAg gene mutations. The study wasperformed on a randomly-selected sample with a viral load >6 log IU/mL for each genotype and on 25HBsAg variants. Each sample was assayed using the Abbott RT assay and with the Roche Cobas AmpliPrep-Cobas TaqMan as a reference method. All HBV genotypes were detected with the Abbott RT assay with anequivalent dynamic range (1–8 log IU/mL). For each genotype, the data suggest that the assay was linearover the entire dilution range (r2: 0.985–0.995). For the 25 HBsAg variants, viral titres determined withthe two assays correlated well (r2: 0.929). The mean difference between the two methods was −0.295

Quantitative PCR

Monitoring (95% CI: −0.520 to −0.071). The difference was lower than 1 log unit in all but two cases. In conclusion, thect andutine

1

ftwaD2siiav0iuh(a

0d

Abbott RT assay can deteand is thus suitable for ro

. Introduction

The hepatitis B virus (HBV) is responsible for one of the mostrequent chronic viral infectious diseases. According to estimates ofhe World Health Organisation, around 350 million people world-ide carry chronic HBV infections and these are responsible for

round one million deaths per year (World Health Organisation andepartment of Communicable Diseases Surveillance and Response,002). The prevalence of chronic hepatitis B infection varies con-iderably around the world, being highest (>8%) in East Asia wheret is endemic, and low in North-Western Europe. Recent stud-es of the prevalence of chronic hepatitis B in France (Meffre etl., 2007) and in England and Wales (Gay et al., 1999) have pro-ided prevalence rates in the adult general population of 0.65% and.4%, respectively. In North-Western Europe, the principal route of

nfection is blood contact, notably from needle-sharing and sex-

al encounter, although immigration of infected individuals fromigh prevalence areas contributes to the pool of chronic infectionDiel et al., 2005; Hahné et al., 2004). Chronic hepatitis B infectionsre associated with significantly increased morbidity and mortality

∗ Corresponding author. Tel.: +33 1 42 17 74 26; fax: +33 1 42 17 74 11.E-mail address: vincent.thibault@psl.aphp.fr (V. Thibault).

166-0934/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.jviromet.2009.04.014

quantify DNA from different HBV variants with equivalent performancemonitoring of patients with chronic HBV infections.

© 2009 Elsevier B.V. All rights reserved.

(Lok and McMahon, 2001) and are a major risk factor for hepato-cellular carcinoma (Chen et al., 2006). A number of genetic variantsof HBV have been identified, which differ in their immunogenicity,viability, virulence or treatment-responsiveness (Buti et al., 2005;Chu and Lok, 2002; Zuckerman and Zuckerman, 2003).

As the most effective way to reduce infection is prevention,control of this infection now relies on worldwide infant vaccina-tion programmes (Lavanchy, 2004). In addition, the introductionof antiviral drugs has had a major impact on the management ofpatients with chronic hepatitis B infections (Zoulim, 2006). Thesedrugs inhibit viral replication at different levels and thus reduceviral load below a clinically relevant threshold, leading in somecases to clearance of HBsAg from the organism. Antiviral therapyfor chronic hepatitis B infections presents two major limitations.Firstly, its limited, if any, capacity to inhibit transcription of super-coiled covalently-closed circular viral DNA (cccDNA) means thattreatment needs to be given for long periods of time (Zoulim,2005). Secondly, resistant HBV strains can emerge due to selectionpressures on the virus population, associated with inefficient proof-

reading activity of the polymerase (Yuan and Lee, 2007; Zoulim,2006). Consequently, the efficacy of both vaccination and antivi-ral therapy are limited. Indeed, selection of vaccine or hepatitis Bimmunoglobulin immune escape variants has been described in thecontext of vaccination programmes in endemic countries (Fujii et

2 logica

aaoouie

otsdgP(orid

ogvfR

2

2

v(ptg(tp26s

itTbr

TC

D

123345567789

N

66 V. Thibault et al. / Journal of Viro

l., 1992), and of liver transplantation in patients with chronic hep-titis B (Protzer-Knolle et al., 1998), as well as in the natural coursef the disease (Zhang et al., 1996). Consequences of the emergencef these variants range from viral breakthrough and treatment fail-re with potentially severe clinical outcomes, to lack of detection of

ndividual variants either at the genomic or the protein level (Lindht al., 2006; Ly et al., 2006).

