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Detection of an acute asymptomatic HBsAg negative hepatitis B virus infection in a blood donor by HBV DNA testing
Journal of Clinical Virology 32 (2005) 67–70
Case report
Detection of an acute asymptomatic HBsAg negative hepatitis B virus infection in a blood donor by HBV DNA testing Bernard Webera,b,∗ , Annelies M¨uhlbacherc , Walter Melchiord a
Laboratoires R´eunis, Junglinster, Centre Langwies, L-6131 Junglinster, Luxembourg b Institute of Medical Virology, Universit¨ atskliniken Frankfurt, Germany c Landesklinik f¨ ur Blutgruppenserologie und Transfusionsmedizin, Salzburg, Austria d Olivierstrasse, Munich, Germany
Received 10 May 2004; received in revised form 15 September 2004; accepted 17 September 2004
Abstract The issue of HBV DNA screening on blood donations is controversially discussed since the economic impact of post-transfusion hepatitis B is expected to be relatively low. We report on a case of HBsAg negative unapparent acute HBV infection, which was detected by HBV NAT testing on 96-member maxi-pools with a commercially available NAT assay, which has a detection threshold of 3 IU/mL of plasma. The presence of an HBsAg escape mutant could be excluded by sequencing the amplified DNA. Follow-up testing showed the presence of an acute HBV infection (anti-HBc-IgM positive) and finally anti-HBs seroconversion. Although the reduction of the diagnostic window with NAT screening on maxi-pools may be relatively low, it may help to improve the residual risk of blood donation, especially in asymptomatic HBV infection, where the HBsAg positive period may be very short and low levels of circulating surface antigen are present. It would also permit to detect occult HBV infection in chronic carriers who are HBsAg negative. Since the viral load in chronic isolated anti-HBc positive carriers is low, there is a potential risk for failure of HBV DNA detection with pool-PCR in blood donors. Anti-HBc screening would reduce the residual risk. © 2004 Elsevier B.V. All rights reserved. Keywords: Hepatitis B virus; Nucleic acid amplification testing (NAT); Isolated anti-HBc positive
1. Introduction The transmission of hepatitis B virus (HBV) by blood transfusion is minimized by HBV surface antigen (HBsAg) and anti-HBc (in some countries) and exclusion of high-risk donors prior to transfusion. There is however still a small residual risk of transmission of HBV from undetected donors with early acute infection, resolving infection, silent (occult) infection or infection with atypical variants or mutants. Many HBV infections are asymptomatic and HBsAg is not detectable or may be present for a very short time interval at very low level. Infected individuals may thus unwittingly present as blood donors. In order to improve the safety of blood donor screening, several approaches to reduce the di∗
Corresponding author. Tel.: +352 788894; fax: +352 7802901. E-mail address: [email protected] (B. Weber).
1386-6532/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2004.09.018
agnostic window period before detection of the first HBV specific marker have been conceived. Screening of blood donations with more sensitive HBsAg assays reduces the diagnostic window period by 2–9 days in comparison to so far licensed tests (Biswas et al., 2003; Weber et al., 1999). The potential benefit of pooled-sample nucleic acid amplification tests (NAT) in comparison to HBsAg screening is relatively moderate, since NAT would further reduce the window period by only 2–3 days depending on the sensitivity of the test protocol and the pool size. NAT screening on individual donations or on 8–16 member mini-pools with highly sensitive single or multiplex NAT may reduce the diagnostic window by 24–36 days. In the USA, it is expected that single sample NAT would increase yield by 13–15 units per 107 donations over pooled-sample NAT and newer assays and by 35–50 units per 107 donations over currently FDA licensed HBsAg assays (Biswas et al., 2003).
68
B. Weber et al. / Journal of Clinical Virology 32 (2005) 67–70
Since post-transfusion hepatitis B has relatively few health or economic repercussions, single-donation HBV NAT would provide a small benefit at a very high cost. Instead, in some circumstances, the cost-effectiveness of enhanced sensitivity HBsAg assays, pooled NAT and anti-HBc screening would be within acceptable ranges for new public health interventions (Pereira, 2003). The present report describes a case of asymptomatic acute HBV infection, which was not detected by HBsAg screening but by pooled NAT. Isolated anti-HBc reactivity and spontaneous resolution with anti-HBs seroconversion was observed during follow-up.
