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Significance of avidity and immunoblot analysis for rubella IgM-positive serum samples in pregnant women
Journal of Virological Methods 130 (2005) 66–71
Significance of avidity and immunoblot analysis for rubella IgM-positive serum samples in pregnant women J. Hofmann, U.G. Liebert ∗ Institute of Virology, University of Leipzig, Johannisallee 30, 04103 Leipzig, Germany Received 13 August 2004; received in revised form 7 June 2005; accepted 9 June 2005 Available online 15 July 2005
Abstract A total of 512 IgM-positive serum samples from 449 pregnant women referred by microbiologists and medical laboratories for additional testing and final interpretation were collected over a 3-year period. Employment of an IgM capture enzyme immunoassay (EIA) confirmed only 31% of the initial EIA-IgM-positive samples. In order to discriminate acute rubella virus infections, which are associated with increased risk of fetal infection and embryopathy, from persistent or non-specific IgM, IgG avidity index and the presence of IgG with specificity to rubella virus E2 glycoprotein was determined by Western immunoblot. In only six patients (1.3%), a primary infection with rubella virus was diagnosed on the basis of IgM positivity, low avidity IgG, absence of E2-specific IgG in immunoblot concordant with clinical findings as well as consistent changes in follow-up samples. The serological results were not compatible with rubella re-infection. The infection status in 14 patients (3.1%) remained inconclusive even when both avidity assay and immunoblot were used, while restriction to either test did not allow a conclusive interpretation in 11.6% of patients. The use of both assays is clearly better, and therefore, recommended in IgM-positive samples. © 2005 Elsevier B.V. All rights reserved.
1. Introduction Post-natal rubella virus infection usually causes a mild disease including mild fever, lymphadenopathy and exanthema, or remains asymptomatic. Within the first 16 weeks of pregnancy, however, the infection can lead in up to 75% of cases to fetal infection and malformations (Chantler et al., 2001). In Germany, approximately, 10% of women in the childbearing age are seronegative despite the availability of a safe and effective vaccine. A further diagnostic problem arises as long-term follow-up studies have demonstrated significantly lower antibody levels in those vaccinated compared to naturally acquired immunity resulting in an increased risk of re-infections following exposure to wild-type rubella virus (Forsgren and Soren, 1985; Mitchell et al., 1999). Therefore, verification of the rubella immune status is included in the guidelines for medical care during pregnancy. Accordingly, determination of rubella-specific IgM and/or followup serology is recommended for persons with suspected ∗
Corresponding author. Tel.: +49 341 9714300; fax: +49 341 9714309. E-mail address: [email protected] (U.G. Liebert).
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acute infection or with contact to rubella or rubella-like illness (Bundes¨arztekammer, 2002). Several laboratories carry out additional IgM determinations or investigate follow-up samples to obtain reliable diagnostic interpretations. This screening reveals approximately, 1% positive IgM results by screening enzyme immunoassays. Since IgM assays may result in non-specific results, additional methods should be used in such cases. These are determination of antibodies to individual viral proteins by immunoblot and avidity of specific IgG antibodies (Pustowoit and Liebert, 1998; Best et al., 2002). While E2-specific antibodies appear with a delay of 3–4 months after primary infection as detected by immunoblots under non-reducing conditions (Mauracher et al., 1993; Pustowoit et al., 1996), E1-specific antibodies may appear as early as 4–6 days post-infection (Hedman and Rousseau, 1989; Nedeljkovic et al., 2001). In a study using well-defined serum samples with follow-up samples up to 12 months from 27 persons, the avidity indices reached 0.21–0.33 within 28–33 days of the rubella rush or after vaccination. Six months after infection or vaccination the avidity indices were 0.54–0.94 with a mean of 0.74 (Nedeljkovic et al., 2001). The present study summarises data of 512 serum
J. Hofmann, U.G. Liebert / Journal of Virological Methods 130 (2005) 66–71
samples that were referred to the Institute of Virology by microbiologists and medical laboratories for further evaluation.
2. Material and methods 2.1. Serological assays (1) An IgM capture (-capture) enzyme immunoassay (Medac, Hamburg, Germany) was used to confirm IgM results obtained in external laboratories. (2) A commercially available enzyme immunoassay (Enzygnost® Dade-Behring, Marburg, Germany, using cell culture derived rubella virus as antigen) was used to determine the quantity of specific IgG (in IU/ml). The avidity indices were determined using the assay with an additional incubation step, which insubstantially elongates the turn-round time of the assay. Briefly, after binding of serum IgG to antigen-coated microtitre plate, wells were incubated in parallel for 5 min either in washing buffer or in washing buffer containing urea/hydrogen peroxide mixture, respectively. Avidity indices were calculated as the ratio of ODs in urea/hydrogen peroxide treated to non-treated sample. Ratios below 0.3 were considered as indicating low avidity IgG, while those above 0.5 as high avidity IgG, and avidity indices between 0.3 and 0.5 are equivocal (Hedman and Rousseau, 1989; Pustowoit and Liebert, 1998). (3) The non-reducing immunoblot (NRIB) was carried out as described previously. Briefly, recombinant rubella-like particles secreted from transfected CHO cells (Hobman et al., 1994), were concentrated and purified as described for cell culture grown virions (Pustowoit et al., 1996). Fifteen micrograms of protein lysate diluted in 270 l loading buffer were separated by 10% SDS-PAGE (using a 1 mm prep/2-D comb with a sample well volume of 400 l in a miniprotean electrophoresis cell (BioRad, M¨unchen, Germany) under non-reducing conditions. The proteins were transferred onto a PVDF-membrane (Millipore, Schwalbach, Germany). Strips cut from the membrane were blocked with 5% milk powder dissolved in PBST (phosphate-buffered saline, pH 7.4 containing 0.5% Tween 20) and incubated subsequently with human sera diluted 1:100 in PBST overnight at room temperature. After washing three times, the strips were incubated with HRP conjugated rabbit anti-human IgG (DAKO, Denmark) diluted 1:2000 in PBST for 2 h at room temperature. For visualisation, Nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate (BioRad, M¨unchen, Germany) have been used. The total time for conducting the test is acceptable in terms of rapid diagnostic requirements. The presence of the viral glycoproteins E1 and E2 in the protein lysate and their location on the PVDF stripes was determined by immunoblot using monoclonal antibodies (M1B9, Roche
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Diagnostics, Mannheim, Germany, and Mab26–24, Viral Antigens Inc., Memphis, TN, USA, respectively, Fig. 2). Selected patient sera were incubated with antigen derived from rubella virus strain Therien-infected BHK-cells. This but not incubation with lysate from non-infected BHK cells resulted in loss of reactivity in immunoblots. Since rubella virus antigen or other necessary prerequisites are not available in each diagnostic laboratory in Germany, samples were usually referred to specialised centers including the laboratory of the University of Leipzig. 2.2. Patient serum samples Five hundred and twelve consecutive serum samples, positive by screening EIA for rubella-specific IgM, were collected from 449 pregnant women over a period of 3 years. The sera were referred by microbiologists and medical laboratories for further evaluation. The mean age of patients was 29.6 ± 5.6 years, approximately, 80% had been vaccinated in the past. These sera had been pre-tested in external laboratories as IgM-positive using different IgM assays “VIDAS Rub IgM” (BioMerieux, -capture), “ETI-RUBEK-M reverse” (DiaSorin, -capture), “IMX Rubella IgM” (Abbott Laboratories, conventional assay format), and others. Clinical data on immunisation history and symptoms compatible with rubella were collected in retrospect from patients, family doctors or referring microbiologists.
