open access

Vol 4, No 2 (2011)
Other materials agreed with the Editors
Published online: 2011-07-07
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Study on polymorphism of the transfusion transmitted viruses in Poland — hepatitis B virus (HBV) and parvovirus B19 (B19V)

Piotr Grabarczyk
Journal of Transfusion Medicine 2011;4(2):45-81.

open access

Vol 4, No 2 (2011)
ROZPRAWA HABILITACYJNA
Published online: 2011-07-07

Abstract

Background: Hepatitis B virus (HBV) and parvovirus B19 (B19V) are tested in blood donors; the markers of the former are screened in all donors, the DNA of the latter is tested in donor plasma for anti-D and anti-HBs immunoglobulin production. The eight HBV genotypes that have been discovered up to date differ in geographical distribution and infection course. Some mutations in gene S are called “escape mutations” and result in decreased reactivity of surface antigen with specific antibodies or even false negative results in diagnostic assays. In the case of B19V, three genotypes have been described. The difficulties related to the diagnosis of genotypes 2 and 3 manifested in false negative results and underestimation of the results of quantitative testing have been described in literature. The course of infection with these genotypes is not well understood.
The aim of the study was to present the results of B19V and HBV polymorphism testing in Poland. Detailed analysis of HBV polymorphism was performed in plasma from donors at various stages of infection. Samples were collected from the territory of the whole country. The HBV genome study involved identification of genotype and specific mutations analysis. Parvovirus B19 genotypes were tested in symptomatic patients. The course of non-genotype 1 infections was described in detail.
Material and methods: HBV infected donors were identified during HBsAg and DNA HBV blood donor screening. For precise discrimination of infection stage additional HBV infection markers were tested (HBe antigen, anti-HBc, anti-HBs and anti-HBe antibodies), HBV DNA level was estimated and when HBsAg was detected in the sample, we also determined the antigen load. The HBV polymorphism analysis was performed in seven donor samples in early infection stage, in 170 samples from HBsAg positive donors and in 40 donors with the so called “occult” HBV infection (OBI). The HBV genotype was determined with sequencing and subsequent phylogenetic analysis or by region pre-S/S analysis with BLAST/Genotyping software. The whole genome (n = 53) or/and specific regions pre-S/S and basic core promoter/pre-core (BCP/PC) were analysed. Patients infected with B19V were identified with real-time PCR (real time polymerase chain reaction), which detects all three B19V genotypes. In the period 2004–2008 sixty nine cases, mostly acute-phase B19V DNA positive, were identified in patients from hematological and obstetric/gynecological wards. Thirty patients were studied in greater detail and genotyping was performed by analysis of the NS1/VP1u region. Follow-up samples were collected from patients with non-genotype 1 infection. Peripheral blood morphology parameters and virological markers (anti-B19V and B19V) were analysed.
Results: Median donor age at early stage of HBV infection was 40; they were HBsAg negative and HBV DNA positive (median 10.12 IU/ml, maximum 140 IU/ml). In one donor, anti-HBs, anti-HBc IgM and anti-HBe were also detected. HBsAg donors were younger (median age 21). In this group we detected anti-HBs, anti-HBe and HBeAg in 5%; 92.4% and 10.5% of donors, respectively. The HBV DNA load ranged between unquantifiable and 3.1 × 1010 IU/ml (median: 4.10 × 103 IU/ml). Donors with OBI were the oldest (median age 47), all anti-HBc positive; in 7 donors a low level of anti-HBs was detected (median 1.45 IU/ml); none of the donors in this group was found HBeAg positive, in 38.1% however we detected antibodies to this antigen.
We identified three genotypes — A, D and H in Polish HBV infected blood donors. The first two genotypes were detected in all three groups of HBV infected donors, genotype H was identified only in two donors with OBI. The distribution of A and D genotypes in HBsAg positive and OBI donors was different. In the former (HBsAg positive) group most donors were infected with genotype A2 (80.5%) while genotype D was almost four fold less frequent (19.5%). In the latter group (OBI) genotype D was most frequent (57,5%) and genotype A was identified only in 37.5% of tested donors. Detailed analysis of MHR of gene S in donors at early infection stage revealed only wild type infection. In HBsAg positive and OBI donors we observed increasing polymorphism at amino acid sequence level. The frequency of HBV donors with amino acid substitutions in MHR was 17.6% and 52.5% of donors with HBsAg and OBI, respectively. In both groups of donors the frequency of this type of substitutions was higher in genotype D infected donors than those infected with genotype A. We identified mutations leading to amino acid substitution which obstracted the recognition of HBsAg by antibodies during diagnostics with serological methods. Detailed phylogenetic analysis of whole genomes from HBsAg positive donors revealed infections with subtypes A2, D1 and D2.
The median HBsAg/HBV DNA ratio expressed in IU/ml was approximately 1 for both genotypes, but extremely low or very high ratios appeared more frequently in genotype D infections. BCP/PC region analysis in HBsAg positive donors revealed the double mutation 1762T/ /1764A in 49/125 (39.2%) genotype A2 and 6/29 (20.7%) genotype D strains (p = 0.08). Mutations in BCP/PC region correlated neither with HBsAg nor HBV DNA levels.
The B19V polymorphism analysis revealed most samples to be infected with genotype 1. Two (6.6%) strains however were identified as genotype 2, associated with high viraemia and identified in a kidney transplant recipient with anemia and a leukemia patient with pancytopenia following chemotherapy. In both cases B19V viraemia was very high. Treatment with intravenous immunoglobulin in the former patient and a change of immunosupression treatment in the latter, resulted in normalization of clinical parameters, and whilst viral loads fell, B19V DNA was still detectable. The kidney transplant recipient subsequently became pregnant with no clinical complications, although persistently infected with B19V genotype 2.
Conclusions: Decision to choose the appropriate diagnostic and screening tests, their validation and quality control should take into account not only the higher frequency of A genotype in HBV and genotype 1 of B19V, but also less frequent genotypes D and H and genotype 2 of respective viruses. The present study is in accordance with up to date information referring to the presence of HBV genotype H in Poland. Our observations do not confirm complete disappearance of genotype 2. in our country. The course of genotype 2 infection in immunosuppresive patients is similar to that observed for genotype 1 B19V infection. Polymorphism in the region which codes epitopes crucial for HBs antigen-binding by antibodies was observed to be higher in donors infected with genotype D HBV compared to donors infected with genotype A This may be significant for proper diagnostics with serological methods and for effectiveness of vaccination and anti-HBs immunoglobulin prophylaxis.
J. Transf. Med. 2011; 2: 45–81

