- Email: [email protected]
Reverse vertical transmission of hepatitis B virus (HBV) infection from a transfusion-infected newborn to her mother
Case Report
Reverse vertical transmission of hepatitis B virus (HBV) infection from a transfusion-infected newborn to her mother Christoph Niederhauser1,⇑, Daniel Candotti2, Tina Weingand3, Andreas Maier3, Caroline Tinguely1, Martin Stolz1, Jean-Pierre Allain4 1
Blood Transfusion Service SRC Berne, Berne, Switzerland; 2National Health Service Blood and Transplant, Cambridge, UK; 3Blood Transfusion Service SRC Central Switzerland, Lucerne, Switzerland; 4Department of Haematology, University of Cambridge, Cambridge, UK
Background & Aims: Clinical cases of viral infections possibly involving the transfusion of blood components are systematically investigated. Methods: Serological and molecular markers of hepatitis B virus were used including HBsAg, anti-HBc, anti-HBs, HBV DNA, and viral load. Full genome sequencing and phylogenetic analyses were performed. Results: An acute HBV infection was diagnosed in the mother of a 16-month-old daughter who had been transfused at age three weeks with one quarter of a regular red cell concentrate (RCC). The repeat donor of the index donation was free of HBV markers in two previous donations but seroconverted to anti-HBc and anti-HBs 3 months post-donation of a unit containing only low level of HBV DNA. One other newborn recipient of the same RCC was asymptomatically HBV infected. A third newborn recipient whose mother had been HBV vaccinated and carried moderate level of anti-HBs was not infected. Full length nucleotide sequence identity between HBV strains from the mother and the two infected transfusion recipients provided evidence of the transfusion origin of all three infections in the absence of donor sequence. Conclusions: Reverse vertical HBV transmission was likely the result of casual mother contact with a baby carrying extremely high viral load. The blood products intended to immunodeficient newborn should be submitted to more thorough viral testing considering their increased susceptibility to infections. Ó 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
and intra-familial, sexual both hetero and homosexual, and through direct blood contact including blood transfusion. To our knowledge, reverse vertical transmission from an externally infected infant to the mother, has not been described. Factors primarily involved in HBV infectivity are, on the virus side, viral load and, in the presence of neutralizing antibodies such as anti-HBs, the selection of infectious escape mutants. On the host side, deficient immune system, whether congenitally or acquired such as with HIV infection, drug induced such as in organ transplantation, or naturally in newborn and infants, was shown to affect the infectious dose being lower, and the natural history by the establishment of chronic infection [1]. In a blood transfusion context, evidence of the shortcomings of screening for the surface antigen, even with the recent most sensitive HBsAg assays, was provided to a little extent clinically but more recently by the introduction of nucleic acid testing (NAT) of blood donations. This approach has identified donors donating blood during the pre-seroconversion window period (WP) or during a late stage of the infection natural history called occult HBV infection or OBI characterized by low viral load, no detectable HBsAg, and usually anti-HBc associated or not with anti-HBs [2]. It was clearly shown that in the WP, the virus produced in the absence of antibodies was extremely infectious (<10 copies) while OBI infectivity was substantially reduced unless in immunodeficient hosts [3].
Materials and methods Case history
Introduction Hepatitis B infection natural history has recognized a large number of possible routes of transmission including vertical from infected mother to child, horizontal between young children
Keywords: Hepatitis B virus; Transfusion; Newborn; Familial transmission. Received 27 June 2011; received in revised form 7 July 2011; accepted 7 July 2011 ⇑ Corresponding author. Address: Blood Transfusion Service SRC Berne, Murtenstrasse 133, 3008 Berne, Switzerland. Tel.: +41 31 384 23 04. E-mail address: [email protected] (C. Niederhauser). Abbreviations: HBV, hepatitis B virus; RCC, red cell concentrate; NAT, nucleic acid testing; WP, window period.
In June 2009 a 40-year-old woman not vaccinated to HBV presented with a clinically acute hepatitis. HBsAg, anti-HBc IgG and IgM, and anti-HBe were positive, confirming a suspected HBV etiology. The viral load was 2.5 105 IU/ml and the alanine aminotransferase level (ALT, Cobas Integra 400, Roche Diagnostics, Rotkreuz, Switzerland) was 2924 IU/l. To determine the origin of this infection, a family investigation was initiated. Both husband and son presented no clinical or serological evidence of HBV infection. However, the 16-month-old daughter was positive for HBsAg and anti-HBc IgG and had a viral load of 1.6 109 IU/ ml. This unexpected discovery in an otherwise clinically healthy baby triggered an investigation leading to the identification of a red cell concentrate transfusion performed three weeks after birth on March 8th, 2009. The donation collected on February 2nd, 2009 was negative for HBsAg and was not tested for either anti-HBc or HBV DNA. The blood had been processed into a quadruple split of red cell concentrates. In addition to the index baby girl,
Journal of Hepatology 2012 vol. 56 j 734–737
JOURNAL OF HEPATOLOGY these blood donation subunits were transfused to two other infant recipients on March 10th (Recipient 2) and 14th (Recipient 3), respectively. The fourth split concentrate was not transfused and discarded when outdated. Retrospective clinical and laboratory examination of these two recipients at age 18 months uncovered the presence of HBsAg, low level anti-HBc and 4.0 103 IU/ml of HBV DNA in Recipient 2 but an absence of HBV markers in Recipient 3. The mother of Recipient 2 did not consent to investigations but the mother of Recipient 3 had been vaccinated to HBV in June 2006, received 2 boosting injections in October 2006 and March 2007, respectively and carried 295 IU/ml of anti-HBs in February 2011, 24 months after delivery.
