Eur J Clin Microbiol Infect Dis DOI 10.1007/s10096-013-1930-9
ARTICLE
Surface antigen-negative hepatitis B virus infection in Dutch blood donors R. W. Lieshout-Krikke & M. W. A. Molenaar-de Backer & P. van Swieten & H. L. Zaaijer
Received: 27 May 2013 / Accepted: 10 July 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Hepatitis B virus (HBV) surface antigen (HBsAg) is a reliable marker for HBV infection, but HBsAg-negative forms of HBV infection occur. The introduction of HBV DNA screening of Dutch blood donors, which were not preselected for absence of HBV core antibodies, enabled the characterization of HBsAg-negative HBV infection in healthy persons and a comparison of the HBV genomes involved. The screening of 4.4 million Dutch blood donations identified 23 HBsAg-negative, HBV DNA-positive persons. Serological testing of the index donations, follow-up samples and archived earlier samples was performed to determine the nature of each HBV DNA-only case. Despite low viral loads HBV DNA could be sequenced in 14 out of 23 donors, allowing HBV genotyping and the analysis of mutations in the HBV surface gene. Four types of HBsAg-negative HBV infection were detected: infection in the early stage before occurrence of HBsAg; suppressed infection after vaccination; HBV genotype G infection with decreased HBsAg production; and chronic occult (HBsAg negative) HBV infection. In the donors with occult HBV genotype D infection the HBV surface gene showed multiple “escape” mutations in the HBsAg adeterminant and CTL epitopes, while in an occult genotype A case the surface gene showed no mutations. HBsAg-negative forms of HBV infection in healthy blood donors explain the
Electronic supplementary material The online version of this article (doi:10.1007/s10096-013-1930-9) contains supplementary material, which is available to authorized users R. W. Lieshout-Krikke : M. W. A. Molenaar-de Backer : P. van Swieten : H. L. Zaaijer (*) Department of Blood-borne Infections, Sanquin Blood Supply Foundation, Sanquin-BOI, Plesmanlaan 125, 1066CX Amsterdam, The Netherlands e-mail: [email protected] H. L. Zaaijer Clinical Virology (CINIMA), Academic Medical Center, Amsterdam, The Netherlands
ongoing transmission of HBV via blood transfusion, if donor screening is limited to HBsAg. The screening of blood donors for HBV DNA and HBV core antibodies seems to cover all stages and variants of HBV infection.
Introduction Despite intensive donor selection and donor testing, transmission of hepatitis B virus (HBV) by blood transfusion still occurs and HBV infection is considered to be the most frequent transfusion-transmitted infection [1]. Since approximately 1973 blood donations in the Western world are screened for hepatitis B surface antigen (HBsAg), eliminating most transfusion-transmitted HBV infections. After exposure to HBV a “window” period occurs. In this phase HBV infection is not yet detectable, but donated blood may already be infectious. For HBV the infectious window period is estimated to be approximately 35.5 days, using a modern HBsAg screening test [1, 2]. To shorten this window period and to detect HBsAg-negative variants of chronic HBV infection, in July 2006 HBV DNA testing was introduced in the Netherlands. The screening of Dutch donors for HBV DNA, in the absence of screening for anti-HBcore antibodies, introduced the detection of HBsAg-negative, HBV DNA-positive donations, enabling the study of mechanisms behind HBsAgnegative HBV infection in healthy persons.
Materials and methods Donor samples, screening, confirmatory testing, and supplemental assays Since 1973 each Dutch blood donation has been tested for the presence of HBsAg. For this study we included all blood donations that were found to be HBsAg-negative but HBV
Eur J Clin Microbiol Infect Dis
DNA-positive, after the introduction of HBV DNA screening in July 2006, until the introduction of anti-HBcore screening in June 2011. In this period 4,389,928 donations from approximately 550,000 different blood donors were screened for HBV DNA as follows. From July 2006 until November 2008 a total of 2,056,942 donations were tested for HBV DNA as part of routine in-process testing for plasma fractionation. For this purpose pools of 480 donations were tested using the COBAS HBV AmpliScreen test (Roche Molecular Diagnostics, Pleasanton, CA, USA; lower limit of detection: approximately 2,500 IU/mL for a pool of 480). From November 2008 until June 2011 all 2,332,986 blood donations were tested for HBV DNA in pools of 6 donations, using the COBAS TaqScreen MPX test on the S201 platform (Roche Molecular Diagnostics; lower limit of detection: 23 IU/mL for a pool of 6), as part of universal donor screening. When a 480-donation plasma pool tested positive for HBV DNA, the pool was deconstructed to identify the individual HBV DNA-positive donation. For each positive donation the plasma bag was retrieved to confirm the presence of HBV DNA (COBAS AmpliScreen, Roche Molecular Diagnostics; lower limit of detection: 5.2 IU/mL) and to test for HBsAg, anti-HBc total (IgG and IgM), anti-HBc IgM, anti-HBs, antiHBe, and HBeAg (Architect or AxSYM assays; Abbott Laboratories, Chicago, IL, USA). Approximately 1 month after the index donation follow-up material was collected to confirm HBV infection. Regarding universal donor screening, reactive pools of 6 donations were deconstructed to identify individual HBV DNA-reactive donations and to perform confirmatory testing. The confirmatory and supplemental tests consisted of HBV DNA PCR (COBAS AmpliScreen, Roche Molecular Diagnostics), anti-HBs and anti-HBc total (Architect, Abbott Laboratories). Approximately 1 month after the index donation follow-up material was collected to confirm HBV infection.
