Diagnosis of recent primary rubella virus infections: Significance of glycoprotein-based IgM serology, IgG avidity and immunoblot analysis

Journal of Virological Methods 174 (2011) 85–93

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Diagnosis of recent primary rubella virus infections: Significance of glycoprotein-based IgM serology, IgG avidity and immunoblot analysis Klaus-Peter Wandinger a , Sandra Saschenbrecker a,∗ , Katja Steinhagen a , Thomas Scheper a , Wolfgang Meyer a , Uwe Bartelt b , Gisela Enders b a b

Institute of Experimental Immunology, Affiliated to EUROIMMUN AG, Seekamp 31, D-23560 Luebeck, Germany Institute for Virology, Infectiology and Epidemiology, Medical Diagnostic Laboratory Prof. Enders and Partners, Rosenbergstrasse 85, D-70193 Stuttgart, Germany

a b s t r a c t Article history: Received 22 November 2010 Received in revised form 15 March 2011 Accepted 5 April 2011 Available online 13 April 2011 Keywords: Rubella virus Glycoprotein Avidity Anti-E2 Immunoblot

Reliable serodiagnosis of rubella virus (RV) infections requires discrimination of specific IgM induced by primary rubella from persistent, reactivated or non-specific IgM reactivity. Sera from 130 pregnant women with recent or past RV infection/vaccination, persistent IgM or negative rubella serology, 26 patients with other acute infections and 5 patients with rheumatoid factor-positivity were analyzed for RV-specific IgM by ELISA coated with whole-virus lysate or native glycoprotein, followed by determination of IgG avidity and E2-specific IgG using lysate-coated ELISA and non-reducing immunoblot. Compared to a reference ␮-capture IgM ELISA, the sensitivity for diagnosing recent rubella infection/vaccination was 90.0% and 100% for the lysate-based and glycoprotein-based IgM ELISA, respectively. With respect to women with past RV infections or negative histories of RV infection/vaccination, both assays were 97.5–100% specific, whereas for patients with other acute infections the glycoprotein substrate provided a specificity of 92.3% compared to only 80.8% using whole-virus antigen. Analyzing anti-RV IgG avidity and anti-E2 IgG reactivity allowed the time point of primary infection to be determined unambiguously in >86% of samples. In conclusion, using RV glycoprotein antigen improves the specificity of indirect IgM ELISA. In cases of RV-specific IgM reactivity, recent primary rubella infection can be confirmed or excluded efficiently by specific IgG avidity and immunoblot analysis. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Rubella (German measles) is usually a mild disease manifesting with a transient maculopapular rash, lymphadenopathy and low fever. However, maternal primary rubella virus (RV) infection in the early months of pregnancy entails a high risk of in utero infection leading to severe developmental abnormalities associated with congenital rubella syndrome (Banatvala and Brown, 2004; Lee and Bowden, 2000; Miller et al., 1982). Laboratory confirmation of recently acquired rubella relies commonly on serum detection of RV-specific IgM antibodies. However, IgM positivity is not invariably indicative of primary infection, but may be due to reinfection (Aboudy et al., 2000; Best et al., 1989; Forsgren and

Abbreviations: ELISA, enzyme-linked immunosorbent assay; HAI, hemagglutination inhibition; IgG, immunoglobulin G; IgM, immunoglobulin M; RAI, relative avidity index; RV, rubella virus. ∗ Corresponding author at: Institute of Experimental Immunology, EUROIMMUN AG, Seekamp 31, Haus 5, D-23560 Luebeck, Germany. Tel.: +49 451 5855 25741; fax: +49 451 5855 591. E-mail address: [email protected] (S. Saschenbrecker). 0166-0934/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2011.04.001

Soren, 1985; Morgan-Capner et al., 1985) or more frequently to IgM persistence, which can last for over 6 years following naturally acquired or vaccine-induced rubella (Al-Nakib et al., 1975; Banatvala et al., 1985; Best et al., 2002; Best and Enders, 2007; Enders, 2005; Pattison, 1975). The presence of long-persisiting IgM antibodies is confirmed when consistently moderate or high positive IgM levels are detected by at least 2 different IgM EIA test methods in consecutive serum samples up to several years after the initial detection. By testing more than 448 mother–infant pairs at the time of delivery it was shown that in cases of correctly defined long-persisiting IgM antibodies there is no risk of congenital infection (Best and Enders, 2007). In addition, infections with other pathogens, such as EBV or human parvovirus B19, are associated with random polyclonal B cell stimulation, which may lead to RVspecific and non-specific (potentially cross-reactive) IgM responses (Dimech et al., 2005; Hudson and Morgan-Capner, 1996; MorganCapner et al., 1983; Nobutoki et al., 1996; Thomas et al., 1999; Tipples et al., 2004). Besides, the presence of IgM class rheumatoid factor can cause false-positive results, while absent, low, transient or delayed IgM appearance might cause false exclusion of primary infection (Hudson and Morgan-Capner, 1996; Meurman and Ziola, 1978; Wolter et al., 1997). In fact, the implementation of

