Rubella in early pregnancy bears a high risk for congenital defects (e.g., cataracts, hearing loss, and heart disease) and for long-term sequelae in the newborn. Despite implementation of vaccination programs in many regions, the threat of devastating consequences from congenital rubella virus infection remains and careful screening of maternal immune status before and during pregnancy helps to reduce the risk. This study compared the performance of the Elecsys Rubella IgG assay with that of other assays routinely used for screening. Samples from 1,090 women undergoing routine antenatal care were tested using the Elecsys and Enzygnost Rubella IgG assays and the hemagglutination inhibition test. Samples with hemagglutination inhibition titers of <32 (n = 148) were additionally tested using the Vidas, AxSYM, Liaison, and Architect Rubella IgG assays. Agreement of qualitative results from the Elecsys, Enzygnost, and hemagglutination inhibition assays was good in all samples. All assays showed 100.0% specificity. In samples with hemagglutination inhibition titers of <32, the Elecsys, AxSYM, and Enzygnost assays showed higher sensitivity (>90.0%) than the other immunoassays (78.6 to 82.4%). The Elecsys assay reported significantly higher rubella virus IgG levels than the other immunoassays across the whole set of 1,090 samples, with the largest bias and deviation from limits of agreement in Bland-Altman analysis. In conclusion, the Elecsys assay is highly sensitive and specific with regard to qualitative results and suitable for routine automated screening. However, given the considerable variation between quantitative results from different immunoassays, testing methods should be documented and the same assay used throughout an individual's antenatal follow-up wherever possible.
The incidence of congenital rubella syndrome as a result of rubella virus infection during early pregnancy has been reduced considerably in many regions due to the implementation of effective vaccination programs. Nevertheless, the possibility of devastating consequences of rubella virus infection remains, due to the presence of unprotected individuals in the population, such as those who have an ethical or religious objection to vaccination, or those who have migrated from regions without adequate vaccination coverage (1). The underlying risk to a pregnant woman and her unborn fetus posed by rubella virus infection can be reduced by careful screening of immune status before and during pregnancy (2–4). Determination of rubella immune status in early pregnancy either by serological screening (2, 5–9) or by control of vaccination status (10–13) is recommended in many countries. In the latter case, rubella antibody testing is required only in the absence of written evidence that an individual has received one (10, 13) or two (11, 12) doses of a rubella virus-containing vaccine.
Different tests are available that can establish whether a woman has had an immune response to rubella in the past through natural infection or vaccination. According to the German Maternity Directives of the Joint Federal Committee (G-BA) (14), it was obligatory until August 2011 to perform a hemagglutination inhibition (HI) test as part of the antenatal care for pregnant women with unknown rubella antibody status prior to pregnancy. If a low positive HI test result was obtained, a second test with an immunoglobulin G (IgG) antibody assay was required to confirm the HI test result. Revised German Maternity Directives issued in August 2011 (12) require the determination of rubella antibody status only in pregnant women who have not tested positive for rubella antibodies prior to pregnancy and do not have two rubella vaccinations documented on their vaccination card. In addition, testing is no longer restricted to the HI test. The rubella test result must be documented either as an HI titer or in international units (IU)/ml.
Over the last few decades, immunoassay techniques have been developed that allow quantification of rubella virus-specific IgG in a standardized and automated manner. These assays generally employ purified viral lysates, recombinant antigens, or recombinant virus-like particles, and results are traceable to a World Health Organization (WHO) International Reference Standard (expressed in IU/ml) to address variations between laboratories. Another potential source of variation is the cutoff level of IgG that defines whether a result is considered positive or negative. Guidance from the National Committee for Clinical Laboratory Standards in 1985 suggested that rubella virus-specific IgG levels of >15 IU/ml protect against reinfection, but revisions in 1992, 1995, and 1997 all suggested a reduction of the cutoff point to 10 IU/ml (15, 16). However, clinical evidence suggests that levels of rubella virus-specific IgG of <15 IU/ml can be protective against reinfection and, conversely, that reinfection can occur even if levels of rubella virus-specific IgG exceed 15 IU/ml (15). These levels take no account of the potential role of cell-mediated immunity in clearance or prevention of rubella virus infection. Other confirmatory assays sometimes used in cases of unresolved immune status include nonreducing immunoblot (NRIB) assays, avidity assays, and cell culture-based neutralization tests (17, 18). The latter are performed only in specialized reference laboratories because facilities for handling viral and cell cultures are required. Given the different assay formats employed and differences between the antigens used, some discrepancy between the results from different assays might be anticipated. In most countries, immunoassays are now used for routine screening during pregnancy, so it is important to understand how the results obtained from different assays can affect clinical decisions. This is particularly important as antenatal guidelines in many countries, such as Germany, do not require that samples taken during the initial and subsequent antenatal appointment are stored, so it is not always possible to retest samples at a later date.
