Multicenter Evaluation of a Semiautomated, Standardized

Assay for Detection of Hepatitis B Virus DNA in Blood

Donations
Luisa Romanò,1 Claudio Velati,2* Lorella Baruffi,2 Laura Fomiatti,2 Giuseppe Colucci,3
Alessandro R. Zanetti,1 and the Italian Group for the Study of Transfusion Transmissible
Diseases

Institute of Virology, University of Milan, Milan,1 Transfusion Medicine and Hematology

Department, Hospital of Sondrio, Sondrio, Italy,2 Scientific Affairs, Roche Molecular Systems,
Rotkreuz, Switzerland3

Received 16 August 2004/ Returned for modification 14 November 2004/ Accepted 1 February
2005

ABSTRACT

Top
We evaluated the COBAS Ampliscreen hepatitis B virus (HBV) test using Abstract
standards, seroconversion panels, consecutive donations, and samples from Text
References
patients with abnormal alanine aminotransferase and chronic hepatitis C.
Specificity was 100% and sensitivity was 20 IU/ml. In seroconversion panels,
HBV DNA was detected up to 4 to 18 days before HBsAg, suggesting that this
assay is useful in shortening the infectious window phase.

TEXT

Top
Over the past decade, the risk of acquiring blood-borne viruses through
Abstract
transfusion has dramatically declined in industrialized countries. A residual risk Text
due to donations of blood during the window period (time from infection to References
reactivity by serological assays) still exists and can be shortened by using nucleic
acid testing (NAT) assays (1-3, 5, 7, 10, 12, 14, 17). Such assays are already
implemented in the United States and in most European countries for the detection of hepatitis C
virus (HCV) RNA and human immunodeficiency virus (HIV) RNA. For hepatitis B virus (HBV),
the estimated probability of having a potentially infectious donation released in the blood supply
is even higher than those expected for HCV and HIV. Hence, the introduction of HBV DNA
assays may be a useful tool in the quest of approaching zero risk (5, 6, 8, 15).

In this study we evaluated a semiautomated PCR test, COBAS Ampliscreen HBV (CAS HBV),
that allows for the detection of HBV DNA in minipools and makes use of the same extract that
can be processed with the respective HCV and HIV assays (16).

The analytical sensitivity of the CAS HBV was assessed using the HBV DNA nucleic acid panel
(NAP; Acrometrix, Benicia, CA) consisting of seven plasma, of which one was negative and six
were positive, with concentrations ranging from 2 x 102 to 2 x 107 IU/ml. Serial dilutions of the 2
x 102 IU/ml sample in HBV-negative human plasma were also tested in duplicate to define the
assay's detection limit.

Five seroconversion panels, PHM 929, PHM 932, PHM 925, PHM 928 (Boston Biomedical Inc.,
West Bridgewater, MA), and SB-0405 (NABI Biochemicals, Bocaraton, FL), comprising serial
plasma collected at close intervals during the seronegative window phase, were used to evaluate
the test's ability to detect HBV DNA. Each of these panels had been previously tested by the
manufacturers using different HBsAg assays.

To assess specificity, plasma samples were tested from individuals (n = 43) with isolated alanine
aminotransferase (ALT) elevation (>60 IU/liter), from patients (n = 23) with chronic hepatitis C,
and from repeat donors (n = 81) previously detected to be negative for both HBsAg and HBV
DNA by an in-house PCR. In addition, 9,547 plasma samples were collected from repeat
consecutive donors and analyzed in five different blood banks.

All specimens were processed according to the CAS HBV Multiprep Sample Processing
procedure, which includes assembling minipools of 24 samples by mixing 100 µl of the sample
under testing and 2.3 ml of HBV-negative human plasma. An aliquot of 1 ml was pelletted by
ultracentrifugation, and HBV DNA was manually extracted by chaotropic lysis. In some
experiments, to increase the test's sensitivity the total volume (2.4 ml) of the minipool was tested.

Extracted samples and controls were then processed for amplification and detection using the
automated system COBAS Amplicor, according to the manufacturer's instructions (4, 9).

The standard sample processing procedure, used for testing of individual samples (volume, 200
µl) according to the manufacturer's instructions, was further added when testing the
seroconversion panels.

