Monika

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

We evaluated the COBAS Ampliscreen hepatitis B virus (HBV) test using standards, seroconversion panels, consecutive donations, and samples from 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.

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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.

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TABLE 1. Timing of detection of HBV DNA in seroconversion panels using COBAS AmpliScreen (CAS HBV)

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. In conclusion, our data showed that the CAS HBV is a reliable assay that can help to improve the safety of blood supplies by shortening the preseroconversion infectious window phase.

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Clinical Chemistry 49: 989-992, 2003; 10.1373/49.6.989 (Clinical Chemistry. 2003;49:989-992.) © 2003 American Association for Clinical Chemistry, Inc.

Technical Briefs

Detection of Mutations in the Hepatitis B Virus Polymerase Gene Harald H. Kessler1,a, Evelyn Stelzl1, Egon Marth1 and Rudolf E. Stauber2

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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 [email protected]

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 laborintensive 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 ).

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Table 1. Patient data, biochemical values, and results obtained by molecular assays.

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 lamivudineresistant 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;25:241244.[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;33:751757.[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, LerouxRoels 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;25:267283.[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;17:165170.[CrossRef][ISI][Medline] [Order article via Infotrieve]

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