2. Case report HBV DNA amplification with a commercially available nucleic acid amplification test (NAT) (Cobas Ampliscreen HBV, Roche Diagnostics, Penzberg, Germany) was performed on 96-member maxi-pools at the Blutzentrale der Landeskrankenanstalten Salzburg, Austria. Sample preparation was performed the Multiprep procedure (Roche Diagnostics) in accordance with the manufacturers’ recommendations. The lower detection-limit for HBV DNA was 3 IU/mL plasma, which corresponds approximately to 300 IU/mL for an individual blood donation in a pool of 96 donations. On November 27th of 2001, one pool was tested positive for HBV DNA. Resolution of the pool by single-donation testing revealed one positive sample. It was from a repeated female blood donor who was asymptomatic, with liver enzyme values within the normal range, and being negative for serological markers for HBV (HBsAg and anti-HBc negative), HCV and HIV. The sample was tested in parallel at the central laboratory of the Landeskrankenanstalten Salzburg with a commercial quantitative NAT assay (Amplicor HBV Monitor V. 2.0, Roche Diagnostics). The detection-limit was 130 IU/ml). The result being negative, hepatitis B infection was ruled out as primary hypothesis and the Ampliscreen HBV result was interpreted as false positive. The blood donation was however discarded. Since anti-HBc-IgM and total anti-HBc-antibody were detected in a follow-up sample obtained 1 week later, acute HBV infection was suspected and the first sample of the donor
was sent for confirmation to the Institute of Virology, University of Regensburg, Germany. HBV DNA was detected at a low copy number (2070 IU/ml) with an in-house PCR protocol (Weinberger et al., 1997). The blood donor remained asymptomatic and showed normal liver enzyme values during the observation period from November 2001 to October 2003. The blood donor denied any risk factor for sexual or parenteral transmission of bloodborne infections. Sequencing of the amplified HBV DNA was performed at the Institute of Virology, University of Regensburg, Germany as described elsewhere (Weinberger et al., 1997). The sequence obtained was compared with published sequences using the HBV sequence database HBV-BLAST program (Los Alamos, National Laboratory). Sequence analysis of HBV DNA revealed that the patient’s HBV DNA was of genotype A, there was no mutation related to so far known diagnostic escape mutants. Additional serological testing for HBV markers (HBsAg, anti-HBc, anti-HBc-IgM, anti-HBs, HBeAg, anti-HBe) was performed with commercially available assays (Enzygnost, Dade-Behring, Marburg, Germany and AxSYM, Abbott Diagnostics, Delkenheim, Germany). All the tests were performed in accordance with the manufacturers’ recommendations. All the serological markers for HBV were negative in the first sample obtained in November 2001 (Table 1). Anti-HBcseroconversion (total anti-HBc and anti-HBc-IgM antibody positive) was observed in a follow-up sample drawn 7 days later. All the other HBV markers were negative. Isolated anti-HBc reactivity (total anti-HBc antibody positive) only was present in the third follow-up sample obtained 45 days later. Anti-HBs seroconversion with a weak antibody titer of 12 IU/L was apparent in a fourth and final follow-up serum sample. No HBsAg and HBV DNA were detected in all the three follow-up samples. HBV DNA amplification was performed on the second sample from the blood donor in a diagnostic laboratory, which used an in-house method with a detection threshold of 300 IU/ml plasma. 3. Discussion In immunocompetent adults, inoculation with a low dose of HBV is associated mostly with asymptomatic self-limiting
Table 1 HBV markers in case of unapparent acute HBsAg negative HBV infection Date of sample collection Results of HBV markers (index values = S/CO (signal/cut-off)) HBsAg 27.11.01 04.12.01 18.01.02 05.04.02 06.11.03 a b
Negativea Negative Negative Negativea Negative
Index Anti-HBc Index Anti-HBc-IgM Index Anti-HBs HBeAg 0.76 NA NA 0.92 0.65
Negativea Positive Positive Positivea Positivea
2.37 NA NA 0.786 0.235
Negativea Positive Negative Negativea Negative
0.148 NA NA 0.132 0.121
<6 IU/L Negative Negative 12 IU/La 10 IU/La
Negativea Negative Negative Negativea Negativea
Index Anti-HBe Index HBV DNA PCR 1.46 NA NA 1.25 1.01
Negativea Negative Negative Negativea Negativea
0.086 NA NA 0.087 0.076
Positive Negativeb Negative Negative Negative
Enzygnost (Dade Behring) and AxSYM (Abbott) HBV serological assays, index values are from the results obtained with Enzygnost HBsAg. Detection-limit 300 IU/ml NA, not available.