Specimen Diluent (Roche Molecular Diagnostics), and sample D23 was resuspended in 100 μL of Specimen Diluent. Ten microliters of the extract were used for full genome PCR, based on Gunther et al. [3]. The full genome PCR used the forward primer HBV-1821Fw and the reverse primer HBV-1825Rv (see Supplementary Material #1), at a concentration of 0.2 μM/reaction, together with 4 mM MgCl2, 0.2 mM of each dNTP and 2.5 units Go Taq DNA polymerase in 1x Go Taq buffer (Promega, Madison, WI, USA). The PCR program was 95 °C for 3 min followed by 29 cycles of 15 s at 95 °C, 15 s at 54 °C, and 4 min at 72 °C, a final elongation step of 72 °C for 10 min and cooling to 4 °C. After the full genome PCR 2 μL from this PCR was taken for nested PCRs of 3–5 different fragments of the HBV DNA (see Supplementary Material #1). The nested PCR mixture consisted of 0.2 μM of a forward and reverse primer, 3.5 mM of MgCl2, 0.2 mM of each dNTP and 2.5 units Go Taq DNA polymerase in 1x Go Taq buffer (Promega). The PCR program was 95 °C for 5 min followed by 34 cycles of 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 90 s, a final elongation step of 72 °C for 10 min followed by cooling to 4 °C. PCR products were cleaned with ExoSAP-IT (Affymetrix, USB products, Santa Clara, CA, USA) and afterwards BigDye cycle sequencing reactions (Applied Biosystems, Carlsbad, CA, USA) were performed with individual primers (see Supplementary Material #1). Sequencing was performed on an ABI PRISM 3130xl Genetic Analyser (Applied Biosystems). The full genome HBV sequence of donor D8 has been described in a separate report and is available in GenBank under accession number GU565217 [4]. Near full genomes (∼3,100 bp, nested fragments 1–5), or Surface and Core gene sequences (∼1,800 bp, nested fragments 2–4) of the other donors are available in GenBank under accession numbers JX310721–JX310735.
HBV DNA sequencing
The HBV sequences were aligned using MUSCLE (Geneious, version 5.6.5; Biomatter Ltd., Auckland, New Zealand). Phylogenetic relations were analyzed using the maximum likelihood method in MEGA5 software [5]. The general time reversible model and gamma-distributed evolution rates among sites (5 discrete gamma categories) were applied in the maximum likelihood method. Bootstrap analysis was performed using 1,000 replicates. HBV reference sequences were used for phylogenetic comparison and for generation of subtype-specific consensus sequences (see Supplementary Material #2). These consensus sequences were used to analyze the donor HBV sequences for the presence of mutations in the HBsAg a-determinant and in the CD8+ cytotoxic T lymphocyte (CTL) epitopes of the S antigen [6, 7]. The nomenclature of amino acid mutations in the HBV genes in this study is in accordance to Stuyver et al. and Desmond et al. [8, 9].
