This case-control study was conducted at the Children’s Hospital of Soochow University. Children with a disease onset > 5 days previously, mixed infections, autoimmune diseases and chronic diseases were excluded. The criteria of IM were as follows : (1) presence of at least three of the following clinical manifestations: fever, pharyngitis, cervical lymphadenopathy, splenomegaly, eyelid edema and hepatomegaly; (2) IgM to EBV viral capsid antigen (VCA-IgM) and IgG to EBV capsid antigen (VCA-IgG) positive, with absence of the antibody to EB nuclear antigen (EBNA); and (3) exclusion of other viral infections such as human immunodeficiency virus, cytomegalovirus, hepatotropic virus and herpes simplex virus. The inclusion criteria of the control group were negative EBV-specific antibody and plasma EBV-DNA polymerase chain reaction (PCR) test results, disease onset < 5 days previously, and no history of chronic infectious diseases, immune system diseases and use of immunomodulators in the past 14 days. On admission to the hospital, the blood samples were collected for testing.
Routine complete blood count, alanine transaminase, adenosine deaminase and immunoglobulin G, M, and A assays
Routine blood count of venous blood from participants was performed on the type BC-5310 instrument (Shenzhen Mindray Biomedical Electronics Co., Ltd). Serum ALT and ADA levels were measured using a lactate dehydrogenase assay (Beijing Strong Biotechnologies, Inc.) and peroxidase assay (test kit from Meikang Biotechnology Co., Ltd), respectively. Both were detected using a HITACHI 7180 biomedical analyzer. Immunoglobulin G (IgG), M (IgM), and A (IgA) were detected using a turbidimetric inhibition immunoassay. Anti-human IgG/IgM/IgA antibody (Orion Diagnostica Oy) was added to a certain proportion of the sample and buffer, which produced an agglutination reaction with IgG/IgM/IgA in the sample, resulting in an increase in the turbidity of the mixture. A Konelab clinical chemistry analyzer was used to detect turbidity at 340 nm wavelength.
Lymphocyte subsets, including T cells (CD3+), helper T cells (CD3+CD4+), killer T cells (CD3+CD8+), natural killer cells (CD3−CD (16+56)+), B cells (CD3−CD19+), and activated B cells (CD19+CD23+) were detected using flow cytometry. Peripheral whole blood samples were labeled with antibodies including anti-CD3-fluorescein isothiocyanate, anti-CD16+56− phycoerythrin, anti-CD45-peridin chlorophyll alpha protein-cyanin5.5, anti-CD4-phycoerythrin cyanin 7, anti-CD19-APC, anti-CD23-Fc EpsilonR II, and anti-CD8-allophycocyanin-cyanin7. The samples were centrifuged and kept in the dark at room temperature for 15 min. Each sample was analyzed using a multi-color flow cytometer (BD FACSCanto II) according to the manufacturer’s instructions.
Indirect immunofluorescence assay (IIF) for EBV-specific antibodies
The anti-EBV-VCA IgG /IgM, anti-EBV-early antigen (EA) IgG and anti-EBNA IgG IIF kits (EUROIMMUN, Lübeck, Germany) was used for testing the EBV-VCA, EA and EBNA in serum. All steps were carried out according to the manufacturer’s instructions. The antibody affinity was determined by comparing the serum fluorescence intensities of children treated with and without urea treatment. The difference in fluorescence intensity was ≥ 2 for low affinity, and < 2 for high affinity.
Plasma Epstein-Barr virus-DNA polymerase chain reaction assay
Plasma EB viral load was determined by polymerase chain reaction, and EBV nucleic acid quantitative detection kit came from Shengxiang Biotechnology Co., Ltd, Hunan, China. Amplification and detection were performed on the LightCycler 480II instrument (Roche, Basel, Switzerland) following the manufacturer’s instructions. Copy numbers were calculated by comparing the cycle threshold (Ct) of the specimens to the standard curve. EBV positivity was defined as a Ct value ≤ 39 (DNA copy number > 400 copies/mL).
All peripheral blood samples were collected within 24 h of admission and sent to the laboratory for analysis immediately.
The Shapiro-Wilk normality test was used to determine whether continuous variables were normally distributed. Values were expressed as the mean ± standard deviation or median and interquartile range. The Mann-Whitney U-test and Kruskal-Wallis test were used for non-normally distributed data. Student’s t-test and analysis of variance (ANOVA) were used for normally distributed variables. Categorical variables were reported as frequency (%) and the frequency in different groups was compared using Chi-squared test or Fisher’s exact test. Spearman correlation analysis was used to determine the correlation between discrete variables. Logistic regression analysis was used to determine odds ratios (ORs) with 95% confidence intervals (CIs). Receiver-operating characteristic (ROC) curve analysis was used to assess the diagnostic accuracy of adenosine deaminase in children with IM. The cut-off value for ADA was determined using Youden’s index. All statistical analyses were performed using SPSS version 25.0 (IBM Corp., Armonk, NY, USA). P-values < 0.05 were considered to be statistically significant.