Multicenter Evaluation of a Semiautomated, Standardized Assay for Detection of Hepatitis B Virus DNA in Blood Donations
http://www.100md.com
微生物临床杂志 2005年第6期
Institute of Virology, University of Milan, Milan
Transfusion Medicine and Hematology Department, Hospital of Sondrio, Sondrio, Italy
Scientific Affairs, Roche Molecular Systems, Rotkreuz, Switzerland
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.
TEXT
Over the past decade, the risk of acquiring blood-borne viruses through transfusion has dramatically declined in industrialized countries. A residual risk due to donations of blood during the window period (time from infection to 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.
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 the procedure are performed in automation by the 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.
ACKNOWLEDGMENTS
We thank Catia Tagliacarne for skillful technical assistance.
Members of the Italian Group for the Study of Transfusion Transmissible Diseases who contributed to the present study. Umberto Bodini (Transfusion Medicine and Hematology Department of Cremona), Mara Mazzucco (Transfusion Medicine and Hematology Department of Pavia), Gianalessandro Moroni (Transfusion Medicine and Hematology Department, S. Paolo Hospital, Milan), Pasqualepaolo Pagliaro (Transfusion Medicine and Hematology Department of Mantova), Daniele Prati (Transfusion Medicine and Hematology Department of Lecco), and Maria Grazia Bellotti (Roche Molecular Systems). L.R., C.V., L.B., L.F., G.C., and A.R.Z. are also members of this study group.
Contributing members of the Italian Group for the Study of Transfusion Transmissible Diseases are listed in Acknowledgments.
REFERENCES
Allain, J. P. 2003. Transfusion risks of yesterday and of today. Transfus. Clin. Biol. 10:1-5.
Alvarez, M., S. Oyonarte, P. M. Rodriguez, and J. M. Hernandez. 2002. Estimated risk of transfusion-transmitted viral infections in Spain. Transfusion 42:994-998.
Busch, M. P., S. H. Kleinman, and G. J. Nemo. 2003. Current and emerging infectious risks of blood transfusions. JAMA 289:959-962.
DiDomenico, N., H. Link, R. Knobel, T. Caratsch, W. Weschler, Z. G. Loewy, and M. Rosenstraus. 1996. COBAS AMPLICOR: fully automated RNA and DNA amplification and detection system for routine PCR. Clin. Chem. 42:1915-1923.
Dodd, R. Y., E. P. Notari IV, and S. L. Stramer. 2002. Current prevalence and incidence of infectious disease markers and estimated window-period risk in the American Red Cross blood donor population. Transfusion 42:975-979.
Glynn, S. A., S. H. Kleinman, D. J. Wright, M. P. Busch, and the NHLBI Retrovirus Epidemiology Donor Study. 2002. International application of the incidence rate/window period model. Transfusion 42:966-972.
Kleinman, S., M. P. Busch, J. J. Korelitz, and G. B. Schreiber. 1997. The incidence/window period model and its use to assess the risk of transfusion-transmitted human immunodeficiency virus and hepatitis C virus infection. Transfus. Med. Rev. 11:155-172.
International Forum. 2002. Implementation of donor screening for infectious agents transmitted by blood by nucleic acid technology. Vox Sang. 82:87-111.
Jungkind, D., S. Direnzo, K. G. Beavis, and N. S. Silverman. 1996. Evaluation of automated COBAS AMPLICOR PCR system for detection of several infectious agents and its impact on laboratory management. J. Clin. Microbiol. 34:2778-2783.
Lackritz, E. M., G. A. Satten, J. Aberle-Grasse, R. Y. Dodd, V. P. Raimondi, R. S. Janssen, W. F. Lewis, E. P. Notari, and L. R. Petersen. 1995. Estimated risk of transmission of the human immunodeficiency virus by screened blood in the United States. N. Engl. J. Med. 333:1721-1725.
Minegishi, K., A. Yoshikawa, S. Kishimoto, H. Yugi, N. Yokoya, M. Sakurada, H. Kiyokawa, K. Nishioka, and Japanese Red Cross NAT Screening Research Group. 2003. Superiority of minipool nucleic acid amplification technology hepatitis B virus over chemiluminescence immunoassay for hepatitis B surface antigen screening. Vox Sang. 84:287-291.