Given these limitations, it is important to monitor treatmentutcome during the course of antiviral therapy using appropriateools (Gish and Locarnini, 2006; Lok et al., 2007). A number of assayystems have been developed for quantitation of HBV DNA, whichiffer in their sensitivity, dynamic range and specificity towardsenomic variants (Pawlotsky et al., 2008). Assays using real timeCR amplification are considered to present the best performanceLaperche et al., 2006). The principal performance characteristicsf the Abbott HBV RealTime M2000sp/rt assay have been reportedecently (Thibault et al., 2007). Given the inherent genetic variabil-ty of the hepatitis B virus, it is important that assay methods canetect different HBV variants accurately and indiscriminately.

The objectives of this study were to evaluate the performancef the Abbott RT assay for quantifying DNA from different HBVenotypes and to determine its ability to detect and quantify HBVariants bearing mutations in the sequence of the open readingrame of the HBsAg gene close to the target sequence for the AbbottT assay.

. Materials and methods

.1. Study samples

Seven samples containing HBV from each genotype (A–G) and airal load above the upper limit of quantitation with the CAP–CTM110,000,000 IU/mL) were selected at random from a cohort of 1300atients with chronic hepatitis B addressed to the La Pitié Hospi-al Virology laboratory for measurement of HBV viral load. HBVenotyping was performed using a previously validated methodBenhamou et al., 2001) based on specific amplification of part ofhe polymerase gene, sequencing of the amplified product and com-arison to reference sequences for each genotype (Stuyver et al.,001). For each genotype A–G, one sample with a viral load abovelog IU/mL was selected at random for assay using the Abbott RT

ystem.Twenty-five HBsAg variants were selected from chronically

nfected patients from two French HBV reference virology labora-ories (Hôpital Paul Brousse in Villejuif and the Institut National deransfusion Sanguine in Paris). HBsAg gene variants were identifiedy specific sequencing of the gene encoding the major hydrophilicegion of HBsAg.

able 1oefficients of variation for triplicate HBV DNA measures using the Abbott RT assay.

Coefficients of variation

ilution (log) A B C

.699 1.033 0.644 0.743

.398 NA NA NA

.097 0.909 0.217 0.295

.796 NA NA NA

.495 0.656 2.177 0.766

.194 2.447 3.356 0.634

.893 0.437 2.648 2.924

.592 0.707 5.737 4.962

.291 2.177 4.684 5.527

.990 8.143 11.558 86.603

.689 173.205 2.725 173.205

.388 ND 86.975 ND

D: not detectable; NA: not assayed. The grey cells indicate determinations falling below

l Methods 159 (2009) 265–270

2.2. Sample handling

All samples were stored at −80 ◦C until assayed. For the dilutionexperiments comparing the different HBV genotypes, one plasmasample from each genotype was selected and then diluted seri-ally (1/5) into pooled plasmas obtained from HIV, HBV and HCVseronegative patients. Serial dilutions were performed on eachsample to below the lower limit of detection of the Abbott assay(10 IU/mL). For the comparison of HBsAg gene variants, sampleswere used without dilution unless the residual volume was insuf-ficient, in which cases they were diluted 1/10 before assay.

2.3. Quantitative PCR detection of HBV DNA

All samples were evaluated prospectively for HBV DNA titrewhen they were first referred to the virology centre using a ref-erence quantitative PCR method, the CAP–CTM (Roche, Meylan,France) (Hochberger et al., 2006). The lower limit of detection of thismethod was 12 IU/mL and the lower limit of quantitation 54 IU/mL.The samples were retested for the purposes of the present studyusing the Abbott RT (Abbott, Les Ulis, France) (Thibault et al., 2007).This assay has a lower limit of quantitation identical to its lowerlimit of detection of 10 IU/mL. Both assays were performed in thesame centre using the manufacturers’ recommended standard pro-tocols. Samples were referenced under a blinded code so that HBVDNA levels determined with the CAP–CTM assay were unknownwhen retested. All samples were assayed in triplicate during thesame experiment.