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vaccination programs has lead to a decrease in rubella incidence, resulting in a decline in the positive predictive value of IgM testing, which entails a considerable risk of false-positive results (Best et al., 2002). Hence, the current primary challenge in rubella serology (in developed countries) is not the diagnosis of recent RV infections itself, but rather the clarification of positive IgM responses. Therefore, reliable diagnosis requires supplemental seroanalyses, such as follow-up serology for evidence of seroconversion or significant titer rises in specific IgG. Moreover, the determination of IgG avidity and the immunoblot detection of IgG against the RV glycoproteins E1 and E2 have proved valuable in narrowing down the time point of infection (Best et al., 2002; Best and Enders, 2007; Hofmann and Liebert, 2005; Mauracher et al., 1992; Pustowoit et al., 1996; Pustowoit and Liebert, 1998; Wolter et al., 1997; Zhang et al., 1992). While low avidity indicates initial stages of IgG maturation following infection or vaccination, high avidity values exclude primary exposure within at least the previous 4–6 weeks up to 3 months, depending on the avidity test applied (Bottiger and Jensen, 1997; Enders and Knotek, 1989; Hedman and Rousseau, 1989). Therefore avidity tests are useful for distinguishing IgM due to primary rubella from long-persisting IgM antibodies (Best and Enders, 2007). Likewise, anti-E1 IgG usually appears within 4–6 days following contact, whereas E2-specific antibodies are not detectable until 3–4 months post-infection and therefore rule out early-stage infection (Pustowoit and Liebert, 1998). The purpose of this study was to analyze the performance of a glycoprotein-based rubella IgM ELISA and to elaborate on the significance of supplemental seroanalyses, i.e. the determination of RV-specific IgG avidity (ELISA) and anti-E2 IgG (immunoblot), for the confirmation of recent primary rubella infection.

2. Materials and methods 2.1. Serum samples A total of 161 sera submitted to the Medical Diagnostic Laboratory of Prof. Enders and Partners (Stuttgart, Germany) were included in this study. Of these samples, 130 were collected from pregnant women (mean age 30.2 years) presenting for antenatal rubella screening or determination of rubella immune status, and were assigned to 5 panels according to clinical information and serological precharacterization: (i) one woman with acute primary RV infection (n = 1, blood taken 2 weeks after rash onset), (ii) recently vaccinated women (n = 9, vaccination 3–16 weeks prior to sampling), (iii) women with persistent anti-RV IgM, i.e. healthy women in early pregnancy whose sera contained RVspecific IgM (detected by at least 2 different IgM tests; see Section 2.2), whereby presence of high avidity IgG and anti-E2 IgG antibodies clearly indicated RV infection/vaccination dating back at least several months (n = 50), (iv) women with past RV infection or vaccination (n = 40), as well as (v) women with a negative history of RV infection/vaccination and with rubella seronegativity (n = 30). The panel (ii) composed of females vaccinated recently was included to serve as a model for acute infections, thus compensating for the lack of a sufficient number of acutely infected pregnant patients. In addition, a panel (vi) of sera from patients with other serologically confirmed acute infections (n = 26, 11 male, 15 female, mean age 27.6 years), including Treponema pallidum (n = 5), mycoplasma (n = 5), Epstein–Barr virus (EBV, n = 5), parvovirus B19 (n = 5), cytomegalovirus (CMV, n = 4) and herpes simplex virus (HSV, n = 2) was examined. A further cohort (vii) was composed of patients with rheumatoid factor positivity (n = 5, 1 male, 4 female, mean age 56.1 years). Samples were included in this study on the basis of a statement from the Central Ethics Committee of Germany on the use of human samples for research

studies. Sera were stored at −20 ◦ C until antibody determination. 2.2. Sample precharacterization All sera were predefined and selected based on clinical information (rubella-like symptoms, vaccination history) and serological preanalysis (Table 1). The latter was performed using an in-house hemagglutination inhibition (HAI) assay (Medical Diagnostic Laboratory Prof. Enders and Partners, Stuttgart, Germany) as well as commercial test systems for the detection of RV-specific IgM (Sorin ETI-RUBEK-M reverse Plus ELISA, LIAISON Rubella IgM CLIA; DiaSorin, Saluggia, Italy) and for the determination of anti-RV IgG by ELISA (Enzygnost Anti-Rubella Virus IgG ELISA, Enzygnost AntiRubella Virus IgG avidity ELISA; Behring, Marburg, Germany) or by immunoblot (RecomBlot Rubella IgG; Mikrogen, Martinsried, Germany). 2.3. Immunoassays All serological analyses in this study were carried out using commercial test systems suitable for the determination of RV-specific IgM, IgG and IgG avidity (EUROIMMUN, Luebeck, Germany). IgM was detected using two test systems based on either whole-RV antigen or highly purified RV glycoproteins. Positive and negative control sera were implemented in all test series. Antibody determinations were performed blinded to clinical data and serological predefinition. 2.3.1. Enzyme-linked immunosorbent assays (ELISA) The Anti-Rubella Virus ELISA (IgG) and Anti-Rubella Virus ELISA (IgM) are based on 96-well microtiter plates (Nunc Polysorb; Thermo Fisher Scientific/Nunc, Roskilde, Denmark) coated with 200 ng/well (IgG) or 50 ng/well (IgM) of lysate of highly purified rubella virions (strain HPV-77), which were obtained from infected Vero cell culture supernatant and isolated by twofold sucrose density gradient centrifugation. The Anti-Rubella Virus Glycoprotein ELISA (IgM) employs the viral glycoprotein fraction (25 ng/well), purified by lectin affinity chromatography and containing predominantly glycoprotein E1 and, to a lesser extent, glycoprotein E2. For the detection of RV-specific IgG antibodies, sera diluted 1:101 in protein-containing PBS/NaN3 buffer were allowed to react with the antigenic solid phase for 30 min at room temperature, and were then washed three times (PBS/Tween buffer) and incubated with peroxidase-labelled rabbit anti-human IgG for 30 min. After a further wash, tetramethylbenzidine/H2 O2 substrate was applied for 15 min, before 0.5 M sulphuric acid stop solution was added. Optical density was determined photometrically at 450 nm (reference 620 nm). Antibody concentrations in international units (IU)/ml were calculated using a standard curve based on the extinction values of 4 calibration sera (1, 10, 50 and 200 IU/ml). Results above or equal to the cut-off value of 11 IU/ml were interpreted as positive, those from 8–11 IU/ml as borderline, and those below 8 IU/ml as negative. Analysis of anti-RV IgG avidity was based on two parallel IgG determinations, one of which included the application of 5 M urea solution after interaction of serum with the antigenic substrate, leading to a detachment of low-avidity antibodies. Avidity was calculated as percent ratio of the extinctions with and without urea treatment, expressed as relative avidity index (RAI). RAI values above 60% indicate the presence of high-avidity antibodies, values between 40% and 60% are considered borderline, and indices below 40% correspond to low avidity. Anti-RV IgM antibody detection was performed essentially as for IgG, except for the use of goat anti-human IgM peroxidase conjugate and an additional initial IgG/rheumatoid factor absorption