B. Weber et al. / Journal of Clinical Virology 32 (2005) 67–70
acute HBV infection with consecutive immunity (Gerlich and Schaefer, 2002). HBsAg is present at low concentrations and during a very short time period in unapparent acute hepatitis B and may therefore not be detected by HBsAg screening in blood donors, as shown by the present case report. In order to increase the safety of blood and blood products, nucleic acid amplification technique (NAT) has been introduced in many industrialised countries for the screening of HIV and HCV in pooled plasma samples and/or individual donations. Technical improvements and automation of all the steps of NAT, including sample preparation, make it possible to perform nucleic acid amplification for the three most-relevant blood-borne infections, HIV, hepatitis B and C in mini-pools or single donations (Meng et al., 2001). While there is an international consensus to perform NAT for HIV and HCV, the utility in terms of cost-effectiveness for HBV NAT screening is still controversially discussed and some European countries are opposed to blood donor screening for HBV DNA. Since about 95% of HBV infections are self-limiting and the mean survival rate of blood recipients is relatively low, due to the severe underlying disease or condition, which necessitates blood transfusion, the incremental costs for reducing the transfusion risk are high but the benefit for the patient in terms of quality of life is relatively poor. HBV DNA is detected 3–5 weeks after infection and 1–2 weeks before the appearance of HBsAg, newer, more sensitive assays are closing this gap (Weber et al., 1999, 2003). HBV DNA in contrast to HIV and HCV RNA rises slowly and circulates at relatively low levels (30–300 IU/mL) during the early HBsAg seronegative window period (Hollinger and Liang, 2001). HBV NAT should have a detection threshold under 30 IU/mL in single donations in order to detect low copy number of virus nucleic acid in the HBsAg negative window phase and to further reduce the NAT diagnostic window period and to detect low-level chronic HBV carriers. Ten- to twenty-fold more sensitive NAT protocols could be used as an alternative for the testing of 8- or 16-member minipools in order to achieve a maximal reduction of the residual risk. In the present case, the goal of reducing the residual risk of HBV infection was achieved by applying a less sensitive protocol with a detection-limit in an individual sample of 300 IU/mL. This finding argues for HBV DNA screening in blood donors in maxi-pools with a sensitive assay. This strategy would permit not only to detect acute HBV infection in asymptomatic blood donors with a very short HBsAg positive period but also occult HBV infection in chronic carriers who are HBsAg negative and may be negative for all the other HBV specific markers or show one of the following possible seroconstellations (isolated anti-HBc positive, with or without anti-HBc-IgM or anti-HBe, anti-HBc and anti-HBs positive, HBeAg positive). Since the viral load in chronic isolated anti-HBc positive carriers is low, there is a potential risk for failure of HBV DNA detection with pool-PCR in blood donors (Kleinman et al., 2003). Anti-HBc screening would reduce the residual
69
risk (Weber et al., 2001a, 2001b; Arraes et al., 2003). There are no clear recommendations for the reintroduction of antiHBc for blood donor screening, although it may be the only positive marker in chronic infectious carriers. The probably relatively poor cost/benefit relation and the crucial issue of increased loss of donors by poor specificity or incorrect result interpretation are major arguments against the routine testing of blood donors, especially in countries with low incidence of HBV infection and where donor screening by pool HBV DNA testing is already performed. Newer anti-HBc assays show an improved sensitivity (Weber et al., 1998). In our present case, asymptomatic HBV infection would have been detected by its isolated anti-HBc reactivity in the second follow-up sample. In order to permit a more cost-effective screening of blood donations combined assays for anti-HBc/HBsAg need to be developed. In the field of HIV screening, combined antigen/antibody assays have achieved now after 5 years of experience and technical improvements a high sensitivity and specificity (Weber et al., 2002).