Donor 15.08.08 02.02.09 (Index)
22.05.09
26.11.09
14.04.10
HBsAg
negative negative
negative
negative
negative
HBV DNA
n.d.
253 IU/ml negative
negative
negative
Anti-HBc
negative n.d.
positive
positive
positive
Anti-HBs
negative n.d.
>1000 IU/L
>1000 IU/L
>1000 IU/L
Recipient 1
Samples examined Plasma donor samples at index, three post-donation and two pre-donation dates were available. Two post-transfusion plasma samples from Recipient 1 and one sample from each Recipients 2 and 3, collected between June and October 2010, were available. Two plasma samples from the mother of Recipient 1 and one sample from mother of Recipient 3 were collected in June 2010. Other samples were collected from several family members but did not carry any HBV markers.
Recipient 3 25.06.10
08.07.10
HBsAg
positive
±
HBsAg
negative
Anti-HBc
positive
positive
Anti-HBc
negative
HBV DNA
n.d.
1.6x109 IU/ml
HBV DNA
negative
Anti-HBs
n.d.
n.d.
Anti-HBs
negative
ALT
44 IU/L
n.d.
ALT
n.d.
08.10.10
Recipient 2 05.10.10
HBV testing HBsAg was tested initially with the Architect i2000SR assay (Abbott AG, Diagnostics Division, Baar, Switzerland). Confirmation was done by alternative assays: Elecsys (Roche, Rotkreuz, Switzerland) and Axsym (Abbott, Delkenheim, Germany). HBeAg, anti-HBc IgM, anti-HBe, anti-HBc total and quantitative anti-HBs were also tested with both Elecsys and Axsym, as previously described [4]. As sample volume permitted, HBV DNA presence and load were tested with the Abbott Real-Time HBV Assay (Abbott, Delkenheim, Germany), the Cobas Amplicor HBV Monitor (Roche Diagnostics, Rotkreuz, Switzerland) and an inhouse real-time PCR in the Cambridge laboratory [5]. Genotyping was performed with the INNO-LiPA HBV Genotype Assay (Innogenetics, Ghent, Belgium) and by phylogenetic analysis of the whole genome, as previously described [6]. HBV strains from the two infected infants and the infected mother of Recipient 1 were sequenced for the HBV regions S, Pre-S/S and the whole HBV genome minus 50 nucleotide of the BCP/PC.
Results The results of the HBV marker investigations in all individuals involved in this case are presented in Fig. 1. The donor details are presented in Table 1. The donor presented low level of HBV DNA but no HBsAg at the time of the index donation. A previous donation taken 4 months before was negative for all markers of HBV, in particular anti-HBc was retrospectively found negative. Three, nine and 10 months after index donation, anti-HBc and high levels of anti-HBs were found, a pattern compatible with recent HBV infection, the index donation being in the window period. The three recipients of the index donation were retrieved and tested for HBV markers. Recipient 1 was tested twice, at twoweek interval at age 17 months. The presence of 1.6 109 IU/ml viral load, HBsAg and anti-HBc 16 months after transfusion with normal ALT level suggests an HBV chronic infection established in an infant with immature immune system. Recipient 2 is most likely in the same situation although a slightly elevated ALT level of 60 IU/L was found. In contrast, Recipient 3, although exposed to the same blood at 2 weeks of age, did not carry markers of HBV infection 20 months after transfusion. None of these infants presented symptoms related to hepatitis virus infection although Recipient 1 presented several weeks of diarrhea at age 1 year and an oozing tear of the anal region. The mother of Recipient 1 was the trigger of the investigation by developing an acute hepatitis B identified 16 months after delivering her last child. The infection was confirmed by high
HBsAg
positive
Anti-HBc
low positive
HBV DNA
4x108 IU/ml
Anti-HBs
negative
ALT
60 IU/L
Mother
Mother 08.10.10
11.06.10
14.6.10
HBsAg
positive
±
HBsAg
negative
Anti-HBc
positive
positive
Anti-HBc
negative
HBV DNA
n.d.