3. Results Of 512 serum samples found initially as IgM-positive by external laboratories, only 159 (31.1%) were confirmed in this study, using a -capture EIA for detection of rubellaspecific IgM antibodies. The concordance of the Medac assay with -capture assays used by external laboratories was determined to 64.7%. Initially, positive results obtained by conventional tests were confirmed in 20.5%. Almost all samples exhibited IgG titres beyond 30 IU/ml (Fig. 1). To characterise the sera further, both avidity of rubella-specific IgG antibodies and immune response to the viral E2 glycoprotein were determined in all samples. The data reveal no correlation between IgG titre and either the avidity index (Fig. 1 and Table 1) or presence of anti-E2 antibodies in immunoblot under non-reducing conditions (Fig. 2 and Table 1). The values of the avidity indices and the presence of anti-E2 antibodies are equally distributed in the samples irrespective of confirmed or unconfirmed IgM positivity (Table 1). Employing the immunoblot assay, E2-specific antibodies were demonstrated in 466 samples (91%, pattern I, Table 2); 426 had high avidity IgG clearly indicating long passed infection or immunisation but no evidence for acute infection (pattern IA), but 40 had low (n = 3) or equivocal avidity index (n = 37, pattern IB). In 34 of them, follow-up samples 2–7 and
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Fig. 1. Correlation between avidity index and quantity (IU/ml) of total rubella-specific IgG (sera with IgG content >300 IU/ml are not shown). All sera (n = 512) were initially tested positive with standard IgM enzyme immunoassays. Of those IgM, reactivity was confirmed using an IgM capture EIA in 31% (n = 159, closed circles). Note equal distribution between avidity and quantity independent on whether the IgM results were confirmed or not confirmed. The dotted horizontal line marks the cut-off for low avidity antibodies, the dotted vertical line the cut-off for IgG positivity of sera.
24 (1 case) weeks later revealed no change in IgG titre or in avidity index. These samples were classified as past infection or the result of immunisation. This interpretation was supported by clinical data including vaccination history that were available in retrospect. Six cases remained uncertain due to lack of clinical information as well as follow-up serology. In 46 sera (9.0%, pattern II), E2-specific IgG was not detected by immunoblot. Sixteen of the samples had avidity indices below 0.5 (pattern IIB, Table 2). In six cases, acute rubella was confirmed by the clinical history and by followup serology (Table 3). Eight samples from three patients were without evidence for acute rubella as there was not any change Table 1 Serologic profile towards rubella immune reactivity in serum samples obtained from pregnant women Total number of sera (%)
IgM capture EIA Positive (%)
Negative (%)
466 (91.0) 46 (9.0)
146 (91.8) 13 (8.2)
320 (90.7) 33 (9.3)
Avidity index >0.5 0.3–0.5 <0.3
456 (89.0) 51 (10.0) 5 (1.0)
149 (93.7) 8 (5.0) 2 (1.3)
308 (87.3) 42 (11.9) 3 (0.8)
Total number
512 (100)
159 (31.1)
353 (68.9)
E2 Detectable Not detectable
The panel of 512 serum samples tested positive in different IgM EIA (>70% with conventional non- capture assays) were referred from external laboratories. The mean IgG content was 153 IU/ml (range 28–540 IU/ml) in samples with capture EIA-confirmed IgM and 144 IU/ml (range 9–710 IU/ml) in those that were IgM capture EIA-negative.
Fig. 2. Immunoblot assay under non-reducing conditions for the detection of antibodies to rubella virus envelope proteins. Monoclonal antibodies to E2 (lane 1) and E1 (lane 2) as well as a serum from a convalescent patient with weak but definite E2 reactivity (lane 3) were used. Homo- and heterodimers of the envelope proteins E1 and E2, respectively, cause bands above 70 kD. The capsid protein forms under non-reducing gel conditions a dimer with a molecular weight of approximately 60 kD and is, therefore, hidden under the dominant E1-specific reactivity.