3. Results

3.1. Study samples

3.1.1. HBV genotypesAll HBV genotypes were detected with the Abbott RT assay. Serial

dilutions of high HBV titre plasma samples within a 10 log unit rangedemonstrated the assay to be linear over the entire dilution rangefor all seven genotypes (Fig. 1). The correlation coefficients for thedifferent genotypes ranged from 0.985 to 0.995. The dynamic rangeof the assay was equivalent for all genotypes and correspondedto 1–8 log IU/mL. Irrespective of genotype, the coefficient of vari-ation between the triplicate determinations remained low (<5%)down to dilutions of 5.893 log units (>750,000-fold), after which itstarted to rise progressively, notably when DNA levels were below

the quantitation limit of the assay (Table 1).

3.1.2. Detection of HBsAg gene variantsThe performance of the Abbott RT assay at detecting HBV DNA

from viral strains bearing HBsAg gene mutations was compared

D E F G

0.326 0.566 1.296 1.3311.465 NA NA NA1.820 1.062 1.074 4.764NA NA 2.377 2.5014.070 1.082 2.156 2.4511.936 2.075 2.438 2.3032.568 2.106 2.467 3.6396.633 4.643 3.456 4.32111.160 8.501 5.654 11.47044.494 5.484 19.578 39.729NA 93.017 ND NDNA ND NA NA

the quantification limit of the assay (10 IU/mL).

V. Thibault et al. / Journal of Virological Methods 159 (2009) 265–270 267

F andom8 cation

wmrwwg

ig. 1. Serial dilution curves for HBV DNA quantified with the Abbott RT assay. R.14 log IU/mL for genotypes A–G, respectively, were diluted serially before quantifi

ith that of the CAP–CTM assay. Twenty-five samples with specific

utations within the HBsAg encoding gene were provided by two

eference laboratories (Roque-Afonso et al., 2007). These samplesere obtained from chronically infected patients and all mutationsere characterised after direct sequencing of the HBsAg gene. The

enotype of all samples and the corresponding amino acid changes

ly selected samples with high viral loads of 8.50, 9.46, 8.24, 8.19, 8.57, 8.76 and.

are listed in Table 2. The correlation between viral titres measured

with the two methods is presented in Fig. 2.

The correlation coefficient determined from linear regressionanalysis of these data was 0.929. The mean difference (Abbott RTvalue—CAP–CTM value) between the two methods was −0.295 (95%CI: −0.520 to −0.071), with a corresponding root mean square of the

268 V. Thibault et al. / Journal of Virological Methods 159 (2009) 265–270

Table 2Viral titres of HBsAg gene variants measured with the CAP–CTM and Abbott methods.

Sample Genotype Amino acid change CAP–CTM log IU/mL ABBOTT log IU/mL Difference

PB V1 D F/Y134H P142L D144E G145R 3.712 2.613 1.099PB V2 A T118V P120Q T126A D144G G145R 2.205 2.623 −0.418PB V3 E G145A 3.974 3.424 0.550PB V4 G/D G112A M133T T140I Y/F161H 3.541 3.281 0.260PB V6 D S143L 2.428 2.699 −0.271PB V7 G P108H S154L 6.735 5.736 0.999PB V8 A G145A E164D 8.343 6.985 1.358PB V9 A P142S G145R 2.617 2.146 0.471PB V10 D Q129H G130E M133T 7.383 6.765 0.618PB V11 D P120L T123N D144G 4.292 4.113 0.179PB V12 D G145R 5.239 4.955 0.284PB V13 D F/Y134N 5.627 5.192 0.435PB V14 E D144E 6.818 6.434 0.384PB V15 D F/Y134N 7.720 7.227 0.493PB V16 E A129V G130R 7.296 6.497 0.799PB V17 F S143L 4.950 5.906 −0.956PB V18 C T126N 7.077 6.144 0.933PB V19 D E164G 6.891 7.24 −0.349PB V20 D C121Y R/K122L T123N G130E M133I D144G G145R 3.521 2.865 0.656INTS V21 D M133I 3.408 3.179 0.230INTS V22 E D144E 3.855 3.676 0.179INTS V23 B G130R 2.602 2.566 0.036III

dbta

4

Awa

FHbT

NTS V24 D T118ANTS V25 C I126NNTS V26 D T131I

ifference of 0.588. The difference between the titres determinedy the two assays was lower than 1 log unit in all cases except forwo patients. Due to the large sample volume needed to run eachssay, insufficient material remained to permit accurate retesting.