All seven NAP samples (Acrometrix) were correctly identified. Analysis of serial dilutions of the
2 x 102 panel members tested in duplicate revealed a detection limit of 20 IU/ml with the
minipool procedure.

When assessing the results obtained on the five seroconversion panels (Table 1), HBV DNA was
positive 4 to 18 days (mean, 10 days) prior to the appearance of HBsAg with the single-sample
procedure and 0 to 11 days (mean, 3.7 days) with the minipool procedure (total volume, 1 ml). A
2-to 3-day shortening of the window phase was observed when samples were analyzed in a 2.4
ml final minipool volume instead of a 1-ml volume. Comparison was made with data reported by
the seroconversion panel manufacturers regarding the first HBsAg detection with the most
sensitive assay.

View this TABLE 1. Timing of detection of HBV DNA in seroconversion panels

table: using COBAS AmpliScreen (CAS HBV)
[in this
window]
[in a new
window]

The CAS HBV test showed 100% specificity, as no reactive samples were detected in samples
from 81 healthy donors and 66 patients with abnormal ALT or chronic hepatitis unrelated to
HBV. Finally, of the 9,547 consecutive donations, one resulted positive for both HBV DNA and
HBsAg.

In recent years a semiautomated PCR system, the COBAS Ampliscreen, was developed for the
detection of HIV and HCV in minipools.

In this study we evaluated the performance of the CAS HBV test, the last to be developed on this
platform for the detection of HBV DNA. Despite the increased sensitivity of serological HBsAg
tests, a residual risk of HBV transmission still exists, and the introduction of HBV NAT in blood
screening may be warranted. Using a real-time PCR-based assay, developed with the same
primer set included in the CAS HBV test, 76 (42%) HBV DNA-positive, HBsAg-negative
donations were detected in minipools of 50 (11). Upon investigation, all samples were traced
back to blood drawn during the seronegative window phases. Sato et al. (13) showed that an
HBsAg chemiluminescence immunoassay had lower sensitivity than a home-brewed nested PCR
with a threshold limit of 100 copies/ml. This value is similar to the 20 IU/ml (approximately 150
copies/ml) detection limit of the CAS HBV observed in our study. In seroconversion panels, the
CAS HBV allowed detection of an active infection on average of 10 and 3.7 days earlier than the
HBsAg test when used in the single-sample format and in the minipool format, respectively. A
certain additional shortening of the window phase was seen with the latter procedure when
samples were analyzed in a 2.4-ml final volume.

Of the 9,547 donations tested, only one was HBV DNA positive in the presence of HBsAg.
Absence of reactivities in the window phase (i.e., HBV DNA positive and HBsAg negative)
might be due to the limited sample size collected in an area where the HBV residual risk is very
small, approximately 13.9 per 106 donations (18).
The potential throughput and flexible configuration of this assay, which allows for different pool
size and sample volume, were appreciated by the participating centers, where its introduction did
not significantly increase the screening workload nor alter the laboratory workflow. CAS HBV
has, in fact, the advantage of using an aliquot of the same extract already obtained for the CAS
HCV and HIV tests, while the remaining steps of This Article
the procedure are performed in automation by the
Extract
COBAS Amplicor.
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Detection of Mutations in the PubMed

Hepatitis B Virus Polymerase PubMed Citation

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Harald H. Kessler1,a, Evelyn Stelzl1, Egon
Molecular Diagnostics and Genetics
Marth1 and Rudolf E. Stauber2
1
Institute of Hygiene, Karl-Franzens-University Graz, A-8010 Graz, Austria
2
Department of Internal Medicine, University Hospital Graz, A-8036 Graz, Austria

a
address correspondence to this author at: Molecular Diagnostics Laboratory, Institute of Hygiene, KF-University
Graz, Universitaetsplatz 4, A-8010 Graz, Austria; fax 43-316-380-9649, e-mail harald.kessler@uni-graz.at

Antiviral treatment of chronic hepatitis B infection aims to reduce viral replication and/or to
affect the immune response to the virus and virus-infected cells. The development of reverse
transcriptase inhibitors such as lamivudine, which has been shown to be a safe and potent
inhibitor of hepatitis B virus (HBV) replication (1)(2), has facilitated major advances in the
antiviral treatment of chronic hepatitis B. Today, lamivudine is a first-line therapy for prophylaxis
of HBV recurrence in decompensated cirrhotic patients and liver transplant recipients.