in quantity or quality of rubella-specific antibodies. The lack of anamnestic information and/or follow-up samples did not allow reliable interpretation for the remaining two specimens in this group. Of those with high avidity index (n = 30, pattern IIA), one sample was obtained from a woman who had acute rubella 5 months previously, and one woman was vaccinated against rubella 4 months before. Six persons from whom 15 samples were available did not show clinical or serological evidence of acute infection. Six samples without anamnestic information or follow-up samples could not be reliably interpreted. For the remaining seven specimens, control samples obtained 3–4 weeks later exhibited weak E2-specific immune-reactivity with consistent dominant E1 reactivity in immunoblot assay suggesting very low amounts of E2-specific IgG at the detection limit. Because of lack of rising IgG and despite non-availability of anamnestic data, these samples were interpreted as not compatible with acute rubella infection but rather indicating infection or immunisation at an undetermined time-point in the past (at least 4 months previously). Four hundred and forty-eight samples with high avidity index (98.2%) and 41 of those with equivocal avidity index (80.4%) were consistent with past infection or immunisation (Table 4). Taking together serological and clinical data including the vaccination history were available, 490 samples were classified as consistent with past infection or vaccination. Eight samples were classified as representing acute infection including those two samples obtained 4 and 5 months following vaccination or acute rubella, respectively. Fourteen samples remained inconclusive due to a lack of clinical information and follow-up serology. The avidity indices (mean range 0.7–0.8) did not decrease with the person’s age (data not shown).
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Table 2 Pattern of IgG reactivity in sera tested IgM-positive irrespective of its confirmation Pattern/subgroup
Immunoblot
I IA
E2-positive
IB IB1
Avidity index
Number of samples (% of total)
>0.5
466 (91.0) 426 (83.2)
≤0.5
40 (7.8)
E2-negative
IIB
>0.5
46 (9.0) 30 (5.9)
≤0.5
16 (3.1)
Interpretation
Number of samples (% of same pattern)
Past infection
426
(91.4)
40
IB2 II IIA
Avidity index
(8.6)
0.3–0.5
Past infection Uncertain
33 4
(7.1) (0.8)
≤0.3
Past infection Uncertain
1 2
(0.2) (0.4)
IIB1
0.3–0.5
IIB2
≤0.3
Past Uncertain Acute
22 6 2
(47.8) (13.0) (4.3)
Past Uncertain Acute Past Uncertain Acute
8 2 6
(17.4) (4.3) (13.0)
Acute
8 2 4
(17.4) (13.0) (8.7)
2
(4.3)
Table 3 Follow-up serology of six acute rubella infections No. of case
1 2 3 4 5 6
1st Sample
2nd Sample
3rd Sample
-Capture IgM
Avidity index
E2-IgGa
Interval to 1st sample in weeksb
-Capture IgM
Avidity index
E2-IgGa
Interval to 1st sample in weeks
-Capture IgM
Avidity index
E2-IgGa
pos pos pos pos pos pos
0.21 0.23 0.31 0.38 0.42 0.48
neg neg neg neg neg neg
3 5 4 5 3 3
pos neg neg neg pos pos
0.54 0.60 0.70 0.65 0.73 0.70
neg neg ± pos pos pos
8 12
neg neg
0.75 0.8
pos pos
All samples except those of case number 2 and 3 were pre-tested with conventional assays by external laboratories. a E2-IgG as detected by non-reducing immunoblot. b The time interval between obtaining serum samples and suspected infection or risk contact was unknown for case no. 3–6 and was 3.6 weeks for case 1 and 2 days for case 2.
4. Discussion The results of avidity assay and immunoblot analysis and the available clinical data described in this report revealed that the vast majority of serum samples were positive for IgM were neither associated with acute rubella nor with reinfection. Only eight (1.6% of all sera or 3.8% of the IgM capture EIA confirmed samples) were obtained from women who experienced an acute rubella infection or a recent vaccination; 490 samples (95.7%) were clear past infections including those seven cases, where the initial sample was E2-negative/high avidity index (pattern IIA) with follow-up samples weakly E2-positive but without rise in IgG. In only 14 samples (2.7%), the time-point of infection could not be determined exactly due to the lack of patient history and follow-up samples. The findings are in agreement with previously published data (Best et al., 2002).
IgM positivity does not reflect always a specific result, since in the present study, only 31% were confirmed by an IgM capture assay. The potential reasons for non-specific immune responses include quality of serum samples that may contain non-specifically reactive factors and non-specific stimulation of antibody producing cells (Thomas et al., 1999). Several laboratories used conventional IgM assays, which are more likely to give non-specific results as described earlier (Hudson and Morgan-Capner, 1996). In addition, IgM responses particularly to rubella could and should be uncommon, as safe and efficient vaccines are available for more than 30 years (Best, 1991). For example, the recommendation for rubella immunisation in Germany includes two vaccinations (the first one at about 13 months of age, the second before 24 months). Despite these rules, a considerable population remains susceptible to rubella infection. The question also arises whether immunity after a long
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Table 4 Interpretation of results according to the results of avidity testing Avidity index
Number of samples (% of total)
>0.5
456 (89.1)
Immunoblot
E2+ E2−
0.3–0.5
≤0.3
Number of samples (% of same pattern)
426 (93.4) 30 (6.6)
51 (10.0)
E2+
37 (72.5)
E2−
14 (27.5)
5 (1.0)
E2+
3
E2−
2