. Discussion

This study demonstrated the satisfactory performance of thebbott RT assay at quantifying viral DNA from patients infectedith HBV strains corresponding to different genotypes and surface

ntigen variants. In a previous characterisation of the performance

ig. 2. Quantification of HBV DNA in plasma samples from 26 patients infected withBV strains bearing mutations in the gene encoding the HBs antigen. Correlationetween measures made using the Abbott RT (Y-axis) and CAP–CTM (X-axis) assays.he best-fit correlation (solid line) is shown (y = 0.8599x + 0.3069; r2 = 0.8952).

2.966 3.026 −0.0602.079 2.597 −0.5174.061 4.068 −0.008

of the Abbott RT assay (Thibault et al., 2007), HBV DNA levels mea-sured with this assay correlated closely with DNA levels obtainedwith the Versant branched-chain DNA assay or the CAP/CTM realtime PCR assay. The present results indicate that the dynamic rangeand linearity of the assay are equivalent across different genotypes,including the F genotype which was under-represented in the pre-vious study. In this respect, the Abbott RT assay is similar to theCAP–CTM assay, which also does not discriminate between HBVgenotypes (Chevaliez et al., 2008; Weiss et al., 2004).

The Abbott RT assay offers some advantages over the CAP–CTMassay in terms of a lower limit of detection and a wider dynamicrange. Although the clinical impact of improved quantitation at thelower limit remains to be demonstrated, recent consensus guide-lines on virological monitoring of HBV treatments recommend thata lower limit of detection of the order of 10 IU/mL may be required,to ensure detection of the emergence of resistance as early as pos-sible (Pawlotsky et al., 2008).

Since the target DNA sequence of the Abbott RT assay lies withinthe open reading frame of the HBsAg gene, it was important toensure that the performance of the assay was not compromisedby mutations in this gene. Such mutations usually arise in thea-determinant sequence, which encodes a highly hydrophilic pep-tide sequence that is critical for the immunogenicity of the surfaceprotein encoded by the HBsAg gene (Zuckerman and Zuckerman,2003). For this reason, infections with strains of HBV bearing HBsAgmutations may be immunosilent (Carman, 1997) and can only bedetected by measuring viral DNA (Scheiblauer et al., 2006). Theseresults suggest that HBsAg variants have little, if any, impact onAbbott HBV DNA quantitation. Indeed, a good overall correlationwas observed between the two assay methods and all tested vari-ants were correctly detected. In this respect, the present findingson S gene variants are comparable to those reported previously, andto those recently published by Ciotti et al. (2008) who reported amean difference of −0.246 log IU/mL between the two assays whentested on unselected chronic carriers. Some variability was however

observed for some variants, when comparing the Abbott RT assayto the CAP–CTM assay, which targets a different 105-bp sequencein the precore–core region of the HBV genome; the source of thisvariation is unexplained and may deserve further investigation. Itis noteworthy that, even though both assays are supposed to be

logica

cltIlahigntootLdtar(fieocbiattc

sgdlrer

2attt

A

AaGrVP

R

B

B

C

C

V. Thibault et al. / Journal of Viro

alibrated against the WHO standard in IU, a higher quantitationevel is always obtained with the assay from Roche compared tohe one from Abbott, independently of the presence of mutations.t is probable that the target sequence of the Abbott RT assay, whichies in a highly-conserved sequence upstream of the a-determinantnd downstream of the pre-S region, is sufficiently remote fromotspots for HBsAg gene mutations, notably sG145R, to preclude

