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], Beijing (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 [
12,
13] and CVA16 [
14] are rapid and convenient for large numbers of specimens. Previously, capture ELISAs for HEV71- and CVA16-IgM were established, which show good efficiency for screening HFMD patients [
12,
14]. An understanding of the kinetic profiles of the IgM antibodies and the diagnostic characteristic of these assays is needed to substantiate their validity. In this study, we aimed to evaluate IgM-capture ELISAs for HEV71 and CVA16 for diagnosis of HFMD in pediatric patients, and to follow the kinetics of IgM antibodies over the course of these infections.
Materials and methods
Patients and clinical samples
HFMD patients with clinic features of herpangina, aseptic meningitis, and encephalitis, hospitalized in Zhujiang Hospital from March 2009 to December 2010, were studied. Laboratory diagnosis of all these patients showed them to be infected with HEV71, CVA16 or other enteroviruses as detected on rectal swabs using real-time RT-PCR plus virus isolation in some cases. Selected cases were confirmed by the neutralization test. The assay results showed 134 HFMD patients (86 male and 48 female, aged 4 months to 14.1 years, median 2.17 years) with HEV71 infection, 67 HFMD patients (49 male and 18 female, aged 6 months to 7.0 years, median 2.17 years) with CVA16 infection, and 29 HFMD patients (21 male and 8 female, aged 5 months to 5.6 years, median 1.83 years) with other enteroviral infections. A total of 434 acute- and convalescent-phase serum specimens were collected between days 1 and 158 after the onset of symptoms from these 230 HFMD patients (a single sample from 69 patients, two from 139 individuals, three from 18 patients, two from four individuals and six from one patient). Nineteen consecutive sera from one patient, confirmed as infected with HEV71 by real-time RT-PCR in combination with virus isolation, were assayed for HEV71-IgM during the course of the disease.
As controls, 105 sera from 75 patients with acute respiratory infections were collected. All these patients had been laboratory-confirmed previously as being infected with respiratory syncytial virus (RSV, 40 patients), adenovirus (9), influenza A virus (5), influenza B virus (2), parainfluenza virus (5), human rhinovirus (3), human metapneumovirus (3) and other respiratory viruses (8) by real-time RT-PCR and/or virus isolation.
Real-time RT-PCR and VP1 semi-nested RT PCR
Viral RNA extraction was performed on swab specimens using the QIAamp Viral RNA Mini Kit (Qiagen). Real-time RT-PCR was performed in a Lightcycler 1.2 (Roche) using Pan-Enterovirus-, HEV71- and CVA16-specific detecting kits (Da An Gene Co., Ltd.). After 25 min of reverse transcription at 40°C and denaturation at 94°C for 3 min, 40 cycles of amplification (denaturation: 93°C, 15 sec; annealing/elongation: 55°C, 45 sec) were used. The semi-nested RT-PCR was as described previously [
15] using RNA extracted from rectal swabs. Sequencing of the amplified VP1 gene product identified the serotype.
Virus Isolation
Viral isolation was attempted on selected rectal swabs that were real-time RT-PCR positive. After shaking vigorously and centrifugation (4°C, 10,000 × g, 20 min), samples were sterilized by filtration (0.22 μm Millipore express® membrane) and used to inoculate human rhabdomyosarcoma (RD) and/or laryngeal carcinoma cells. Once a complete cytopathic effect (CPE) was noted, cultures were harvested and viral identification was performed by real-time RT-PCR as described above.
Neutralization test
HEV71 and CVA16 specific neutralizing antibodies were detected according to a standard protocol [
16]. Briefly, 50 μl two-fold serially diluted serum was mixed with an equal volume of HEV71 or CVA16 (100 TCID
50/50 μl) and incubated at 34°C for 2 h. The mixtures were added to replicate microplate cultures of RD cells and incubated at 34°C for 7 days. CPE was observed under a microscope after 2 to 7 days. The highest dilution that prevented the occurrence of the CPE was designated as the neutralizing antibody titer.
IgM-capture ELISA
The IgM-capture ELISA for HEV71 and CVA16 has been described previously [
12,
14]. The cutoff value was set as 0.1 plus mean OD
450 value of negative controls. An S/CO (sample/cutoff) value greater than 1.0 indicated a positive result.
Statistical analysis
Sensitivity and specificity were calculated from the ELISA and real-time RT-PCR results. Differences between the proportions of positive results were compared by McNemar's chi-square test using SPSS software (version.13.0) and considered to be significant when P < 0.05.
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 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 [
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 OD
450 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.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
NY performed the real-time RT-PCR and VP1 RT-semi-nest-PCR assays, analyzed the data and drafted the manuscript. MG collected most serum samples and performed the capture ELISA assays for both HEV71 and CVA16 specific IgM. SH and YP jointly performed virus isolation and identification. XC made clinical diagnoses and helped in collecting the clinical samples. XD and WH jointly performed PCR for respiratory virus infected patients and collected the serum samples. YW partly performed the real-time RT-PCR assay. SG and NX optimized the capture ELISA for HEV71 and CVA16 specific IgM, respectively, and jointly analyzed the data. XC conceived and designed the study, and drafted the manuscript. All authors read and approved the final manuscript.