A major problem with lamivudine treatment is the emergence of drug resistance, which increases
with extended duration of therapy (3). Resistant variants have been localized in the reverse
transcriptase (rt) region of the HBV polymerase gene. Lamivudine-resistant amino acids have
been described at positions rt180 (rtL180M) and rt204 (rtM204V/I/S) (4)(5). Methods for the
identification of mutations in the HBV polymerase gene include conventional DNA sequencing,
restriction fragment length polymorphism analysis, and reverse hybridization (6)(7). In the past,
conventional direct DNA sequencing, which is the gold standard method, was the most labor-
intensive and time-consuming method (6). Recently, however, a standardized and largely
automated HBV polymerase gene-sequencing assay, the TrugeneTM HBV Genotyping Kit,
version 1.0 (Bayer/Visible Genetics, Toronto, Ontario), became commercially available. This
assay may be suitable for routine diagnostic laboratory work and clinical trial applications. In this
preliminary study, we evaluated the performance of the new HBV genotyping assay. Patients
undergoing lamivudine treatment were retrospectively investigated for emergence of specific
mutations.

Serum samples from five HBV DNA-positive (serum load >1000 HBV DNA copies/mL)
patients undergoing lamivudine therapy for more than 6 months were analyzed retrospectively.
Blood had been collected in 9.0-mL tubes (VacuetteTM; Greiner Bio-one GmbH), and after
centrifugation, sera had been aliquoted and stored at -70 °C. Alanine aminotransferase (ALT)
concentrations had been determined with the ALT assay for Roche/Hitachi analyzers (Roche
Diagnostics), and aspartate aminotransferase (AST) concentrations had been determined with the
AST assay (Roche). If the ALT concentration exceeded 23 U/L, it was considered abnormal, and
the corresponding value for AST was 19 U/L. Serum HBV load had previously been measured
with the Cobas AmplicorTM HBV Monitor Test (Roche Diagnostic Systems) according to the
manufacturer’s instructions. This molecular assay has a detection limit of 2.0 x 102 HBV DNA
copies/mL. Testing had routinely been done at 3-month intervals beginning at month 9 after the
start of therapy. Between months 9 and 15 after the start of therapy, all patients had an increase in
HBV DNA load of at least 1 log.

For testing mutations in the HBV polymerase gene, an aliquot was thawed, and HBV DNA was
obtained according to the extraction protocol included in the Cobas Amplicor HBV Monitor
Test protocol. Subsequent steps were done according to the manufacturer’s protocol for the
TrugeneTM HBV Genotyping Kit, version 1.0. Initially, a 1.2-kb sequence of the HBV
polymerase gene, representing the central portion of the rt domain, was amplified by PCR, and
sequencing reactions were then performed on this amplification product with the CLIPTM
sequencing (Visible Genetics) technology. CLIP sequencing allows both directions of the
amplification products to be sequenced simultaneously in the same tube with use of two different
dye-labeled primers for each of the four sequencing reactions.

Electrophoresis and subsequent data analysis were performed automatically with the automated
OpenGeneTM and GeneObjectsTM DNA sequence analysis system (Bayer/Visible Genetics). Data
were acquired with the GeneLibrarian module of GeneObjects software by combination of the
forward and reverse sequences. The query sequence was compared with the consensus sequences
of HBV genotypes A to G in the Trugene HBV Module of the OpenGene software to determine
the HBV genotype of the sample. Mutations in the rt gene as well as in the overlapping surface
antigen (HBsAg) gene were also automatically detected and reported. According to the
manufacturer, the detection limit of this system is 2.0 x 103 HBV DNA copies/mL, and all viral
variants present at concentrations 20% of the total can be detected.