interval for single immunisation is still protective. Nonetheless, fetal damage after maternal re-infection with rubella virus is an extremely rare event (Bullens et al., 2000). Reinfection would provide a hazard to the fetus if associated with viraemia. The frequency of re-infections is difficult to determine, as it is usually clinically inapparent and not associated with viraemia (Best and Banatvala, 2000). Therefore, evidence of re-infection depends on serological data. Re-infection should be considered only if a pre-infection serum sample or a sample close to the putative time-point of re-infection is available. Alternatively, a significant rise in antibody titre (four-fold) and high avidity index may provide evidence for re-exposure and possible re-infection even if viraemia cannot be detected (Best et al., 1989; Pustowoit and Liebert, 1998). Since in the present study, the avidity indices as well as the presence of E2-specific IgG did not correlate either with the conventional or with the -capture EIA, a second IgM testing cannot be recommended any longer. A serum sample with a positive IgM result (irrespective of the assay format used), low avidity index (≤0.3) and absence of specific antibodies to the viral E2 protein permits unequivocal diagnosis of acute rubella. Samples with positive capture IgM result and absence of anti-E2-specific antibodies but avidity index above 0.3 require a follow-up serology to verify a possible acute infection. Maturation of IgG as expressed by the avidity index has been suggested as a useful marker for an acute infection (Hedman and Rousseau, 1989; Thomas et al., 1993; Nedeljkovic et al., 2001). Of the 512 serum samples in this paper, 11% did not exceed avidity indices of 0.5; thus, acute
Interpretation
Number of samples (% of same pattern)
Past infection Uncertain Acute Past infection Past infection Uncertain Acute
448 6 2
Past infection Uncertain Acute Past infection Uncertain Acute Past infection Uncertain Acute
41 6 4
Past infection Uncertain Acute Past infection Uncertain Acute
1 2 2
(98.2) (1.3) (0.4) 426 22 6 2
(93.4) (4.8) (1.3) (0.4) (80.4) (11.8) (7.8)
33 4 0 8 2 4
(64.7) (7.8) (15.7) (3.9) (7.8)
1 2 2
or recently experienced infection could not be excluded without follow-up samples and clinical data. The determination of the avidity of rubella-specific IgG reduces substantially per se the number of suspected acute infections. Similarly, the immunoblot test resulted in 91% samples with detectable E2-specific antibody. Under the conditions used for the experiments in this study the immunoblot allows the detection of antibodies to the rubella virus envelope proteins in samples containing less than 5 IU/ml IgG (Mauracher et al., 1993; Pustowoit et al., 1996). However, the absence of E2specific antibodies does not necessarily mean recent infection during the previous 3–4 months but might reflect loss of antibodies after long time intervals since the last exposure to rubella virus. In this study, 30 of 46 E2-negative sera (65.2%) were in fact consistent with past infection or vaccination, and only 8 (17.4%) were associated with acute rubella. Surprisingly, there was no unequivocal case that met the criteria for re-infection. This finding is particularly significant, as the serum panel of this study is selected with only those sera included that were tested IgM-positive at any one time-point. The data suggest that rubella virus does not circulate extensively in a population with a seroprevalence of >90% for rubella IgG. Moreover, although the risk of fetal infection had been calculated to approximately 8% following maternal re-infection during the first 16 weeks of pregnancy, fetal malformations are very rare (Morgan-Capner et al., 1991). The calculated figure for fetal infection is probably too large since in several of the reported cases the data are incomplete, and therefore, not all cases after potential re-infection appear as documented unequivocally congenital
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rubella (Morgan-Capner, 1986; Bullens et al., 2000). Conceivably, rubella virus re-infections can occur after immunisation in the past or naturally acquired infection at a timepoint when the immune reactivity had declined and became insufficient to prevent viraemia. This may also include rare cases with partial defects of the immune system such as an inability to produce antibodies to the protective epitopes of the virus. Nonetheless, considering an IgM positivity rate of 1% in pregnant women, and a re-infection rate of less than 1%, a maximum of eight fetal infections in one million gestations were to be expected based exclusively on serological data. The risk of congenital malformations following reinfection is not well known but considered very small as hazardous viraemia is uncommon during re-infection (Enders and Pustowoit, personal communication). The combination of immunoblot and antibody avidity reduces the rate of inconclusive serology results to approximately 3% even in a panel that was selected because of initial putative IgM reactivity. In countries without a vaccination programme and significant case numbers of congenital rubella the diagnostic should include avidity testing and immunoblot.
Acknowledgements The authors are grateful to those colleagues referring their unclear serum samples to the Institute of Virology, particularly, Drs. Weichsel (Geesthacht), Peters (Schwerin), Berthold (Frankfurt/Oder), Lehmann (Leipzig), Jung (Cologne), R¨ocker (Berlin), Gahner (Bautzen), and several other colleagues. Without their continuous referrals and contributing anamnestic data of patients involved, this study would not have been possible.
References Best, J.M., 1991. Rubella vaccines: past, present and future. Epidemiol. Infect. 107, 17–30. Best, J.M., Banatvala, J.E., 2000. Rubella. In: Zuckerman, A.J., Banatvala, J.E., Pattison, J.R. (Eds.), Principles and Practice of Clinical Virology, fourth ed. John Wiley & Sons, Chichester, pp. 387–418. Best, J.M., Banatvala, J.E., Morgan-Capner, P., Miller, E., 1989. Fetal infection after maternal reinfection with rubella: criteria for defining reinfection. BMJ 299, 773–775.
