The Prevalence of Antibody to Parvovirus B19 in Hemophiliacs and in the General Population

Zbl. Bakt. 284, 232-240 (1996) © Gustav Fischer Verlag, Stuttgart· Jena . New York

The Prevalence of Antibody to Parvovirus B19 in Hemophiliacs and in the General Population * A. M. EIS-HOBINGER 1, J. OLDENBURG l , H.-H. BRACKMANN l , B. MATZt, and K. E. SCHNEWEIS 1 I 2

Institute of Medical Microbiology and Immunology, University of Bonn, Germany Institute of Experimental Hematology and Transfusion Medicine, University of Bonn, Germany

Received March 5, 1996 . Accepted March 13, 1996

Summary The prevalence of antibodies to parvovirus B19 (B19) was measured in the sera of 566 hemophiliacs and 524 individuals of the general population by immunofluorescence assays, using antigen expressed by the baculovirus system. In the general population, antiB19 IgG seroprevalence was found to continuously decline from 64 percent at birth to 0 percent in the age of 9-11 months and thereupon to increase to 61 percent in the age of 12 years. In younger adults and older people, IgG seroprevalence only slowly increased with age, reaching 77 percent in people aged 60 and above. In contrast, in hemophilic children treated exclusively with virally inactivated clotting factor concentrates, neither decrease nor increase of B19 IgG antibody was detectable and the overall seroprevalence was 92 percent. In the group of hemophiliacs older than 12 years and treated before 1984 with non-inactivated clotting factor concentrates, 98 percent showed antibody to B19. AntiB19 IgM seroprevalence was significantly higher in hemophilic than in non-hemophilic individuals older than 12 years. Since it seems to be unlikely that the high seroprevalence in hemophiliacs is acquired by immunization with inactivated viral antigen, the results suggest that infection with B19 is transmitted by clotting factor concentrates, even if subjected to virucidal methods. Introduction In 1983, parvovirus B19 (B19) has been recognized as the etiological agent of erythema infectiosum, a mild febrile illness with a maculopapular rash of variable intensity (1). In the meantime, the spectrum of diseases caused by B19 includes red blood cell aplasia leading to anemia (22, 31), hydrops fetalis or fetal loss after intrauterine infection (5, 20), hand and foot purpura ("glove and sock" syndrome) (11, 18), and

* Dedicated to Prof. Dr. H. Brandis on occasion of his 80 th birthday.

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various forms of arthropathy (32, 42, 45). Furthermore, meningitis and encephalopathy (7, 40, 43, 44), cardiomyopathy (25) and systemic necrotising vasculitis (12) have been associated with B19 infection. Particularly in immunocompromised patients, chronic and persistent infections have been recognized (13,14,15,19,21,30). The broad spectrum of diseases seems to be consistent with the distribution of the cellular receptor of the virus, the blood-group P antigen (globoside) (6), which is not only present on erythrocytes and erythroblasts but also on megakaryocytes, endothelial cells, and placenta, fetal liver, and heart cells (4, 34). Effective replication of B19, however, has only been demonstrated in the erythroid progenitor cells. The natural route of transmission of B19 is through respiratory droplets. However, blood-borne transmissions have been reported (2, 3, 24), since infection can be asymptomatic and viremia is high during the acute stage (10 11 _10 14 genome copies/mL serum) (14). In particular, clotting factor concentrates prepared from large plasma pools are at a high risk of transmitting B19. Although virucidal treatment of coagulation factor concentrates is now routinely practised and clotting factors are considered to be safe with regard to enveloped viruses (HIV, HBV, HeV), it is not clear whether the non-enveloped and thermoresistant B19 will be inactivated by these procedures. We therefore investigated, by an indirect immunofluorescence assay, the anti-Bl9 IgG and IgM seroprevalence in hemophilic children, treated only with virally inactivated concentrates, hemophilic adults treated before 1984 with non-inactivated concentrates, and in age-matched non-hemophilic individuals.