The HBV DNA load in HBsAg-negative samples is often very low. Therefore, the sequencing of HBV genomes was attempted using high input volumes, as follows. For 20 donors 4 mL of plasma was used to extract HBV DNA, for donor D23 (see Table 1) only 1.5 mL was available. Two donations (D16 and D20) could not be sequenced owing to a lack of material. The plasma was centrifuged in 2-mL Eppendorf tubes for 60– 65 min at 23,000 g and 4 °C to pellet HBV. Subsequently, the supernatant was removed and HBV was lysed with 600 μL/ tube of MultiPrep Lysis Reagent (Roche Molecular Diagnostics). After 10-min incubation at room temperature 700 μL isopropanol was added to each tube and the samples were centrifuged for 15 min at 1,750 g at room temperature. The pellet was washed with 1 mL of 70 % ethanol. After removal of the wash buffer, 20 samples were resuspended in 200 μL of
Phylogenetic analysis
Eur J Clin Microbiol Infect Dis Table 1 Classification and characteristics of 23 HBsAg-negative, HBV DNA-positive blood donors Nature of HBV infection
Donor Male/ Age Index donation female (years) AntiAntiHBs HBc (IU/L)
Early D1 preseroconversion infection D2 D3 D4 Suppressed infection D5a after HBV vaccination D6 D7
HBV genotype G
D8 a
Occult chronic HBV D9 infection D10 D11 D12
Follow-up samples HBV HBV DNA Genbank genotype load (IU/ accession ml) number
Days after index
HBsAg
AntiHBs (IU/L)
AntiHBc
Negative Positive (105) Negative Negative Negative Positive (250) Positive (122) Positive (570) Positive (118) Negative Positive (162) Negative Negative Positive (78) Positive (141) Positive (104) Positive (19) Negative Positive (18) Positive (25) Negative Negative Negative Negative Negative Positive (32) Positive (96) Positive (14) Negative
Negative Positive
Male
30
Negative Negative A2
2870
JX310722
14 49
Positive
Female Male Male Male
27 33 41 44
A2 A2 A2 A2
nd 205 66 nd
JX310724 JX310725 JX310732 JX310723
9 14 30 22
Positive Positive Positive Negative
Male
43
Negative Negative Negative Positive (241) Positive (90) Negative
Negative A2
26
JX310731
8
Negative
Negative A2
201
JX310730
14
Negative
134
Negative
Female 54
Male
42
Male 46 Female 63 Female 61 Male
62
D13
Male
64
D14 D15
Male Male
58 67
Negative Negative Negative Negative
Negative Negative G
Negative Negative Positive (31) Positive (86)
nd
GU565217
37 159
Negative Negative
JX310728 JX310726 JX310721
17 8 14
Negative Negative Negative
15
Negative
50
Negative
15
Negative
15 11
Negative Negative
28
Negative
13 37 15 14 11 24
Negative Negative Negative Negative Negative Negative
16
Negative
21
Negative
12
Negative
Positive Positive Positive
D2 D7 A2
ARTICLE
Surface antigen-negative hepatitis B virus infection in Dutch blood donors R. W. Lieshout-Krikke & M. W. A. Molenaar-de Backer & P. van Swieten & H. L. Zaaijer
Received: 27 May 2013 / Accepted: 10 July 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Hepatitis B virus (HBV) surface antigen (HBsAg) is a reliable marker for HBV infection, but HBsAg-negative forms of HBV infection occur. The introduction of HBV DNA screening of Dutch blood donors, which were not preselected for absence of HBV core antibodies, enabled the characterization of HBsAg-negative HBV infection in healthy persons and a comparison of the HBV genomes involved. The screening of 4.4 million Dutch blood donations identified 23 HBsAg-negative, HBV DNA-positive persons. Serological testing of the index donations, follow-up samples and archived earlier samples was performed to determine the nature of each HBV DNA-only case. Despite low viral loads HBV DNA could be sequenced in 14 out of 23 donors, allowing HBV genotyping and the analysis of mutations in the HBV surface gene. Four types of HBsAg-negative HBV infection were detected: infection in the early stage before occurrence of HBsAg; suppressed infection after vaccination; HBV genotype G infection with decreased HBsAg production; and chronic occult (HBsAg negative) HBV infection. In the donors with occult HBV genotype D infection the HBV surface gene showed multiple “escape” mutations in the HBsAg adeterminant and CTL epitopes, while in an occult genotype A case the surface gene showed no mutations. HBsAg-negative forms of HBV infection in healthy blood donors explain the
Electronic supplementary material The online version of this article (doi:10.1007/s10096-013-1930-9) contains supplementary material, which is available to authorized users R. W. Lieshout-Krikke : M. W. A. Molenaar-de Backer : P. van Swieten : H. L. Zaaijer (*) Department of Blood-borne Infections, Sanquin Blood Supply Foundation, Sanquin-BOI, Plesmanlaan 125, 1066CX Amsterdam, The Netherlands e-mail: [email protected] H. L. Zaaijer Clinical Virology (CINIMA), Academic Medical Center, Amsterdam, The Netherlands
ongoing transmission of HBV via blood transfusion, if donor screening is limited to HBsAg. The screening of blood donors for HBV DNA and HBV core antibodies seems to cover all stages and variants of HBV infection.