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Best, J.M., O’Shea, S., Tipples, G., Davis, N., Al-Khusaiby, S.M., Krause, A., Hesket, L.M., Jin, L., Enders, G., 2002. Interpretation of rubella serology in pregnancy-pitfalls and problems. BMJ 325, 147–148. Bullens, D., Smets, K., Vanhaesebrouck, P., 2000. Congenital rubella syndrome after maternal reinfection. Clin. Pediatr. 39, 113–116. Bundes¨arztekammer, 2002. Directives of the German Federal Joint Committee concerning the Medical Care during Pregnancy and after Childbirth, published under the title “Mutterschaftsrichtlinie R¨oteln”. Bundesanzeiger, No. 242. Chantler, J.K., Wolinsky, J., Tingle, A., 2001. Rubella virus. In: Knipe, D.M., Howley, P.M. (Eds.), Fields Virology, fourth ed. Raven Press, NY, pp. 963–990. Forsgren, M., Soren, L., 1985. Subclinical rubella reinfection in vaccinated women with rubella-specific IgM response during pregnancy and transmission of virus to the fetus. Scand. J. Infect. Dis. 17, 337–341. Hedman, K., Rousseau, S.A., 1989. Measurement of avidity of specific IgG for verification of recent primary rubella. J. Med. Virol. 27, 288–292. Hobman, T.C., Lundstrom, M.L., Mauracher, C.A., Woodward, L., Gillam, S., Farquhar, M.G., 1994. Assembly of rubella virus structural proteins into virus-like particles in transfected cells. Virology 202, 574–585. Hudson, P., Morgan-Capner, P., 1996. Evaluation of fifteen commercial enzyme immunoassays for the detection of rubella-specific IgM. Clin. Diagn. Virol. 5, 21–26. Mauracher, C.A., Mitchell, L.A., Tingle, A.J., 1993. Selective tolerance to the E1 protein of rubella virus in congenital rubella syndrome. J. Immunol. 151, 2041–2049. Mitchell, L.A., Tingle, A.J., Decarie, D., Shukin, R., 1999. Identification of rubella virus T-cell epitopes recognized in anamnestic response to RA27/3 vaccine: associations with boost in neutralizing antibody titer. Vaccine 17, 2356–2365. Morgan-Capner, P., 1986. Does rubella reinfection matter?. In: Public Health Virology Report. Public Health Laboratory Service, London, pp. 50–62. Morgan-Capner, P., Miller, E., Vurdien, J.E., Ramsay, M.E., 1991. Outcome of pregnancy after maternal reinfection with rubella. Commun. Dis. Rep. 1, 57–59. Nedeljkovic, J., Jovanovic, T., Oker-Blom, C., 2001. Maturation of IgG avidity to individual rubella virus structural proteins. J. Clin. Virol. 22, 47–54. Pustowoit, B., Liebert, U.G., 1998. Predictive value of serological tests in rubella virus infection during pregnancy. Intervirology 41, 170–177. Pustowoit, B., Grangeot-Keros, L., Hobman, T.C., Hofmann, J., 1996. Evaluation of recombinant rubella-like particles in a commercial immunoassay for the detection of anti-rubella IgG. Clin. Diagn. Virol. 5, 13–20. Thomas, H.I., Morgan-Capner, P., Cradock-Watson, J.E., Enders, G., Best, J.M., O’Shea, S., 1993. Slow maturation of IgG1 avidity and persistence of specific IgM in congenital rubella: implications for diagnosis and immunopathology. J. Med. Virol. 41, 196–200. Thomas, H.I., Barret, E., Hesket, L.M., Wynne, A., Morgan-Capner, P., 1999. Simultaneous IgM reactivity by EIA against more than one virus in measles, parvovirus B19 and rubella infection. J. Clin. Virol. 14, 107–118.
Significance of avidity and immunoblot analysis for rubella IgM-positive serum samples in pregnant women J. Hofmann, U.G. Liebert ∗ Institute of Virology, University of Leipzig, Johannisallee 30, 04103 Leipzig, Germany Received 13 August 2004; received in revised form 7 June 2005; accepted 9 June 2005 Available online 15 July 2005
Abstract A total of 512 IgM-positive serum samples from 449 pregnant women referred by microbiologists and medical laboratories for additional testing and final interpretation were collected over a 3-year period. Employment of an IgM capture enzyme immunoassay (EIA) confirmed only 31% of the initial EIA-IgM-positive samples. In order to discriminate acute rubella virus infections, which are associated with increased risk of fetal infection and embryopathy, from persistent or non-specific IgM, IgG avidity index and the presence of IgG with specificity to rubella virus E2 glycoprotein was determined by Western immunoblot. In only six patients (1.3%), a primary infection with rubella virus was diagnosed on the basis of IgM positivity, low avidity IgG, absence of E2-specific IgG in immunoblot concordant with clinical findings as well as consistent changes in follow-up samples. The serological results were not compatible with rubella re-infection. The infection status in 14 patients (3.1%) remained inconclusive even when both avidity assay and immunoblot were used, while restriction to either test did not allow a conclusive interpretation in 11.6% of patients. The use of both assays is clearly better, and therefore, recommended in IgM-positive samples. © 2005 Elsevier B.V. All rights reserved.
1. Introduction Post-natal rubella virus infection usually causes a mild disease including mild fever, lymphadenopathy and exanthema, or remains asymptomatic. Within the first 16 weeks of pregnancy, however, the infection can lead in up to 75% of cases to fetal infection and malformations (Chantler et al., 2001). In Germany, approximately, 10% of women in the childbearing age are seronegative despite the availability of a safe and effective vaccine. A further diagnostic problem arises as long-term follow-up studies have demonstrated significantly lower antibody levels in those vaccinated compared to naturally acquired immunity resulting in an increased risk of re-infections following exposure to wild-type rubella virus (Forsgren and Soren, 1985; Mitchell et al., 1999). Therefore, verification of the rubella immune status is included in the guidelines for medical care during pregnancy. Accordingly, determination of rubella-specific IgM and/or followup serology is recommended for persons with suspected ∗
Corresponding author. Tel.: +49 341 9714300; fax: +49 341 9714309. E-mail address: [email protected] (U.G. Liebert).
0166-0934/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2005.06.004
acute infection or with contact to rubella or rubella-like illness (Bundes¨arztekammer, 2002). Several laboratories carry out additional IgM determinations or investigate follow-up samples to obtain reliable diagnostic interpretations. This screening reveals approximately, 1% positive IgM results by screening enzyme immunoassays. Since IgM assays may result in non-specific results, additional methods should be used in such cases. These are determination of antibodies to individual viral proteins by immunoblot and avidity of specific IgG antibodies (Pustowoit and Liebert, 1998; Best et al., 2002). While E2-specific antibodies appear with a delay of 3–4 months after primary infection as detected by immunoblots under non-reducing conditions (Mauracher et al., 1993; Pustowoit et al., 1996), E1-specific antibodies may appear as early as 4–6 days post-infection (Hedman and Rousseau, 1989; Nedeljkovic et al., 2001). In a study using well-defined serum samples with follow-up samples up to 12 months from 27 persons, the avidity indices reached 0.21–0.33 within 28–33 days of the rubella rush or after vaccination. Six months after infection or vaccination the avidity indices were 0.54–0.94 with a mean of 0.74 (Nedeljkovic et al., 2001). The present study summarises data of 512 serum
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samples that were referred to the Institute of Virology by microbiologists and medical laboratories for further evaluation.