ND ND ND ND ND

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Fig. 1. Reactivity of RV envelope glycoproteins E1 and E2 in non-reducing immunoblot assay. Blot strips contain electrophoretically separated whole-RV antigen. Incubation with (A) mouse monoclonal antibody against E1 (58 kDa), (B) mouse monoclonal antibody against E2 (42–47 kDa), (C) serum collected from a person with negative RV serology (anti-E1 negative, anti-E2 negative), (D) serum from a patient with recent primary RV infection (anti-E1 positive, anti-E2 negative) and (E) serum from a late convalescent patient (anti-E1 positive, anti-E2 positive).

step, as provided and recommended by the assay manufacturer (EUROIMMUN). Preabsorption is necessary in order to prevent IgM displacement from the antigen by specifically binding IgG (leading to false negative IgM results), and to prevent possibly present IgM class rheumatoid factors from reacting with substrate-bound IgG (leading to false positive IgM reactivity). IgM results were evaluated semiquantitatively by calculating the ratio between the extinction of the patient sample and the extinction of the calibrator serum. Ratios above or equal to 1.1 were interpreted as positive, those from 0.8–1.1 as borderline, and those below 0.8 as negative. +, positive; +/−, borderline; −, negative; ND, not determined. a Patients seropositive for Treponema pallidum (n = 5), mycoplasma (n = 5), EBV (n = 5), parvovirus B19 (n = 5), CMV (n = 4) or HSV (n = 2). b Analysis included only a subset of sera.

ND ND ND ND ND ND

ND ND ND ND ND

26 (100%) 5 (100%) ND ND ND

25 (96.2%) 5 (100%) ND

ND

1 (3.8%) 0 (0%)

ND ND ND

0 (0%) 0 (0%)

0 (0%) 0 (0%)

ND

ND ND ND ND ND ND

ND ND ND ND ND

39 (97.5%) ND ND

40 (100%) 0 (0%) 40 (100%) 0 (0%)

0 (0%) 30 (100%)

0 (0%) 30 (100%)

ND

ND

0 (0%) ND

1 (2.5%) ND

ND

1 (100%) 8 (88.9%) 0 (0%) 0 (0%) 1 (11.1%) 0 (0%) 0 (0%) 0 (0%) 50 (100%) 0 (0%) 0/8b (0%) 0 (0%) 0 (0%) 0/8b (0%) 6 (12%)

− +/− + − +/−

1 (100%) 8/8b (100%) 44 (88%) 0 (0%) 0 (0%) 0 (0%)

+ − +/−

0 (0%) 1 (11.1%) 2 (4%)

+

1 (100%) 8 (88.9%) 48 (96%) 1 (100%) 7 (77.8%) 0 (0%)

Moderate Low

0 (0%) 2 (22.2%) 0 (0%) 0 (0%) 0 (0%) 50 (100%)

High −

0 (0%) 0 (0%) 0 (0%)

+

1 (100%) 9 (100%) 50 (100%)

−

0 (0%) 0 (0%) 0 (0%)

+

1 (100%) 9 (100%) 50 (100%)

Acute primary RV infection (n = 1) Recent vaccination (n = 9) Past RV infection or vaccination, persistent anti-RV IgM (n = 50) Past RV infection or vaccination (n = 40) Negative history of RV infection/vaccination (n = 30) Other acute infections (n = 26)a Rheumatoid factor (n = 5)

Hemagglutination inhibition (HAI, Enders) Cohort

Table 1 Precharacterization of sera (n = 161).