References Arraes LC, Ximenes R, Andrieu JM, Wei L, Barreto S, Pereira LMMB, et al. The biological meaning of anti-HBc positive result in blood donors: relation to HBV-DNA and to other serological markers. Rev Inst Med Trop Sau Paulo 2003;45(3):137–40 (ISSN 0036-4665). Biswas R, Tabor E, Hsia CC, Wright DJ, Laycock ME, Fiebig EW, et al. Comparative sensitivity of HBV NATs and HBsAg assays for detection of acute HBV infection. Transfusion 2003;43:788– 98. Gerlich WH, Schaefer S. Hepadnaviren: hepatitis-B-virus. In: Doerr HW, Gerlich HW, editors. Medizinische Virologie. Stuttgart: Georg Thieme Verlag; 2002. p. 191–210. Hollinger FB, Liang TJ. Hepatitis B virus. In: Knipe DM, Howley PM, editors. Fields Virology, 4th ed.. Philadelphia: Lippincott Williams & Wilkins; 2001. p. 2971–3036. Kleinman SH, Kuhns MC, Todd DS, Glynn Sa, McNamara A, DiMarco A, et al. Retrovirus epidemiology donor study. Frequency of HBV DNA detection in US blood donors testing positive for the presence of anti-HBc: implications for transfusion transmission and donor screening. Transfusion 2003;43:696–704. Meng Q, Wong C, Rangachari A, Tamatsukuri S, Sasaki M, Fiss E, et al. Automated multiplex assay system for simultaneous detection of hepatitis B virus DNA, hepatitis C virus RNA, and human immunodeficiency virus type 1 RNA. J Clin Microbiol 2001;39:2937–45. Pereira A. Health and economic impact of posttransfusion hepatitis B and cost-effectiveness analysis of expanded HBV testing protocols of blood donors: a study focused on the European Union. Transfusion 2003;43:192–201. Weber B, Bayer A, Kirch P, Schl¨uter V, Schlieper D, Melchior W. Improved detection of hepatitis B virus surface antigen by a new rapid automated assay. J Clin Microbiol 1999;37:2639–47. Weber B, Dengler T, Berger A, Doerr HW, Rabenau H. Evaluation of two new automated assays for hepatitis B virus surface antigen (HBsAg) detection: IMMULITE HBsAg and IMMULITE 2000 HBsAg. J Clin Microbiol 2003;41:135–43. Weber B, Gurtler L, Thorstensson R, Michl U, Muhlbacher A, Burgisser P, et al. Multicenter evaluation of a new automated fourthgeneration human immunodeficiency virus screening assay with a sensitive antigen detection module and high specificity. J Clin Microbiol 2002;40:1938–46.
70
B. Weber et al. / Journal of Clinical Virology 32 (2005) 67–70
Weber M, Hedges D, Seifried E, Roth WK. Low level HBV DNA carriers detected among HBsAg negative anti-HBc positive blood donors. 34. Jahreskongress der Deutschen Gesellschaft f¨ur Transfusionsmedizin und Immunh¨amatologie. Hamburg 25–28 September 2001. Infusionsther Transfusionsmed 2001b;28:45. Weinberger KM, Kreuzpaintner EA, Hottentr¨ager B, Neifer S, Jilg W. Mutations in the S-gene of hepatitis B virus isolates from chronic carriers with anti-HBc as the only serological marker of HBV infection. In: Rizetto M, Purcell RH, Gerin JL, Verne G, editors. Viral
Hepatitis and Liver Disease. Torino: Edizioni Minerva Medica; 1997. p. 138–43. Weber B, Melchior W, Gehrke R, Berger A, Rabenau HF. Hepatitis B virus (HBV) markers in isolated anti-HBc positive individuals. J Med Virol 2001a;64:312–9. Weber B, Michl U, M¨uhlbacher A, Paggi G, Bossi V. Evaluation of The New Automated Enzymun-Test® Anti-HBc Plus for the detection of hepatitis B virus (HBV) core antibody. Intervirology 1998;41:17– 23.
