Rubella and measles infections are usually diagnosed in the laboratory by RT-PCR detection of the virus genomes during the first four days of the disease. However, samples taken at the appropriate time for making a direct diagnosis are not always available, so the detection of IgM-class antibodies is used as a fundamental tool for correct case characterization.1 Using a combination of the two approaches greatly improves the performance of measles2,3 and rubella2 diagnosis. In a situation of elimination, such as the current one, serological tests with good sensitivity and specificity characteristics are required. Siemens Healthcare Diagnostics (Marburg, Germany) (ELISA-Siemens) currently supplies the reference methods (ELISA assays, Enzygnost®) for determining IgM against rubella (RuV) and measles (MeV) viruses. The Enzygnost® kits have been used by a large number of network laboratories for many years.4,5 However, they will soon be withdrawn from the market, so the identification of alternative methods with acceptable performance characteristics is a challenge for clinical laboratories.

The aim of this study is to assess the operating characteristics of ELISA assays, based on indirect or μ chain capture methodologies, and chemiluminescence (CLIA) (capture), all for the purpose of determining the levels of IgM against RuV and MeV, and of electrochemiluminescence (ECLIA) (capture), for determining IgM versus RuV. Seven assays were compared for RuV IgM and six for MeV IgM.

In 2018, measles and rubella were eliminated in 35 (66%) and 39 (73%) of the Member States of the WHO European Region.6 This was made possible by adequate vaccine coverage and surveillance systems, and the availability of sensitive and specific diagnostic methods, using both direct detection by PCR and serological determination of specific IgMs. In recent years, the most commonly used methods for determining specific IgMs against RuV and MeV have been those of Enzygnost® Siemens. These methods have been used as reference.5 However, the company has ceased the production of these reagents, so the need to identify suitable alternative, sensitive and specific methods has become an important matter. Here, we compared seven methods for determining IgM against RuV and six methods for determining MeV-IgM, including Enzygnost® assays. Despite the consideration of the Siemens assays as reference5 we have used a consensus criterion for classification of the samples, due to the assay from Siemens may show negative results in very early samples at least for measles, as previously reported.7 Thus, we think that by using a consensus criterion, the case classification is strongly improved.

Given the lack of a gold standard, the criterion of the majority of results obtained in the compared assays was taken as the reference for classifying our results. For rubella, the best agreement, sensitivity and specificity figures were obtained by the RuV-CLIA and RuV-ECLIA methods, both of which are based on μ chain capture methods. Three of the ELISAs (RuV-Euroimmun-1, RuV-Euroimmun-2 and RuV-Diesse) yielded better values than those obtained by RuV-Siemens, the best of these being RuV-Euroimmun-2, which uses GP as the antigen, thereby making it a suitable alternative for detecting RuV IgM.

Several comparative studies have been published in recent years, some of which have involved the methods examined here. The RuV-ECLIA assay (Elecsys, Roche) shows good agreement (98.0%) with another CLIA (Architect, Abbott).8 RuV-EuroImmun-1 shows good comparability with RuV-Siemens in the diagnosis of suspected cases of congenital rubella syndrome, with an agreement of 94.7%.9 The RuV-ECLIA showed a sensitivity of 79.8–96.0% in a multicenter study, which is a lower range than that noted in the present study, with high specificity (98.7–99.0%).10

The best specificity values obtained in this study were those of RuV-CLIA and RuV-ECLIA. Moreover, RuV-EuroImmun-2 has been shown to be the most specific of the ELISA-based methods when compared with an ELISA method using whole-virus antigen.11 The majority of the discrepancies occurred in the RuV-Virion assay when measles samples were tested (35/72, 48%), resulting in a very low specificity for this method.

In relation to measles assays, the best values of sensitivity, specificity, and agreement were obtained with MeV-CLIA (98.5%, 100% and 99.4%, respectively). The only assay that produced low values for agreement and specificity (73.0% and 53.3%) was MeV-Diesse; the remaining assays yielded acceptable (MeV-Virion and MeV-Euroimmun-2) or excellent (MeV-Siemens and MeV-Euroimmun-2) overall values. The assays with more discrepant results were MeV-Diesse (33 indeterminate and 9 positive samples) and MeV-Virion (8 indeterminate and 4 positive samples) (Table 4), resulting in a very low value of specificity in both assays.