The aim of this study was to assess the performance of the Elecsys Rubella IgG assay for routine assessment of immune status in comparison with the established HI assay and the Enzygnost Anti-Rubella-Virus IgG assay. Samples with low HI titers (8 and 16) could be expected to give the most discordant results. As these are important cases where reporting of the correct result is critical for appropriate management, samples in this study with HI titers of <32 were tested with additional comparator assays.
MATERIALS AND METHODS
Remnants of consecutive specimens obtained during routine antenatal care that would normally have been discarded were used for the present study. The only selection criteria were that the woman was at ≤14 weeks of gestation when testing was carried out and that the sample was of sufficient volume. All samples were collected between 29 September 2009 and 31 October 2009. Sera were coded and patient data anonymized prior to study commencement.
All commercial immunoassays, including the immunoblot assays, were performed according to manufacturers' instructions, and results were reported with reference to manufacturer-specified cutoffs (Table 1) unless specified otherwise.
Table 1 Cutoff levels for each assay used in the study
Cutoff values represent titers except where otherwise indicated. ODcorr, corrected optical density value; ODuncorr, uncorrected optical density value.
Interpretation of HI test may differ between laboratories.
1st International Standard for Anti-Rubella Immunoglobulin, Human.
2nd International Reference Preparation of Anti-Rubella Serum, Human.
Samples with rubella virus IgG concentrations ranging within ±10% of the cutoff should be retested in order to confirm the initial result.
Hemagglutination inhibition test.
For the HI test, a heparin-MnCl2 procedure was used (19). Dextrose gelatin veronal (DGV) buffer at pH 7.2 served as a diluent for all test components. Briefly, sera were absorbed with heparin-MnCl2 buffer (containing 1 part heparin sodium [5,000 IU/ml], 1 part MnCl2 [0.5 M in distilled water], and 4 parts DGV buffer) and a 30% suspension of chicken erythrocytes. After centrifugation, HI tests were performed in microtiter “U” disposable plastic plates (Greiner Bio-One, Frickenhausen, Germany). Serial 2-fold dilutions of pretreated sera were made in DGV buffer containing 0.2% bovine serum albumin and then mixed with equal volumes of a rubella virus hemagglutinin antigen (HA; Siemens, Marburg, Germany) suspension containing 4 HA units. After incubation for 20 min at 37°C, an equal volume of a 0.25% suspension of 1-day-old chicken erythrocytes (Dr. Merk & Kollegen GmbH, Ochsenhausen, Germany) was added followed by incubation for 2 h at 2 to 8°C. An HI titer of <8 was considered negative.