Case report
Detection of an acute asymptomatic HBsAg negative hepatitis B virus infection in a blood donor by HBV DNA testing Bernard Webera,b,∗ , Annelies M¨uhlbacherc , Walter Melchiord a
Laboratoires R´eunis, Junglinster, Centre Langwies, L-6131 Junglinster, Luxembourg b Institute of Medical Virology, Universit¨ atskliniken Frankfurt, Germany c Landesklinik f¨ ur Blutgruppenserologie und Transfusionsmedizin, Salzburg, Austria d Olivierstrasse, Munich, Germany
Received 10 May 2004; received in revised form 15 September 2004; accepted 17 September 2004
Abstract The issue of HBV DNA screening on blood donations is controversially discussed since the economic impact of post-transfusion hepatitis B is expected to be relatively low. We report on a case of HBsAg negative unapparent acute HBV infection, which was detected by HBV NAT testing on 96-member maxi-pools with a commercially available NAT assay, which has a detection threshold of 3 IU/mL of plasma. The presence of an HBsAg escape mutant could be excluded by sequencing the amplified DNA. Follow-up testing showed the presence of an acute HBV infection (anti-HBc-IgM positive) and finally anti-HBs seroconversion. Although the reduction of the diagnostic window with NAT screening on maxi-pools may be relatively low, it may help to improve the residual risk of blood donation, especially in asymptomatic HBV infection, where the HBsAg positive period may be very short and low levels of circulating surface antigen are present. It would also permit to detect occult HBV infection in chronic carriers who are HBsAg negative. Since the viral load in chronic isolated anti-HBc positive carriers is low, there is a potential risk for failure of HBV DNA detection with pool-PCR in blood donors. Anti-HBc screening would reduce the residual risk. © 2004 Elsevier B.V. All rights reserved. Keywords: Hepatitis B virus; Nucleic acid amplification testing (NAT); Isolated anti-HBc positive
1. Introduction The transmission of hepatitis B virus (HBV) by blood transfusion is minimized by HBV surface antigen (HBsAg) and anti-HBc (in some countries) and exclusion of high-risk donors prior to transfusion. There is however still a small residual risk of transmission of HBV from undetected donors with early acute infection, resolving infection, silent (occult) infection or infection with atypical variants or mutants. Many HBV infections are asymptomatic and HBsAg is not detectable or may be present for a very short time interval at very low level. Infected individuals may thus unwittingly present as blood donors. In order to improve the safety of blood donor screening, several approaches to reduce the di∗
Corresponding author. Tel.: +352 788894; fax: +352 7802901. E-mail address: [email protected] (B. Weber).
1386-6532/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2004.09.018
agnostic window period before detection of the first HBV specific marker have been conceived. Screening of blood donations with more sensitive HBsAg assays reduces the diagnostic window period by 2–9 days in comparison to so far licensed tests (Biswas et al., 2003; Weber et al., 1999). The potential benefit of pooled-sample nucleic acid amplification tests (NAT) in comparison to HBsAg screening is relatively moderate, since NAT would further reduce the window period by only 2–3 days depending on the sensitivity of the test protocol and the pool size. NAT screening on individual donations or on 8–16 member mini-pools with highly sensitive single or multiplex NAT may reduce the diagnostic window by 24–36 days. In the USA, it is expected that single sample NAT would increase yield by 13–15 units per 107 donations over pooled-sample NAT and newer assays and by 35–50 units per 107 donations over currently FDA licensed HBsAg assays (Biswas et al., 2003).
68
B. Weber et al. / Journal of Clinical Virology 32 (2005) 67–70
Since post-transfusion hepatitis B has relatively few health or economic repercussions, single-donation HBV NAT would provide a small benefit at a very high cost. Instead, in some circumstances, the cost-effectiveness of enhanced sensitivity HBsAg assays, pooled NAT and anti-HBc screening would be within acceptable ranges for new public health interventions (Pereira, 2003). The present report describes a case of asymptomatic acute HBV infection, which was not detected by HBsAg screening but by pooled NAT. Isolated anti-HBc reactivity and spontaneous resolution with anti-HBs seroconversion was observed during follow-up.
