Improved Diagnosis of Gestational Parvovirus B19 Infection at the Time of Nonimmune Fetal Hydrops

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1 MAJOR ARTICLE Improved Diagnosis of Gestational Parvovirus B19 Infection at the Time of Nonimmune Fetal Hydrops Martin Enders, 1 Andrea Weidner, 1 Tessa Rosenthal, 1 Carola Baisch, 1 Lea Hedman, 2 Maria Söderlund-Venermo, 2 and Klaus Hedman 2 1 Labor Enders und Partner und Institut für Virologie, Infektiologie und Epidemiologie e.v., Stuttgart, Germany; 2 Department of Virology, Haartman Institute, University of Helsinki, and Helsinki University Central Hospital Laboratory Division, Helsinki, Finland Background. In the diagnosis of parvovirus B19 infection, the detection of virus-specific IgG in the absence of virus-specific IgM is considered to indicate past immunity. Methods. We determined the diagnostic value of a high-quality B19 IgM EIA, compared with that of a VP1 IgG avidity EIA, a VP2 IgG epitope-type specificity (ETS) EIA, and real-time polymerase chain reaction (PCR) in the diagnosis of maternal B19 infection during nonimmune fetal hydrops. Results. Serum samples from 101 pregnant women with confirmed B19 induced fetal hydrops were collected at the time of invasive prenatal diagnosis. The samples were investigated for B19 IgM, VP1 IgG avidity, and VP2 IgG ETS. With the B19 IgM EIA, 78 women (77.2 %) showed positive results, 15 (14.9%) showed negative results, and 8 (7.9 %) showed equivocal results. According to the combined B19 IgG avidity and IgG ETS EIA results, only 5 (5%) of 101 women were classified as having past immunity. Available serum samples (n 24) that had nondiagnostic results in the antibody assays were further investigated by PCR. All were B19 DNA positive (mean load, genome equivalents/ml; range, ). Conclusions. At the time of B19 induced hydrops, detection of B19 DNA in maternal blood had the best diagnostic sensitivity for identifying maternal B19 infection. However, given the long persistence of B19 DNAemia, supplementary measurement of VP1 IgG avidity and VP2 IgG ETS improves the precision of diagnosis and management of pregnant women affected by the B19 virus. Human parvovirus B19 (B19) causes a common exanthematous disease of childhood, erythema infectiosum (fifth disease) [1]. Close and frequent contact with young children is a major risk factor for acquiring B19 infection during pregnancy [2 5]. Acute B19 infection is associated with an increased risk of fetal hydrops and fetal loss, especially if the maternal infection occurs during the first half of pregnancy [6 12]. During pregnancy, determination of B19 antibody status is recommended after contact with a patient with erythema infectiosum or with other B19-related symptoms [13 Received 30 April 2007; accepted 9 July 2007; electronically published 10 December Potential conflicts of interest: none reported. Financial support: Helsinki University Central Hospital Research and Education Fund (TYH6101); Sigrid Jusélius Foundation ( ); Finnish Funding Agency for Technology and Innovation ( ); Academy of FInland ( ). Reprints or correspondence: Dr. Martin Enders, Labor Enders und Partner und Institut für Virologie, Infektiologie und Epidemiologie e.v., Stuttgart, Germany (menders@labor-enders.de). The Journal of Infectious Diseases 2008; 197: by the Infectious Diseases Society of America. All rights reserved /2008/ $15.00 DOI: / ]. Measurement of maternal B19 IgM is a highly sensitive and specific marker for acute B19 infection [17, 18]. However, maternal IgM antibodies may be undetectable at the time of fetal sampling for nonimmune fetal hydrops [16]. The purpose of our investigation was to evaluate the diagnostic value of supplementary molecular (quantitative polymerase chain reaction [PCR]) or serodiagnostic(vp1 IgG avidity and VP2 IgG epitope type specificity [ETS]) assays [19 22] in the diagnosis of maternal B19 infection at the time of B19-induced fetal hydrops. MATERIALS AND METHODS Specimens During an 11-year period from 1995 to 2005, parvovirus B19-induced fetal hydrops was confirmed in 156 pregnancies by detection of B19 DNA in fetal specimens (e.g., amniotic fluid and fetal blood) by PCR. PCR was performed as described elsewhere [23, 24]. Maternal serum samples were obtained concomitantly at the time of fetal sampling from 134 patients and studied for B19 IgG 58 JID 2008:197 (1 January) Enders et al.