Materials and Methods Study population Hemophilic patients. From September 1993 to August 1994, a total of 1151 serum samples was obtained from 566 hemophiliacs receiving coagulation factor concentrates prepared from blood. 460 hemophiliacs suffered from hemophilia A, 70 from hemophilia B, and 26 patients from von WiJlebrand's disease. Hemophilia was severe in 74 percent of these patients, 1 patient suffered from a severe lack of factor V, 2 patients from a severe lack of factor VII, 2 patients from a severe lack of factor XIII and 5 patients suffered from an autoimmune disease with severe coagulopathy. 16 patients were female. 13 hemophiliacs under the age of 2 years and 125 hemophiliacs between 2 and 12 years of age were exclusively treated with virally inactivated concentrates. 428 hemophiliacs were between 13 and 79 years of age (age distribution described in more detail in Table 1), and were treated before 1984 with non-inactivated concentrates. All hemophiliacs were outpatients, living in different districts of Germany. 75 of them were related or lived in the same household (35 pairs of brothers, once 3 brothers; one married couple). There was no indication of an epidemic outbreak of B19 infections among the hemophiliacs. Non-hemophilic population. Between September 1993 and August 1994, a total of 524 sera from 524 non-hemophilic subjects (control group) was examined. No person had clinical signs of a B19 infection. 18 individuals were female. 130 serum specimens were from children between 0 to 23 months of age, and 240 sera from children between 2 and 12 years of age. 51 sera were obtained from persons between 20 and 25 years, and 51 sera from persons between 26 and 30 vears of age. 52 sera were received from persons over 60 years of age. Serological assays. Sera were tested for anti-Bl9 IgG and IgM antibodies by an indirect immunofluorescence assay (IFA) using recombinant B19 84 kDa coat protein VPl produced in a baculovirus expression system (Biotrin, Dublin, Ireland). Except for preabsorption of the sera with Rheuma Faktor-Absorbens® (Behringwerke, Marburg, Germany) for B19 IgM

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assay to avoid false-positive results, the assay was performed according to the instructions of the manufacturer. Serum dilution for the anti-B19 IgG assay was 1: 64, and 1: 16 for the IgM assay. Statistical analysis. Prevalence rates of the groups examined were compared for significant differences by the the chi-square test. Results Anti-B 19 IgG seroprevalence in children

The anti-B19 IgG serostatus was based on the result obtained with the first specimen of an individual, although these results were always reproducible with follow-up sera except for four hemophilic children who seroconverted. Figure 1 shows the prevalence of anti-B19 IgG in non-hemophilic (controls) and hemophilic children up to the age of 12 years. In the control group, seroprevalence continuously decreased from 64 percent at birth to 0 percent at the age of 9-11 months and thereupon increased to 61 percent at the age of 12 years. In the group of hemophiliacs, neither decrease nor increase of B19 IgG antibody was detectable. With one exception, all hemophilic children aged 9 months (under 9 months of age, maternally-derived antibodies cannot be

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Fig. 1. Prevalence of anti-B19 IgG in 138 hemophilic and 370 non-hemophilic children. Since the group of hemophilic children below two years of age has been rather small, the number of positive (+) or negative (-) sera per age-group is presented. The small numbers beside the curves represent the number of sera tested per age group. Symbols: Cb, cord blood;.e, hemophiliacs; ~,non-hemophilic individuals.

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235

excluded) to 4 years were anti-B19 seropositive. All these seropositive hemophiliacs suffered from a severe coagulopathy requiring a continuous and high substitution therapy. The lower prevalence of anti-B19 IgG in some of the higher age-groups was due to the fact that 6 out of 9 seronegative patients suffered from a mild coagulopathy, requiring only sporadic therapy. In 4 of these, seroconversion occurred when replace-

Table 1. Anti-B19 IgG seroprevalence in adult non-hemophilic and hemophilic individuals No. of individuals showing anti-B19 IgG

Group

Age (years)

Non-hemophilic individuals

20-25 26-30 ::0- 60 total

32/51 (62.7) 34/51 (66.7) 40152 (76.9)

13-19 20-30 31-40 41-79 total

47151 (92.2) 166/169 (98.2) 99199 (100)

Hemophiliacs

Ino. of investigated individuals (%)

1061154 (68.8)

1091109 (100) 4211428 (98.4)a

a p < 0.001 vs. non-hemophilic individuals.

