Regions involved in maintaining measles elimination refer specimens from many cases of rash illness and fever for laboratory confirmation of measles, typically by IgM enzyme immunoassay and less frequently by other serological or molecular-based tests (4
). However, due to inherent limitations of the methods used in measles diagnostics, not all cases can be resolved (1
). For confirmed cases in vaccinees, vaccine failure classification would normally ensue by determination of IgG avidity. In these regions, the primary use of avidity testing is in the classification of vaccine failures. However, a well-validated avidity assay can also support confirmation of clinically ambiguous cases.
As an approach to solving some of the current problems with measles diagnostics, a rapid (<2 h) and accurate measles-specific avidity assay was developed by adding a DEA wash step to the protocol of a commercially available platform. The utility of this measles avidity assay was illustrated by the analysis of samples collected during a measles outbreak in an elimination setting. This outbreak was originally confirmed by the detection of measles-specific IgM, the preferred confirmatory test. However, with the current paucity of commercial IgM assays and the limitations of current confirmatory assays, there is a need to find alternatives to help confirm the diagnosis of measles and help detect and control measles outbreaks. In our study, the majority of low-avidity results were obtained in immunologically naive IgM-positive individuals during the first 3 weeks after exposure to measles virus. Thus, in the event that IgM assays are not commercially available, this IgG-based avidity method could help confirm measles in those suspected cases with sufficient measles-specific IgG for testing. Furthermore, because specific antibodies are of low avidity for several weeks after classic rash onset, low-avidity control samples should be easier to obtain after recovery from classic measles and are expected to be more readily available than controls for IgM assays.
The central role of avidity testing is vaccine failure classification, which is important for both control and surveillance purposes (3
). In current elimination settings, where surveillance is case based, vaccine failure classification can be helpful in characterizing the frequency and the symptoms of modified measles and in investigating the role of SVF cases in measles transmission. Additionally, cases with unknown vaccination status and high-avidity results can be classified as suspect SVF. High measles IgG avidity and very high neutralization titers appear to correlate with SVF cases upon recent exposure to measles. Together, these two parameters have recently been proposed as biomarkers for the confirmation of SVF cases, and with appropriate validation, they could be used to confirm suspected SVF cases (19
). The everyday application of this diagnostic approach was further investigated with the analysis of suspected cases referred to our laboratory from 2009 to 2011 (data not shown). Among 15 confirmed cases, avidity testing classified 10 as SVF and 5 as suspected SVF. Of the 10 SVF cases, 3 had PRN titers of ≥81,916 mIU/ml, or 54 to 163 times the mean (1,525 mIU/ml) observed in unexposed vaccinated individuals (the mean was calculated by averaging the PRN geometric mean titer values of four previously described studies in unexposed vaccinated [1 or 2 doses] individuals) (19
). One of these three SVF had not been confirmed by IgM or reverse transcription (RT)-PCR assays, only by an epidemiological link, and it is an example of the potential application of elevated high-avidity IgG titers as a biomarker for SVF. Interestingly, none of these 3 cases transmitted measles to others (34
). Furthermore, although widespread 2-dose coverage has made PVF a rarity, identifying PVF cases in young children vaccinated once can expedite control measures.