Recently published information is available from measles IgM assays. In general, capture ELISA methods are more sensitive than indirect ones.12

The MeV-CLIA method has been compared with the MeV-Siemens method in some reports, in which it showed sensitivity and specificity of 94.0% and 100%,13 and 93.8% and 98.8%,14 respectively. Compared with capture ELISA (Microimmune), MeV-CLIA showed 92.0% sensitivity and 100% specificity, with a small proportion of indeterminate results.15 In an additional report, MeV-CLIA showed improved corresponding values of a different CLIA (Vircell), although the difference was not statistically significant.16

Most assays for MeV IgM, except MeV-Diesse and, to a lesser extent, MeV-Virion, gave excellent specificity values. Therefore, it does not appear that the specificity of the assays is related to the antigen used (NP in the cases of MeV-Euroimmun-2 and MeV-CLIA, and extract from infected cells in the cases of MeV-Siemens and MeV-Euroimmun-1). In the current situation of elimination of measles and rubella, when the number of cases is declining, the positive predictive value of IgM determination decreases, so confirmatory methods are necessary. Detection of seroconversion or a significant increase in antibody titer in two paired samples indicates an acute infection.1 On the other hand, IgG avidity assays allow primary and secondary infections to be differentiated, this being applicable to measles cases in which the characterization of vaccine failure is possible,17 and to rubella,9 mainly to rule out primary infection in pregnancy, of devastating teratogenic effect. Commercial methods for characterizing IgG avidity against both viruses are available, including ELISA and CLIA.8,11,17,18 However, in the current circumstances, comparative elimination studies are necessary to ensure the quality of the serological methods used in clinical laboratories.5,19

Although the simultaneous reactivity against measles and rubella seems not be frequent,20 it can occur in subjects recently immunized by using measles-mumps-rubella (MMR) vaccine. In the present study no data about antecedents of recent vaccination was recorded. This situation could explain the finding of positive IgM to both viruses. According to our experience, in the context of measles and rubella outbreaks, MMR vaccination is implemented, as a Public Health measure. That is the reason why, in such situations, a number of vaccine related cases could be expected. To further explore this point, we have tested for mumps IgM the three samples classified as double infections, trying to confirm a recent MMR vaccination. One of them showed a positive result in the mumps IgM assay, confirming a recent vaccination, the remaining two being negative (results not shown).

The study has some weaknesses. First, samples were taken in 1991 and 1996, in an epidemic context. Although the medical doctors who sent the samples clinically characterized the patients, no additional information was available about the time of bleeding after the appearance of the exanthema. This information is important for the correct interpretation of discrepant results, which are mainly false negatives. Second, we tried to establish the performance characteristics of assays for RuV- and MeV-IgM, using samples from both diseases. However, samples of other infections that may cause differential diagnostic problems, mainly human parvovirus B19 (HPVB19), as well as cytomegalovirus and Epstein Barr virus, should also been included. This aspect is of great interest in the context of the elimination of measles and rubella, as is consistently evident for both RuV and MeV.1,21 The same occurs in travelers with cases acquired in countries with endemic circulation of arboviruses (Zika virus [ZikV], dengue and chikungunya). A certain degree of false-positive heterologous reactivity has recently been described, whereby the LIAISON assay for ZikV in cases of measles and HPVB19 gave positive results that were not confirmed in immunofluorescence assays.22 Finally, positive heterologous results due to polyclonal stimulation of B lymphocytes in both rubella and measles cases could not be excluded, which could compromise the specificity of measles and rubella assays. For these reasons, further studies are required to determine the importance of this phenomenon.

In conclusion, the information reported here will help clinical laboratories to improve the serological diagnosis of measles and rubella infections.

The authors declare that they have no conflict of interest.