nterference with the assay. Moreover, the Abbott RT assay tar-ets a sequence in the N-terminal part of the S gene, which doesot tolerate deletions, in contrast to the pre-S region where dele-ions are often found (Chen et al., 2007). As for any assay basedn specific hybridisation of nucleotide sequences, the emergencef a specific variant that could interfere with the performance ofhe assay cannot be excluded. A case in point was reported byindh et al. (2006), who described the emergence under lamivu-ine therapy of a mismatch within the probe hybridisation regionhat led to dramatic underquantitation of HBV DNA. A remark-ble mismatch on a genotype D strain located in the 3′ end of theeverse primer of the Cobas Amplicor assay has also been identifiedThibault, unpublished results) which, fortunately, could be quanti-ed correctly with the Cobas Taqman assay. As has been pointed outlsewhere (Roque-Afonso et al., 2007; Sloan et al., 2008), emergencef variants should be considered as a natural phenomenon in theourse of chronic hepatitis B infections, which may be facilitatedy any therapeutic intervention. It is noteworthy that experiencen lamivudine-resistant patients, carrying HBV strains with aminocid changes commonly associated with lamivudine-resistance andreated with an adefovir–lamivudine combination, also indicatehat the performance of the Abbott RT assay is not affected byommon resistance mutations (data not shown).

While some genomic regions seem more affected than others byuch variations, it may be wise to resort to a second-line assay tar-eting a different region of the genome whenever an unexplainediscrepancy between clinical manifestations and measured viral

oad occurs. Faced with this kind of unpredictable event, it is theesponsibility of virologists to make use of all tools available tonsure correct quantitation of HBV DNA and to be vigilant withespect to the emergence of any new variant.

In conclusion, and in support of previous findings (Thibault et al.,007), the Abbott RT real time PCR amplification assay can detectnd quantify HBV DNA from different genetic variants of the hepati-is B virus with equivalent performance. It is therefore suitable forhe routine monitoring of patients with chronic hepatitis B infec-ions treated with antiviral drugs.

cknowledgements

This work was supported in part by a grant from the Nationalgency for Research against AIDS and Hepatitis (ANRS) to VT. Theuthors wish to express their gratitude to Sven Thamm (Abbott,ermany) and Eric Gaillard (Abbott, France) for providing the

eagents and for many helpful discussions, as well as to C. Aymé,. Boutonnet and N. Hamm (Laboratoire de Virologie, Hôpital de laitié-Salpêtrière) for their technical expertise.

eferences

enhamou, Y., Bochet, M., Thibault, V., Calvez, V., Fievet, M.H., Vig, P., Gibbs, C.S.,Brosgart, C., Fry, J., Namini, H., Katlama, C., Poynard, T., 2001. Safety and efficacyof adefovir dipivoxil in patients co-infected with HIV-1 and lamivudine-resistanthepatitis B virus: an open-label pilot study. Lancet 358, 718–723.

uti, M., Rodriguez-Frias, F., Jardi, R., Esteban, R., 2005. Hepatitis B virus genome

variability and disease progression: the impact of pre-core mutants and HBVgenotypes. J. Clin. Virol. 34 (Suppl. 1), S79–82.

arman, W.F., 1997. The clinical significance of surface antigen variants of hepatitisB virus. J. Viral Hepat. 4 (Suppl. 1), 11–20.

hen, C.H., Hung, C.H., Lee, C.M., Hu, T.H., Wang, J.H., Wang, J.C., Lu, S.N., Changchien,C.S., 2007. Pre-S deletion and complex mutations of hepatitis B virus related

l Methods 159 (2009) 265–270 269

to advanced liver disease in HBeAg-negative patients. Gastroenterology 133,1466–1474.

Chen, C.J., Yang, H.I., Su, J., Jen, C.L., You, S.L., Lu, S.N., Huang, G.T., Iloeje, U.H., 2006.Risk of hepatocellular carcinoma across a biological gradient of serum hepatitisB virus DNA level. JAMA 295, 65–73.