Abstract

Background: Hepatitis B virus (HBV) and parvovirus B19 (B19V) are tested in blood donors; the markers of the former are screened in all donors, the DNA of the latter is tested in donor plasma for anti-D and anti-HBs immunoglobulin production. The eight HBV genotypes that have been discovered up to date differ in geographical distribution and infection course. Some mutations in gene S are called “escape mutations” and result in decreased reactivity of surface antigen with specific antibodies or even false negative results in diagnostic assays. In the case of B19V, three genotypes have been described. The difficulties related to the diagnosis of genotypes 2 and 3 manifested in false negative results and underestimation of the results of quantitative testing have been described in literature. The course of infection with these genotypes is not well understood.
The aim of the study was to present the results of B19V and HBV polymorphism testing in Poland. Detailed analysis of HBV polymorphism was performed in plasma from donors at various stages of infection. Samples were collected from the territory of the whole country. The HBV genome study involved identification of genotype and specific mutations analysis. Parvovirus B19 genotypes were tested in symptomatic patients. The course of non-genotype 1 infections was described in detail.
Material and methods: HBV infected donors were identified during HBsAg and DNA HBV blood donor screening. For precise discrimination of infection stage additional HBV infection markers were tested (HBe antigen, anti-HBc, anti-HBs and anti-HBe antibodies), HBV DNA level was estimated and when HBsAg was detected in the sample, we also determined the antigen load. The HBV polymorphism analysis was performed in seven donor samples in early infection stage, in 170 samples from HBsAg positive donors and in 40 donors with the so called “occult” HBV infection (OBI). The HBV genotype was determined with sequencing and subsequent phylogenetic analysis or by region pre-S/S analysis with BLAST/Genotyping software. The whole genome (n = 53) or/and specific regions pre-S/S and basic core promoter/pre-core (BCP/PC) were analysed. Patients infected with B19V were identified with real-time PCR (real time polymerase chain reaction), which detects all three B19V genotypes. In the period 2004–2008 sixty nine cases, mostly acute-phase B19V DNA positive, were identified in patients from hematological and obstetric/gynecological wards. Thirty patients were studied in greater detail and genotyping was performed by analysis of the NS1/VP1u region. Follow-up samples were collected from patients with non-genotype 1 infection. Peripheral blood morphology parameters and virological markers (anti-B19V and B19V) were analysed.
Results: Median donor age at early stage of HBV infection was 40; they were HBsAg negative and HBV DNA positive (median 10.12 IU/ml, maximum 140 IU/ml). In one donor, anti-HBs, anti-HBc IgM and anti-HBe were also detected. HBsAg donors were younger (median age 21). In this group we detected anti-HBs, anti-HBe and HBeAg in 5%; 92.4% and 10.5% of donors, respectively. The HBV DNA load ranged between unquantifiable and 3.1 × 1010 IU/ml (median: 4.10 × 103 IU/ml). Donors with OBI were the oldest (median age 47), all anti-HBc positive; in 7 donors a low level of anti-HBs was detected (median 1.45 IU/ml); none of the donors in this group was found HBeAg positive, in 38.1% however we detected antibodies to this antigen.