In the measles laboratory diagnostic toolbox, the measles avidity assay is a specialized tool that can be used to help confirm suspected measles cases when information from routine assays is inconclusive (). Avidity results can be applied as follows to rule in cases. First, low-avidity results provide support to confirm measles cases, similar to the identification of recent primary infections with rubella virus, cytomegalovirus, human herpesvirus 6 and 7, and HIV (5
). In measles, low-avidity results are especially useful to rule in sporadic cases appearing outside an outbreak and when a false IgM-positive result is suspected. For example, two cases were described with IgM-positive and low-avidity IgG results. They involved a person born before the vaccine era who would generally be assumed immune and a person born outside the United States who was unvaccinated. Moreover, low-avidity results are helpful when a serum specimen is collected late and a false IgM-negative result is suspected. The high sensitivity of this measles avidity assay in the determination of primary infections provides reassurance of IgM-positive results obtained from sporadic cases, especially those suspected cases of children presenting with febrile rash illness, unknown exposure to confirmed cases, unknown vaccination status, and absence of international travel. Second, high-avidity results support the notion that elevated PRN titers in modified measles cases derive from the activation of memory responses to measles virus. In the future, high-avidity results, together with elevated measles antibody titers, could assist in confirming SVF (19
). In investigating SVF cases, intermediate-avidity results could be used to support the confirmation of some cases (see the supplemental material). In contrast to congenital rubella, cytomegalovirus infection, and toxoplasmosis diagnostics, high-avidity results cannot be applied to rule out cases (5
). In our laboratory, high-avidity results have been observed in an SVF case with classic measles disease and in an unvaccinated confirmed case with modified measles (data not shown) (19
Interpretation and application of measles avidity results obtained in the absence of vaccination within 10 days of rash onset and from samples collected within the first 8 weeks
To correctly interpret the results of measles avidity assays, it is critical to have good records of vaccination status, with the number of doses and dates of administration, date of birth, time of rash onset and sample collection, and symptoms. This information is especially useful when investigating cases with modified-measles presentations. Additionally, results obtained from young children can be misinterpreted due to the presence of maternal IgG, which is generally of high avidity. For example, avidity results for a nonimmune infant infected with measles virus can initially be of high avidity. As the infant's immune response to measles virus progresses, maternal high-avidity IgG antibodies will be replaced with low-avidity antibodies from the infant, which will likely be detected in a second sample collected later. In this study, this was observed in a 9-month-old measles case with a high-avidity result in a sample collected 5 days after rash onset and a low-avidity result in a sample collected 6 days later. It was also observed in two infants recently vaccinated with their first measles-mumps-rubella vaccine (MMR) dose ().
The described measles avidity assay has limitations. First, a minimum level of measles virus-specific IgG is required; samples must be IgG positive by the Capita assay. Second, the results of the assay cannot be used in isolation; the interpretation of the results relies on accurate medical history and epidemiological information and should be considered together with other laboratory results. Third, during our validation, low-avidity results were observed in sera collected up to 9 weeks after vaccination with a first dose of MMR but were not observed later (data not shown). Therefore, low-avidity results are difficult to interpret if vaccination has occurred recently, and the results cannot be used to distinguish vaccine from wild-type infections. Fourth, intermediate-avidity results are complex to interpret, and more data are needed to understand their diagnostic relevance and ultimate value. Finally, the assay cannot rule out false IgM-positive results; the presence of high-avidity measles virus IgG does not rule out measles as a diagnosis.
The evaluation of the assay was limited in that samples were selected according to the clinical course of measles alone without considering underlying conditions, as in HIV infection, that appear to have slower measles-specific IgG avidity maturation (28
). A low-avidity result in samples collected in a timely fashion from HIV-infected unvaccinated individuals would still confirm measles (6
). Because an evaluation was not performed on known HIV-infected measles cases, this avidity assay should not be used in the classification of vaccine failures in HIV-infected, measles-vaccinated cases.
In conclusion, this paper introduces a new measles avidity assay that is ready to be used in elimination settings. It is highly accurate, precise, and reproducible, as well as sensitive and specific. The assay is able to detect measles-specific antibody maturation over time, in line with previous observations (14
). Besides vaccine failure classification, avidity testing can provide very valuable information in confirming those cases with RT-PCR-negative results or with questionable IgM results. It must be emphasized that the assay can only be used to help rule in measles cases, but not to rule them out. With appropriate assay evaluation, it may be possible to adapt other commercially available measles IgG platforms to avidity testing by using the DEA elution method presented here. This avidity method worked well with another commercial plate coated with whole measles virus antigen (data not shown). Finally, the endpoint titration method was used because it is independent of the IgG concentration and it is considered superior to the single-dilution method. However, in situations where time or resources are limited, the avidity assay could be performed using a single dilution (see the supplemental material).