Four patients were found infected with HBV genotype A and one patient with HBV genotype D.
In four of the five patients, one or more characteristic mutations were detected in the rt region of
the viral polymerase gene (Table 1 ). In two of the four patients, mutations had developed
within the first year of lamivudine therapy; in the remaining two patients, mutations had
developed within the second year of lamivudine therapy. Mutations were found at positions 173
(V173L), 180 (L180M), 204 (M204I and M204V), and 207 (V207I). In three patients, mutations
appeared during lamivudine therapy together with a significant increase (minimum of 3 logs) in
serum HBV load. In the fourth patient, the M204I mutation was found although viral load was
rather low (month 9 after start of therapy; Fig. 1 ). In this patient, lamivudine therapy had been
continued, and by month 12, the serum HBV load had increased by 1 log. Analysis at this time
point revealed the appearance of the V207I mutation in addition to the existing M204I mutation.
After discontinuation of therapy, serum HBV load increased by 3 logs within 3 months, and the
mutant HBV strains had almost disappeared, whereas the wild-type virus had reappeared.
Although the M204I mutation was no longer detectable, the V207I mutation was still detectable
but showed an R (G or A with 25% A) instead of the expected G at this position (Fig. 1 ).

View this Table 1. Patient data, biochemical values, and results

table: obtained by molecular assays.
[in this
window]
[in a new
window]
 Figure 1. Appearance of resistance mutations in
the rt region of the HBV polymerase gene in a
patient (patient 4) with HBV genotype A
infection.

View larger version

(15K):
[in this window]
[in a new window]

The Trugene HBV Genotyping assay could be performed within 6 h. Amplification of the
polymerase gene of HBV took 2 h, followed by a 2.5-h sequencing reaction including a 0.5-h
manual pipetting. Finally, 1.5 h was needed for electrophoresis and analysis of data.

In this preliminary study, the Trugene HBV Genotyping assay was used in a routine diagnostic
laboratory. The assay is mainly automated and can easily be performed by a trained medical
technologist. In contrast to conventional direct DNA sequencing, this sequencing assay provides
automated generation of the genotyping report. Manual sequence analysis is time-consuming and
difficult, especially if detection of both the rt and HBsAg mutations is required in addition to the
genotyping. Moreover, because of the sensitive CLIP technology, the Trugene HBV Genotyping
assay does not require a nested PCR step, which might be prone to contamination. The Trugene
HBV Genotyping assay thus meets standardization requirements of the routine diagnostic
laboratory.

Methods such as restriction fragment length polymorphism analysis and reverse hybridization
have been proposed to identify mutations in the HBV genome (6)(7). Both methods seem to be
sensitive but identify only known variants. In contrast, sequencing is the only method currently
available that enables identification of new mutants that could be related to resistance (5).
Because it is possible that more variants will arise during lamivudine therapy, sequence analysis
should always be one of the diagnostic tools.

In this study, we found mutations at position rt204 in all patients with one or more characteristic
mutations and a mutation at position rt180 in one of those patients. Both of these mutations have
been associated with lamivudine resistance (4)(5). Mutations at positions rt207 (in two patients)
and rt173 (in one patient) were also found. Both of these are secondary mutations and have
previously been described as associated with famciclovir treatment (4)(8). Although lamivudine-
resistant HBV strains have been shown to have impaired replication capacity compared with the
wild type, their clinical emergence often leads to deterioration of liver function, which
occasionally may be severe or even fatal. It is therefore of major importance to detect mutations
as soon as possible. This could be guaranteed by frequent (every 3 months) determinations of
serum HBV load and sequence analysis in the case of a significant increase. If one or more
characteristic mutations are present, alternative therapies such as adefovir dipivoxil may be
indicated (9).
The region of the HBV genome that is associated with the development of lamivudine resistance
is also classically used to differentiate HBV genotypes. The Trugene HBV Genotyping assay
automatically analyzes the sequence and compares it with genomic reference sequences; it is
therefore able to provide HBV genotype and resistance information from the same data. The
HBV genotype may correlate with different clinical features of HBV infection. Recent data
suggest that Eastern Asian patients with HBV genotype C are more likely to have severe liver
disease, whereas those with genotype B are more likely to develop hepatocellular carcinoma
(10)(11). In India, HBV genotypes A and D were found to be predominant, and HBV genotype D
is associated with more severe liver disease and may predict occurrence of hepatocellular
carcinoma in younger patients (12).