The microneutralization (MN) assay was carried out as a microfocus reduction neutralization test according to a recently described method for detection of measles antibodies (20). Rubella virus strain M-33 (ATCC VR-315) was used as a challenge virus. Serial 2-fold dilutions of heat-inactivated sera from 1:10 to 1:1,280, each 100 μl, were mixed with an equal volume of virus containing between 10 and 100 focus-forming units (FFU) and incubated for 90 min at room temperature. A 100-μl volume of this mixture was inoculated into wells of a 96-well plate with Vero cells (European Collection of Cell Cultures [ECACC] no. 688020401) from which the medium had been removed shortly beforehand. After incubation for 36 to 48 h, the monolayers of inoculated cells were fixed for 10 min with acetone/methanol (40:60) and stained by the immunoperoxidase method using rubella virus-specific monoclonal antibodies (MAb 1-6, anti-E1; MAb 26-24, anti-E2; MAb 2-36, anti-C; Viral Antigen Inc., Memphis, TN). The stained foci of infected cells were counted using an EliSpot Reader image analyzer (AID Diagnostika GmbH, Straßberg, Germany). The neutralizing titer of a sample was assigned as the highest dilution producing a ≥66% reduction of foci compared with an average number of foci in an untreated virus control. All sera were investigated using two replicates. The assay run was accepted as valid when the results of both replicates were within a 2-fold dilution. The serum titer was expressed as the lower result of two replicates; titers of <8 were considered negative.
All samples were tested with the HI test, Elecsys Rubella IgG assay (Roche Diagnostics GmbH, Mannheim, Germany), and Enzygnost Anti-Rubella-Virus IgG assay (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany). Samples that gave HI titers of >16 and positive results with both immunoassays were designated positive. Samples that gave HI titers of <32 (n = 152) underwent additional testing using the Vidas RUB IgG assay (bioMérieux, Marcy-l'Étoile, France), AxSYM Rubella IgG assay (Abbott Laboratories, Wiesbaden, Germany), Liaison Rubella IgG assay (DiaSorin, Saluggia, Italy), and Architect Rubella IgG assay (Abbott Laboratories, Wiesbaden, Germany). A rubella immunoassay IgG status (positive or negative) was assigned if at least four concordant immunoassay results were recorded.
If the HI test result and concordant immunoassay status agreed, the result was confirmed as positive or negative. Samples with a discordant immunoassay status (n = 19) or discordance between the HI test result and immunoassay status (n = 23) were further resolved using confirmatory tests (IgG NRIB [recomBlot Rubella IgG; Mikrogen GmbH, Neuried, Germany] and anti-rubella virus microneutralization test [in-house]). A result was finally confirmed if concordant results were obtained from the two confirmatory tests; otherwise, samples were classed indeterminate.
Demographic data and qualitative test results of pregnant women were analyzed using descriptive statistical methods (JMP version 10). Four samples that gave indeterminate test results according to the diagnostic algorithm were excluded from the analyses, meaning n = 1,090 for all analyses unless stated otherwise. The distribution of HI titers for all women was described by a zero-inflated log-normal distribution with parameters estimated by maximum likelihood (21). Sensitivity and specificity values for the immunoassays were determined with regard to the assigned rubella virus IgG status with equivocal results interpreted as negative; 95% confidence intervals (CI) were based on the binomial distribution. The extent of agreement between rubella immunoassays in 131 samples with low HI titers (8 and 16) was assessed by the Bland-Altman method (22). Antilogs of the log differences were plotted against the geometric means of all 15 pairs of the six tests to determine whether the slopes depend significantly on the geometric means. The significance level for the number of tests was adjusted according to the Bonferroni-Holm method (23). Antilog values with deviations between 0.5-fold and 2.0-fold from the mean ratio were considered within acceptance limits as differences of more than 1 log2-dilution step are generally considered to be clinically relevant. The limits of agreement were determined as mean ± 1.96 times the standard deviation of the residuals on the log scale (22). A z-test was used to assess whether more than 5% of the observations were outside the acceptance limits. The extent of agreement between immunoassays across the whole sample set was estimated by the concordance correlation coefficient (24) using the classification proposed by McBride (25). Results from the six immunoassays were compared by analysis of variance (ANOVA) and post hoc analysis using Tukey's HSD test.
Demographic data and rubella HI titers of pregnant women.