We identified three genotypes — A, D and H in Polish HBV infected blood donors. The first two genotypes were detected in all three groups of HBV infected donors, genotype H was identified only in two donors with OBI. The distribution of A and D genotypes in HBsAg positive and OBI donors was different. In the former (HBsAg positive) group most donors were infected with genotype A2 (80.5%) while genotype D was almost four fold less frequent (19.5%). In the latter group (OBI) genotype D was most frequent (57,5%) and genotype A was identified only in 37.5% of tested donors. Detailed analysis of MHR of gene S in donors at early infection stage revealed only wild type infection. In HBsAg positive and OBI donors we observed increasing polymorphism at amino acid sequence level. The frequency of HBV donors with amino acid substitutions in MHR was 17.6% and 52.5% of donors with HBsAg and OBI, respectively. In both groups of donors the frequency of this type of substitutions was higher in genotype D infected donors than those infected with genotype A. We identified mutations leading to amino acid substitution which obstracted the recognition of HBsAg by antibodies during diagnostics with serological methods. Detailed phylogenetic analysis of whole genomes from HBsAg positive donors revealed infections with subtypes A2, D1 and D2.
The median HBsAg/HBV DNA ratio expressed in IU/ml was approximately 1 for both genotypes, but extremely low or very high ratios appeared more frequently in genotype D infections. BCP/PC region analysis in HBsAg positive donors revealed the double mutation 1762T/ /1764A in 49/125 (39.2%) genotype A2 and 6/29 (20.7%) genotype D strains (p = 0.08). Mutations in BCP/PC region correlated neither with HBsAg nor HBV DNA levels.
The B19V polymorphism analysis revealed most samples to be infected with genotype 1. Two (6.6%) strains however were identified as genotype 2, associated with high viraemia and identified in a kidney transplant recipient with anemia and a leukemia patient with pancytopenia following chemotherapy. In both cases B19V viraemia was very high. Treatment with intravenous immunoglobulin in the former patient and a change of immunosupression treatment in the latter, resulted in normalization of clinical parameters, and whilst viral loads fell, B19V DNA was still detectable. The kidney transplant recipient subsequently became pregnant with no clinical complications, although persistently infected with B19V genotype 2.
Conclusions: Decision to choose the appropriate diagnostic and screening tests, their validation and quality control should take into account not only the higher frequency of A genotype in HBV and genotype 1 of B19V, but also less frequent genotypes D and H and genotype 2 of respective viruses. The present study is in accordance with up to date information referring to the presence of HBV genotype H in Poland. Our observations do not confirm complete disappearance of genotype 2. in our country. The course of genotype 2 infection in immunosuppresive patients is similar to that observed for genotype 1 B19V infection. Polymorphism in the region which codes epitopes crucial for HBs antigen-binding by antibodies was observed to be higher in donors infected with genotype D HBV compared to donors infected with genotype A This may be significant for proper diagnostics with serological methods and for effectiveness of vaccination and anti-HBs immunoglobulin prophylaxis.
J. Transf. Med. 2011; 2: 45–81
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Keywords

hepatitis B virus; HBV; parvovirus B19; B19V; polymorphism; genotypes; mutants; variants; transmission; epidemiology; blood donors

About this article
Title

Study on polymorphism of the transfusion transmitted viruses in Poland — hepatitis B virus (HBV) and parvovirus B19 (B19V)

Journal

Journal of Transfusion Medicine

Issue

Vol 4, No 2 (2011)

Article type

Other materials agreed with the Editors

Pages

45-81

Published online

2011-07-07

Bibliographic record

Journal of Transfusion Medicine 2011;4(2):45-81.

Keywords

hepatitis B virus
HBV
parvovirus B19
B19V
polymorphism
genotypes
mutants
variants
transmission
epidemiology
blood donors

Authors

Piotr Grabarczyk

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