Table 2. Anti-B19 IgM seroprevalence in non-hemophilic and hemophilic children and adults. Results refer to the first serum specimen collected from an individual Group

Age

No. of B19 IgM positive sera Ina. of investigated sera (%)

Non-hemophilic children

9-11 to 21-23 months 2 to 12 years total

0158 (0) 7/240 (2.9) 7/298 (2.3)

Hemophilic children

9-11 to 21-23 months 2 to 12 years total

1110 (10) 31125 (2.4) 41135 (3.0)"

Non-hemophilic adults

20 to 30 years ::0- 60 years total

0152 (0)

Hemophilic adults

13 to 19 years 20 years total ::0-

a, b not significant vs. non-hemophilic children. C p < 0.001 vs. non-hemophilic adults. d p < 0.003 vs. non-hemophilic adults.

01102 (0) 01154 (0)

3151 (5.9) 301377 (8.0) 33/428 (7.7)C

Ina. of B19 IgG positive sera (%)

011 (0)

7/82 (8.5) 7/83 (8.4) 1/9 (11.1)

31116 (2.6) 4/125 (3.2)b 0166 (0) 0/40 (0)

01105 (0) 3/47 (6.4) 301374 (8.0) 33/421 (7.8)d

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ment therapy had to be intensified. The difference in anti-B19 IgG seroprevalence between the hemophilic children ~9 months old (1251135=93%) and age- and sexmatched controls (83/298 = 28%) was highly significant (p < 0.001).

Anti-B19 JgG seroprevalence in adults Table 1 shows that the anti-B19 IgG seroprevalence in hemophiliacs, infused before 1984 with non-inactivated clotting factor concentrates was found to be significantly higher than in the adult controls (p < 0.001).

Anti-B19 IgM seroprevalence Anti-B19 IgM reactivity was always associated with anti-B19 IgG reactivity. No significant difference in anti-B19 IgM seroprevalence was noted between non-hemophilic and hemophilic children ~ 9 months old (Table 2). In contrast, anti-B19 IgM was detected in 8 per cent (33/428) of the first serum specimens collected from hemophiliacs older than 12 years, whereas in the control group, none of the sera showed reactivity (p < 0.001). Furthermore, 31 out of 240 hemophiliacs ~ 20 years and negative for anti-B19 IgM in the first serum specimen showed a positive IgM reaction in one or more follow-up sera. 10 hemophiliacs showed positive IgM reactions in at least two consecutive sera. Discussion The anti-B19 IgG seroprevalence in the general German population, measured by the indirect immunofluorescence technique using antigen expressed by the baculovirus system in insect cells, was higher than hitherto expected from studies with bacterially expressed or even natural B19 antigen and the enzyme immunoassay method. The latter suggested that up to a maximum of 47 percent, adults were anti-B19 IgG-positive (17, 37, 39). Like in the case of air-borne virus infections, the higher rate of seroprevalence seems to be plausible. Furthermore, the prevalence of anti-B19 IgG determined by Cohen and Buckley (8) with an antibody-capture radioimmunoassay was similar to our results. The results further show that the anti-B19 seroprevalence in hemophiliacs exposed to clotting factor concentrates extracted from blood is significantly higher than in nonhemophilic individuals. This agrees with previous studies from the United States (28), Italy (3), England (46) and Norway (33). Since seroprevalence after natural infection gradually increases, the most striking difference in seroprevalence between non-hemophilic and hemophilic humans exists during childhood. This finding suggests that although having been subjected to virucidal methods clotting factor concentrates are contaminated with B19. It is most likely that the high anti-B19 prevalence in hemophiliacs results from B19 infection. However, the possibility cannot absolutely be excluded that seroprevalence derives from immunization with inactivated virus. Detection and persistence of antiB19 IgM could be consistent with continuous immunization, though repeated exposure to inactivated virus usually does not induce an IgM response. Moreover, B19 DNA detected in plasma or serum of hemophiliacs (17) could represent virus passively acquired by the coagulation factor product, and detection of B19 DNA by peR or other techniques in 20 and 36 per cent of clotting factor concentrates (23,48) does al-