2. Case report HBV DNA amplification with a commercially available nucleic acid amplification test (NAT) (Cobas Ampliscreen HBV, Roche Diagnostics, Penzberg, Germany) was performed on 96-member maxi-pools at the Blutzentrale der Landeskrankenanstalten Salzburg, Austria. Sample preparation was performed the Multiprep procedure (Roche Diagnostics) in accordance with the manufacturers’ recommendations. The lower detection-limit for HBV DNA was 3 IU/mL plasma, which corresponds approximately to 300 IU/mL for an individual blood donation in a pool of 96 donations. On November 27th of 2001, one pool was tested positive for HBV DNA. Resolution of the pool by single-donation testing revealed one positive sample. It was from a repeated female blood donor who was asymptomatic, with liver enzyme values within the normal range, and being negative for serological markers for HBV (HBsAg and anti-HBc negative), HCV and HIV. The sample was tested in parallel at the central laboratory of the Landeskrankenanstalten Salzburg with a commercial quantitative NAT assay (Amplicor HBV Monitor V. 2.0, Roche Diagnostics). The detection-limit was 130 IU/ml). The result being negative, hepatitis B infection was ruled out as primary hypothesis and the Ampliscreen HBV result was interpreted as false positive. The blood donation was however discarded. Since anti-HBc-IgM and total anti-HBc-antibody were detected in a follow-up sample obtained 1 week later, acute HBV infection was suspected and the first sample of the donor
was sent for confirmation to the Institute of Virology, University of Regensburg, Germany. HBV DNA was detected at a low copy number (2070 IU/ml) with an in-house PCR protocol (Weinberger et al., 1997). The blood donor remained asymptomatic and showed normal liver enzyme values during the observation period from November 2001 to October 2003. The blood donor denied any risk factor for sexual or parenteral transmission of bloodborne infections. Sequencing of the amplified HBV DNA was performed at the Institute of Virology, University of Regensburg, Germany as described elsewhere (Weinberger et al., 1997). The sequence obtained was compared with published sequences using the HBV sequence database HBV-BLAST program (Los Alamos, National Laboratory). Sequence analysis of HBV DNA revealed that the patient’s HBV DNA was of genotype A, there was no mutation related to so far known diagnostic escape mutants. Additional serological testing for HBV markers (HBsAg, anti-HBc, anti-HBc-IgM, anti-HBs, HBeAg, anti-HBe) was performed with commercially available assays (Enzygnost, Dade-Behring, Marburg, Germany and AxSYM, Abbott Diagnostics, Delkenheim, Germany). All the tests were performed in accordance with the manufacturers’ recommendations. All the serological markers for HBV were negative in the first sample obtained in November 2001 (Table 1). Anti-HBcseroconversion (total anti-HBc and anti-HBc-IgM antibody positive) was observed in a follow-up sample drawn 7 days later. All the other HBV markers were negative. Isolated anti-HBc reactivity (total anti-HBc antibody positive) only was present in the third follow-up sample obtained 45 days later. Anti-HBs seroconversion with a weak antibody titer of 12 IU/L was apparent in a fourth and final follow-up serum sample. No HBsAg and HBV DNA were detected in all the three follow-up samples. HBV DNA amplification was performed on the second sample from the blood donor in a diagnostic laboratory, which used an in-house method with a detection threshold of 300 IU/ml plasma. 3. Discussion In immunocompetent adults, inoculation with a low dose of HBV is associated mostly with asymptomatic self-limiting
Table 1 HBV markers in case of unapparent acute HBsAg negative HBV infection Date of sample collection Results of HBV markers (index values = S/CO (signal/cut-off)) HBsAg 27.11.01 04.12.01 18.01.02 05.04.02 06.11.03 a b
Negativea Negative Negative Negativea Negative
Index Anti-HBc Index Anti-HBc-IgM Index Anti-HBs HBeAg 0.76 NA NA 0.92 0.65
Negativea Positive Positive Positivea Positivea
2.37 NA NA 0.786 0.235
Negativea Positive Negative Negativea Negative
0.148 NA NA 0.132 0.121
<6 IU/L Negative Negative 12 IU/La 10 IU/La
Negativea Negative Negative Negativea Negativea
Index Anti-HBe Index HBV DNA PCR 1.46 NA NA 1.25 1.01
Negativea Negative Negative Negativea Negativea
0.086 NA NA 0.087 0.076
Positive Negativeb Negative Negative Negative
Enzygnost (Dade Behring) and AxSYM (Abbott) HBV serological assays, index values are from the results obtained with Enzygnost HBsAg. Detection-limit 300 IU/ml NA, not available.