2. Material and methods 2.1. Serological assays (1) An IgM capture (-capture) enzyme immunoassay (Medac, Hamburg, Germany) was used to confirm IgM results obtained in external laboratories. (2) A commercially available enzyme immunoassay (Enzygnost® Dade-Behring, Marburg, Germany, using cell culture derived rubella virus as antigen) was used to determine the quantity of specific IgG (in IU/ml). The avidity indices were determined using the assay with an additional incubation step, which insubstantially elongates the turn-round time of the assay. Briefly, after binding of serum IgG to antigen-coated microtitre plate, wells were incubated in parallel for 5 min either in washing buffer or in washing buffer containing urea/hydrogen peroxide mixture, respectively. Avidity indices were calculated as the ratio of ODs in urea/hydrogen peroxide treated to non-treated sample. Ratios below 0.3 were considered as indicating low avidity IgG, while those above 0.5 as high avidity IgG, and avidity indices between 0.3 and 0.5 are equivocal (Hedman and Rousseau, 1989; Pustowoit and Liebert, 1998). (3) The non-reducing immunoblot (NRIB) was carried out as described previously. Briefly, recombinant rubella-like particles secreted from transfected CHO cells (Hobman et al., 1994), were concentrated and purified as described for cell culture grown virions (Pustowoit et al., 1996). Fifteen micrograms of protein lysate diluted in 270 l loading buffer were separated by 10% SDS-PAGE (using a 1 mm prep/2-D comb with a sample well volume of 400 l in a miniprotean electrophoresis cell (BioRad, M¨unchen, Germany) under non-reducing conditions. The proteins were transferred onto a PVDF-membrane (Millipore, Schwalbach, Germany). Strips cut from the membrane were blocked with 5% milk powder dissolved in PBST (phosphate-buffered saline, pH 7.4 containing 0.5% Tween 20) and incubated subsequently with human sera diluted 1:100 in PBST overnight at room temperature. After washing three times, the strips were incubated with HRP conjugated rabbit anti-human IgG (DAKO, Denmark) diluted 1:2000 in PBST for 2 h at room temperature. For visualisation, Nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate (BioRad, M¨unchen, Germany) have been used. The total time for conducting the test is acceptable in terms of rapid diagnostic requirements. The presence of the viral glycoproteins E1 and E2 in the protein lysate and their location on the PVDF stripes was determined by immunoblot using monoclonal antibodies (M1B9, Roche
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Diagnostics, Mannheim, Germany, and Mab26–24, Viral Antigens Inc., Memphis, TN, USA, respectively, Fig. 2). Selected patient sera were incubated with antigen derived from rubella virus strain Therien-infected BHK-cells. This but not incubation with lysate from non-infected BHK cells resulted in loss of reactivity in immunoblots. Since rubella virus antigen or other necessary prerequisites are not available in each diagnostic laboratory in Germany, samples were usually referred to specialised centers including the laboratory of the University of Leipzig. 2.2. Patient serum samples Five hundred and twelve consecutive serum samples, positive by screening EIA for rubella-specific IgM, were collected from 449 pregnant women over a period of 3 years. The sera were referred by microbiologists and medical laboratories for further evaluation. The mean age of patients was 29.6 ± 5.6 years, approximately, 80% had been vaccinated in the past. These sera had been pre-tested in external laboratories as IgM-positive using different IgM assays “VIDAS Rub IgM” (BioMerieux, -capture), “ETI-RUBEK-M reverse” (DiaSorin, -capture), “IMX Rubella IgM” (Abbott Laboratories, conventional assay format), and others. Clinical data on immunisation history and symptoms compatible with rubella were collected in retrospect from patients, family doctors or referring microbiologists.
3. Results Of 512 serum samples found initially as IgM-positive by external laboratories, only 159 (31.1%) were confirmed in this study, using a -capture EIA for detection of rubellaspecific IgM antibodies. The concordance of the Medac assay with -capture assays used by external laboratories was determined to 64.7%. Initially, positive results obtained by conventional tests were confirmed in 20.5%. Almost all samples exhibited IgG titres beyond 30 IU/ml (Fig. 1). To characterise the sera further, both avidity of rubella-specific IgG antibodies and immune response to the viral E2 glycoprotein were determined in all samples. The data reveal no correlation between IgG titre and either the avidity index (Fig. 1 and Table 1) or presence of anti-E2 antibodies in immunoblot under non-reducing conditions (Fig. 2 and Table 1). The values of the avidity indices and the presence of anti-E2 antibodies are equally distributed in the samples irrespective of confirmed or unconfirmed IgM positivity (Table 1). Employing the immunoblot assay, E2-specific antibodies were demonstrated in 466 samples (91%, pattern I, Table 2); 426 had high avidity IgG clearly indicating long passed infection or immunisation but no evidence for acute infection (pattern IA), but 40 had low (n = 3) or equivocal avidity index (n = 37, pattern IB). In 34 of them, follow-up samples 2–7 and
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Fig. 1. Correlation between avidity index and quantity (IU/ml) of total rubella-specific IgG (sera with IgG content >300 IU/ml are not shown). All sera (n = 512) were initially tested positive with standard IgM enzyme immunoassays. Of those IgM, reactivity was confirmed using an IgM capture EIA in 31% (n = 159, closed circles). Note equal distribution between avidity and quantity independent on whether the IgM results were confirmed or not confirmed. The dotted horizontal line marks the cut-off for low avidity antibodies, the dotted vertical line the cut-off for IgG positivity of sera.