Median maternal age at the first antenatal visit was 29 years (interquartile range, 26 to 33 years; range, 15 to 44 years). The HI titers showed a zero-inflated lognormal distribution (Fig. 1A), and 78% of samples had HI titers of 32, 64, or 128. Only a very small proportion of samples (1.6%) had HI titers of <8. The proportion of women with HI titers of <32 was highest (33%; 95% CI, 20 to 50%) in the youngest age group (<20 years; Fig. 1B) and decreased linearly to the lowest value in the age group 30 to 35 years (7%; 95% CI, 4 to 10%). For women aged 36 to 40 years and >40 years, the proportions with HI titers of <32 were 11% (95% CI, 6 to 17%) and 21% (95% CI, 10 to 36%), respectively.
Using the diagnostic algorithm described previously, 17 negative (HI of <8) and 1,073 positive results were assigned. Comparison of the diagnostic status of samples with the HI test results indicated that HI titers of 8 or 16 were all true positive results in the present study. Sensitivity and specificity of the HI test were 100.0% (95% CI, 99.7 to 100.0%) and 100.0% (95% CI, 80.5 to 100.0%), respectively, using the final assigned status as the “gold standard.”
The Elecsys and Enzygnost assays recorded similar numbers of positive results (Table 2), and there was excellent agreement between the assays with regard to the overall numbers of positive and negative results reported. Both immunoassays agreed with the HI test in ≥99.0% of samples, with less than 1% discordance between the HI test and Elecsys assay (Table 2). In samples that reported a HI titer of ≥32, there was 100.0% agreement between the Elecsys and Enzygnost results.
Table 2 Agreement of the HI, Elecsys, and Enzygnost assays with respect to assay results in all samples (n = 1,090)
The samples that reported HI test titers of <32 (n = 148) were additionally tested with the Vidas, AxSYM, Liaison, and Architect Rubella IgG assays (Table 3). Within this group, the Elecsys, Enzygnost, and AxSYM assays reported similar numbers of positive results (121, 123, and 119, respectively), with the Vidas, Liaison, and Architect assays reporting fewer positives (108, 108, and 103, respectively). The Architect assay reported 22 equivocal results, twice as many as the Vidas, AxSYM, and Enzygnost assays, which also employ a gray zone.
Table 3 Immunoassay results in samples with low HI titersa
<8 (n = 17)
8 (n = 20)
16 (n = 111)
Data represent results for samples with HI titers of <32 (n = 148).
In the samples that had a HI titer of 16, there were very few negative results from the immunoassays, with the exception of the Liaison assay, which reported 10 of 111 samples as negative (Table 3). However, in the 20 samples with HI titers of 8, the immunoassays reported between 0 and 13 negative results.
Regarding sensitivity and specificity assessed in the low HI titer range, all assays showed 100.0% specificity (95% CI, 80.5 to 100.0%). However, there was more variation between the immunoassays with regard to sensitivity; the Elecsys, AxSYM, and Enzygnost assays showed sensitivity values of >90%, while the remaining assays showed much lower sensitivity (78.6 to 82.4%; Table 4).
Data represent results for samples with HI titers of <32 (n = 148). CI, confidence interval.
The distribution of results with respect to rubella virus IgG level in IU/ml (n = 1,090; see Fig. S1 in the supplemental material) showed that the Elecsys assay reported fewer results ≤100 IU/ml and more results >100 IU/ml than the Enzygnost assay. The largest difference between the two tests was found for values between 35.1 and 100 IU/ml: 17.2% in Elecsys compared with 42.5% in Enzygnost. A similar pattern was observed when the distributions of results from all six immunoassays were compared in the samples with HI titers of <32 (n = 148; Fig. 2). The Elecsys assay reported 45% of values as >60 IU/ml, whereas the other five tests reported <5% of results in this category. The variabilities of the quantitative results reported by each of the tests differed considerably in these samples (see Fig. S2 in the supplemental material). The Elecsys assay results showed the lowest median value in samples with HI titers of <8 but, conversely, the highest median value in samples with HI titers of 8 and 16 compared with the other immunoassays. The extent of agreement between results from the six immunoassays in 131 samples with HI titers of 8 and 16 was estimated using the Bland-Altman method (Fig. 3). For seven pairs (indicated in red), the slopes were significantly different from zero (adjusted for multiple testing) (23) and the number of observations outside the limits of acceptability was significantly higher than 5% of the total (P < 0.0004). For three pairs (indicated in black), the slopes were significantly different from zero but the number of observations outside the limits of acceptability was compatible with the expected 5%. For five pairs (indicated in green), the slopes were not significantly different from zero, with the