Pillonel, J., S. Laperche, C. Saura, J. C. Desenclos, A. M. Courouce, and Transfusion-Transmissible Agents Working Group of the French Society of Blood Transfusion. 2002. Trends in residual risk of transfusion-transmitted viral infections in France between 1992 and 2000. Transfusion 42:980-988.
Sato, S., W. Ohhashi, H. Ihara, S. Sakaya, T. Kato, and H. Ikeda. 2001. Comparison of the sensitivity of NAT using pooled donor samples for HBV and that of a serologic HBsAg assay. Transfusion 41:1107-1113.
Schreiber, G. B., M. P. Busch, S. H. Kleinman, J. J. Korelitz, and the NHLBI Retrovirus Epidemiology Blood Donor Study. 1996. The risk of transfusion-transmitted viral infections. N. Engl. J. Med. 11:155-172.
Seed, C. R., Cheng, A., S. L. Ismay, W. V. Bolton, P. Kiely, T. J. Cobain, A. J. Keller, and Virology Subcommittee of the National Donor and Product Safety Committee, Australian Red Cross Blood Service. 2002. Assessing the accuracy of three viral risk models in predicting the outcome of implementing HIV and HCV NAT donor screening in Australia and the implications for future HBV NAT. Transfusion 42:1365-1372.
Sun, R., W. Schilling, H. Jayakar, J. Ku, J. Wang, M. Rosenstraus, and J. Spadoro. 1999. Simultaneous extraction of hepatitis C virus (HCV), hepatitis B virus, and HIV-1 from plasma and detection of HCV RNA by a reverse transcriptase-polymerase chain reaction assay designed for screening pooled units of donated blood. Transfusion 39:1111-1119.
Velati, C., L. Romanò, L. Baruffi, M. Pappalettera, V. Carreri, and A. R. Zanetti. 2002. Residual risk of transfusion-transmitted HCV and HIV infections by antibody-screened blood in Italy. Transfusion 42:989-993.
Velati, C., L. Baruffi, L. Romanò, L. Fomiatti, V. Carreri, A. Zanetti, and the Italian Group for the Study of Transfusion Transmissible Diseases. 2003. The introduction of a NAT method of screening blood donors in Italy: a report of the first two year's experience and a re-evaluation of residual risk. Blood Transfus. 4:368-378.(Luisa Romanò, Claudio Vel)
Transfusion Medicine and Hematology Department, Hospital of Sondrio, Sondrio, Italy
Scientific Affairs, Roche Molecular Systems, Rotkreuz, Switzerland
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.
TEXT
Over the past decade, the risk of acquiring blood-borne viruses through transfusion has dramatically declined in industrialized countries. A residual risk due to donations of blood during the window period (time from infection to 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.
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 the procedure are performed in automation by the 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.
ACKNOWLEDGMENTS
We thank Catia Tagliacarne for skillful technical assistance.
Members of the Italian Group for the Study of Transfusion Transmissible Diseases who contributed to the present study. Umberto Bodini (Transfusion Medicine and Hematology Department of Cremona), Mara Mazzucco (Transfusion Medicine and Hematology Department of Pavia), Gianalessandro Moroni (Transfusion Medicine and Hematology Department, S. Paolo Hospital, Milan), Pasqualepaolo Pagliaro (Transfusion Medicine and Hematology Department of Mantova), Daniele Prati (Transfusion Medicine and Hematology Department of Lecco), and Maria Grazia Bellotti (Roche Molecular Systems). L.R., C.V., L.B., L.F., G.C., and A.R.Z. are also members of this study group.
Contributing members of the Italian Group for the Study of Transfusion Transmissible Diseases are listed in Acknowledgments.
REFERENCES
Allain, J. P. 2003. Transfusion risks of yesterday and of today. Transfus. Clin. Biol. 10:1-5.
Alvarez, M., S. Oyonarte, P. M. Rodriguez, and J. M. Hernandez. 2002. Estimated risk of transfusion-transmitted viral infections in Spain. Transfusion 42:994-998.
Busch, M. P., S. H. Kleinman, and G. J. Nemo. 2003. Current and emerging infectious risks of blood transfusions. JAMA 289:959-962.
DiDomenico, N., H. Link, R. Knobel, T. Caratsch, W. Weschler, Z. G. Loewy, and M. Rosenstraus. 1996. COBAS AMPLICOR: fully automated RNA and DNA amplification and detection system for routine PCR. Clin. Chem. 42:1915-1923.