B. Weber et al. / Journal of Clinical Virology 32 (2005) 67–70
acute HBV infection with consecutive immunity (Gerlich and Schaefer, 2002). HBsAg is present at low concentrations and during a very short time period in unapparent acute hepatitis B and may therefore not be detected by HBsAg screening in blood donors, as shown by the present case report. In order to increase the safety of blood and blood products, nucleic acid amplification technique (NAT) has been introduced in many industrialised countries for the screening of HIV and HCV in pooled plasma samples and/or individual donations. Technical improvements and automation of all the steps of NAT, including sample preparation, make it possible to perform nucleic acid amplification for the three most-relevant blood-borne infections, HIV, hepatitis B and C in mini-pools or single donations (Meng et al., 2001). While there is an international consensus to perform NAT for HIV and HCV, the utility in terms of cost-effectiveness for HBV NAT screening is still controversially discussed and some European countries are opposed to blood donor screening for HBV DNA. Since about 95% of HBV infections are self-limiting and the mean survival rate of blood recipients is relatively low, due to the severe underlying disease or condition, which necessitates blood transfusion, the incremental costs for reducing the transfusion risk are high but the benefit for the patient in terms of quality of life is relatively poor. HBV DNA is detected 3–5 weeks after infection and 1–2 weeks before the appearance of HBsAg, newer, more sensitive assays are closing this gap (Weber et al., 1999, 2003). HBV DNA in contrast to HIV and HCV RNA rises slowly and circulates at relatively low levels (30–300 IU/mL) during the early HBsAg seronegative window period (Hollinger and Liang, 2001). HBV NAT should have a detection threshold under 30 IU/mL in single donations in order to detect low copy number of virus nucleic acid in the HBsAg negative window phase and to further reduce the NAT diagnostic window period and to detect low-level chronic HBV carriers. Ten- to twenty-fold more sensitive NAT protocols could be used as an alternative for the testing of 8- or 16-member minipools in order to achieve a maximal reduction of the residual risk. In the present case, the goal of reducing the residual risk of HBV infection was achieved by applying a less sensitive protocol with a detection-limit in an individual sample of 300 IU/mL. This finding argues for HBV DNA screening in blood donors in maxi-pools with a sensitive assay. This strategy would permit not only to detect acute HBV infection in asymptomatic blood donors with a very short HBsAg positive period but also occult HBV infection in chronic carriers who are HBsAg negative and may be negative for all the other HBV specific markers or show one of the following possible seroconstellations (isolated anti-HBc positive, with or without anti-HBc-IgM or anti-HBe, anti-HBc and anti-HBs positive, HBeAg positive). Since the viral load in chronic isolated anti-HBc positive carriers is low, there is a potential risk for failure of HBV DNA detection with pool-PCR in blood donors (Kleinman et al., 2003). Anti-HBc screening would reduce the residual
69
risk (Weber et al., 2001a, 2001b; Arraes et al., 2003). There are no clear recommendations for the reintroduction of antiHBc for blood donor screening, although it may be the only positive marker in chronic infectious carriers. The probably relatively poor cost/benefit relation and the crucial issue of increased loss of donors by poor specificity or incorrect result interpretation are major arguments against the routine testing of blood donors, especially in countries with low incidence of HBV infection and where donor screening by pool HBV DNA testing is already performed. Newer anti-HBc assays show an improved sensitivity (Weber et al., 1998). In our present case, asymptomatic HBV infection would have been detected by its isolated anti-HBc reactivity in the second follow-up sample. In order to permit a more cost-effective screening of blood donations combined assays for anti-HBc/HBsAg need to be developed. In the field of HIV screening, combined antigen/antibody assays have achieved now after 5 years of experience and technical improvements a high sensitivity and specificity (Weber et al., 2002).
