2.1 Case selection
In this study, we firstly obtained informed consent from 399 Chinese children in the Affiliated Hospital of Qingdao University and grouped them according to different clinical diagnostic criteria. EV71 infection was diagnosed by both clinical features and reverse transcription polymerase chain reaction (RT-PCR). RNA was extracted from the cerebrospinal fluid (CSF), throat swabs, stool specimens, and any other tissues obtained from the patients being studied the day after admission. EV71 encephalitis was confirmed by brain parenchymal damages, CSF examination (white blood cell count >5/mm3), and parenchymal lesions as identified by brain computed tomography (CT) or magnetic resonance imaging (MRI).
EV71 encephalitis patients with the following characteristics were regarded as severe cases: (1) focal or diffuse parenchymal lesions of the brain parenchyma, (2) abnormal MRI showing edema in the adjacent brain parenchyma and destruction of the brain parenchyma, (3) prolonged coma and confusion, and (4) other clinical characteristics (including oral ulcers and skin eruption on the hands and feet) [21]. All the subjects in this study were analyzed by clinical interview, physical routine examination, serological test, MRI, CSF analysis, and other auxiliary checks. Moreover, corresponding records were made.
2.2 Data collection
Clinical and laboratory data were collected from June 2011 to December 2012. In this study, we recorded demographic data such as sex and age and the results of laboratory examinations such as white blood cell (WBC) count, C-reactive protein (CRP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase-myocardial isozyme (CK-MB), electroencephalogram (EEG), electrocardiogram (ECG), CT of the brain, and myelencephalon MRI of the brain or spine.
A commercial kit (Qiagen) was used to extract the genomic DNA from WBCs. To detect the –607C/A polymorphism in IL–18, the improved multiplex ligation detection reaction (iMLDR) technique developed by Genesky Biotechnologies Inc. (Shanghai, China) was used for genotyping at position –607 of IL–18 on chromosome 11q22.2–22.3. The product size was 261 bp. The primers designed for the PCR are shown in Table 1. The PCR conditions were as follows: 95 °C for 2 min; 11 cycles at 94 °C for 20 s, 65 °C–0.5 °C/cycle for 40 s, and 72 °C for 1 min 30 s; and 24 cycles at 94 °C for 20 s, 59 °C for 30 s, and 72 °C for 1 min 30 s, followed by 72 °C for 2 min and holding at 4°C. The PCR products were purified by digestion with 1 U of shrimp alkaline phosphatase at 37 °C for 1 h and at 75 °C for 15 min.
The ligation reaction mixture contained 2 μL 10 × ligase buffer, 0.2 μL Taq DNA ligase, 1 μL probe mixture, and 3 μL purified PCR product mixture. In a double connection reaction, each site contained two 5′ ends of allele-specific probes and following a 3′end of an allele-specific probe. Each allele-specific connection product was distinguished by its fluorescence, while different loci were distinguished by the different lengths added to the 3′end of the allele-specific probe. The probes were TAM-TTTGGTATCCCTCTCC-MGB and TET-TTTGGTAGCCCTCTV-MGB. The ligation cycling program consisted of 35 cycles at 94 °C for 1 min and 56 °C for 4 min, and then holding at 4 °C. The SNPs were genotyped using a 3130xl Genetic Analyzer (ABI), and the raw data was validated using the Gene Mapper 4.0 (Applied Biosystems, USA).
2.3 Estimation of IFN-γ levels
Plasma concentrations of IFN-γ were detected using ELISA kits (R&D Systems) according to the manufacturer’s instructions. The sensitivity of detection is 8 pg/mL as indicated by the manufacturer. Each sample was repeated, and these values were in the linear part of the standard curve.
2.4 Statistical analysis
The Chi-square test was used to compare frequencies of the three genotypes (CC, CA, and AA) and two alleles (C and A) in different groups as well as demographics (such as sex) and clinical findings and symptoms (such as abnormal ECG or MRI, temperature >39 ℃, vomiting, and mental change after convulsions) among different genotype groups, respectively. The differences in demographics and clinical manifestation such as age, duration of fever, WBC count, CRP, ALT, AST, CK-MB, and BG of the different genotype groups are presented as means ± standard deviation (SD) or median values (25th–75th percentage values) and analyzed using a one-way analysis of variance (ANOVA).
The concentration of IFN-γ is presented as means ± SD and was analyzed using the t-test. The relationship of IL–18–607C/A polymorphism to the susceptibility and severity of EV71-related encephalitis was evaluated by calculating the odds ratio (OR) and 95% confidence intervals (95%CI) using logistic regression. Pearson’s collection statistical analysis was performed using the Statistical Package for the Social Science (SPSS) version 21.0 IBM SPSS software, USA) and significance was set at P<0.05.