24 (1 case) weeks later revealed no change in IgG titre or in avidity index. These samples were classified as past infection or the result of immunisation. This interpretation was supported by clinical data including vaccination history that were available in retrospect. Six cases remained uncertain due to lack of clinical information as well as follow-up serology. In 46 sera (9.0%, pattern II), E2-specific IgG was not detected by immunoblot. Sixteen of the samples had avidity indices below 0.5 (pattern IIB, Table 2). In six cases, acute rubella was confirmed by the clinical history and by followup serology (Table 3). Eight samples from three patients were without evidence for acute rubella as there was not any change Table 1 Serologic profile towards rubella immune reactivity in serum samples obtained from pregnant women Total number of sera (%)
IgM capture EIA Positive (%)
Negative (%)
466 (91.0) 46 (9.0)
146 (91.8) 13 (8.2)
320 (90.7) 33 (9.3)
Avidity index >0.5 0.3–0.5 <0.3
456 (89.0) 51 (10.0) 5 (1.0)
149 (93.7) 8 (5.0) 2 (1.3)
308 (87.3) 42 (11.9) 3 (0.8)
Total number
512 (100)
159 (31.1)
353 (68.9)
E2 Detectable Not detectable
The panel of 512 serum samples tested positive in different IgM EIA (>70% with conventional non- capture assays) were referred from external laboratories. The mean IgG content was 153 IU/ml (range 28–540 IU/ml) in samples with capture EIA-confirmed IgM and 144 IU/ml (range 9–710 IU/ml) in those that were IgM capture EIA-negative.
Fig. 2. Immunoblot assay under non-reducing conditions for the detection of antibodies to rubella virus envelope proteins. Monoclonal antibodies to E2 (lane 1) and E1 (lane 2) as well as a serum from a convalescent patient with weak but definite E2 reactivity (lane 3) were used. Homo- and heterodimers of the envelope proteins E1 and E2, respectively, cause bands above 70 kD. The capsid protein forms under non-reducing gel conditions a dimer with a molecular weight of approximately 60 kD and is, therefore, hidden under the dominant E1-specific reactivity.
in quantity or quality of rubella-specific antibodies. The lack of anamnestic information and/or follow-up samples did not allow reliable interpretation for the remaining two specimens in this group. Of those with high avidity index (n = 30, pattern IIA), one sample was obtained from a woman who had acute rubella 5 months previously, and one woman was vaccinated against rubella 4 months before. Six persons from whom 15 samples were available did not show clinical or serological evidence of acute infection. Six samples without anamnestic information or follow-up samples could not be reliably interpreted. For the remaining seven specimens, control samples obtained 3–4 weeks later exhibited weak E2-specific immune-reactivity with consistent dominant E1 reactivity in immunoblot assay suggesting very low amounts of E2-specific IgG at the detection limit. Because of lack of rising IgG and despite non-availability of anamnestic data, these samples were interpreted as not compatible with acute rubella infection but rather indicating infection or immunisation at an undetermined time-point in the past (at least 4 months previously). Four hundred and forty-eight samples with high avidity index (98.2%) and 41 of those with equivocal avidity index (80.4%) were consistent with past infection or immunisation (Table 4). Taking together serological and clinical data including the vaccination history were available, 490 samples were classified as consistent with past infection or vaccination. Eight samples were classified as representing acute infection including those two samples obtained 4 and 5 months following vaccination or acute rubella, respectively. Fourteen samples remained inconclusive due to a lack of clinical information and follow-up serology. The avidity indices (mean range 0.7–0.8) did not decrease with the person’s age (data not shown).
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Table 2 Pattern of IgG reactivity in sera tested IgM-positive irrespective of its confirmation Pattern/subgroup
Immunoblot
I IA
E2-positive
IB IB1
Avidity index
Number of samples (% of total)
>0.5
466 (91.0) 426 (83.2)
≤0.5
40 (7.8)
E2-negative
IIB
>0.5
46 (9.0) 30 (5.9)
≤0.5
16 (3.1)
Interpretation
Number of samples (% of same pattern)
Past infection
426
(91.4)
40
IB2 II IIA
Avidity index
(8.6)
0.3–0.5
Past infection Uncertain
33 4
(7.1) (0.8)
≤0.3
Past infection Uncertain
1 2
(0.2) (0.4)
IIB1
0.3–0.5
IIB2
≤0.3
Past Uncertain Acute
22 6 2
(47.8) (13.0) (4.3)
Past Uncertain Acute Past Uncertain Acute
8 2 6
(17.4) (4.3) (13.0)
Acute
8 2 4
(17.4) (13.0) (8.7)
2
(4.3)
Table 3 Follow-up serology of six acute rubella infections No. of case
1 2 3 4 5 6
1st Sample
2nd Sample
3rd Sample
-Capture IgM
Avidity index
E2-IgGa
Interval to 1st sample in weeksb
-Capture IgM
Avidity index
E2-IgGa
Interval to 1st sample in weeks
-Capture IgM
Avidity index
E2-IgGa
pos pos pos pos pos pos
0.21 0.23 0.31 0.38 0.42 0.48
neg neg neg neg neg neg
3 5 4 5 3 3
pos neg neg neg pos pos
0.54 0.60 0.70 0.65 0.73 0.70
neg neg ± pos pos pos
8 12
neg neg
0.75 0.8
pos pos
All samples except those of case number 2 and 3 were pre-tested with conventional assays by external laboratories. a E2-IgG as detected by non-reducing immunoblot. b The time interval between obtaining serum samples and suspected infection or risk contact was unknown for case no. 3–6 and was 3.6 weeks for case 1 and 2 days for case 2.
4. Discussion The results of avidity assay and immunoblot analysis and the available clinical data described in this report revealed that the vast majority of serum samples were positive for IgM were neither associated with acute rubella nor with reinfection. Only eight (1.6% of all sera or 3.8% of the IgM capture EIA confirmed samples) were obtained from women who experienced an acute rubella infection or a recent vaccination; 490 samples (95.7%) were clear past infections including those seven cases, where the initial sample was E2-negative/high avidity index (pattern IIA) with follow-up samples weakly E2-positive but without rise in IgG. In only 14 samples (2.7%), the time-point of infection could not be determined exactly due to the lack of patient history and follow-up samples. The findings are in agreement with previously published data (Best et al., 2002).