Introduction Despite intensive donor selection and donor testing, transmission of hepatitis B virus (HBV) by blood transfusion still occurs and HBV infection is considered to be the most frequent transfusion-transmitted infection [1]. Since approximately 1973 blood donations in the Western world are screened for hepatitis B surface antigen (HBsAg), eliminating most transfusion-transmitted HBV infections. After exposure to HBV a “window” period occurs. In this phase HBV infection is not yet detectable, but donated blood may already be infectious. For HBV the infectious window period is estimated to be approximately 35.5 days, using a modern HBsAg screening test [1, 2]. To shorten this window period and to detect HBsAg-negative variants of chronic HBV infection, in July 2006 HBV DNA testing was introduced in the Netherlands. The screening of Dutch donors for HBV DNA, in the absence of screening for anti-HBcore antibodies, introduced the detection of HBsAg-negative, HBV DNA-positive donations, enabling the study of mechanisms behind HBsAgnegative HBV infection in healthy persons.
Materials and methods Donor samples, screening, confirmatory testing, and supplemental assays Since 1973 each Dutch blood donation has been tested for the presence of HBsAg. For this study we included all blood donations that were found to be HBsAg-negative but HBV
Eur J Clin Microbiol Infect Dis
DNA-positive, after the introduction of HBV DNA screening in July 2006, until the introduction of anti-HBcore screening in June 2011. In this period 4,389,928 donations from approximately 550,000 different blood donors were screened for HBV DNA as follows. From July 2006 until November 2008 a total of 2,056,942 donations were tested for HBV DNA as part of routine in-process testing for plasma fractionation. For this purpose pools of 480 donations were tested using the COBAS HBV AmpliScreen test (Roche Molecular Diagnostics, Pleasanton, CA, USA; lower limit of detection: approximately 2,500 IU/mL for a pool of 480). From November 2008 until June 2011 all 2,332,986 blood donations were tested for HBV DNA in pools of 6 donations, using the COBAS TaqScreen MPX test on the S201 platform (Roche Molecular Diagnostics; lower limit of detection: 23 IU/mL for a pool of 6), as part of universal donor screening. When a 480-donation plasma pool tested positive for HBV DNA, the pool was deconstructed to identify the individual HBV DNA-positive donation. For each positive donation the plasma bag was retrieved to confirm the presence of HBV DNA (COBAS AmpliScreen, Roche Molecular Diagnostics; lower limit of detection: 5.2 IU/mL) and to test for HBsAg, anti-HBc total (IgG and IgM), anti-HBc IgM, anti-HBs, antiHBe, and HBeAg (Architect or AxSYM assays; Abbott Laboratories, Chicago, IL, USA). Approximately 1 month after the index donation follow-up material was collected to confirm HBV infection. Regarding universal donor screening, reactive pools of 6 donations were deconstructed to identify individual HBV DNA-reactive donations and to perform confirmatory testing. The confirmatory and supplemental tests consisted of HBV DNA PCR (COBAS AmpliScreen, Roche Molecular Diagnostics), anti-HBs and anti-HBc total (Architect, Abbott Laboratories). Approximately 1 month after the index donation follow-up material was collected to confirm HBV infection.
Specimen Diluent (Roche Molecular Diagnostics), and sample D23 was resuspended in 100 μL of Specimen Diluent. Ten microliters of the extract were used for full genome PCR, based on Gunther et al. [3]. The full genome PCR used the forward primer HBV-1821Fw and the reverse primer HBV-1825Rv (see Supplementary Material #1), at a concentration of 0.2 μM/reaction, together with 4 mM MgCl2, 0.2 mM of each dNTP and 2.5 units Go Taq DNA polymerase in 1x Go Taq buffer (Promega, Madison, WI, USA). The PCR program was 95 °C for 3 min followed by 29 cycles of 15 s at 95 °C, 15 s at 54 °C, and 4 min at 72 °C, a final elongation step of 72 °C for 10 min and cooling to 4 °C. After the full genome PCR 2 μL from this PCR was taken for nested PCRs of 3–5 different fragments of the HBV DNA (see Supplementary Material #1). The nested PCR mixture consisted of 0.2 μM of a forward and reverse primer, 3.5 mM of MgCl2, 0.2 mM of each dNTP and 2.5 units Go Taq DNA polymerase in 1x Go Taq buffer (Promega). The PCR program was 95 °C for 5 min followed by 34 cycles of 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 90 s, a final elongation step of 72 °C for 10 min followed by cooling to 4 °C. PCR products were cleaned with ExoSAP-IT (Affymetrix, USB products, Santa Clara, CA, USA) and afterwards BigDye cycle sequencing reactions (Applied Biosystems, Carlsbad, CA, USA) were performed with individual primers (see Supplementary Material #1). Sequencing was performed on an ABI PRISM 3130xl Genetic Analyser (Applied Biosystems). The full genome HBV sequence of donor D8 has been described in a separate report and is available in GenBank under accession number GU565217 [4]. Near full genomes (∼3,100 bp, nested fragments 1–5), or Surface and Core gene sequences (∼1,800 bp, nested fragments 2–4) of the other donors are available in GenBank under accession numbers JX310721–JX310735.