2.5x105 IU/ml
HBV DNA
n.d.
Anti-HBs
n.d.
negative
Anti-HBs
295 IU/L
ALT
2942 IU/L n.d.
ALT
n.d.
Fig. 1. General summary of the case. Dates on top of each box indicate the time of sample collection. The flow chart goes logically from infected blood donation to recipients to mother of Recipients 1 and Recipient 2, although the flow of HBV infection discovery went in the reverse order. n.d., test not done; ± indicates weak signal in the grey zone of the assay; ALT, alanine amino transferase is expressed in IU/l. HBV DNA level is expressed in IU/ml and anti-HBs level is expressed in IU/L.
level of ALT, high viral load, positive anti-HBc, and declining HBsAg. In order to determine whether the donor was the potential origin of the three infections (Recipient 1 and his mother, Recipient 2), full genome sequencing of HBV was performed separately for each sample in order to exclude the risk of laboratory cross-contamination. As shown in Fig. 2, all three strains were genotype A2 and identical. The amino acid sequences of the pre-S/S proteins and of the major hydrophilic region were identical to the consensus sequence of the sub-genotype indicating wild type strains. Direct evidence of a common blood donation origin could not be obtained since no blood donor material was available. However, the identity of nucleotide sequence between the two unrelated recipients living in distant Swiss towns strongly suggests such relationship. Evidence that an upstream infection from baby to mother occurred was investigated. She did not breast feed at any time. The acute clinical maternal infection occurring 16 months after delivery suggested an infection when the infant was 13–14 months. At that time, the mother reports that the infant had diarrhea occasionally streaked with blood. The mother noticed that she had very dry skin with small crevices but she did not wear gloves when taking care of her baby.
Journal of Hepatology 2012 vol. 56 j 734–737
735
Case Report Table 1. Donor data in index, pre- and post-index transfusion samples.
1 2
Donor
Index donation
Date
09.06.08
15.10.08
02.02.09
22.05.09
26.11.09
HBsAg screening
Negative
Negative
Negative
Negative
Negative
Negative
HBV NAT screening
n.d.2
n.d.
n.d.
n.d.
Negative1
Negative1
Anti-HBc IgG/IgM
Negative
Negative
n.d.
Positive
Positive
Positive
Anti-HBs (IU/L)
Negative
Negative
n.d.
>1000
>1000
>1000
Anti-HBe
n.d.
n.d.
n.d.
n.d.
n.d.
Positive
HBV DNA quantitative
n.d.
n.d.
253 IU/ml
n.d.
n.d.
n.d.
14.04.10
HBV NAT screening in pools of six samples with the Roche s201 system was instituted in September 2009. n.d., not done.
The mother of Recipient 3 had been vaccinated to HBV and, when tested in February 2011 when the child was 24-monthold, her anti-HBs level was 295 IU/L, more than 3.5 years after a vaccine boosting injection.
Discussion The interpretation of the data collected on this extraordinary case is of an adult, asymptomatic, blood donor whose split red cell concentrate transmitted HBV to two susceptible recipients, one of them infecting her mother whose clinical expression of the infection triggered an investigation leading to the discovery of asymptomatic chronic infections in two immunodeficient infants and a recovered asymptomatic infection in an immunocompetent adult (Fig. 1). In the absence of a donor HBV sequence due to lack of available sample from the incriminated blood donation, this scenario is only supported by the identity of sequences between two silently infected unconnected newborns (Fig. 2). This identity extended to the sequence obtained from the mother of newborn Recipient 1, virtually excluding a laboratory contamination from Recipient 2 to Recipient 1. Nosocomial infection can be excluded by the fact that both babies were hospitalized in different hospitals, the only link between the two being the transfusion of one third of the index blood unit. The third baby, Recipient 3, was not infected although receiving the same amount of blood containing approximately 5 ml of plasma. His mother had been vaccinated to HBV and carried 295 IU/ml of anti-HBs 24 months after delivery suggesting 300–400 IU/L at the time of delivery. Although her child did not carry detectable levels of anti-HBs at age 20 months (Fig. 1), the presence of approximately the same level of passively acquired antibodies from his mother anti-HBs at the time of transfusion [7] was effective to prevent infection from low titer HBV (1500 IU infectious dose). Vaccine-induced IgG 1 and 3 are readily transferred to the fetus during the last trimester of pregnancy [8]. The trigger of this investigation was the symptomatic acute HBV infection of Recipient 1 whose virus Pre-S/S sequence was identical to her transfused daughter, strongly suggesting daughter to mother infection. Considering the 16-month interval between the likely transfusion-related infection of the newborn daughter and the mother acute HBV infection, it is likely that the transmission took place 13–14 month after delivery when the HBV load of the daughter was at least as high as it was when