IgM positivity does not reflect always a specific result, since in the present study, only 31% were confirmed by an IgM capture assay. The potential reasons for non-specific immune responses include quality of serum samples that may contain non-specifically reactive factors and non-specific stimulation of antibody producing cells (Thomas et al., 1999). Several laboratories used conventional IgM assays, which are more likely to give non-specific results as described earlier (Hudson and Morgan-Capner, 1996). In addition, IgM responses particularly to rubella could and should be uncommon, as safe and efficient vaccines are available for more than 30 years (Best, 1991). For example, the recommendation for rubella immunisation in Germany includes two vaccinations (the first one at about 13 months of age, the second before 24 months). Despite these rules, a considerable population remains susceptible to rubella infection. The question also arises whether immunity after a long
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Table 4 Interpretation of results according to the results of avidity testing Avidity index
Number of samples (% of total)
>0.5
456 (89.1)
Immunoblot
E2+ E2−
0.3–0.5
≤0.3
Number of samples (% of same pattern)
426 (93.4) 30 (6.6)
51 (10.0)
E2+
37 (72.5)
E2−
14 (27.5)
5 (1.0)
E2+
3
E2−
2
interval for single immunisation is still protective. Nonetheless, fetal damage after maternal re-infection with rubella virus is an extremely rare event (Bullens et al., 2000). Reinfection would provide a hazard to the fetus if associated with viraemia. The frequency of re-infections is difficult to determine, as it is usually clinically inapparent and not associated with viraemia (Best and Banatvala, 2000). Therefore, evidence of re-infection depends on serological data. Re-infection should be considered only if a pre-infection serum sample or a sample close to the putative time-point of re-infection is available. Alternatively, a significant rise in antibody titre (four-fold) and high avidity index may provide evidence for re-exposure and possible re-infection even if viraemia cannot be detected (Best et al., 1989; Pustowoit and Liebert, 1998). Since in the present study, the avidity indices as well as the presence of E2-specific IgG did not correlate either with the conventional or with the -capture EIA, a second IgM testing cannot be recommended any longer. A serum sample with a positive IgM result (irrespective of the assay format used), low avidity index (≤0.3) and absence of specific antibodies to the viral E2 protein permits unequivocal diagnosis of acute rubella. Samples with positive capture IgM result and absence of anti-E2-specific antibodies but avidity index above 0.3 require a follow-up serology to verify a possible acute infection. Maturation of IgG as expressed by the avidity index has been suggested as a useful marker for an acute infection (Hedman and Rousseau, 1989; Thomas et al., 1993; Nedeljkovic et al., 2001). Of the 512 serum samples in this paper, 11% did not exceed avidity indices of 0.5; thus, acute
Interpretation
Number of samples (% of same pattern)
Past infection Uncertain Acute Past infection Past infection Uncertain Acute
448 6 2
Past infection Uncertain Acute Past infection Uncertain Acute Past infection Uncertain Acute
41 6 4
Past infection Uncertain Acute Past infection Uncertain Acute
1 2 2
(98.2) (1.3) (0.4) 426 22 6 2
(93.4) (4.8) (1.3) (0.4) (80.4) (11.8) (7.8)
33 4 0 8 2 4
(64.7) (7.8) (15.7) (3.9) (7.8)
1 2 2
or recently experienced infection could not be excluded without follow-up samples and clinical data. The determination of the avidity of rubella-specific IgG reduces substantially per se the number of suspected acute infections. Similarly, the immunoblot test resulted in 91% samples with detectable E2-specific antibody. Under the conditions used for the experiments in this study the immunoblot allows the detection of antibodies to the rubella virus envelope proteins in samples containing less than 5 IU/ml IgG (Mauracher et al., 1993; Pustowoit et al., 1996). However, the absence of E2specific antibodies does not necessarily mean recent infection during the previous 3–4 months but might reflect loss of antibodies after long time intervals since the last exposure to rubella virus. In this study, 30 of 46 E2-negative sera (65.2%) were in fact consistent with past infection or vaccination, and only 8 (17.4%) were associated with acute rubella. Surprisingly, there was no unequivocal case that met the criteria for re-infection. This finding is particularly significant, as the serum panel of this study is selected with only those sera included that were tested IgM-positive at any one time-point. The data suggest that rubella virus does not circulate extensively in a population with a seroprevalence of >90% for rubella IgG. Moreover, although the risk of fetal infection had been calculated to approximately 8% following maternal re-infection during the first 16 weeks of pregnancy, fetal malformations are very rare (Morgan-Capner et al., 1991). The calculated figure for fetal infection is probably too large since in several of the reported cases the data are incomplete, and therefore, not all cases after potential re-infection appear as documented unequivocally congenital
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rubella (Morgan-Capner, 1986; Bullens et al., 2000). Conceivably, rubella virus re-infections can occur after immunisation in the past or naturally acquired infection at a timepoint when the immune reactivity had declined and became insufficient to prevent viraemia. This may also include rare cases with partial defects of the immune system such as an inability to produce antibodies to the protective epitopes of the virus. Nonetheless, considering an IgM positivity rate of 1% in pregnant women, and a re-infection rate of less than 1%, a maximum of eight fetal infections in one million gestations were to be expected based exclusively on serological data. The risk of congenital malformations following reinfection is not well known but considered very small as hazardous viraemia is uncommon during re-infection (Enders and Pustowoit, personal communication). The combination of immunoblot and antibody avidity reduces the rate of inconclusive serology results to approximately 3% even in a panel that was selected because of initial putative IgM reactivity. In countries without a vaccination programme and significant case numbers of congenital rubella the diagnostic should include avidity testing and immunoblot.
Acknowledgements The authors are grateful to those colleagues referring their unclear serum samples to the Institute of Virology, particularly, Drs. Weichsel (Geesthacht), Peters (Schwerin), Berthold (Frankfurt/Oder), Lehmann (Leipzig), Jung (Cologne), R¨ocker (Berlin), Gahner (Bautzen), and several other colleagues. Without their continuous referrals and contributing anamnestic data of patients involved, this study would not have been possible.
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