Background

Hand-foot-and-mouth disease (HFMD), characterized by fever and acute vesicular eruptions of palms, soles of the feet and mouth (herpangina), is a common exanthema in young children. It is caused by members of the non-polio Enterovirus genus (family Picornoviridae), such as Coxsackievirus A (CVA) and B, Echovirus 4, 6 and 7, particularly CVA16 and human enterovirus (HEV) 71. Outbreaks have occurred recently in the Asia-Pacific region: Malaysia (2000-2003) [1], Taiwan (1998-2005) [2, 3], Singapore (2000) [4], Brunei (2006) [5], Thailand (2008-2009) [6], Korea (2008-2009) [7], and Hong Kong (2008) [8]. In mainland China, large epidemics of HFMD have been reported: Shenzhen (1999-2004) [9], Bei**g (2008) [10], and Fuyang city (2008) [11]. Surveillance studies have indicated that HEV71 and CVA16 circulate widely in central and southern China. The severe complications and even fatal cases in young children associated with HEV71 make HFMD an important health concern. With large outbreaks occurring frequently and the increased concern of fatal HFMD caused by HEV71, a rapid, specific, and cost-effective assay to identify the HFMD-causing enterovirus is of great importance. Recognition of the causative agent for HFMD mainly relies on laboratory identification of the virus so that treatment and effective public health measures can be taken early.

Diagnostic techniques include time consuming and labor intensive methods such as virus isolation, a neutralization test, and RT-PCR for viral RNA detection. In contrast, newly developed IgM-capture ELISAs for HEV71 [15] of the 29 other enterovirus infected patients showed CVA6 in four individuals, CVA10 in two, one each of CVA21, CVB2, Echo 16 or Echo 25, and 19 untyped; the latter were further confirmed as neither HEV71 nor CVA16 infected by the neutralization test. HEV71-IgM was positive in 38 of 122 CVA16, 14 of 49 other enterovirus and two (both from one patient co-infected with RSV subgroup A and B) of 105 respiratory virus infected sera. The specificity was 69.6% (52/171) compared to other enteroviruses and 98.1% (2/105) compared to other respiratory viruses (Table 1). CVA16-IgM was apparently positive in 58 of 211 HEV71, 16 of 48 other enterovirus and 3 (from two patients co-infected with RSV subgroup A and B and one with RSV subgroup B) of 105 respiratory virus infected sera, giving a specificity of 71.4% (74/259) compared to other enteroviruses and 97.1% (3/105) compared to other respiratory viruses. The positive and negative predictive values were 81.3% (95% confidence interval: 76.9-85.1%) and 91.0% (95% confidence interval: 87.5-93.8%) for HEV71, 54.4% (95% confidence interval: 48.3-60.9%) and 88.6% (95% confidence interval: 85.1-91.4%) for CVA16, respectively (Table 1).

To further analyze the cross-reactivity of the assays, a total of 206 sera from 134 HEV71 patients and 119 sera from 66 CVA16 infected patients were tested for both HEV71- and CVA16-IgM simultaneously (Figure 2). For the HEV71-infected patients (Figure 2A), 199 of 206 samples were positive for HEV71-IgM (95.7%) while the cross-reactivity towards CVA16-IgM was 28.2% (58/206). However, of the 58 CVA16-IgM positive sera, the ratio of OD450 value for HEV71-IgM divided by that for CVA16-IgM greater than 1.0 can successfully identify 56 (96.6%) HEV71 infections (Figure 2A, inset). For the CVA16 patients (Figure 2B), CVA16-IgM was detected in only 83 of 119 samples (69.7%) while cross-reactivity towards HEV71-IgM was seen in 30.3% (36/119). While of the 36 HEV71-IgM positive sera, the ratio of OD450 value for CVA16-IgM divided by that for HEV71-IgM greater than 1.0 can successfully identify 33 (91.7%) CVA16 infections (Figure 2B, inset).

Figure 2
figure 2

OD 450 values and ratio for HEV71-IgM and CVA16-IgM in HEV71 or CVA16 infected sera collected at various days after onset of symptoms. Panel A shows the OD450 values for HEV71 infected patients; values greater than cutoff (horizontal line) represent a positive result. The inset shows the ratio of OD450 values for HEV71-IgM divided by that for CVA16-IgM. Panel B shows the OD450 values for CVA16 infected patients; values greater than 1.0 (horizontal line) represent a positive result. The inset shows the ratio of OD450 values for CVA16-IgM divided by that for HEV71-IgM.

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Correlation between real-time RT-PCR and IgM-capture ELISA results

In all patients, HEV71 or CVA16 was identified by real-time RT-PCR of a rectal swab, but this was not always taken on the same day that serum was collected. Subsets of the data, where both samples (111 HEV71 and 53 CVA16) were collected within 24 hours of one another were analyzed. For HEV71, the difference between ELISA and real-time RT-PCR was not statistically significant (McNemar's chi-square test exact P = 0.648) and the measure agreement was high (Kappa value = 0.729). For CVA16, the difference between ELISA and real-time RT-PCR was not statistically significant (McNemar's chi-square test exact P = 0.885) but the measure agreement was relatively low (Kappa value = 0.300). In other respects, the data are similar to those in Figure 1 in that IgM increased during the very early acute phase and reached the 100% positive rate by day 4 (HEV71) or 8 (CVA16). The sensitivity for HEV71-IgM was 90.1% (95% confidence interval: 84.1-94.4%) while that for CVA16-IgM was 56.6% (95% confidence interval: 44.5-67.9%).

Discussion

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 develo** 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 [13] 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 [17]. 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%) [12], while that for CVA16 IgM (72.8%) was somewhat lower than that found earlier (84.6%) [14]. 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 [18]. 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 [19] so early diagnosis of the infecting virus is crucial.

Conclusions

This study represents the first report of the kinetics of IgM in HEV71 and CVA16 infections. The IgM-capture ELISAs for HEV71 and CVA16 were found to be highly effective in correctly identifying the infecting virus. ELISAs have the advantage over RT-PCR to provide a convenient and relatively rapid diagnostic tool for HFMD infections. Assaying for both HEV71-IgM and CVA1-IgM can be deployed successfully as a cost-effective diagnosis of HFMD in clinical and public health laboratories.

Ethical approval

Approval was obtained from the Ethics Committee of Zhujiang Hospital. The parents of each subject gave informed written consent before collection of rectal swabs and serum samples.