corresponding fitted ratios constant for all values on the horizontal axis and the number of observations outside the limits of acceptability compatible with the expected 5% of the total. The smallest deviation from the limits of agreement was observed for the AxSYM assay (factor of 1.1), while the largest deviation was observed for the Elecsys assay (factor of 2.1). Comparisons that included the Elecsys Rubella IgG assay showed the greatest bias and deviation from the limits of agreement. The ratio of the two measurements was significantly associated with increasing IgG antibody concentrations in 10 of 15 pairs of tests. The best agreement between immunoassays was observed for the following test pairs: Vidas and AxSYM; Vidas and Enzygnost; and Enzygnost and Liaison (see Fig. S3 in the supplemental material). The concordance correlation coefficients were classified into four categories ranging from “almost perfect” to “poor,” with the lowest concordance correlation coefficients observed for pairs involving Elecsys and highest for pairs involving Vidas. However, it is interesting that even for assay pairs with good agreement, individual samples within the data set still showed unacceptable ratios with regard to quantitative test results.
When the means of the results of pairs of the six tests for the 131 samples with HI titers of 8 or 16 were compared, there were significant differences for 13 of 15 comparisons (P < 0.05 for all 13 comparisons, P < 0.0001 for 10 comparisons). The only exceptions are AxSYM and Enzygnost (P = 0.6198) and Enzygnost and Liaison (P = 0.7877; see Fig. S4 in the supplemental material).
Testing of a single blood specimen is generally sufficient to assess rubella immune status. Qualitative and quantitative tests are acceptable for this purpose (16). Diagnosis of acute rubella virus infection or reinfection can be made by detection of rubella virus-specific IgM and the detection of a clinically relevant rise in the rubella virus-specific IgG concentration (4, 17). Hence, quantitative IgG tests are essential for a correct diagnosis and the adequate management of pregnant women who may have been exposed to rubella virus.
In the current study, we have compared the performance of the Elecsys Rubella IgG assay, a fully automated random-access assay, with other immunoassays and a HI test. The diagnostic algorithm employed in this study successfully resolved all but four samples. With regard to qualitative results, i.e., positive (previous exposure to rubella) or negative (no previous exposure), there was a very high level of agreement between the HI test and Enzygnost and Elecsys assays over the whole set of 1,090 unselected samples from routine testing. However, it is particularly interesting to consider the performance of an immunoassay at low antibody levels. Approximately 1.6% of the samples from the study population had HI titers of <8 and 12% had HI titers between 8 and 16. None of the commercial immunoassays (Elecsys, Enzygnost, Vidas, AxSYM, Liaison, and Architect) reported false-positive results using the manufacturers' cutoffs or a cutoff of 10 IU/ml. Assay sensitivity ranged from 78.6 to 92.4% in the low antibody concentration range.
The cutoff levels selected by manufacturers to give the best possible discrimination between positive and negative results differ. The majority of assays also employ an equivocal or gray zone. In clinical practice, samples that are classified within this gray zone must be retested. The Elecsys assay does not use a gray zone, which has the potential to reduce the number of confirmatory tests or repeat serum samplings required in routine practice. False-negative rubella test results are a lesser issue, as vaccination of an already immune individual causes no adverse effects, and pregnant women found to be rubella antibody negative during initial antenatal testing are followed up later in pregnancy. Therefore, pregnant women with equivocal test results are generally regarded as seronegative. On the other hand, elimination of false-positive results is very important, as they can lead to the conclusion that a woman is already immune to rubella virus infection when, in reality, she is not. False-positive IgG results have been reported for the HI test and other rubella immunoassays (26–29). In general, HI assays are considered sensitive and specific, provided that natural agglutinins and nonspecific serum inhibitors are efficiently removed. In the current study, our HI test was at least as sensitive and specific as the immunoassays, including the very sensitive Elecsys assay. All sera with HI titers of 8 or 16 that tested negative for rubella virus-specific IgG with one of the other immunoassays (Table 3) were confirmed positive by immunoblot and neutralization testing. It should, however, be considered that the HI test requires rigorous quality control for all reagents, is technically more demanding, and should be performed only by experienced technicians.