Enzygnost Anti-Rubella Virus IgG (ELISA, Behring)

Enzygnost Anti-Rubella Virus IgG avidity (ELISA, Behring)

Sorin ETI-RUBEK-M reverse Plus (ELISA, qual., DiaSorin)

LIAISON Rubella IgM (CLIA, quant., DiaSorin)

Anti-E2 IgG RecomBlot Rubella IgG (Immunoblot, Mikrogen)

K.-P. Wandinger et al. / Journal of Virological Methods 174 (2011) 85–93

2.3.2. Immunoblot assay For the Anti-Rubella virus immunoblot assay (IgG), whole antigen extract of RV (strain HPV-77) was obtained from infected Vero cell culture, separated according to molecular mass by discontinuous non-reducing SDS-PAGE, and Western blotted to nitrocellulose membranes (110 ng antigen per test strip). Diagnostically relevant antigen bands were characterized using the mouse monoclonal antibodies MAB925 and MAB927 (Chemicon-Millipore, Schwalbach, Germany), which are directed against the RV envelope glycoproteins E1 (58 kDa) and E2 (42–47 kDa), respectively (OkerBlom et al., 1983) (Fig. 1). Blot strips were incubated for 30 min at room temperature with sera diluted 1:51 in Tris-buffered saline. After a wash step with the same buffer, specific antibody binding was detected by addition of alkaline phosphatase-conjugated goat anti-human IgG for 30 min and exposure to NBT/BCIP for 10 min. Blot strips were digitalized using a flatbed scanner, and band intensities were quantified automatically by the EUROLineScan software (EUROIMMUN). Signal strengths up to 12 intensity units were regarded as negative, those between 13 and 20 units as borderline and those above a cutoff of 20 units as positive. In the evaluation of band patterns, anti-E1 positive/anti-E2 negative results indicate early-stage rubella infection or recent vaccination (however, a late infection state cannot be ruled out due to the possibility of retarded, reduced or absent anti-E2 formation). Simultaneous detection of anti-E1 and anti-E2 IgG is characteristic for infection or vaccination, with the presence of anti-E2 ruling out early-stage infection. 2.4. Statistical analysis Analytical sensitivity and specificity were calculated with reference to the samples’ predefined serostatus in two ways, that is by considering sera with borderline results as either positive or negative. Confidence intervals (CI 95%) were calculated according to the modified Wald method. Pearson’s correlation coefficient (r) was calculated to determine the degree of linear correlation between two test systems. Statistical analyses were carried out using the EUROStat statistical package (EUROIMMUN) and GraphPad QuickCalcs (GraphPad Software Inc., La Jolla, CA, USA).

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Figure 2. RV-specific IgG avidity and anti-E2 IgG reactivity. 131 serum samples (predefinition: acute infection, n = 1; recent vaccination, n = 9; past RV infection or vaccination with persistent anti-RV IgM, n = 50; past RV infection or vaccination, n = 40; other acute infections, n = 26; rheumatoid factor positivity, n = 5) were analyzed for anti-RV IgM (A, ELISA based on whole-RV antigen; B, ELISA based on purified RV glycoprotein), RV-specific IgG avidity (A–C, ELISA coated with whole-virus lysate) and anti-E2 IgG (C, immunoblot based on whole-RV antigen). The determination of anti-RV IgM antibodies included initial IgG/rheumatoid factor absorption. Dashed lines indicate avidity cut-off values: relative avidity index (RAI) <40%, low avidity; RAI 40–60%, borderline avidity; >60%, high avidity. The asterisk indicates an RV-seronegative sample.

3. Results 3.1. ELISA determination of RV-specific IgM antibodies using whole-virus lysate or purified glycoprotein as antigenic substrate In order to compare the performance of the whole-virus lysatebased anti-RV IgM ELISA with the glycoprotein-based anti-RV IgM ELISA, all serum samples (n = 161) were analyzed using both assays, as detailed in Table 2A and B. Notably, using the lysate-based test, 1 out of 9 (11.1%) pregnant women vaccinated recently was anti-

RV IgM negative (recently vaccinated women were examined as substitutes for acutely infected women from whom samples were not available readily). In sera pre-characterized as containing persistent RV-specific IgM, this test was borderline in 13/50 (26.0%) and negative in 7/50 (14.0%) cases (Fig. 2A). Employing highly purified RV glycoprotein as antigenic substrate, 2/9 (22.2%) postvaccination sera were classified as borderline. Among samples with persistent IgM, 7/50 (14.0%) were borderline and 8/50 (16.0%) were negative (Fig. 2B). According to these data, the lysate-based and the glycoprotein-based ELISA each yielded an overall analytical sensi-

Table 2 Determination of anti-RV IgM antibodies. Cohorta

Anti-Rubella Virus ELISA IgM, lysate-based (EUROIMMUN) No. of sera with result

No. of sera with result

% Sensitivity (CI 95%), with borderline results considered

Positive

Borderline

Negative

Positive

Negative

Positive

Borderline

Negative

Positive

Negative

1

0

0

100 (16.8–100)

100 (16.8–100)

1

0

0

100 (16.8–100)

100 (16.8–100)

8

0

1

88.9 (54.3–99.9) 88.9 (54.3–99.9)

7

2

0

100 (65.5–100)

77.8 (44.3–94.7)

9 30

0 13

1 7

90.0 (57.4–99.9) 90.0 (67.9–100) 86.0 (73.5–93.4) 60.0 (46.2–72.4)

8 35

2 7

0 8

100 (67.9–100) 80.0 (47.9–95.4) 84.0 (71.2–91.3) 70.0 (56.2–81.0)

0

0

40

100 (89.6–100)

100 (89.6–100)

0

1

39

97.5 (86.0–99.9) 100 (89.6–100)

