Ethic Statement

Written informed consent was obtained from each subject. Independent Ethics Committee approval was obtained from the Ethics Committee of the National Institute of Diagnostics and Vaccine Development in infectious diseases.

Study design

The sensitivity of the IgM anti-EV71 assay was evaluated in HFMD patients who were confirmed to be recently EV71 infection, and was classified by the days apart from the onset. The specificity of the assay was evaluated in children patients with confirming diagnosis of other respiratory diseases. The cross-reactivity of the assay was evaluated in HFMD patients infected by other enteroviruses.

Serum samples

A total of 376 serum samples were collected from HFMD patients, herpangina, aseptic meningitis, or encephalitis between March and September 2008. Of these samples, 221 were collected from 165 EV71-infected patients with the mean age of 2.6±2.1, 155 were from CA16–infected patients with the mean age of 2.7±2.5. The infection of EV71 or CA16 among these patients was determined by detection of the viral RNA by reverse transcript PCR. Twelve serum samples collected from patients infected by other enteroviruses (4 coxsackievirus A2, 1 coxsackievirus A4, 1 coxsackievirus B3, 2 coxsackievirus B4, 2 coxsackievirus B5, and 2 echovirus 6) were gifts from Dr. P. J. Chen of National Taiwan University, which were determined by virus isolation.

Control samples for this study included three groups. The first group included 128 sera from children patients with the following clinical features: Pneumonia (83 cases), Bronchitis (18), acute upper respiratory infections (15), and Influenza (12). The second group included 1907 stored sera from healthy children who received health examinations in with the mean age of 2.1±2.7. The third group included 807 sera from healthy adult blood donors. The EV71 neutralizing antibody titers of all control samples were less than 1∶100. Twenty serum samples positive with rheumatoid factor were also used to evaluate the possible disturbance to IgM testing. All serum samples were kept in aliquots at −20°C until use.

Viral RNA extraction and PCR amplification

Viral RNA was extracted from the clinical specimens using a QIAamp Mini viral RNA Extraction Kit (Qiagen). The primers used for RT-PCR are listed in Table 1. RT-PCR amplification was performed using AccessQuickTM RT-PCR kit (Promega). Conditions for RT-PCR amplification were: 45 min of reverse transcription at 45°C; 5 min denaturation at 94°C; 35 cycles of 95°C for 40 sec, 53°C for 40 sec, 72°C for 40 sec; and then a final elongation step of 72°C for 5 min. The second round amplification was performed in 25 µl volumes, which contains 2.5 µl 10x PCR reaction buffer, 1 µl 10 mM dNTP, 0.5 µl 10 mM each primer, 0.5 µl Taq (TaKaRa), 1 µl of the first-round PCR product, and 19 µl Nuclease-Free Water, under the same conditions as the first-round PCR, except reverse transcription step. The PCR products were examined by electrophoresis with a 3.0% agarose gel.

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Table 1. List of primers designed for the specific amplification of EV71 and CA16.

https://doi.org/10.1371/journal.pone.0011388.t001

Virus Isolation

Clinical specimens (including throat swabs and rectal swabs) were submitted for virus isolation. Samples were inoculated into RD and human laryngeal carcinoma (Hep-2) cell cultures. Cultures that exhibited a characteristic enterovirus CPE were further evaluated by RT-PCR and sequencing.

Neutralization test

Laboratory methods for measuring EV71 neutralizing antibody followed standard protocol for the neutralization test on microtiter plates [17], [18]. Serum specimens were serially diluted two-fold (from 8 to 2048) and mixed with equal volume of EV71 (100 TCID₅₀/50 µl) at 37°C for 60 min. The mixtures were incubated in replicate microplate cultures of human embryo rhabdomyosarcoma (RD) cells. Cytopathic effects (CPE) were read under an inverted microscope after 2 to 7 days. Neutralizing antibody titer was defined as the highest dilution of serum that prevented the occurrence of CPE.

Detection of IgM anti-EV71

IgM anti-EV71 was detected using an IgM μ-chain capture enzyme-linked immunoabsorbant assay (ELISA) (Beijing Wantai Biological Pharmacy Enterprise Co., Ltd., China) according to manufacturer’s instruction. Briefly, 100 µl of dilution buffer were added to microplate wells pre-coated with anti-human IgM μ-chain, then 10 µl of serum samples were added and mixed tenderly, incubated at 37°C for 30 min, washed 5 times with washing buffer. Then 50 µl of the antigen solution, prepared by inactivating and fracturing of the cell free culture supernatant of an EV71 isolate JS/52-3/06, was added followed by 50 µl of a horseradish peroxidase (HRP)-conjugated mouse anti-EV71 monoclonal antibody, mixed, incubated for 30 min and then washed 5 times. 100 µl of TMB substrate was added then incubated at 37°C for 15 min before adding 50 µl 2N H₂SO₄ for terminating the reaction. The optical density (OD) was read at a wavelength of 450 nm with a reference filter of 620 nm. The cutoff value was calculated as 0.1+ mean OD value of the negative control. If the mean absorbance value of the negative control was lower than 0.05, this was treated as 0.05. Levels of anti-EV71 IgM was expressed in S/CO value, which was calculated as the ratio of the OD value obtained with the test sample to the cutoff value determined concurrently. A S/CO value not less than 1.0 indicated a positive result.

Statistical analysis

The detection rate was compared between groups used two-sided Fishers exact test and 95% confidence intervals (CI) were calculated with the use of SPSS program (v. 11.5).