2 and IgM antibodies. Of the 134 serum samples, a total of 103 samples were available for retrospective analysis of VP1 IgG avidity and VP2 IgG ETS. Information on maternal age, week of gestation at prenatal diagnosis, ultrasound findings, and pregnancy outcome were reported by the referring gynecologists. Serological Analysis B19 IgG and IgM. VP2 IgM antibodies were identified by use of a -capture EIA (Biotrin International), in accordance with the manufacturer s instructions. VP2 IgG antibodies were identified by use of a sandwich IgG EIA (Biotrin International), as described elsewhere [25]. VP1 IgG avidity and VP2 IgG ETS. Blinded and coded serum samples were studied for VP1 IgG avidity and VP2 IgG ETS. VP1 IgG avidity was determined with a protein-denaturing EIA that used a prokaryotic fusion protein containing the VP1 unique region as antigen [19, 26]. The avidity index values were calculated after serum titration and washes in the presence and absence of 8 mol/l urea, from 2 2 data points (sample dilutions) per sample, by use of curve-fitting software [27]. An IgG avidity index of 16 (low avidity) indicates acute infection (within 50 days after the onset of symptoms), and an IgG avidity index of 25 (high avidity) indicates past infection ( 100 days after onset) [19, 21]. The second-generation ETS EIA was performed as described elsewhere [20], with VP2 capsids (10 ng/microwell) and a synthetic peptide (16 ng/microwell) containing the VP2 IgG epitope (KYVTGIN) in linear, monomeric form as antigens in separate wells. The ratio of IgG absorbances obtained with these 2 antigens (the ETS index ) was calculated; a cutoff value of 10 has been shown to separate the IgG of acute infection ( 10) from the IgG of past immunity ( 10) [20, 21]. LightCycler PCR (LC PCR) and Melting Temperature (Tm) Analysis The semiquantitative LC-PCR assay and Tm analysis were performed as described elsewhere [24]. In brief, 200 L of each sample was subjected to nucleic acid extraction on a MagNA Pure LC automated instrument (Roche Diagnostics). Master mixes were based on a ready-to-use kit (LC FastStart DNA Master Hyb Probes; Roche Diagnostics) containing FastStart Taq Polymerase and dntp mix. The mix was supplemented with MgCl 2, uracil-n-glycosylase, primers, and probes. After pipetting 15 L of this mixture into LC capillaries, 5 L of the crude nucleic acid preparation was added. Immediately after LC-PCR, an additional analytical cycle for Tm analysis was included. The resulting melting curves were automatically converted by the LC software into melting peaks. Isolates with a Tm value differing significantly from that determined for B19 genotype 1 are considered to belong to B19 genotypes 2 or 3 [28, 29]. The DNA concentrations given are the mean geometric values of those that were recorded. Statistical Analysis Differences between B19 IgM EIA results for patients with acute infection and patients with past immunity were analyzed according to VP1 IgG avidity or VP2 IgG ETS EIA results by 1-way analysis of variance; Dunn s posttest correction was used for multiple comparisons (SigmaStat; Systat Software). RESULTS The median maternal age was 31.5 years (range, years; interquartile range [IQR], years). At the time of sampling for nonimmune fetal hydrops, the median gestational age was 22 weeks (range, weeks; IQR, weeks). Most cases of parvovirus B19 associated hydrops fetalis occurred between 17 and 28 weeks gestation, with a peak incidence at weeks gestation. Of the 103 patients studied retrospectively with a VP1 IgG avidity EIA, 101 were VP1 IgG positive; 54.5% (55/101) showed low avidity, 5.0% (5/101) showed high avidity, and 40.6% (41/ 101) showed borderline avidity results. Of the 101 VP1 IgG positive serum samples, 77.2% (78/101) were B19 IgM positive, 14.9% (15/101) were B19 IgM negative, and 7.9% (8/101) showed equivocal B19 IgM test results. None of the 31 mothers with onset of fetal hydrops before 21 weeks gestation showed high avidity (figure 1). In the VP2 IgG ETS EIA, 60.2% (62/103) of the serum samples showed diagnostic results (i.e., acute infection), and 39.8% (41/103) showed nondiagnostic results (i.e., past immunity). A total of 76 patients (75%) had low avidity or diagnostic ETS results. In tandem use of the IgG avidity and IgG ETS EIAs, 45 of 101 serum samples (44.5%) gave concordant diagnostic results; the stage of infection was classified as acute