0

0

30

100 (86.5–100)

100 (86.5–100)

0

0

30

100 (86.5–100)

3

2

21

80.8 (61.7–92.0) 88.5 (70.2–96.8)

1

1

24

92.3 (74.7–99.0) 96.2 (79.6–99.9)

0

0

5

100 (51.1–100)

0

0

5

100 (51.1–100)

3

2

96

95.0 (88.7–98.2) 97.0 (91.3–99.4)

1

2

98

97.0 (91.3–99.4) 99.0 (94.1–99.9)

100 (51.1–100)

100 (86.5–100)

100 (51.1–100)

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(A) analytical sensitivity Acute primary RV infection (n = 1) Recent vaccination (n = 9) Total (n = 10) Past RV infection or vaccination, persistent anti-RV IgM (n = 50) (B) analytical specificity Past RV infection (n = 40) Negative history of RV infection/vaccination (n = 30) Other acute infections (n = 26)b Rheumatoid factor (n = 5) Total (n = 101)

Anti-Rubella Virus Glycoprotein ELISA IgM (EUROIMMUN) % Sensitivity (CI 95%), with borderline results considered

CI, confidence interval. a Classification of patients according to clinical and serological precharacterization. b Patients seropositive for Treponema pallidum (n = 5), mycoplasma (n = 5), EBV (n = 5), parvovirus B19 (n = 5), CMV (n = 4) or HSV (n = 2).

89

90

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Figure 4. Immune response following primary RV infection or vaccination. The relative avidity index (RAI) of RV-specific IgG (ELISA coated with whole-virus lysate) and the presence of anti-E2 IgG (immunoblot) were determined in sera collected after indicated time intervals from pregnant women with acute primary RV infection (n = 1, sample taken 2 weeks after rash) or after vaccination (n = 9, samples taken 3–16 weeks post-vaccination). The dashed line represents the cut-off between low avidity (RAI < 40%) and borderline avidity (RAI 40–60%).

Both of these glycoprotein-reactive sera were drawn from patients with mycoplasma or EBV infection and also reacted in the lysatebased ELISA (Fig. 3B). Altogether, analytical specificities determined in the cohorts of women with past RV infection, with a negative history of RV infection/vaccination or with rheumatoid factor positivity were 100% for the lysate-based and 97.5–100% for the glycoprotein-based test (borderline results considered as positive) (Table 2B). For samples with potential interference due to other acute infections, the values amounted to 80.8% (lysate-based) and 92.3% (glycoprotein-based). Summarizing the control panels, overall specificities amounted to 95% and 97%, respectively. For all samples analyzed, overall comparison of the two IgM ELISA systems revealed a correlation coefficient (r) of 0.915 and (borderline results considered as positive) absolute concordance in 90.1% of cases. Figure 3. Rubella serology in patients with other acute infections or with rheumatoid factor positivity. 26 sera from individuals with acute infectious diseases (Treponema pallidum, n = 5; mycoplasma, n = 5; EBV, n = 5; parvovirus B19, n = 5; CMV, n = 4; HSV, n = 2), as well as 5 rheumatoid factor positive sera were examined for anti-RV IgM using ELISA based on (A) whole-RV antigen or (B) RV glycoprotein. The determination of anti-RV IgM antibodies included initial IgG/rheumatoid factor absorption. Results for anti-RV IgM ELISA: (+) IgM positive; (+/−) IgM borderline; (−) IgM negative. Additionally, the relative avidity index (RAI) of anti-RV IgG was determined by whole-virus lysate-coated ELISA, and the presence of anti-E2 IgG was analyzed using immunoblot based on whole-RV antigen. Dashed lines indicate cut-off values for IgG avidity: RAI < 40%, low avidity; RAI 40–60%, borderline avidity; >60%, high avidity. The asterisk indicates a RV-seronegative sample.

tivity of 86.7% (borderline results considered as positive), which improved to 90.0% and 100%, respectively, if sera predefined to contain persistent anti-RV IgM were not considered (Table 2A). With regard to specificity, only 1/40 (2.5%) sera from women with past RV infections was borderline in the glycoprotein-based test (Fig. 2A and B). In a panel of sera from cases of recent infections with other agents, all of which were predefined as anti-RV IgM negative, the lysate-based assay yielded borderline results in 2/26 (7.7%) and positive results in 3/26 (11.5%) patients infected with mycoplasma, EBV or parvovirus B19 (Fig. 3A). In contrast, using the glycoprotein-based ELISA in the same panel, borderline and positive IgM titers were found in only 1 (3.8%) serum each.

3.2. Determination of RV-specific IgG avidity (ELISA) and anti-E2 reactivity (immunoblot) for confirming or refuting primary infection in IgM positive sera 3.2.1. Primary infection sera In the cohort comprising recently infected or vaccinated pregnant women (n = 10), only low avidity IgG was detected. Avidity indices increased from 3% to 38% with time (2–16 weeks) after infection, indicating progressive IgG maturation (Fig. 4). Anti-E2 reactivity was not detected in 9 (90%) specimens. One of the women (10%), whose vaccination dated back 16 weeks, showed a borderline anti-E2 titer. Thus, correlation between low avidity values and the absence of E2-specific IgG was observed in 9 (90%) samples. RAI values below 40% and anti-E2 negativity correlated with the detection of RV-specific IgM in 89% (8/9) or 100% (9/9 including 1 borderline) of samples when analyzed using the lysate-based or the glycoprotein-based test, respectively (Fig. 2). All 3 sera with borderline or negative IgM titers were characterized by low avidity IgG, while anti-E2 reactivity was negative in 2 and borderline in 1 of these cases. Thus, serological results were consistent with recent primary infection/vaccination for all women in this cohort.