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so not necessarily indicate infectivity. On the other hand, it must be taken into account that inactive B19 transmitted to the hemophiliacs by clotting factor concentrates would probably not be detectable in a small blood specimen and should be eliminated quickly. Since suitable in-vitro culture systems are lacking, successful attempts to detect infectious B19 in clotting factor concentrates have not yet been carried out. Though infectivity of clotting factor concentrates cannot be proved in a strict sense there is a considerable number of findings suggesting blood-borne infections in hemophiliacs. B19 is highly resistant to the chemical and physical inactivation procedures now routinely used for virus inactivation. While solvent/detergent treatment is effective in the inactivation of enveloped viruses, it is ineffective for non-enveloped viruses such as B19. Heating procedures may be able to reduce infectivity of B19. However, in a study using PCR, two of five initially PCR-positive plasma pools destinated for preparation of clotting factors remained PCR-positive after dry-heating (80°C for 72 hours) (26). Apparently, the effect of the inactivation procedure seems to be dependent on the amount of B19 contamination, since evaluation of the procedure by spiking batches with B19 revealed a reduction of B19 DNA of about 2 logs only (26). In agreement with these results, we found a seroprevalence in hemophilic children receiving only heat-treated products that was slightly, but not significantly lower, than in children substituted exclusively with solvent/detergent-treated concentrates (17). Moreover, removal of B19 by about 2 logs by ion-exchange chromatography (38) or purification of clotting factors by monoclonal antibodies (48) combined with inactivation procedures proved not to be satisfactory. Azzi et al. (2) detected seroconversion in 9 out of 20 B19-susceptible, previously untreated hemophiliacs within 12 weeks after infusion with such high-purity factor concentrates. The most convincing argument suggesting infection by clotting factor concentrates is given by reports documenting symptomatic B19 infections with rash or anemia in patients with little or no previous exposure to blood products after infusion of solvent! detergent- andlor heat-treated factor concentrates (24, 27, 35, personal observations in patients of the Hemophilia Center of Bonn). Blood-borne transmission of B19 seems to be minimized when clotting factor concentrates are prepared from small plasma pools (33). Considering the incidence of viremic blood donors, the pooling of thousands of plasma donations for manufacturing clotting factor concentrates inevitably results in B19 contamination of many lots. In a study involving 20000 Scottish volunteer blood donors, B19 DNA was detected by PCR in 6 donors (0.03%) (26), and in a more recent Japanese study involving 1000 blood donors, 6 (0.6%) viremic individuals were found (47). Using antigen detection methods the rate of viremic donors is probably underestimated, being approximately 0.003 to 0.004 per cent (9, 41). Seasonal accumulation of B19 infections and a certain periodicity of epidemics can influence the rate of viremic donors (36, 41). The concentration of B19 DNA in donations from healthy blood donors ranged from 2.4 X 104 to 5 X 10 10 copies of viral DNA per mL (26), and the amount of antibody in the plasma pool is probably not sufficient to neutralize infectivity. With regard to the stability of B19 to physical and chemical inactivation procedures, one single contaminated blood donation within a large plasma pool used for manufacturing clotting factors, can cause a high number of B19 infections, particularly, when the plasma pool originates from plasmapheresis donors, contributing 600 to 700 mL of plasma to the pool at short intervals. The anti-B19 IgM reactivity in the adult hemophilic population may be explained as a booster after repeated virus contact, most probably mediated by clotting factor