736
quantified at age 17 months (1.9 109 IU/ml). This mother did not breastfeed at any time but took care of her baby. She reported that when her daughter was approximately 1 year old (winter 2009–2010) she presented dry skin and crevices of her hands. It has been previously reported that saliva and urine from chronic HBV infected individuals contain viral DNA and can be infectious [9–13]. Saliva was likely partially contaminated with serum when the child’s teeth were coming out and saliva exchange might have taken place during kissing. Changing urine-contaminated nappies without gloves protecting breached skin might be an alternative, although less likely, route of infection. The main factor in this case is the extremely high viral load observed in a baby infected during a period of relative immunodeficiency following birth. The evidence supporting the reality of this series of infections originating from the blood donor, although indirect, is rather strong. In addition to the patients’ HBV sequence identity indicated above, the pattern of absence of HBV markers in samples taken 9 and 4 months prior to the index donation, low level HBV DNA without detectable HBsAg in the index donation, followed 100 days later by the presence of anti-HBc and high level of anti-HBs strongly suggest the occurrence of an asymptomatic acute infection followed by full recovery; the index donation corresponding to the early stages of the window period (Table 1). Upon questioning, however, the donor denied any risk factors, such has sexual activity with a known carrier of HBV, promiscuous homo- or heterosexual behavior, tattooing or skin piercing. This report illustrates several important points regarding HBV epidemiology. Low levels of HBV DNA, particularly during the window period, are highly infectious especially by transfusion received by immunodeficient infants. The systematic testing of HBV DNA recently instituted nationwide in Switzerland should therefore prevent the occurrence of such infections [3]. Red cell concentrates split for pediatric use should be the object of special safety measures including single unit nucleic acid testing. The infectivity of blood or body fluids from HBV infected infants or young children is high in proportion to their extremely high viral load, explaining the high level of horizontal transmission observed in sub-Saharan Africa [14,15]. The same mechanisms here applied to a caring mother. Low levels of vaccine-acquired, transmitted through placenta, anti-HBs have been protective of low level though highly infectious HBV.
Journal of Hepatology 2012 vol. 56 j 734–737
JOURNAL OF HEPATOLOGY Conflict of interest
AY090457H X69798F
100
The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
X75658F X75663F AB036910F AB036920F M38636C
100
D23680C D50519C M54923B D23678B D50522B
100
AB194949-A3
99
AB194950-A3 AB116077-A2 100
100 100 100
10501 10255 10499 Z72478-A2 AY233286-A2 X70185-A2 Z35717-A2 V00866-A2 S50225-A2
100
100 100
M57663-A1 AY233290-A1 AY934772-A1 AY934773-A1 U9551D Z35716D M32138D X02496D AB091256E
100
AB091255E AB106564E X75664E X75657E
100
AB056515G AB056513G AB064311G
AY090454H 0.02 Fig. 2. Phylogenetic tree of the HBV full genome of three related infections. The Pre-S/S region sequenced includes 1190 nucleotides. 10,501, 10,255, and 10,499 correspond to the sequences of Recipient 1, her mother and Recipient 2, respectively. GenBank accession number followed by the hyphenated genotype identifies the reference sequences. The numbers on top of bars indicate the bootstrap value.
References [1] Wursthorn K, Wedemeyer H, Manns MP. Managing HBV in patients with impaired immunity. Gut 2010;59:1430–1445. [2] Raimondo G, Allain JP, Brunetto MR, Buendia MA, Chen DS, Colombo M, et al. Statements from the Taormina expert meeting on occult hepatitis B virus infection. J Hepatol 2008;49:652–657. [3] Kleinman SH, Lelie N, Busch MP. Infectivity of human immunodeficiency virus-1, hepatitis C virus, and hepatitis B virus and risk of transmission by transfusion. Transfusion 2009;49:2454–2489. [4] Niederhauser C, Weingand T, Candotti D, Maier A, Tinguely C, Wuillemin WA, et al. Fatal outcome of a hepatitis B virus transfusion-transmitted infection. Vox Sang 2010;98:504–507. [5] Allain JP, Candotti D, Soldan K, Sarkodie F, Phelps B, Giachetti C, et al. The risk of hepatitis B virus infection by transfusion in Kumasi, Ghana. Blood 2003;101:2419–2425. [6] Zahn A, Li C, Danso K, Candotti D, Owusu-Ofori S, Temple J, et al. Molecular characterization of occult hepatitis B virus in genotype E-infected subjects. J Gen Virol 2008;89:409–418. [7] Simister NE. Placental transport of immunoglobulin G. Vaccine 2003;21:3365–3369. [8] Gregorek H, Madalin´ski K, Woynarowski M, Mikołajewicz J, Syczewska M, Socha J. The IgG subclass profile of anti-HBs response in vaccinated children and children seroconverted after natural infection. Vaccine 2000;18:1210–1217. [9] Kidd-Ljunggren K, Holmberg A, Blackberg