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3.2.2. Sera containing persistent IgM In the panel of sera (n = 50) with IgM persistence according to precharacterization, 48 (96%) samples contained RV-specific IgG of high avidity, while borderline or low avidity was found in only one serum each. Anti-E2 IgG was positive in 44 (88%) cases, borderline in 5 (10%) and negative in 1 (2%). Both sera with borderline or low avidity IgG were anti-E2 positive, while all 6 samples with borderline or negative anti-E2 titers showed high RAI values. In total, agreement between high avidity and anti-E2 positivity occurred in 42 (84%) samples (Fig. 2). The finding of high avidity values and/or positive anti-E2 reactivity supported the interpretation as past RV infection in all samples of this cohort and excluded the possibility of the IgM reactivity being a consequence of primary exposure. 3.2.3. Past infection sera In the group of sera (n = 40) representing past rubella infection, avidity was high in 39 (97.5%) cases and borderline in 1 (2.5%). Without exception, all sera were positive for anti-E2 IgG. Thus, high avidity indices and the presence of anti-E2 IgG coincided in 39 (97.5%) samples, and were in diagnostic agreement with negative IgM reactivity in all (100%) sera (Fig. 2). Consequently, the predefined past infection status was confirmed for the entire cohort. 3.2.4. Sera from patients with acute infections other than rubella or with rheumatoid factor positivity Among 31 sera with potential interference (e.g. polyclonal stimulation, cross-reactivity, false IgM positivity) due to recent non-RV infections (n = 26) or presence of rheumatoid factor (n = 5), 30 were found to be anti-RV IgG positive. Of these 30 sera, 28 (93.3%) contained specific IgG of high avidity, while in the remaining 2 (7.6%) borderline avidity was measured. E2-specific IgG reactivity was positive in 23 (76.7%) sera, borderline in 4 (13.3%) and negative in 3 (10%). Among the 7 samples showing borderline or negative anti-E2 titers, 6 contained high avidity IgG and 1 borderline avidity IgG. In 22 (73.3%) patients, high avidity and anti-E2 positivity were detected in parallel (Fig. 2). Of the 5 samples that were positive or borderline by either of the two IgM ELISA, all had high avidity indices together with positive (4/5) or borderline (1/5) anti-E2 IgG levels, thus indicating RV infection/vaccination in the past. 3.2.5. Overall correlation between RV-specific IgG avidity and anti-E2 reactivity Taking into account all anti-RV IgG positive samples (n = 130), agreement between high avidity and anti-E2 positivity occurred in 103 (79.2%) cases, while low avidity coincided with anti-E2 negativity in 9 (6.9%) samples, giving a total concordance rate of 86.2%. Discordance was observed in 3.8%, i.e. high (low) avidity was determined in parallel with anti-E2 negativity (positivity) in 4(1) specimens. Borderline results for either (or both) of the parameters were determined in 12(1) sera (Fig. 2C). 4. Discussion Obstetric care includes routinely testing for rubella immunity as well as seromonitoring of susceptible women to ensure rapid diagnosis of primary rubella. The determination of RV-specific IgM antibodies is the primary laboratory approach to confirm acute infection. However, problems associated with IgM serology may lead to the misinterpretation of serological results and erroneous diagnosis, with potentially serious consequences for the outcome of pregnancy. Therefore, it is crucial to distinguish reliably IgM reactivity elicited by recent primary infection from that resulting from persistence, reinfection, polyclonal B-cell stimulation, crossreactivity or other factors interfering with the detection system