in 40 patients (39.6%) and as past in 5 patients (5.0%). The stage of infection could not be classified in the remaining patients (n 56) either because of borderline results (n 41) for the IgG avidity EIA or because of discrepant results (n 15) for the avidity EIA and the ETS EIA (i.e., low IgG avidity and nondiagnostic ETS results) (table 1). We furthermore observed significantly higher B19 IgM EIA indices in the groups of patients with either diagnostic IgG avidity or diagnostic IgG ETS values, compared with those in the groups of patients with borderline or nondiagnostic values by the 2 latter antibody assays (figure 2). Available serum samples (n 24) that showed past immunity by both the IgG avidity and IgG ETS EIAs and samples that had negative or equivocal results for the IgM EIA at the diagnosis of nonimmune fetal hydrops were retrospectively investigated by real-time PCR. All the serum samples were B19 DNA positive, with a mean viral DNA load of genome equivalents (geq) per ml (range, geq/ml). We observed no difference in pregnancy outcome with respect to the B19 DNA levels in these patients. According to Tm analysis, all the B19 findings were of genotype 1. Diagnosis of Parvovirus B19 Infection during Pregnancy JID 2008:197 (1 January) 59

3 Figure 1. Frequency of fetal hydrops cases according to gestational age at the time of invasive prenatal diagnosis and distribution of IgG avidity EIA results for respective maternal serum samples (n 101). DISCUSSION During pregnancy, the laboratory diagnosis of parvovirus B19 infection relies primarily on IgG and IgM antibody tests [16]. However, at the time of fetal sampling for nonimmune hydrops, maternal B19 IgM levels may already have dropped below the detection limit. This caveat has been brought out in several studies in which maternal B19 IgM seronegativity rates (in the presence of B19 IgG antibodies) at the time intrauterine B19 infection was diagnosed varied widely: 62.5% (5/8) [30], 38.5% (5/ 13) [31], 18.7% (3/16) [32], and 16.6% (3/18) [33]. In our series, the proportion of B19 IgM negative patients at the time of diagnosis was 14.9% (15/101). Therefore, reliance on IgM serological results alone may not justify ruling out maternal B19 infection. The inappropriately negative IgM serological results can, in our experience, cause delay or denial of intrauterine transfusion therapy and inadvertent fetal loss. At the time of fetal hydrops, PCR analysis of maternal blood samples appears to identify B19 infection with greater diagnostic sensitivity. Our findings confirm a number of reports on the presence of B19 DNAemia and the absence of B19 IgM in maternal blood at the time of invasive prenatal diagnosis of nonimmune fetal hydrops [32, 34, 35]. However, the use of this technique to ascertain recent maternal B19 infection has some limitations. Because PCR is notoriously prone to contamination, proper sampling methods and careful handling of samples in the laboratory are required to obtain reliable results. Furthermore, according to our findings, the DNA levels in maternal blood at the onset of hydrops ranged from to geq/ml. Hence, B19-induced hydrops is not invariably accompanied by high B19 DNA levels in maternal blood. In our study, this finding is reflected by the detection of B19 DNAemia in serum samples with nondiagnostic EIA results (by use of supplementary IgG assays or B19 IgM EIA). We have previously shown that maternal DNAemia may exceed concentrations of geq/ml during the very acute stage of gestational B19 infection, and it then often persists for several months, mostly at low levels [22]. Long-term persistence of B19 DNA in nonpregnant, immunocompetent individuals Table 1. VP2 IgG epitope-type specificity (ETS) EIA and VP1-IgG avidity EIA test results for maternal serum samples collected at the time of fetal sampling for nonimmune hydrops (n 101 patients). VP1 IgG avidity b EIA results VP2 IgG ETS a EIA values Low (index 16) Borderline (index 16 25) High (index 25) Specimens tested, no Total NOTE. Blinded and coded serum samples were retrospectively investigated with the supplementary IgG assays. a ETS EIA values 10 are diagnostic (i.e., indicate acute infection), and ETS EIA values 10 are nondiagnostic (i.e., indicate past infection). b Avidity EIA indices 16 indicate acute infection, and avidity EIA indices 25 indicate past infection. 60 JID 2008:197 (1 January) Enders et al.

