Conventional methods for diagnosis of HEV71 and CVA16 infection (virus isolation, neutralization or RT-PCR) are slow, complex and/or costly, do not lend themselves to large number of specimens and are, therefore, unsuited to the clinics of developing countries. IgM-capture ELISA, with its notable advantages of convenience and low cost, provides a potentially frontline assay for diagnosis of HFMD.
We mapped, for the first time, the kinetics of IgM in HEV71 and CVA16 infection. In 138/153 sera of HEV71 and 66/97 sera of CVA16, IgM was detected during the acute phase (within 7 days after symptom onset), consistent with Wang' s study  for HEV71. The positive rate reached 100% at day five and eight, somewhat later than that of nucleic acid detection of HEV71 in throat and fecal samples from HFMD patients. However, the IgM is maintained for several months while the detection rate of nucleic acid fell markedly during 9-12 days after onset of disease . For example, IgM was detected by Wang on day 94, while we found two cases, one each of HEV71 and CVA16 infection, where the corresponding IgM was detectable on day 74 and 87 respectively. However, 3-4 months after onset, both IgMs had largely declined to undetectable levels. Nevertheless, it should be noted that these results were obtained from multiple individuals and need to be confirmed using consecutive specimens from individual patients.
Recent results may be compared with those reported previously. The sensitivity for HEV71 (93.6%) is consistent with that reported (94.1%) , while that for CVA16 IgM (72.8%) was somewhat lower than that found earlier (84.6%) . The discrepancy may due to the time when the sera were collected; our results show that CVA16 IgM is detectable in only 68% of patients in days 1-7 of illness, but rises to 100% on days 8-11. When blood and rectal swabs were collected on the same day, the agreement between capture-ELISA and real-time RT-PCR in both HEV71 and CVA16 infections suggested both capture-ELISAs perform as well as RT-PCR in diagnosing HFMD and could be deployed successfully in clinical and public health laboratories. Because the sample size was relatively small, particularly for CVA16, it is difficult to compare the sensitivity results with our larger data set.
We observed significant cross-reactivity between HEV71- and CVA16-IgM ELISAs and several reasons can be advanced for this apparent lack of specificity. First, co-infection by the two viruses could occur, leading to simultaneous production of both HEV71- and CVA16-IgMs. This is ruled out by the real-time RT-PCR results, which never detected both of these two viruses. Second, there may have been prior infection with the other virus. If this prior infection had been several months before clinical presentation, the dominant immunoglobulin isotype would be IgG, with the level of IgM low or undetectable. More recent prior infection could be the explanation; although the virus itself would have been cleared and not detected, the corresponding IgM can persist for several weeks. In this case, it would be expected that the cross-reactive IgM would have been detected in the earliest samples that were collected. Figure 2 shows that cross-reactivity is delayed, taking a few days to become evident.
The third hypothesis, which we favor, is that the IgMs may recognize a common epitope between these two related viruses. Homology between HEV71 and CVA16 is 77% at the genome level and 89% for amino acid sequences . The resulting antigenic similarity means that infection with one virus could elicit antibodies against a second enterovirus serotype. This hypothesis is supported by the observed cross-reactivity with other enteroviruses (Table 1).
For example, 38 of 122 (31.1%) CVA16 infected samples were positive for HEV71-IgM, a value comparable to the 14 of 49 (28.6%) samples from other enteroviral infections. In contrast, only 2 of 105 (1.9%) respiratory virus infected sera were HEV71-IgM positive. This is strong evidence against the hypothesis that this cross-reactivity is due to a recent prior infection to HEV71. It seems unlikely that about 30% of the patients infected with CVA16 or with other enteroviruses were previously infected by HEV71, while only 2% of the respiratory virus infected patients had this prior infection. Similarly, CVA16-IgM was apparently positive in 58 of 211 (27.5%) HEV71 infected samples, 16 of 48 (33.3%) of other enterovirus infections, but only 3 of 105 (2.9%) for other respiratory virus infected sera. It was demonstrated, by virus neutralization tests, that none of the patients infected with other enteroviruses or other respiratory viruses, was virus-positive for HEV71 or CVA16.
We suggest that infection with either HEV71 or CVA16 results in several IgMs, some that are specific for the infecting virus and others that cross-react with related enteroviruses. From a practical standpoint, ELISA yielding the higher OD450 value was successful in identifying whether the enteroviral infection was by HEV71 or CVA16 in most cases. This is an important result because it can be used as a predictor to distinguish these two causes HFMD. A small proportion of HEV71-infected children develop severe and sometimes fatal neurological and systemic complications over days or even hours  so early diagnosis of the infecting virus is crucial.