Chevaliez, S., Bouvier-Alias, M., Laperche, S., Pawlotsky, J.M., 2008. Performance ofthe Cobas AmpliPrep/Cobas TaqMan real-time PCR assay for hepatitis B virusDNA quantification. J. Clin. Microbiol. 46, 1716–1723.

Chu, C.J., Lok, A.S., 2002. Clinical significance of hepatitis B virus genotypes. Hepa-tology 35, 1274–1276.

Ciotti, M., Marcuccilli, F., Guenci, T., Prignano, M.G., Perno, C.F., 2008. Evaluation of theAbbott RealTime HBV DNA assay and comparison to the Cobas AmpliPrep/CobasTaqMan 48 assay in monitoring patients with chronic cases of hepatitis B. J. Clin.Microbiol. 46, 1517–1519.

Diel, R., Helle, J., Gottschalk, R., 2005. Transmission of hepatitis B in Hamburg,Germany, 1998–2002: a prospective, population-based study. Med. Microbiol.Immunol. 194, 193–199.

Fujii, H., Moriyama, K., Sakamoto, N., Kondo, T., Yasuda, K., Hiraizumi, Y., Yamazaki,M., Sakaki, Y., Okochi, K., Nakajima, E., 1992. Gly145 to Arg substitution in HBsantigen of immune escape mutant of hepatitis B virus. Biochem. Biophys. Res.Commun. 184, 1152–1157.

Gay, N.J., Hesketh, L.M., Osborne, K.P., Farrington, C.P., Morgan-Capner, P., Miller, E.,1999. The prevalence of hepatitis B infection in adults in England and Wales.Epidemiol. Infect. 122, 133–138.

Gish, R.G., Locarnini, S.A., 2006. Chronic hepatitis B: current testing strategies. Clin.Gastroenterol. Hepatol. 4, 666–676.

Hahné, S., Ramsay, M., Balogun, K., Edmunds, W.J., Mortimer, P., 2004. Incidence androutes of transmission of hepatitis B virus in England and Wales, 1995–2000:implications for immunisation policy. J. Clin. Virol. 29, 211–220.

Hochberger, S., Althof, D., Gallegos de Schrott, R., Nachbaur, N., Rock, H., Ley-ing, H., 2006. Fully automated quantitation of hepatitis B virus (HBV) DNA inhuman plasma by the COBAS AmpliPrep/COBAS TaqMan system. J. Clin. Virol.35, 373–380.

Laperche, S., Thibault, V., Bouchardeau, F., Alain, S., Castelain, S., Gassin, M., Gueudin,M., Halfon, P., Larrat, S., Lunel, F., Martinot-Peignoux, M., Mercier, B., Pawlotsky,J.M., Pozzetto, B., Roque-Afonso, A.M., Roudot-Thoraval, F., Saune, K., Lefrere, J.J.,2006. Expertise of laboratories in viral load quantification, genotyping, and pre-core mutant determination for hepatitis B virus in a multicenter study. J. Clin.Microbiol. 44, 3600–3607.

Lavanchy, D., 2004. Hepatitis B virus epidemiology, disease burden, treatment,and current and emerging prevention and control measures. J. Viral Hepat. 11,97–107.

Lindh, M., Hannoun, C., Malmstrom, S., Lindberg, J., Norkrans, G., 2006. Lamivudineresistance of hepatitis B virus masked by coemergence of mutations in proberegion of the COBAS AMPLICOR assay. J. Clin. Microbiol. 44, 2587–2589.

Lok, A.S., McMahon, B.J., 2001. Chronic hepatitis B. Hepatology 34, 1225–1241.Lok, A.S., Zoulim, F., Locarnini, S., Bartholomeusz, A., Ghany, M.G., Pawlotsky, J.M.,

Liaw, Y.F., Mizokami, M., Kuiken, C., 2007. Antiviral drug-resistant HBV: stan-dardization of nomenclature and assays and recommendations for management.Hepatology 46, 254–265.

Ly, T.D., Servant-Delmas, A., Bagot, S., Gonzalo, S., Ferey, M.P., Ebel, A., Dussaix, E.,Laperche, S., Roque-Afonso, A.M., 2006. Sensitivities of four new commercialhepatitis B virus surface antigen (HBsAg) assays in detection of HBsAg mutantforms. J. Clin. Microbiol. 44, 2321–2326.