In many cases, the rubella immune status of a pregnant woman can be identified from a qualitative test; however, there are instances where a quantitative rubella IgG test result is needed (e.g., diagnosis of rubella virus reinfection). One criterion for rubella virus reinfection in persons with preexisting immunity (i.e., documented vaccination or rubella antibodies) is a clinically relevant rise in the antibody concentration, a rubella virus-specific IgM response, or both (30). Often, serum samples obtained before reinfection are not available for retesting so previously documented laboratory results must be used. However, although most current rubella immunoassays are standardized against a WHO International Reference Standard and test results are reported in IU/ml, previous observations suggest that there can still be differences between results reported by different assays. In this study, we noted that the Elecsys assay reported considerably higher levels of rubella virus IgG in IU/ml than those reported by the other immunoassays (see Fig. S2 in the supplemental material). Bland-Altman analysis showed that assay comparisons that included the Elecsys assay most frequently showed the greatest bias, a clear association between the antibody concentration and the magnitude of the ratio between two measurements, and a large deviation from the limits of agreement. Even though the extent of agreement was better, but not always acceptable, for the remaining assay combinations, there were still some individual samples that gave unacceptable ratios with regard to quantitative results. So unless the testing method is recorded, laboratory results, whether documented as HI titer or as IU/ml, have to be regarded with caution when diagnosing possible rubella virus reinfection.
The different components and technologies that are employed in the immunoassays used in this study may explain the differences observed between the Elecsys assay and other immunoassays for quantifying rubella virus IgG antibodies. All but the Elecsys assay include purified rubella virus antigen; the Elecsys assay contains recombinant rubella virus-like particles (RLP) and recombinant E1 antigen. As a large portion of the rubella virus-specific IgG response is directed toward the E1 antigen, this may be a factor in the increased antibody values observed with the Elecsys assay. There are also differences in standardization; the Elecsys, Vidas, Liaison, and Architect assays are all traceable to the 1st International Standard Anti Rubella Immunoglobulin, Human (31), whereas the older AxSYM and Enzygnost assays are standardized to the 2nd International Reference Preparation Anti-Rubella Serum. It should also be noted that the Elecsys assay reports very low results in samples with HI titers of <8, indicating that this assay discriminates very well between negative and positive results.
According to our observations, greater than 97% of pregnant women in the present cohort had detectable HI antibody to rubella virus, suggesting that immunity levels in pregnant women are currently high. Nevertheless, we observed a relevant proportion of women with low HI titers (<32) in the younger age groups which is likely to be because rubella immunity is vaccine induced in most of these women and natural boosters are less likely to occur due to improving vaccination coverage of children in recent years in Germany (32, 33). It remains to be determined whether these women are at increased risk of rubella virus viremia during reinfection.
In conclusion, this study showed the Elecsys assay to be highly sensitive and specific. As a gray zone is not employed in this assay, there is no need for time-consuming and costly retesting of equivocal samples. Rubella antibody concentrations reported by the Elecsys assay were considerably higher than those from other assays, and our findings indicate a lack of agreement between the Elecsys assay and other rubella virus immunoassays with respect to quantitative test results. This observation has also been made for other test pairs in our study, despite the fact that all assays are calibrated against an international standard. This disparity between the quantitative results of the different immunoassays suggests that the same assay should be used throughout a pregnant woman's care, particularly if samples are not stored during pregnancy for later retesting. Furthermore, if quantitative test results are recorded, the type of assay and manufacturer should also be documented.
We thank Walter Melchior for the study setup, technical discussion, and review of the manuscript.
Medical writing assistance was provided by Elements Communications Ltd. (Westerham, United Kingdom) with financial support from Roche Diagnostics, Rotkreuz, Switzerland.
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