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(Best et al., 2002; Wolter et al., 1997). Given high vaccination and seroprevalence rates (≥90%) in most developed countries (Nardone et al., 2008) and considering previous reports on rubella serology, in daily practice most anti-RV IgM positive results are not associated with acute primary RV infection but instead are mainly due to longpersisting IgM or false positivity, resulting in a decrease of the IgM positive predictive value (Best et al., 2002; Hofmann and Liebert, 2005). This substantiates the demand not only for IgM assays providing high specificity, but also for follow-up serology and above all supplemental immunoanalyses, such as determination of IgG avidity and anti-E1/anti-E2 reactivity (Best and Enders, 2007; Bottiger and Jensen, 1997; Enders and Knotek, 1989; Hedman and Rousseau, 1989; Hofmann and Liebert, 2005; Pustowoit and Liebert, 1998). In this study, a well-characterized panel of sera was analyzed for anti-RV IgM, avidity and anti-E2 IgG employing EUROIMMUN tests (EUROIMMUN, Luebeck, Germany) in order to compare the antigenic performance of RV glycoprotein with that of whole-virus lysate in ELISA IgM detection, and to elaborate on the diagnostic significance provided by multi-parametric rubella testing. Commercial assays for anti-rubella IgM serology differ in diagnostic performance, as demonstrated by comparative studies revealing sensitivities (specificities) in the range of 63–92% (82–98%) (Hudson and Morgan-Capner, 1996), 82.7–100% (98.3–100%) (Grangeot-Keros and Enders, 1997), 66.4–78.9% (85.6–97.2%) (Tipples et al., 2004) or 84.2–96.5% (96.8–99.9%) (Dimech et al., 2005). These discordances between test systems could be due to different detection principles and/or the diversity of antigenic substrates. Notably, the diagnostic performance of assays with capture format was in many cases superior to that of solid-phase antigen formats, particularly in terms of specificity. In the present study, the performance of both IgM assays (solid-phase antigen format) corresponded to that reported for other commercial assays. In relation to the capture reference test (Sorin ETI-RUBEK-M reverse Plus ELISA; DiaSorin, Saluggia, Italy) (Grangeot-Keros and Enders, 1997; Matter et al., 1994; Revello et al., 1987), the lysate-based and glycoprotein-based IgM ELISA (EUROIMMUN) revealed sensitivities of 90% and 100%, respectively, in the cohorts of acutely rubella-infected or recently vaccinated women. However, it is relevant to note that these sensitivity values represent only 10 serum samples, thus limiting comparison with other studies examining larger numbers of acutely infected and/or recently vaccinated pregnant women. Including sera predefined to contain persistent anti-RV IgM, the lysate-based and the glycoprotein-based ELISA each yielded a sensitivity of 86.7%. Overall specificities amounted to 95.0% and 97.0%, respectively, reaching 97.5–100% when analyzing only past infection samples, RV negative subjects and rheumatoid factor-positive sera. For all sera collected from recently infected or vaccinated women, the detection of low IgG avidity (in 100% of samples) in addition to the absence of anti-E2 IgG (90% negative, 10% borderline) was indicative of primary infection. This also applies to the 3 post-vaccination samples with borderline or negative IgM titers (however, avidity and blot analyses are not performed commonly in IgM negative cases). Among the latter, there was one serum whose partly inconclusive serological results (borderline reactivity in the glycoprotein-based IgM assay, borderline antiE2 titers, RAI 31%) were likely due to the fact that it was taken as late as 16 weeks post vaccination. In fact, the time course of IgG maturation demonstrated in this study for 9 sera obtained at various time points following vaccination, reaching almost borderline avidity and anti-E2 reactivity about 3–4 months after primary exposure, is generally in accordance with data on post-vaccination serology reported elsewhere (Best and Enders, 2007; Enders and Knotek, 1989; Hedman et al., 1989; Hofmann and Liebert, 2005). The observed anti-E2 reactivity is in agreement with the findings of Pustowoit and Liebert (1998), who reported that anti-E2 IgG was

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not detected until at least 12 weeks after infection. Regarding IgG avidity, however, direct comparison of results obtained with different test systems is not possible without reservations, since the increase in the avidity index is strongly dependent on the particular assay method used. In the cohort of sera with persistent IgM reactivity, exclusion of recently acquired rubella was indicated for all women examined based on the finding of high avidity IgG, anti-E2 positivity or both, thus reflecting the significance of avidity and immunoblot analysis for the confirmation of antibody persistence in cases of positive IgM responses. However, in the 8 samples which yielded inconsistent results or minor deviations (n = 2, RAI < 60%, anti-E2 positive; n = 1, RAI > 60%, anti-E2 negative; n = 5, RAI > 60%, anti-E2 borderline), the optimal way to confirm a past infection would be by taking into account clinical information, vaccination history and/or follow-up serological findings (Hofmann and Liebert, 2005). In the panel of sera from patients with other acute infections, the analytical specificity of the lysate-based IgM ELISA amounted to 80.8%. In contrast, using only the glycoprotein fraction as antigenic substrate, specificity increased by 11.5% to reach 92.3%. Importantly, all 5 anti-RV IgM reactive sera in this cohort were shown to contain RV-specific IgG of high avidity in addition to anti-E2 IgG. Thus, IgM responses due to primary rubella infection could be excluded and other causes for IgM detection have to be assumed. For example, positive anti-RV IgM results induced not by recent primary infection but by polyclonal B cell activation and IgM cross-reactivity in other acute infections (e.g. parvovirus B19, EBV, CMV, mycoplasma, measles, toxoplasma) have been reported repeatedly (Arneborn et al., 1983; Dimech et al., 2005; Hudson and Morgan-Capner, 1996; Morgan-Capner et al., 1983; Nobutoki et al., 1996; Revello et al., 1987; Tipples et al., 2004). As a further cause of interference, IgM class rheumatoid factors can react with specifically bound IgG antibodies, thus leading to false-positive IgM results (Hudson and Morgan-Capner, 1996; Meurman and Ziola, 1978). However, none of the rheumatoid factor-positive sera analyzed here proved anti-RV IgM reactive, indicating complete IgG/rheumatoid factor absorption prior to IgM detection. Considering the importance of utmost assay specificity, data obtained in the group of patients with other acute infections substantiate that the use of RV-specific glycoproteins (predominantly E1 as earlyphase antigen) has the potential to overcome specificity problems in IgM detection, and is thus preferable to whole-virus antigen. Moreover, the results in this cohort suggest that the specificity of both ELISA with solid-phase antigen is inferior to that of the ␮capture approach. However, due to the lack of a true gold standard laboratory test, it is speculative if specificity and sensitivity adjustment of the reference capture assay is, in general, diagnostically preferable. With respect to all 130 anti-RV IgG positive samples examined in this study, the correlation between avidity and anti-E2 reactivity amounted to 86.2%, while discordance was observed in only 3.8% of specimens. On the whole, avidity and immunoblot analysis complemented each other and proved efficient in reducing the number of suspected acute infections, as became particularly obvious in the cohorts of sera with IgM persistence or with potential interference. As demonstrated by Hofmann and Liebert, the employment of both methods allows a conclusive interpretation in more pregnant women than possible using either method alone (Hofmann and Liebert, 2005). In that study, serological results remained ambiguous for only 2.7% of samples. Concerning inconsistent results for avidity and anti-E2 analysis, two aspects require consideration. On the one hand, slow maturation of IgG avidity has been observed not only in cases of congenital rubella (Thomas et al., 1993) but also following vaccination (Best and Enders, 2007; Vauloup-Fellous and Grangeot-Keros, 2007), leading to persistence of low or moderate avidity values for several months or longer than one year. This may

