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Journal of Virological Methods
j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j v i r o m e t
Impact of hepatitis B virus genotypes and surface antigen variants on the performance of HBV real time PCR quantification
Vincent Thibault
a,∗, Syria Laperche
b, Sepideh Akhavan
a, Annabelle Servant-Delmas
b, Dalila Belkhiri
c, Anne-Marie Roque-Afonso
c,d,eaLaboratoire de Virologie, AP-HP Hôpital de la Pitié-Salpêtrière, Paris, France
bCentre National de Référence pour les Hépatites B et C en Transfusion, Institut National de la Transfusion Sanguine, Paris, France
cAP-HP Hôpital Paul Brousse, Virologie, Villejuif, France
dInserm, Unité 785, Villejuif, France
eUniversité Paris-Sud, UMR-S 785, Villejuif, France
Article history:
Received 26 February 2009
Received in revised form 10 April 2009 Accepted 20 April 2009
Available online 3 May 2009
Keywords:
Hepatitis B virus Genotypes Quantitative PCR Monitoring
a b s t r a c t
Quantitative PCR assays used to monitor hepatitis B virus (HBV) load differ in their ability to detect different HBV variants. This study evaluated the performance of the Abbott RT PCR assay for quantitating DNA from different HBV genotypes and from HBV variants bearing HBsAg gene mutations. The study was performed on a randomly-selected sample with a viral load >6 log IU/mL for each genotype and on 25 HBsAg 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 an equivalent dynamic range (1–8 log IU/mL). For each genotype, the data suggest that the assay was linear over the entire dilution range (r2: 0.985–0.995). For the 25 HBsAg variants, viral titres determined with the two assays correlated well (r2: 0.929). The mean difference between the two methods was−0.295 (95% CI:−0.520 to−0.071). The difference was lower than 1 log unit in all but two cases. In conclusion, the Abbott RT assay can detect and quantify DNA from different HBV variants with equivalent performance and is thus suitable for routine monitoring of patients with chronic HBV infections.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
The hepatitis B virus (HBV) is responsible for one of the most frequent chronic viral infectious diseases. According to estimates of the World Health Organisation, around 350 million people world- wide carry chronic HBV infections and these are responsible for around one million deaths per year (World Health Organisation and Department of Communicable Diseases Surveillance and Response, 2002). The prevalence of chronic hepatitis B infection varies con- siderably around the world, being highest (>8%) in East Asia where it is endemic, and low in North-Western Europe. Recent stud- ies of the prevalence of chronic hepatitis B in France (Meffre et al., 2007) and in England and Wales (Gay et al., 1999) have pro- vided prevalence rates in the adult general population of 0.65% and 0.4%, respectively. In North-Western Europe, the principal route of infection is blood contact, notably from needle-sharing and sex- ual encounter, although immigration of infected individuals from high prevalence areas contributes to the pool of chronic infection (Diel et al., 2005; Hahné et al., 2004). Chronic hepatitis B infections are 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).
(Lok and McMahon, 2001) and are a major risk factor for hepato- cellular carcinoma (Chen et al., 2006). A number of genetic variants of 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 introduction of antiviral drugs has had a major impact on the management of patients with chronic hepatitis B infections (Zoulim, 2006). These drugs inhibit viral replication at different levels and thus reduce viral load below a clinically relevant threshold, leading in some cases to clearance of HBsAg from the organism. Antiviral therapy for 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 that treatment needs to be given for long periods of time (Zoulim, 2005). Secondly, resistant HBV strains can emerge due to selection pressures 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 B immunoglobulin immune escape variants has been described in the context of vaccination programmes in endemic countries (Fujii et 0166-0934/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.jviromet.2009.04.014
tools (Gish and Locarnini, 2006; Lok et al., 2007). A number of assay systems have been developed for quantitation of HBV DNA, which differ in their sensitivity, dynamic range and specificity towards genomic variants (Pawlotsky et al., 2008). Assays using real time PCR amplification are considered to present the best performance (Laperche et al., 2006). The principal performance characteristics of the Abbott HBV RealTime M2000sp/rt assay have been reported recently (Thibault et al., 2007). Given the inherent genetic variabil- ity of the hepatitis B virus, it is important that assay methods can detect different HBV variants accurately and indiscriminately.
The objectives of this study were to evaluate the performance of the Abbott RT assay for quantifying DNA from different HBV genotypes and to determine its ability to detect and quantify HBV variants bearing mutations in the sequence of the open reading frame of the HBsAg gene close to the target sequence for the Abbott RT assay.
2. Materials and methods
2.1. Study samples
Seven samples containing HBV from each genotype (A–G) and a viral load above the upper limit of quantitation with the CAP–CTM (110,000,000 IU/mL) were selected at random from a cohort of 1300 patients with chronic hepatitis B addressed to the La Pitié Hospi- tal Virology laboratory for measurement of HBV viral load. HBV genotyping was performed using a previously validated method (Benhamou et al., 2001) based on specific amplification of part of the polymerase gene, sequencing of the amplified product and com- parison to reference sequences for each genotype (Stuyver et al., 2001). For each genotype A–G, one sample with a viral load above 6 log IU/mL was selected at random for assay using the Abbott RT system.