Meffre, C., Le Strat, Y., Delarocque-Astagneau, E., Antona, D., Desenclos, J., 2007. Pré-valence des hépatites B et C en France en 2004. Institut de Veille Sanitaire, SaintMaurice.

Pawlotsky, J.M., Dusheiko, G., Hatzakis, A., Lau, D., Lau, G., Liang, T.J., Locarnini, S.,Martin, P., Richman, D.D., Zoulim, F., 2008. Virologic monitoring of hepatitis Bvirus therapy in clinical trials and practice: recommendations for a standardizedapproach. Gastroenterology 134, 405–415.

Protzer-Knolle, U., Naumann, U., Bartenschlager, R., Berg, T., Hopf, U., Meyer zumBuschenfelde, K.H., Neuhaus, P., Gerken, G., 1998. Hepatitis B virus with anti-genically altered hepatitis B surface antigen is selected by high-dose hepatitis Bimmune globulin after liver transplantation. Hepatology 27, 254–263.

Roque-Afonso, A.M., Ferey, M.P., Ly, T.D., Graube, A., Costa-Faria, L., Samuel, D., Dus-saix, E., 2007. Viral and clinical factors associated with surface gene variantsamong hepatitis B virus carriers. Antivir. Ther. 12, 1255–1263.

Scheiblauer, H., Soboll, H., Nick, S., 2006. Evaluation of 17 CE-marked HBsAg assayswith respect to clinical sensitivity, analytical sensitivity, and hepatitis B virusmutant detection. J. Med. Virol. 78 (Suppl. 1), S66–70.

Sloan, R.D., Ijaz, S., Moore, P.L., Harrison, T.J., Teo, C.G., Tedder, R.S., 2008. Antivi-ral resistance mutations potentiate hepatitis B virus immune evasion throughdisruption of its surface antigen a determinant. Antivir. Ther. 13, 439–447.

Stuyver, L.J., Locarnini, S.A., Lok, A., Richman, D.D., Carman, W.F., Dienstag, J.L., Schi-nazi, R.F., 2001. Nomenclature for antiviral-resistant human hepatitis B virusmutations in the polymerase region. Hepatology 33, 751–757.

Thibault, V., Pichoud, C., Mullen, C., Rhoads, J., Smith, J.B., Bitbol, A., Thamm, S.,Zoulim, F., 2007. Characterization of a new sensitive PCR assay for quantifica-tion of viral DNA isolated from patients with hepatitis B virus infections. J. Clin.

Microbiol. 45, 3948–3953.

Weiss, J., Wu, H., Farrenkopf, B., Schultz, T., Song, G., Shah, S., Siegel, J., 2004. Realtime TaqMan PCR detection and quantitation of HBV genotypes A–G with theuse of an internal quantitation standard. J. Clin. Virol. 30, 86–93.

World Health Organisation and Department of Communicable Diseases Surveillanceand Response, 2002. Hepatitis B, World Health Organisation, Geneva.

2 logica

Y

Z

70 V. Thibault et al. / Journal of Viro

uan, H.J., Lee, W.M., 2007. Molecular mechanisms of resistance to antiviral therapyin patients with chronic hepatitis B. Curr. Mol. Med. 7, 185–197.

hang, Y.Y., Nordenfelt, E., Hansson, B.G., 1996. Increasing heterogeneity of the ‘a’determinant of HBsAg found in the presumed late phase of chronic hepatitis Bvirus infection. Scand. J. Infect. Dis. 28, 9–15.

l Methods 159 (2009) 265–270

Zoulim, F., 2005. New insight on hepatitis B virus persistence from the study ofintrahepatic viral cccDNA. J. Hepatol. 42, 302–308.

Zoulim, F., 2006. Antiviral therapy of chronic hepatitis B. Antiviral Res. 71, 206–215.Zuckerman, J.N., Zuckerman, A.J., 2003. Mutations of the surface protein of hepatitis

B virus. Antiviral Res. 60, 75–78.