4 Figure 2. A, Parvovirus B19 IgM antibody response in patients with acute B19 infection or with past immunity according to VP1 IgG avidity EIA results (acute infection [low avidity], n 55; borderline, n 41; past immunity [high avidity], n 5). A statistically significant difference was observed between patients with low and high avidity EIA results, as well as between patients with low and borderline avidity EIA results (P.001). B, B19 IgM antibody response in patients with acute B19 infection or with past immunity according to VP2 IgG epitope-type specificity (ETS) EIA results (diagnostic [i.e., acute infection], n 62; nondiagnostic [past immunity], n 41). A statistically significant difference was observed between patients with diagnostic and nondiagnostic ETS EIA results (P.002). Reference values for the B19 IgM EIA (index) are 0.9, negative; , equivocal; 1.1, positive. has also been reported by others [36 38]. Given that the B19- associated fetal complications occur mostly within 12 weeks of maternal infection [8, 11, 39], many women will remain B19 DNA positive beyond the time of greatest risk for B19-induced hydrops. A general recommendation for B19 DNA testing of pregnant women at risk for B19 infection who are B19 IgG positive and B19 IgM negative would be very likely to increase the referrals for ultrasound examinations, especially during periods of high B19 activity. Moreover, many pregnant women would be unnecessarily concerned. On the other hand, when fetal anemia or hydrops is evident on Doppler or ultrasound examination, invasive prenatal diagnosis is often recommended to rule out intrauterine B19 infection, irrespective of maternal B19 IgM EIA results. In this context, B19 PCR analysis of maternal blood might be a useful approach. To establish the negative predictive value of PCR diagnostic techniques for maternal B19 infection, even larger clinical studies (preferably follow-up studies) might be warranted, in addition to general standardization of the methods used. In the present study, we further investigated the value of supplementary serodiagnostic techniques (VP1 IgG avidity and VP2 IgG ETS EIAs) among pregnant women. Overall, we found a close association between the B19 IgM concentration and the IgG avidity or IgG ETS values. Of note, the 2 latter assays measure a parameter distinct from IgM and in other contexts have been shown to improve the diagnosis of B19 infection [21, 40]. Recently, we demonstrated that the combined occurrence of high IgG avidity and nondiagnostic IgG ETS results ruled out the possibility of B19 infection having occurred within 12 weeks of sampling, with a high negative predictive value [22]. In the current investigation, only 5.0% (5/101) of the women showed such a marker of past infection at the time of hydrops. This figure agrees with the observation by Yaegashi et al. [39] that 5.5% (3/55) of hydrops cases occur 12 weeks after the corresponding maternal infection. In our study, the proportion of pregnant women with negative B19 IgM EIA results at the time of diagnosis of fetal hydrops was 14.9% (15/101). The use of supplementary IgG avidity and IgG ETS diagnostic techniques, differing slightly from each other in maturation kinetics, at this time will reduce the rate of false-negative results with respect to B19 infection. None of the 31 pregnant women who presented with fetal hydrops before 21 weeks gestation showed high IgG avidity (figure 1). We therefore suggest that in the absence of B19 IgM, in the presence of high IgG avidity and nondiagnostic IgG-ETS EIA results, and in the presence of normal findings on ultrasound before 16 weeks gestation, the risk for B19-associated fetal hydrops is extremely small even in the presence