explain the above-reported sporadic coincidence of low/borderline avidity and anti-E2 positivity. On the other hand, anti-E2 IgG can be absent, delayed (e.g. following vaccination), or (if the last exposure dates back a long period of time) reduced (Best and Enders, 2007; Cusi et al., 1989; Dorsett et al., 1985; Pustowoit and Liebert, 1998). Therefore, the finding of anti-E2 negativity does not necessarily indicate infection during the previous 3–4 months, and might coincide with borderline or positive IgG avidity. This situation could apply to some of the few discordant findings observed in this study. Importantly, specific IgM positivity may also be observed in cases of reinfection. Although the risk to the fetus is considered low (Cradock-Watson et al., 1981), some earlier studies provide evidence of fetal infection and malformation caused by maternal reinfection during pregnancy (Best et al., 1989; Enders et al., 1984; Gilbert and Kudesia, 1989). Criteria for the classification of reinfection based on anamnestic and serological data have been described (Best et al., 1989; Enders, 2005), but were not assigned to any of the specimens examined in the present study. In routine laboratory testing, IgM positive sera may be subjected to reliable avidity testing (Mubareka et al., 2007), while only problematic sera undergo immunoblot analysis in addition. However, the results of the present study demonstrate that, optimally, both supplemental approaches should be applied in parallel to ensure correct interpretation. In conclusion, this study demonstrates the superiority of glycoprotein over whole-virus lysate as antigenic substrate for RVspecific IgM detection, as expressed by higher assay specificity. The results also provide evidence that the determination of both IgG avidity and anti-E2 immunoblot reactivity are of utmost importance for confirming or refuting recent primary RV infection in pregnant women with positive or ambiguous IgM serology. Acknowledgement Critical review and editing of the manuscript by J. Gosink and S. Simon is gratefully acknowledged. References Aboudy, Y., Barnea, B., Yosef, L., Frank, T., Mendelson, E., 2000. Clinical rubella reinfection during pregnancy in a previously vaccinated woman. J. Infect. 41, 187–189. Al-Nakib, W., Best, J.M., Banatvala, J.E., 1975. Rubella-specific serum and nasopharygeal immunoglobulin responses following naturally acquired and vaccine-induced infection. Prolonged persistence of virus-specific IgM. Lancet 1, 182–185. Arneborn, P., Biberfeld, G., Forsgren, M., von Stedingk, L.V., 1983. Specific and non-specific B cell activation in measles and varicella. Clin. Exp. Immunol. 51, 165–172. Banatvala, J.E., Best, J.M., O’Shea, S., Dudgeon, J.A., 1985. Persistence of rubella antibodies after vaccination: detection after experimental challenge. Rev. Infect. Dis. 1 (7 Suppl), S86–S90. Banatvala, J.E., Brown, D.W., 2004. Rubella. Lancet 363, 1127–1137. 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. Best, J.M., Enders, G., 2007. Laboratory diagnosis of rubella and congenital rubella. In: Banatvala, J., Peckham, C. (Eds.), Rubella Viruses. Perspectives in Medical Virology, 15, first ed. Elsevier Life Sciences, London, pp. 39–77. Best, J.M., O’Shea, S., Tipples, G., Davies, N., Al-Khusaiby, S.M., Krause, A., Hesketh, L.M., Jin, L., Enders, G., 2002. Interpretation of rubella serology in pregnancy — pitfalls and problems. BMJ 325, 147–148. Bottiger, B., Jensen, I.P., 1997. Maturation of rubella IgG avidity over time after acute rubella infection. Clin. Diagn. Virol. 8, 105–111. Cradock-Watson, J.E., Ridehalgh, M.K., Anderson, M.J., Pattison, J.R., 1981. Outcome of asymptomatic infection with rubella virus during pregnancy. J. Hyg. (Lond) 87, 147–154. Cusi, M.G., Metelli, R., Valensin, P.E., 1989. Immune responses to wild and vaccine rubella viruses after rubella vaccination. Arch. Virol. 106, 63–72. Dimech, W., Panagiotopoulos, L., Marler, J., Laven, N., Leeson, S., Dax, E.M., 2005. Evaluation of three immunoassays used for detection of anti-rubella virus immunoglobulin M antibodies. Clin. Diagn. Lab. Immunol. 12, 1104–1108. Dorsett, P.H., Miller, D.C., Green, K.Y., Byrd, F.I., 1985. Structure and function of the rubella virus proteins. Rev. Infect. Dis. 7 (Suppl 1), S150–S156.

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