J, Lindqvist B. High levels of hepatitis B virus DNA in body fluids from chronic carriers. J Hospital Infect 2006;64:352–357. [10] Knuttson M, Kidd-Ljunggren K. Urine from chronic hepatitis B virus carriers: implications for infectivity. J Med Virol 2000;60:17–20. [11] Thakur V, Kazim SN, Guptan RC, Malhotra V, Sarin SK. Molecular epidemiology and transmission of hepatitis B virus in close family contacts of HBV-related chronic liver disease patients. J Med Virol 2003;70: 520–528. [12] Zampino R, Lobello S, Chiaramonte M, Venturi-Pasini C, Dumpis U, Thursz M, et al. Intra-familial transmission of hepatitis B virus in Italy: phylogenetic sequence analysis and amino-acid variation of the core gene. J Hepatol 2002;36:248–253. [13] van der Eijk AA, Niesters HG, Götz HM, Janssen HL, Schalm SW, Osterhaus AD, et al. Paired measurements of quantitative hepatitis B virus DNA in saliva and serum of chronic hepatitis B patients: implications for saliva as infectious agent. J Clin Virol 2004;29:92–94. [14] Hadziyannis SJ. Natural history of chronic hepatitis B in Euro-Mediterranean and African Countries. J Hepatol 2011, [Epub ahead of print]. [15] Norkrans G. Epidemiology of hepatitis B virus (HBV) infections with particular regard to current routes of transmission and development of cirrhosis and malignancy. Scand J Infect Dis Suppl 1990;69:43–47.
Journal of Hepatology 2012 vol. 56 j 734–737
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Reverse vertical transmission of hepatitis B virus (HBV) infection from a transfusion-infected newborn to her mother Christoph Niederhauser1,⇑, Daniel Candotti2, Tina Weingand3, Andreas Maier3, Caroline Tinguely1, Martin Stolz1, Jean-Pierre Allain4 1
Blood Transfusion Service SRC Berne, Berne, Switzerland; 2National Health Service Blood and Transplant, Cambridge, UK; 3Blood Transfusion Service SRC Central Switzerland, Lucerne, Switzerland; 4Department of Haematology, University of Cambridge, Cambridge, UK
Background & Aims: Clinical cases of viral infections possibly involving the transfusion of blood components are systematically investigated. Methods: Serological and molecular markers of hepatitis B virus were used including HBsAg, anti-HBc, anti-HBs, HBV DNA, and viral load. Full genome sequencing and phylogenetic analyses were performed. Results: An acute HBV infection was diagnosed in the mother of a 16-month-old daughter who had been transfused at age three weeks with one quarter of a regular red cell concentrate (RCC). The repeat donor of the index donation was free of HBV markers in two previous donations but seroconverted to anti-HBc and anti-HBs 3 months post-donation of a unit containing only low level of HBV DNA. One other newborn recipient of the same RCC was asymptomatically HBV infected. A third newborn recipient whose mother had been HBV vaccinated and carried moderate level of anti-HBs was not infected. Full length nucleotide sequence identity between HBV strains from the mother and the two infected transfusion recipients provided evidence of the transfusion origin of all three infections in the absence of donor sequence. Conclusions: Reverse vertical HBV transmission was likely the result of casual mother contact with a baby carrying extremely high viral load. The blood products intended to immunodeficient newborn should be submitted to more thorough viral testing considering their increased susceptibility to infections. Ó 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
and intra-familial, sexual both hetero and homosexual, and through direct blood contact including blood transfusion. To our knowledge, reverse vertical transmission from an externally infected infant to the mother, has not been described. Factors primarily involved in HBV infectivity are, on the virus side, viral load and, in the presence of neutralizing antibodies such as anti-HBs, the selection of infectious escape mutants. On the host side, deficient immune system, whether congenitally or acquired such as with HIV infection, drug induced such as in organ transplantation, or naturally in newborn and infants, was shown to affect the infectious dose being lower, and the natural history by the establishment of chronic infection [1]. In a blood transfusion context, evidence of the shortcomings of screening for the surface antigen, even with the recent most sensitive HBsAg assays, was provided to a little extent clinically but more recently by the introduction of nucleic acid testing (NAT) of blood donations. This approach has identified donors donating blood during the pre-seroconversion window period (WP) or during a late stage of the infection natural history called occult HBV infection or OBI characterized by low viral load, no detectable HBsAg, and usually anti-HBc associated or not with anti-HBs [2]. It was clearly shown that in the WP, the virus produced in the absence of antibodies was extremely infectious (<10 copies) while OBI infectivity was substantially reduced unless in immunodeficient hosts [3].