of lowlevel B19 DNAemia. The use of supplementary diagnostic techniques improves the management of B19 infection during pregnancy and facilitates counselling pregnant women with respect to the necessity and timing of ultrasound and Doppler examinations. Acknowledgments We thank Sabine Helbig and Andrea Hunjet for excellent technical assistance as well as Frank Knotek for database management (Stuttgart). References 1. Anderson MJ, Jones SE, Fisher-Hoch SP, et al. Human parvovirus, the cause of erythema infectiosum (fifth disease)? Lancet 1983; 1: Harger JH, Adler SP, Koch WC, Harger GF. Prospective evaluation of 618 pregnant women exposed to parvovirus B19: risks and symptoms. Obstet Gynecol 1998; 91: Kelly HA, Rae PB, Donnelly JK, Leydon JA. Fifth disease in a small rural Diagnosis of Parvovirus B19 Infection during Pregnancy JID 2008:197 (1 January) 61

5 community: what are the consequences? Aust Fam Physician 1999; 28: Valeur-Jensen AK, Pedersen CB, Westergaard T, et al. Risk factors for parvovirus B19 infection in pregnancy. JAMA 1999; 281: Enders M, Weidner A, Enders G. Current epidemiological aspects of human parvovirus B19 infection during pregnancy and childhood in the western part of Germany. Epidemiol Infect 2007; 135: Brown T, Anand A, Ritchie LD, Clewley JP, Reid TM. Intrauterine parvovirus infection associated with hydrops fetalis. Lancet 1984; 2: Prospective study of human parvovirus (B19) infection in pregnancy. Public Health Laboratory Service Working Party on Fifth Disease. BMJ 1990; 300: Miller E, Fairley CK, Cohen BJ, Seng C. Immediate and long term outcome of human parvovirus B19 infection in pregnancy. Br J Obstet Gynaecol 1998; 105: Yaegashi N, Niinuma T, Chisaka H, et al. Serologic study of human parvovirus B19 infection in pregnancy in Japan. J Infect 1999; 38: Nunoue T, Kusuhara K, Hara T. Human fetal infection with parvovirus B19: maternal infection time in gestation, viral persistence and fetal prognosis. Pediatr Infect Dis J 2002; 21: Enders M, Weidner A, Zoellner I, Searle K, Enders G. Fetal morbidity and mortality after acute human parvovirus B19 infection in pregnancy: prospective evaluation of 1018 cases. Prenat Diagn 2004; 24: Chisaka H, Ito K, Niikura H, et al. Clinical manifestations and outcomes of parvovirus B19 infection during pregnancy in Japan. Tohoku J Exp Med 2006; 209: Rodis JF, Quinn DL, Gary GW Jr, et al. Management and outcomes of pregnancies complicated by human B19 parvovirus infection: a prospective study. Am J Obstet Gynecol 1990; 163: Crane J. Parvovirus B19 infection in pregnancy. J Obstet Gynaecol Can 2002; 24: Morgan-Capner P, Crowcroft NS. Guidelines on the management of, and exposure to, rash illness in pregnancy (including consideration of relevant antibody screening programmes in pregnancy). Commun Dis Public Health 2002; 5: de Jong EP, de Haan TR, Kroes AC, Beersma MF, Oepkes D, Walther FJ. Parvovirus B19 infection in pregnancy. J Clin Virol 2006; 36: Doyle S, Kerr S, O Keeffe G, O Carroll D, Daly P, Kilty C. Detection of parvovirus B19 IgM by antibody capture enzyme immunoassay: receiver operating characteristic analysis. J Virol Methods 2000; 90: Beersma MF, Claas EC, Sopaheluakan T, Kroes AC. Parvovirus B19 viral loads in relation to VP1 and VP2 antibody responses in diagnostic blood samples. J Clin Virol 2005; 34: Söderlund M, Brown CS, Cohen BJ, Hedman K. Accurate serodiagnosis of B19 parvovirus infections by measurement of IgG avidity. J Infect Dis 1995; 171: Kaikkonen L, Lankinen H, Harjunpää I, et al. Acute-phase-specific heptapeptide epitope for diagnosis of parvovirus B19 infection. J Clin Microbiol 1999; 37: Kaikkonen L, Söderlund-Venermo M, Brunstein J, et al. Diagnosis of human parvovirus B19 infections