HBV DNA sequencing
The HBV sequences were aligned using MUSCLE (Geneious, version 5.6.5; Biomatter Ltd., Auckland, New Zealand). Phylogenetic relations were analyzed using the maximum likelihood method in MEGA5 software [5]. The general time reversible model and gamma-distributed evolution rates among sites (5 discrete gamma categories) were applied in the maximum likelihood method. Bootstrap analysis was performed using 1,000 replicates. HBV reference sequences were used for phylogenetic comparison and for generation of subtype-specific consensus sequences (see Supplementary Material #2). These consensus sequences were used to analyze the donor HBV sequences for the presence of mutations in the HBsAg a-determinant and in the CD8+ cytotoxic T lymphocyte (CTL) epitopes of the S antigen [6, 7]. The nomenclature of amino acid mutations in the HBV genes in this study is in accordance to Stuyver et al. and Desmond et al. [8, 9].
The HBV DNA load in HBsAg-negative samples is often very low. Therefore, the sequencing of HBV genomes was attempted using high input volumes, as follows. For 20 donors 4 mL of plasma was used to extract HBV DNA, for donor D23 (see Table 1) only 1.5 mL was available. Two donations (D16 and D20) could not be sequenced owing to a lack of material. The plasma was centrifuged in 2-mL Eppendorf tubes for 60– 65 min at 23,000 g and 4 °C to pellet HBV. Subsequently, the supernatant was removed and HBV was lysed with 600 μL/ tube of MultiPrep Lysis Reagent (Roche Molecular Diagnostics). After 10-min incubation at room temperature 700 μL isopropanol was added to each tube and the samples were centrifuged for 15 min at 1,750 g at room temperature. The pellet was washed with 1 mL of 70 % ethanol. After removal of the wash buffer, 20 samples were resuspended in 200 μL of
Phylogenetic analysis
Eur J Clin Microbiol Infect Dis Table 1 Classification and characteristics of 23 HBsAg-negative, HBV DNA-positive blood donors Nature of HBV infection
Donor Male/ Age Index donation female (years) AntiAntiHBs HBc (IU/L)
Early D1 preseroconversion infection D2 D3 D4 Suppressed infection D5a after HBV vaccination D6 D7
HBV genotype G
D8 a
Occult chronic HBV D9 infection D10 D11 D12
Follow-up samples HBV HBV DNA Genbank genotype load (IU/ accession ml) number
Days after index
HBsAg
AntiHBs (IU/L)
AntiHBc
Negative Positive (105) Negative Negative Negative Positive (250) Positive (122) Positive (570) Positive (118) Negative Positive (162) Negative Negative Positive (78) Positive (141) Positive (104) Positive (19) Negative Positive (18) Positive (25) Negative Negative Negative Negative Negative Positive (32) Positive (96) Positive (14) Negative
Negative Positive
Male
30
Negative Negative A2
2870
JX310722
14 49
Positive
Female Male Male Male
27 33 41 44
A2 A2 A2 A2
nd 205 66 nd
JX310724 JX310725 JX310732 JX310723
9 14 30 22
Positive Positive Positive Negative
Male
43
Negative Negative Negative Positive (241) Positive (90) Negative
Negative A2
26
JX310731
8
Negative
Negative A2
201
JX310730
14
Negative
134
Negative
Female 54
Male
42
Male 46 Female 63 Female 61 Male
62
D13
Male
64
D14 D15
Male Male
58 67
Negative Negative Negative Negative
Negative Negative G
Negative Negative Positive (31) Positive (86)
nd
GU565217
37 159
Negative Negative
JX310728 JX310726 JX310721
17 8 14
Negative Negative Negative
15
Negative
50
Negative
15
Negative
15 11
Negative Negative
28
Negative
13 37 15 14 11 24
Negative Negative Negative Negative Negative Negative
16
Negative
21
Negative
12
Negative
Positive Positive Positive
D2 D7 A2