Twenty-five HBsAg variants were selected from chronically infected patients from two French HBV reference virology labora- tories (Hôpital Paul Brousse in Villejuif and the Institut National de Transfusion Sanguine in Paris). HBsAg gene variants were identified by specific sequencing of the gene encoding the major hydrophilic region of HBsAg.
were 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 titre when 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 this method was 12 IU/mL and the lower limit of quantitation 54 IU/mL.
The samples were retested for the purposes of the present study using the Abbott RT (Abbott, Les Ulis, France) (Thibault et al., 2007).
This assay has a lower limit of quantitation identical to its lower limit of detection of 10 IU/mL. Both assays were performed in the same centre using the manufacturers’ recommended standard pro- tocols. Samples were referenced under a blinded code so that HBV DNA levels determined with the CAP–CTM assay were unknown when retested. All samples were assayed in triplicate during the same experiment.
3. Results
3.1. Study samples 3.1.1. HBV genotypes
All HBV genotypes were detected with the Abbott RT assay. Serial dilutions of high HBV titre plasma samples within a 10 log unit range demonstrated the assay to be linear over the entire dilution range for all seven genotypes (Fig. 1). The correlation coefficients for the different genotypes ranged from 0.985 to 0.995. The dynamic range of the assay was equivalent for all genotypes and corresponded to 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 it started to rise progressively, notably when DNA levels were below the quantitation limit of the assay (Table 1).
3.1.2. Detection of HBsAg gene variants
The performance of the Abbott RT assay at detecting HBV DNA from viral strains bearing HBsAg gene mutations was compared
Table 1
Coefficients of variation for triplicate HBV DNA measures using the Abbott RT assay.
Coefficients of variation
Dilution (log) A B C D E F G
1.699 1.033 0.644 0.743 0.326 0.566 1.296 1.331
2.398 NA NA NA 1.465 NA NA NA
3.097 0.909 0.217 0.295 1.820 1.062 1.074 4.764
3.796 NA NA NA NA NA 2.377 2.501
4.495 0.656 2.177 0.766 4.070 1.082 2.156 2.451
5.194 2.447 3.356 0.634 1.936 2.075 2.438 2.303
5.893 0.437 2.648 2.924 2.568 2.106 2.467 3.639
6.592 0.707 5.737 4.962 6.633 4.643 3.456 4.321
7.291 2.177 4.684 5.527 11.160 8.501 5.654 11.470
7.990 8.143 11.558 86.603 44.494 5.484 19.578 39.729
8.689 173.205 2.725 173.205 NA 93.017 ND ND
9.388 ND 86.975 ND NA ND NA NA
ND: not detectable; NA: not assayed. The grey cells indicate determinations falling below the quantification limit of the assay (10 IU/mL).
Fig. 1.Serial dilution curves for HBV DNA quantified with the Abbott RT assay. Randomly selected samples with high viral loads of 8.50, 9.46, 8.24, 8.19, 8.57, 8.76 and 8.14 log IU/mL for genotypes A–G, respectively, were diluted serially before quantification.
with that of the CAP–CTM assay. Twenty-five samples with specific mutations within the HBsAg encoding gene were provided by two reference laboratories (Roque-Afonso et al., 2007). These samples were obtained from chronically infected patients and all mutations were characterised after direct sequencing of the HBsAg gene. The genotype of all samples and the corresponding amino acid changes
are listed inTable 2. The correlation between viral titres measured with the two methods is presented inFig. 2.
The correlation coefficient determined from linear regression analysis of these data was 0.929. The mean difference (Abbott RT value—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
PB V9 A P142S G145R 2.617 2.146 0.471
PB V10 D Q129H G130E M133T 7.383 6.765 0.618
PB V11 D P120L T123N D144G 4.292 4.113 0.179
PB V12 D G145R 5.239 4.955 0.284
PB V13 D F/Y134N 5.627 5.192 0.435
PB V14 E D144E 6.818 6.434 0.384
PB V15 D F/Y134N 7.720 7.227 0.493
PB V16 E A129V G130R 7.296 6.497 0.799
PB V17 F S143L 4.950 5.906 −0.956
PB V18 C T126N 7.077 6.144 0.933
PB V19 D E164G 6.891 7.24 −0.349
PB V20 D C121Y R/K122L T123N G130E M133I D144G G145R 3.521 2.865 0.656
INTS V21 D M133I 3.408 3.179 0.230
INTS V22 E D144E 3.855 3.676 0.179
INTS V23 B G130R 2.602 2.566 0.036
INTS V24 D T118A 2.966 3.026 −0.060
INTS V25 C I126N 2.079 2.597 −0.517
INTS V26 D T131I 4.061 4.068 −0.008
difference of 0.588. The difference between the titres determined by the two assays was lower than 1 log unit in all cases except for two patients. Due to the large sample volume needed to run each assay, insufficient material remained to permit accurate retesting.