Dodd, R. Y., E. P. Notari IV, and S. L. Stramer. 2002. Current prevalence and incidence of infectious disease markers and estimated window-period risk in the American Red Cross blood donor population. Transfusion 42:975-979.
Glynn, S. A., S. H. Kleinman, D. J. Wright, M. P. Busch, and the NHLBI Retrovirus Epidemiology Donor Study. 2002. International application of the incidence rate/window period model. Transfusion 42:966-972.
Kleinman, S., M. P. Busch, J. J. Korelitz, and G. B. Schreiber. 1997. The incidence/window period model and its use to assess the risk of transfusion-transmitted human immunodeficiency virus and hepatitis C virus infection. Transfus. Med. Rev. 11:155-172.
International Forum. 2002. Implementation of donor screening for infectious agents transmitted by blood by nucleic acid technology. Vox Sang. 82:87-111.
Jungkind, D., S. Direnzo, K. G. Beavis, and N. S. Silverman. 1996. Evaluation of automated COBAS AMPLICOR PCR system for detection of several infectious agents and its impact on laboratory management. J. Clin. Microbiol. 34:2778-2783.
Lackritz, E. M., G. A. Satten, J. Aberle-Grasse, R. Y. Dodd, V. P. Raimondi, R. S. Janssen, W. F. Lewis, E. P. Notari, and L. R. Petersen. 1995. Estimated risk of transmission of the human immunodeficiency virus by screened blood in the United States. N. Engl. J. Med. 333:1721-1725.
Minegishi, K., A. Yoshikawa, S. Kishimoto, H. Yugi, N. Yokoya, M. Sakurada, H. Kiyokawa, K. Nishioka, and Japanese Red Cross NAT Screening Research Group. 2003. Superiority of minipool nucleic acid amplification technology hepatitis B virus over chemiluminescence immunoassay for hepatitis B surface antigen screening. Vox Sang. 84:287-291.
Pillonel, J., S. Laperche, C. Saura, J. C. Desenclos, A. M. Courouce, and Transfusion-Transmissible Agents Working Group of the French Society of Blood Transfusion. 2002. Trends in residual risk of transfusion-transmitted viral infections in France between 1992 and 2000. Transfusion 42:980-988.
Sato, S., W. Ohhashi, H. Ihara, S. Sakaya, T. Kato, and H. Ikeda. 2001. Comparison of the sensitivity of NAT using pooled donor samples for HBV and that of a serologic HBsAg assay. Transfusion 41:1107-1113.
Schreiber, G. B., M. P. Busch, S. H. Kleinman, J. J. Korelitz, and the NHLBI Retrovirus Epidemiology Blood Donor Study. 1996. The risk of transfusion-transmitted viral infections. N. Engl. J. Med. 11:155-172.
Seed, C. R., Cheng, A., S. L. Ismay, W. V. Bolton, P. Kiely, T. J. Cobain, A. J. Keller, and Virology Subcommittee of the National Donor and Product Safety Committee, Australian Red Cross Blood Service. 2002. Assessing the accuracy of three viral risk models in predicting the outcome of implementing HIV and HCV NAT donor screening in Australia and the implications for future HBV NAT. Transfusion 42:1365-1372.
Sun, R., W. Schilling, H. Jayakar, J. Ku, J. Wang, M. Rosenstraus, and J. Spadoro. 1999. Simultaneous extraction of hepatitis C virus (HCV), hepatitis B virus, and HIV-1 from plasma and detection of HCV RNA by a reverse transcriptase-polymerase chain reaction assay designed for screening pooled units of donated blood. Transfusion 39:1111-1119.
Velati, C., L. Romanò, L. Baruffi, M. Pappalettera, V. Carreri, and A. R. Zanetti. 2002. Residual risk of transfusion-transmitted HCV and HIV infections by antibody-screened blood in Italy. Transfusion 42:989-993.
Velati, C., L. Baruffi, L. Romanò, L. Fomiatti, V. Carreri, A. Zanetti, and the Italian Group for the Study of Transfusion Transmissible Diseases. 2003. The introduction of a NAT method of screening blood donors in Italy: a report of the first two year's experience and a re-evaluation of residual risk. Blood Transfus. 4:368-378.(Luisa Romanò, Claudio Vel)