Public health agencies have been persistently frustrated by the challenges of using standard laboratory diagnostic methods to reliably confirm mumps (re)infection of individuals who have been previously vaccinated or naturally infected. This is perhaps best illustrated by two reports which describe the use of ELISA to detect mumps-specific IgM and real-time RT-PCR to detect the viral RNA from suspected mumps cases during outbreaks at the University of Kansas (5
) and the University of Virginia (38
) during 2006. All of the patients with suspect cases from the University of Virginia had previously received two doses of MMR vaccine, and 97% of the patients with cases from the University of Kansas had two doses; the remainder received one dose. In both studies, the rate of IgM detection among suspect cases of infection was 12 to 13%. Similarly, the rate of detection by RT-PCR was 31 to 35%. Furthermore, the University of Kansas study (5
) suggested that virus shedding is minimal after the first 3 days of symptom onset. In many cases, it is not possible or practical to obtain specimens for RT-PCR in this brief time frame. Even if samples are collected during the first 3 days of onset, RT-PCR requires careful shipping, storage, and processing of the specimen to prevent RNA degradation. Although detection of a 4-fold rise in IgG titer between acute- and convalescent-phase serum specimens has also been used to diagnose recent infection, we have found this method to be similarly unreliable as acute-phase specimens from vaccinated individuals typically have intermediate to high levels of IgG (our unpublished observations). Resolution of these diagnostic challenges is of the utmost importance because it confounds basic prevention measures (such as the quarantine of cases) and limits the accuracy of the epidemiologic assessment of outbreaks. A high number of false-negative results will lead to an underestimation of disease incidence. Moreover, asymptomatic spread among case contacts may expand and prolong an outbreak.
Based on several published observations that antibody-secreting B cells (ASCs/plasmablasts) are detectable in circulation only following recent activation by antigen (4
), we hypothesized that mumps-specific ELISPOT detection of these cells could be used as a more reliable alternative to standard diagnostic methods. In the absence of a sustained mumps outbreak, we first examined the numbers and kinetics of ASCs produced by volunteers who were recently vaccinated with MMR-II. This dose of vaccine was the third dose of MMR received by 10 of the 16 volunteers. We found that mumps-specific ASCs could be detected in 12 of 16 (68%) individuals from 1 to 3 weeks postvaccination. Volunteer O, who was mumps IgG negative at the prevaccination time point, had 4-fold more mumps ASCs present at 3 weeks postvaccination than the next-highest response from any other person (volunteer B) at any time point and was the only person to have a 4-fold rise in IgG titer between the prevaccination and day-21-postvaccination samples (data not shown). Other than this one example, the number of mumps-specific ASCs detectable following vaccination did not appear to correlate with IgG titer, age, or any other known parameter. The timing and duration of the rubella- and measles-specific ASC responses were similar to those for mumps. However, the average number of rubella-specific ASCs was approximately 5-fold higher per volunteer than that observed for both measles and mumps. Statistical analysis indicated that the detection of virus-specific ASCs has predictive value for confirming recent exposure to mumps, measles, and rubella.
It is unclear why certain individuals did not have a detectable ASC response to vaccine, but this could be explained in a number of ways. First, it is possible that the numbers of their ASCs peaked and declined between specimen collection time points. Alternatively, the attenuated viruses in the vaccine may have been neutralized before a measurable ASC response could be elicited or measured. However, it was generally noted that individuals who responded well to one component of the vaccine also responded relatively well to the other two components. Although two separate cohorts of individuals were used to quantify memory B-cell numbers and the ASC response to a third dose of MMR, it is interesting that the hierarchy of memory B-cell levels mirrored that of the ASC response to vaccine. We observed more rubella-specific memory B cells and ASCs than those specific for measles, and there were more measles-specific cells observed than for mumps. Thus, the level of ASC response following infection may likely be directly related to the number of memory B cells that are available to respond in a given individual. While it may be expected that the B-cell response will vary significantly among viruses/antigens, these data could suggest that mumps elicits poor B-cell memory. If this is true, it may be a basis for susceptibility to reinfection, especially if the majority of the memory response is directed to nonneutralizing virus epitopes.
Given the frequency with which we were able to detect mumps-specific ASC responses among individuals who had been recently vaccinated and the relatively broad window of time during which these cells were present, we suspected that mumps-specific ASC detection following wild-type virus infection of previously vaccinated individuals would be possible. Furthermore, we anticipated that this method might be particularly useful when these cases cannot be confirmed by conventional testing (4
). This was demonstrated by testing samples that were obtained from seven two-dose vaccinees who were recently infected. Mumps ASCs were detected in all seven cases, but only three of them were positive by standard testing. It is possible that the difference between rate of ASC detection among the naturally infected individuals (100%) and the rate of ASC detection among the individuals who were challenged with the vaccine (68%) could be attributed to the force and/or route of infection. These rates of ASC detection by ELISPOT assay compared very favorably with the RT-PCR and IgM serology results from both studies described here and published results for IgM (12 to 13%) and RT-PCR (31 to 35%), as noted above (5
In contrast to virus shedding, which rapidly diminishes (within 3 days) after symptom onset (5
), mumps-specific ASCs were present and detectable out to 28 days post-symptom onset in one individual and ASCs were easily detected in individuals who were 7 to 12 days post-symptom onset. It is worth noting, however, that the timing of the peak of the ASC response to mumps infection has not yet been well established and may vary considerably among individuals. In one study (47
), the ASC response to influenza vaccine peaked at day 7 postvaccination and was barely detectable by day 14. While it is important to more accurately determine the optimal timing for sample collection, these data suggest that 5 to 10 days after onset may provide the best opportunity for detection. Although these results are encouraging and provide proof of concept, additional testing is needed to more carefully determine the best cutoff value and to further validate this method before it is implemented as a diagnostic tool.
Unlike testing for 4-fold rises in IgG between paired acute- and convalescent-phase serum specimens, which requires a significant time delay, ASC detection by ELISPOT assay requires only one sample and is extraordinarily sensitive. The lower limit of detection will vary among donors in accordance with the total number of cells assayed and the number of cells that respond to the negative control antigen. However, we have routinely observed limits of detection as low as 1 cell per 107 PBMCs.
Overall, we have found the ELISPOT method for detecting virus-specific ASCs to be more sensitive throughout a longer period of time than RT-PCR or IgM ELISA for detecting recent mumps infection in individuals who have been either previously vaccinated or naturally infected. While this is not intended as a replacement for IgM and RT-PCR testing, it may be useful for diagnosing cases that cannot be otherwise confirmed by standard methods, for initially confirming cases in an outbreak, and for testing asymptomatic case contacts. As previously reviewed (4
), this assay can be easily adapted for use with other infectious agents. Similar diagnostic challenges exist for varicella-zoster (29
) and pertussis (45
), and the assay can be completed in as few as 16 to 24 h.
Although this method is promising, some potential limitations exist. First, ASCs do not survive for an extended length of time after blood collection. This may require processing and testing of the blood in a timely manner after phlebotomy to maintain ASC viability. Second, the method may not be well suited for use on infants or small children because they may be unable to initiate a mature B-cell response and because the minimum volume of blood required (approximately 5 ml) may not be acceptable. However, the very young are less likely to be vaccinated, in which case standard diagnostics would be the better choice. Finally, there exists a possibility of cross-reactivity between B cells specific for mumps and other viruses such as parainfluenza viruses 2 and 4. This might pose a challenge particularly for the diagnosis of sporadic suspect cases, but could potentially be addressed by the use of recombinant antigens.