Materials and methods Case history
Introduction Hepatitis B infection natural history has recognized a large number of possible routes of transmission including vertical from infected mother to child, horizontal between young children
Keywords: Hepatitis B virus; Transfusion; Newborn; Familial transmission. Received 27 June 2011; received in revised form 7 July 2011; accepted 7 July 2011 ⇑ Corresponding author. Address: Blood Transfusion Service SRC Berne, Murtenstrasse 133, 3008 Berne, Switzerland. Tel.: +41 31 384 23 04. E-mail address: [email protected] (C. Niederhauser). Abbreviations: HBV, hepatitis B virus; RCC, red cell concentrate; NAT, nucleic acid testing; WP, window period.
In June 2009 a 40-year-old woman not vaccinated to HBV presented with a clinically acute hepatitis. HBsAg, anti-HBc IgG and IgM, and anti-HBe were positive, confirming a suspected HBV etiology. The viral load was 2.5 105 IU/ml and the alanine aminotransferase level (ALT, Cobas Integra 400, Roche Diagnostics, Rotkreuz, Switzerland) was 2924 IU/l. To determine the origin of this infection, a family investigation was initiated. Both husband and son presented no clinical or serological evidence of HBV infection. However, the 16-month-old daughter was positive for HBsAg and anti-HBc IgG and had a viral load of 1.6 109 IU/ ml. This unexpected discovery in an otherwise clinically healthy baby triggered an investigation leading to the identification of a red cell concentrate transfusion performed three weeks after birth on March 8th, 2009. The donation collected on February 2nd, 2009 was negative for HBsAg and was not tested for either anti-HBc or HBV DNA. The blood had been processed into a quadruple split of red cell concentrates. In addition to the index baby girl,
Journal of Hepatology 2012 vol. 56 j 734–737
JOURNAL OF HEPATOLOGY these blood donation subunits were transfused to two other infant recipients on March 10th (Recipient 2) and 14th (Recipient 3), respectively. The fourth split concentrate was not transfused and discarded when outdated. Retrospective clinical and laboratory examination of these two recipients at age 18 months uncovered the presence of HBsAg, low level anti-HBc and 4.0 103 IU/ml of HBV DNA in Recipient 2 but an absence of HBV markers in Recipient 3. The mother of Recipient 2 did not consent to investigations but the mother of Recipient 3 had been vaccinated to HBV in June 2006, received 2 boosting injections in October 2006 and March 2007, respectively and carried 295 IU/ml of anti-HBs in February 2011, 24 months after delivery.
Donor 15.08.08 02.02.09 (Index)
22.05.09
26.11.09
14.04.10
HBsAg
negative negative
negative
negative
negative
HBV DNA
n.d.
253 IU/ml negative
negative
negative
Anti-HBc
negative n.d.
positive
positive
positive
Anti-HBs
negative n.d.
>1000 IU/L
>1000 IU/L
>1000 IU/L
Recipient 1
Samples examined Plasma donor samples at index, three post-donation and two pre-donation dates were available. Two post-transfusion plasma samples from Recipient 1 and one sample from each Recipients 2 and 3, collected between June and October 2010, were available. Two plasma samples from the mother of Recipient 1 and one sample from mother of Recipient 3 were collected in June 2010. Other samples were collected from several family members but did not carry any HBV markers.
Recipient 3 25.06.10
08.07.10
HBsAg
positive
±
HBsAg
negative
Anti-HBc
positive
positive
Anti-HBc
negative
HBV DNA
n.d.
1.6x109 IU/ml
HBV DNA
negative
Anti-HBs
n.d.
n.d.
Anti-HBs
negative
ALT
44 IU/L
n.d.
ALT
n.d.
08.10.10
Recipient 2 05.10.10
HBV testing HBsAg was tested initially with the Architect i2000SR assay (Abbott AG, Diagnostics Division, Baar, Switzerland). Confirmation was done by alternative assays: Elecsys (Roche, Rotkreuz, Switzerland) and Axsym (Abbott, Delkenheim, Germany). HBeAg, anti-HBc IgM, anti-HBe, anti-HBc total and quantitative anti-HBs were also tested with both Elecsys and Axsym, as previously described [4]. As sample volume permitted, HBV DNA presence and load were tested with the Abbott Real-Time HBV Assay (Abbott, Delkenheim, Germany), the Cobas Amplicor HBV Monitor (Roche Diagnostics, Rotkreuz, Switzerland) and an inhouse real-time PCR in the Cambridge laboratory [5]. Genotyping was performed with the INNO-LiPA HBV Genotype Assay (Innogenetics, Ghent, Belgium) and by phylogenetic analysis of the whole genome, as previously described [6]. HBV strains from the two infected infants and the infected mother of Recipient 1 were sequenced for the HBV regions S, Pre-S/S and the whole HBV genome minus 50 nucleotide of the BCP/PC.