In summary, patients undergoing lamivudine therapy who show a significant increase in serum
HBV load should be tested for the emergence of drug resistance. The Trugene HBV Genotyping
Kit is useful for the routine diagnostic laboratory and provides important molecular information
to allow optimal therapeutic management of patients with chronic HBV infection.

Acknowledgments

This project was supported in part by a grant from Visible Genetics, Inc. We gratefully
acknowledge Anne Beyou and Berwyn Clarke for technical assistance and stimulating
discussions.

References

1. Lai CL, Ching CK, Tung AK, Li E, Young J, Hill A, et al. Lamivudine is
effective in suppressing hepatitis B virus DNA in Chinese hepatitis B surface
antigen carriers: a placebo-controlled trial. Hepatology 1997;25:241-
244.[CrossRef][ISI][Medline] [Order article via Infotrieve]

2. Dienstag JL, Schiff ER, Wright TL, Perrillo RP, Hann HW, Goodman Z, et
al. Lamivudine as initial treatment for chronic hepatitis B in the United
States. N Engl J Med 1999;341:1256-1263.[Abstract/Free Full Text]

3. Leung NW, Lai CL, Chang TT, Guan R, Lee CM, Ng KY, et al. Extended
lamivudine treatment in patients with chronic hepatitis B enhances hepatitis
B e antigen seroconversion rates: results after 3 years of therapy. Hepatology
2001;33:1527-1532.[CrossRef][ISI][Medline] [Order article via Infotrieve]

4. Stuyver LJ, Locarnini SA, Lok A, Richman DD, Carman WF, Dienstag JL,
et al. Nomenclature for antiviral-resistant human hepatitis B virus mutations
in the polymerase region [Review]. Hepatology 2001;33:751-
757.[CrossRef][ISI][Medline] [Order article via Infotrieve]
5. Niesters HGM, De Man RA, Pas SD, Fries E, Osterhaus ADME.
Identification of a new variant in the YMDD motif of the hepatitis B virus
polymerase gene selected during lamivudine therapy. J Med Microbiol
2002;51:695-699.[Abstract/Free Full Text]

6. Allen MI, Gauthier J, Deslauriers M, Bourne EJ, Carrick KM, Baldanti F, et

al. Two sensitive PCR-based methods for detection of hepatitis B virus
variants associated with reduced susceptibility to lamivudine. J Clin
Microbiol 1999;37:3338-3347.[Abstract/Free Full Text]

7. Stuyver L, Van Geyt C, De Gendt S, Van Reybroeck G, Zoulim F, Leroux-

Roels G, et al. Line probe assay for monitoring drug resistance in hepatitis B
virus-infected patients during antiviral therapy. J Clin Microbiol
2000;38:702-707.[Abstract/Free Full Text]

8. Zoulim F. Detection of hepatitis B virus resistance to antivirals. J Clin Virol

2001;21:243-253.[CrossRef][Medline] [Order article via Infotrieve]

9. Feld J, Locarnini S. Antiviral therapy for hepatitis B virus infections: new

targets and technical challenges. J Clin Virol 2002;25:267-
283.[CrossRef][Medline] [Order article via Infotrieve]

10.Kao JH, Chen PJ, Lai MY, Chen DS. Hepatitis B genotypes correlate with
clinical outcomes in patients with chronic hepatitis B. Gastroenterology
2000;118:554-559.[CrossRef][ISI][Medline] [Order article via Infotrieve]

11.Fujie H, Moriya K, Shintani Y, Yotsuyanagi H, Iino S, Koike K. Hepatitis B

virus genotypes and hepatocellular carcinoma in Japan. Gastroenterology
2001;120:1564-1565.[ISI][Medline] [Order article via Infotrieve]

12.Thakur V, Guptan RC, Kazim SN, Malhotra V, Sarin SK. Profile, spectrum
and significance of HBV genotypes in chronic liver disease patients in the
Indian subcontinent. J Gastroenterol Hepatol 2002;17:165-
170.[CrossRef][ISI][Medline] [Order article via Infotrieve]
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