References Arraes LC, Ximenes R, Andrieu JM, Wei L, Barreto S, Pereira LMMB, et al. The biological meaning of anti-HBc positive result in blood donors: relation to HBV-DNA and to other serological markers. Rev Inst Med Trop Sau Paulo 2003;45(3):137–40 (ISSN 0036-4665). Biswas R, Tabor E, Hsia CC, Wright DJ, Laycock ME, Fiebig EW, et al. Comparative sensitivity of HBV NATs and HBsAg assays for detection of acute HBV infection. Transfusion 2003;43:788– 98. Gerlich WH, Schaefer S. Hepadnaviren: hepatitis-B-virus. In: Doerr HW, Gerlich HW, editors. Medizinische Virologie. Stuttgart: Georg Thieme Verlag; 2002. p. 191–210. Hollinger FB, Liang TJ. Hepatitis B virus. In: Knipe DM, Howley PM, editors. Fields Virology, 4th ed.. Philadelphia: Lippincott Williams & Wilkins; 2001. p. 2971–3036. Kleinman SH, Kuhns MC, Todd DS, Glynn Sa, McNamara A, DiMarco A, et al. Retrovirus epidemiology donor study. Frequency of HBV DNA detection in US blood donors testing positive for the presence of anti-HBc: implications for transfusion transmission and donor screening. Transfusion 2003;43:696–704. Meng Q, Wong C, Rangachari A, Tamatsukuri S, Sasaki M, Fiss E, et al. Automated multiplex assay system for simultaneous detection of hepatitis B virus DNA, hepatitis C virus RNA, and human immunodeficiency virus type 1 RNA. J Clin Microbiol 2001;39:2937–45. Pereira A. Health and economic impact of posttransfusion hepatitis B and cost-effectiveness analysis of expanded HBV testing protocols of blood donors: a study focused on the European Union. Transfusion 2003;43:192–201. Weber B, Bayer A, Kirch P, Schl¨uter V, Schlieper D, Melchior W. Improved detection of hepatitis B virus surface antigen by a new rapid automated assay. J Clin Microbiol 1999;37:2639–47. Weber B, Dengler T, Berger A, Doerr HW, Rabenau H. Evaluation of two new automated assays for hepatitis B virus surface antigen (HBsAg) detection: IMMULITE HBsAg and IMMULITE 2000 HBsAg. J Clin Microbiol 2003;41:135–43. Weber B, Gurtler L, Thorstensson R, Michl U, Muhlbacher A, Burgisser P, et al. Multicenter evaluation of a new automated fourthgeneration human immunodeficiency virus screening assay with a sensitive antigen detection module and high specificity. J Clin Microbiol 2002;40:1938–46.
70
B. Weber et al. / Journal of Clinical Virology 32 (2005) 67–70
Weber M, Hedges D, Seifried E, Roth WK. Low level HBV DNA carriers detected among HBsAg negative anti-HBc positive blood donors. 34. Jahreskongress der Deutschen Gesellschaft f¨ur Transfusionsmedizin und Immunh¨amatologie. Hamburg 25–28 September 2001. Infusionsther Transfusionsmed 2001b;28:45. Weinberger KM, Kreuzpaintner EA, Hottentr¨ager B, Neifer S, Jilg W. Mutations in the S-gene of hepatitis B virus isolates from chronic carriers with anti-HBc as the only serological marker of HBV infection. In: Rizetto M, Purcell RH, Gerin JL, Verne G, editors. Viral
Hepatitis and Liver Disease. Torino: Edizioni Minerva Medica; 1997. p. 138–43. Weber B, Melchior W, Gehrke R, Berger A, Rabenau HF. Hepatitis B virus (HBV) markers in isolated anti-HBc positive individuals. J Med Virol 2001a;64:312–9. Weber B, Michl U, M¨uhlbacher A, Paggi G, Bossi V. Evaluation of The New Automated Enzymun-Test® Anti-HBc Plus for the detection of hepatitis B virus (HBV) core antibody. Intervirology 1998;41:17– 23.