Results The results of the HBV marker investigations in all individuals involved in this case are presented in Fig. 1. The donor details are presented in Table 1. The donor presented low level of HBV DNA but no HBsAg at the time of the index donation. A previous donation taken 4 months before was negative for all markers of HBV, in particular anti-HBc was retrospectively found negative. Three, nine and 10 months after index donation, anti-HBc and high levels of anti-HBs were found, a pattern compatible with recent HBV infection, the index donation being in the window period. The three recipients of the index donation were retrieved and tested for HBV markers. Recipient 1 was tested twice, at twoweek interval at age 17 months. The presence of 1.6 109 IU/ml viral load, HBsAg and anti-HBc 16 months after transfusion with normal ALT level suggests an HBV chronic infection established in an infant with immature immune system. Recipient 2 is most likely in the same situation although a slightly elevated ALT level of 60 IU/L was found. In contrast, Recipient 3, although exposed to the same blood at 2 weeks of age, did not carry markers of HBV infection 20 months after transfusion. None of these infants presented symptoms related to hepatitis virus infection although Recipient 1 presented several weeks of diarrhea at age 1 year and an oozing tear of the anal region. The mother of Recipient 1 was the trigger of the investigation by developing an acute hepatitis B identified 16 months after delivering her last child. The infection was confirmed by high
HBsAg
positive
Anti-HBc
low positive
HBV DNA
4x108 IU/ml
Anti-HBs
negative
ALT
60 IU/L
Mother
Mother 08.10.10
11.06.10
14.6.10
HBsAg
positive
±
HBsAg
negative
Anti-HBc
positive
positive
Anti-HBc
negative
HBV DNA
n.d.
2.5x105 IU/ml
HBV DNA
n.d.
Anti-HBs
n.d.
negative
Anti-HBs
295 IU/L
ALT
2942 IU/L n.d.
ALT
n.d.
Fig. 1. General summary of the case. Dates on top of each box indicate the time of sample collection. The flow chart goes logically from infected blood donation to recipients to mother of Recipients 1 and Recipient 2, although the flow of HBV infection discovery went in the reverse order. n.d., test not done; ± indicates weak signal in the grey zone of the assay; ALT, alanine amino transferase is expressed in IU/l. HBV DNA level is expressed in IU/ml and anti-HBs level is expressed in IU/L.
level of ALT, high viral load, positive anti-HBc, and declining HBsAg. In order to determine whether the donor was the potential origin of the three infections (Recipient 1 and his mother, Recipient 2), full genome sequencing of HBV was performed separately for each sample in order to exclude the risk of laboratory cross-contamination. As shown in Fig. 2, all three strains were genotype A2 and identical. The amino acid sequences of the pre-S/S proteins and of the major hydrophilic region were identical to the consensus sequence of the sub-genotype indicating wild type strains. Direct evidence of a common blood donation origin could not be obtained since no blood donor material was available. However, the identity of nucleotide sequence between the two unrelated recipients living in distant Swiss towns strongly suggests such relationship. Evidence that an upstream infection from baby to mother occurred was investigated. She did not breast feed at any time. The acute clinical maternal infection occurring 16 months after delivery suggested an infection when the infant was 13–14 months. At that time, the mother reports that the infant had diarrhea occasionally streaked with blood. The mother noticed that she had very dry skin with small crevices but she did not wear gloves when taking care of her baby.
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Case Report Table 1. Donor data in index, pre- and post-index transfusion samples.
1 2
Donor
Index donation
Date
09.06.08
15.10.08
02.02.09
22.05.09
26.11.09
HBsAg screening
Negative
Negative
Negative
Negative
Negative
Negative
HBV NAT screening
n.d.2
n.d.
n.d.
n.d.
Negative1
Negative1
Anti-HBc IgG/IgM
Negative
Negative
n.d.
Positive
Positive
Positive
Anti-HBs (IU/L)
Negative
Negative
n.d.
>1000
>1000
>1000
Anti-HBe
n.d.
n.d.
n.d.
n.d.
n.d.
Positive
HBV DNA quantitative
n.d.
n.d.
253 IU/ml
n.d.
n.d.
n.d.
14.04.10
HBV NAT screening in pools of six samples with the Roche s201 system was instituted in September 2009. n.d., not done.
The mother of Recipient 3 had been vaccinated to HBV and, when tested in February 2011 when the child was 24-monthold, her anti-HBs level was 295 IU/L, more than 3.5 years after a vaccine boosting injection.