by detection of epitope-type-specific VP2 IgG. J Med Virol 2001; 64: Enders M, Schalasta G, Baisch C, et al. Human parvovirus B19 infection during pregnancy value of modern molecular and serological diagnostics. J Clin Virol 2006; 35: Searle K, Guilliard C, Wallat S, Schalasta G, Enders G. Acute parvovirus B19 infection in pregnant women an analysis of serial samples by serological and semi-quantitative PCR techniques. Infection 1998; 26: Schalasta G, Schmid M, Lachmund T, Enders G. Light Cycler consensus PCR for rapid and differential detection of human erythrovirus B19 and V9 isolates. J Med Virol 2004; 73: Searle K, Guilliard C, Enders G. Parvovirus B19 diagnosis in pregnant women quantification of IgG antibody levels (IU/ml) with reference to the international parvovirus B19 standard serum. Infection 1997; 25: Söderlund M, Brown KE, Meurman O, Hedman K. Prokaryotic expression of a VP1 polypeptide antigen for diagnosis by a human parvovirus B19 antibody enzyme immunoassay. J Clin Microbiol 1992; 30: Korhonen MH, Brunstein J, Haario H, Katnikov A, Rescaldani R, Hedman K. A new method with general diagnostic utility for the calculation of immunoglobulin G avidity. Clin Diagn Lab Immunol 1999; 6: Servant A, Laperche S, Lallemand F, et al. Genetic diversity within human erythroviruses: identification of three genotypes. J Virol 2002; 76: Hokynar K, Norja P, Laitinen H, et al. Detection and differentiation of human parvovirus variants by commercial quantitative real-time PCR tests. J Clin Microbiol 2004; 42: Wattre P, Dewilde A, Subtil D, Andreoletti L, Thirion V. A clinical and epidemiological study of human parvovirus B19 infection in fetal hydrops using PCR Southern blot hybridization and chemiluminescence detection. J Med Virol 1998; 54: Zerbini M, Musiani M, Gentilomi G, Venturoli S, Gallinella G, Morandi R. Comparative evaluation of virological and serological methods in prenatal diagnosis of parvovirus B19 fetal hydrops. J Clin Microbiol 1996; 34: Dieck D, Schild RL, Hansmann M, Eis-Hübinger AM. Prenatal diagnosis of congenital parvovirus B19 infection: value of serological and PCR techniques in maternal and fetal serum. Prenat Diagn 1999; 19: Török TJ, Wang QY, Gary GW Jr, Yang CF, Finch TM, Anderson LJ. Prenatal diagnosis of intrauterine infection with parvovirus B19 by the polymerase chain reaction technique. Clin Infect Dis 1992; 14: Knöll A, Louwen F, Kochanowski B, et al. Parvovirus B19 infection in pregnancy: quantitative viral DNA analysis using a kinetic fluorescence detection system (TaqMan PCR). J Med Virol 2002; 67: de Haan TR, Beersma MF, Oepkes D, de Jong EP, Kroes AC, Walther FJ. Parvovirus B19 infection in pregnancy: maternal and fetal viral load measurements related to clinical parameters. Prenat Diagn 2007; 27: Musiani M, Zerbini M, Gentilomi G, Plazzi M, Gallinella G, Venturoli S. Parvovirus B19 clearance from peripheral blood after acute infection. J Infect Dis 1995; 172: Lefrère JJ, Servant-Delmas A, Candotti D, et al. Persistent B19 infection in immunocompetent individuals: implications for transfusion safety. Blood 2005; 106: Lindblom A, Isa A, Norbeck O, et al. Slow clearance of human parvovirus B19 viremia following acute infection. Clin Infect Dis 2005; 41: Yaegashi N, Niinuma T, Chisaka H, et al. The incidence of, and factors leading to, parvovirus B19-related hydrops fetalis following maternal infection: report of 10 cases and meta-analysis. J Infect 1998; 37: Söderlund M, Ruutu P, Ruutu T, Asikainen K, Franssila R, Hedman K. Primary and secondary infections by human parvovirus B19 following bone marrow transplantation: characterization by PCR and B-cell molecular immunology. Scand J Infect Dis 1997; 29: JID 2008:197 (1 January) Enders et al.