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concentrates, or as chronic or persistent infection. There is an increasing number of reports describing persistence of B19, especially in hemophiliacs concomitantly infected with HIV (13, 14,30,16). Musiani et al. (29) detected viral persistence for 40 months in hemophilic patients, accompanied by acute manifestations of B19 infection (aplastic crisis, fever) when the viral titre had risen. In conclusion, it seems to be difficult to develop effective methods to inactivate B19 in clotting factor concentrates. Therefore, screening of concentrates (48) or blood donations for parvovirus B19 by immunoelectrophoresis (9), peR methods (48,26), or as described very recently, by a hemagglutination assay, which detects at least donations containing high-titre B19 virus (10, 36) was suggested to reduce the risk of bloodborne B19 infection. References 1. Anderson, M.]., S. E. jones, S. P. Fisher-Hoch, E. Lewis, S. M. Hall, C. L. R. Bartlett, B. j. Cohen, P. P. Mortimer, and M. S. Pereira: Human parvovirus, the cause of erythema infectiosum (fifth disease)? Lancet (1983) 1378 2. Azzi, A., S. Ciappi, K. Zakvrzewska, M. Morfini, G. Mariani, and P. M. Mannucci: Human parvovirus B19 infection in hemophiliacs first infused with two high-purity, virally attenuated factor VIII concentrates. Amer. J. Hematol. 39 (1992) 228-230 3. Bartolomei Corsi, 0., A. Azzi, M. Morfini, R. Fanci, and P. Rossi Ferrini: Human parvovirus infection in haemophiliacs first infused with treated clotting factor concentrates. J. Med. Virol. 25 (1988) 165-170 4. von dem Borne, A. E. G. K., M.]. E. Bas, N.joustra-Maas, ]. R Tromp, R. van Wijngaarden-Du Bois, and P. A. T. Tetteroo: A murine monoclonal IgM antibody specific for blood group P antigen (globoside). Br. J. Haematol. 63 (1986) 35-46 5. Brown, T., A. Anand, L. D. Ritchie,]. P. Clewley, and T. M. S. Reid: Intrauterine parvovirus infection associated with hydrops fetalis. Lancet (1984) 1033-1034 6. Brown, K. E., S. M. Anderson, and N. S. Young: Erythrocyte P antigen: cellular receptor for B19 parvovirus. Science 262 (1993) 114-117 7. Cassinotti, P., M. Weitz, and G. Siegl: Human parvovirus B19 infections: routine diagnosis by a new nested polymerase chain reaction assay. J. Med. Virol. 40 (1993) 228-243 8. Cohen, B.}. and M. M. Buckley: The prevalence of antibody to human parvovirus B19 in England and Wales. J. Med. Microbiol. 25 (1988) 151-153 9. Cohen, B.} .. A. M. Field, S. Gudnadottir, S. Beard, and]. A.]. Barbara: Blood donor screening for parvovirus B19. J. Virol. Methods 30 (1990) 233-238 10. Cohen, B., A. Millar, and P. Schwind: Screening blood donations for parvovirus B19. Lancet 346 (1995) 1631 11. Feldmann, R., M. Harms, and ].-H. Saurat: Papular-purpuric 'gloves and socks' syndrome: not only parvovirus B19. Dermat. 188 (1994) 85-87 12. Finkel, T. H .. T.]. Torok, P.]. Ferguson, E. L. Durigon, S. R. Zaki, D. Y. M. Leung, R.]. Harbeck, E. W. Gelfand, R T. Saulsbury,}. R. Hollister, and L.]. Anderson: Chronic parvovirus B19 infection and systemic necrotising vasculitis: opportunistic infection or aetiologic agent? Lancet 343 (1994) 1255-1208 13. Foto, F., K. G. Saag, L. L. Scharosch, E.]. Howard, and S.j. Naides: Parvovirus B19specific DNA in bone marrow from B19 arthropathy patients: evidence for B19 virus persistence . ./. Infect. Dis. 167 (1993) 744-748 14. Frickhofen, N. and N. S. Young: Persistent parvovirus B19 infections in humans. Microbial. Pathog. 7 (1989) 319-327 15. Gahr, M., A. Pekrun, and H. Eiffert: Persistence of parvovirus B19-DNA in blood of a child with severe combined immunodeficiency associated with chronic pure red cell aplasia. Eur. J. Pediatr. 150 (1991) 470-472

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Dr. Anna Maria Eis-Hiibinger, Institute of Medical Microbiology and Immunology, University Bonn, Sigmund-Freud-Strage 25, D-53127 Bonn, Germany