Discussion The interpretation of the data collected on this extraordinary case is of an adult, asymptomatic, blood donor whose split red cell concentrate transmitted HBV to two susceptible recipients, one of them infecting her mother whose clinical expression of the infection triggered an investigation leading to the discovery of asymptomatic chronic infections in two immunodeficient infants and a recovered asymptomatic infection in an immunocompetent adult (Fig. 1). In the absence of a donor HBV sequence due to lack of available sample from the incriminated blood donation, this scenario is only supported by the identity of sequences between two silently infected unconnected newborns (Fig. 2). This identity extended to the sequence obtained from the mother of newborn Recipient 1, virtually excluding a laboratory contamination from Recipient 2 to Recipient 1. Nosocomial infection can be excluded by the fact that both babies were hospitalized in different hospitals, the only link between the two being the transfusion of one third of the index blood unit. The third baby, Recipient 3, was not infected although receiving the same amount of blood containing approximately 5 ml of plasma. His mother had been vaccinated to HBV and carried 295 IU/ml of anti-HBs 24 months after delivery suggesting 300–400 IU/L at the time of delivery. Although her child did not carry detectable levels of anti-HBs at age 20 months (Fig. 1), the presence of approximately the same level of passively acquired antibodies from his mother anti-HBs at the time of transfusion [7] was effective to prevent infection from low titer HBV (1500 IU infectious dose). Vaccine-induced IgG 1 and 3 are readily transferred to the fetus during the last trimester of pregnancy [8]. The trigger of this investigation was the symptomatic acute HBV infection of Recipient 1 whose virus Pre-S/S sequence was identical to her transfused daughter, strongly suggesting daughter to mother infection. Considering the 16-month interval between the likely transfusion-related infection of the newborn daughter and the mother acute HBV infection, it is likely that the transmission took place 13–14 month after delivery when the HBV load of the daughter was at least as high as it was when
736
quantified at age 17 months (1.9 109 IU/ml). This mother did not breastfeed at any time but took care of her baby. She reported that when her daughter was approximately 1 year old (winter 2009–2010) she presented dry skin and crevices of her hands. It has been previously reported that saliva and urine from chronic HBV infected individuals contain viral DNA and can be infectious [9–13]. Saliva was likely partially contaminated with serum when the child’s teeth were coming out and saliva exchange might have taken place during kissing. Changing urine-contaminated nappies without gloves protecting breached skin might be an alternative, although less likely, route of infection. The main factor in this case is the extremely high viral load observed in a baby infected during a period of relative immunodeficiency following birth. The evidence supporting the reality of this series of infections originating from the blood donor, although indirect, is rather strong. In addition to the patients’ HBV sequence identity indicated above, the pattern of absence of HBV markers in samples taken 9 and 4 months prior to the index donation, low level HBV DNA without detectable HBsAg in the index donation, followed 100 days later by the presence of anti-HBc and high level of anti-HBs strongly suggest the occurrence of an asymptomatic acute infection followed by full recovery; the index donation corresponding to the early stages of the window period (Table 1). Upon questioning, however, the donor denied any risk factors, such has sexual activity with a known carrier of HBV, promiscuous homo- or heterosexual behavior, tattooing or skin piercing. This report illustrates several important points regarding HBV epidemiology. Low levels of HBV DNA, particularly during the window period, are highly infectious especially by transfusion received by immunodeficient infants. The systematic testing of HBV DNA recently instituted nationwide in Switzerland should therefore prevent the occurrence of such infections [3]. Red cell concentrates split for pediatric use should be the object of special safety measures including single unit nucleic acid testing. The infectivity of blood or body fluids from HBV infected infants or young children is high in proportion to their extremely high viral load, explaining the high level of horizontal transmission observed in sub-Saharan Africa [14,15]. The same mechanisms here applied to a caring mother. Low levels of vaccine-acquired, transmitted through placenta, anti-HBs have been protective of low level though highly infectious HBV.
Journal of Hepatology 2012 vol. 56 j 734–737
JOURNAL OF HEPATOLOGY Conflict of interest
AY090457H X69798F
100
The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
X75658F X75663F AB036910F AB036920F M38636C
100
D23680C D50519C M54923B D23678B D50522B
100
AB194949-A3
99
AB194950-A3 AB116077-A2 100
100 100 100
10501 10255 10499 Z72478-A2 AY233286-A2 X70185-A2 Z35717-A2 V00866-A2 S50225-A2
100
100 100
M57663-A1 AY233290-A1 AY934772-A1 AY934773-A1 U9551D Z35716D M32138D X02496D AB091256E
100
AB091255E AB106564E X75664E X75657E
100
AB056515G AB056513G AB064311G
AY090454H 0.02 Fig. 2. Phylogenetic tree of the HBV full genome of three related infections. The Pre-S/S region sequenced includes 1190 nucleotides. 10,501, 10,255, and 10,499 correspond to the sequences of Recipient 1, her mother and Recipient 2, respectively. GenBank accession number followed by the hyphenated genotype identifies the reference sequences. The numbers on top of bars indicate the bootstrap value.
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