4. Discussion
This study demonstrated the satisfactory performance of the Abbott RT assay at quantifying viral DNA from patients infected with HBV strains corresponding to different genotypes and surface antigen variants. In a previous characterisation of the performance
Fig. 2.Quantification of HBV DNA in plasma samples from 26 patients infected with HBV strains bearing mutations in the gene encoding the HBs antigen. Correlation between measures made using the Abbott RT (Y-axis) and CAP–CTM (X-axis) assays.
The best-fit correlation (solid line) is shown (y= 0.8599x+ 0.3069;r2= 0.8952).
of the Abbott RT assay (Thibault et al., 2007), HBV DNA levels mea- sured with this assay correlated closely with DNA levels obtained with the Versant branched-chain DNA assay or the CAP/CTM real time PCR assay. The present results indicate that the dynamic range and 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 the CAP–CTM assay, which also does not discriminate between HBV genotypes (Chevaliez et al., 2008; Weiss et al., 2004).
The Abbott RT assay offers some advantages over the CAP–CTM assay in terms of a lower limit of detection and a wider dynamic range. Although the clinical impact of improved quantitation at the lower limit remains to be demonstrated, recent consensus guide- lines on virological monitoring of HBV treatments recommend that a 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 within the open reading frame of the HBsAg gene, it was important to ensure that the performance of the assay was not compromised by mutations in this gene. Such mutations usually arise in the a-determinant sequence, which encodes a highly hydrophilic pep- tide sequence that is critical for the immunogenicity of the surface protein encoded by the HBsAg gene (Zuckerman and Zuckerman, 2003). For this reason, infections with strains of HBV bearing HBsAg mutations may be immunosilent (Carman, 1997) and can only be detected by measuring viral DNA (Scheiblauer et al., 2006). These results suggest that HBsAg variants have little, if any, impact on Abbott HBV DNA quantitation. Indeed, a good overall correlation was observed between the two assay methods and all tested vari- ants were correctly detected. In this respect, the present findings on S gene variants are comparable to those reported previously, and to those recently published byCiotti et al. (2008)who reported a mean difference of−0.246 log IU/mL between the two assays when tested on unselected chronic carriers. Some variability was however observed for some variants, when comparing the Abbott RT assay to the CAP–CTM assay, which targets a different 105-bp sequence in the precore–core region of the HBV genome; the source of this variation is unexplained and may deserve further investigation. It is noteworthy that, even though both assays are supposed to be
calibrated against the WHO standard in IU, a higher quantitation level is always obtained with the assay from Roche compared to the one from Abbott, independently of the presence of mutations.
It is probable that the target sequence of the Abbott RT assay, which lies in a highly-conserved sequence upstream of the a-determinant and downstream of the pre-S region, is sufficiently remote from hotspots for HBsAg gene mutations, notably sG145R, to preclude interference with the assay. Moreover, the Abbott RT assay tar- gets a sequence in the N-terminal part of the S gene, which does not tolerate deletions, in contrast to the pre-S region where dele- tions are often found (Chen et al., 2007). As for any assay based on specific hybridisation of nucleotide sequences, the emergence of a specific variant that could interfere with the performance of the assay cannot be excluded. A case in point was reported by Lindh et al. (2006), who described the emergence under lamivu- dine therapy of a mismatch within the probe hybridisation region that led to dramatic underquantitation of HBV DNA. A remark- able mismatch on a genotype D strain located in the 3end of the reverse primer of the Cobas Amplicor assay has also been identified (Thibault, unpublished results) which, fortunately, could be quanti- fied correctly with the Cobas Taqman assay. As has been pointed out elsewhere (Roque-Afonso et al., 2007; Sloan et al., 2008), emergence of variants should be considered as a natural phenomenon in the course of chronic hepatitis B infections, which may be facilitated by any therapeutic intervention. It is noteworthy that experience in lamivudine-resistant patients, carrying HBV strains with amino acid changes commonly associated with lamivudine-resistance and treated with an adefovir–lamivudine combination, also indicate that the performance of the Abbott RT assay is not affected by common resistance mutations (data not shown).
While some genomic regions seem more affected than others by such variations, it may be wise to resort to a second-line assay tar- geting a different region of the genome whenever an unexplained discrepancy between clinical manifestations and measured viral load occurs. Faced with this kind of unpredictable event, it is the responsibility of virologists to make use of all tools available to ensure correct quantitation of HBV DNA and to be vigilant with respect to the emergence of any new variant.
In conclusion, and in support of previous findings (Thibault et al., 2007), the Abbott RT real time PCR amplification assay can detect and quantify HBV DNA from different genetic variants of the hepati- tis B virus with equivalent performance. It is therefore suitable for the routine monitoring of patients with chronic hepatitis B infec- tions treated with antiviral drugs.
Acknowledgements
This work was supported in part by a grant from the National Agency for Research against AIDS and Hepatitis (ANRS) to VT. The authors wish to express their gratitude to Sven Thamm (Abbott, Germany) and Eric Gaillard (Abbott, France) for providing the reagents and for many helpful discussions, as well as to C. Aymé, V. Boutonnet and N. Hamm (Laboratoire de Virologie, Hôpital de la Pitié-Salpêtrière) for their technical expertise.
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