Infants with bronchiolitis were enrolled from November 2016 through March 2017. We also enrolled 24 healthy children with no history of wheezing from the department of children's health prevention as a control group. Nasopharyngeal aspirates were obtained for detecting respiratory virus and analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR) and direct immunofluorescent assay. Serum cytokines including TSLP, IL2, IL13, TIMP-1, MMP-9, IL33, IL5, IL4, IL25, TNF- α and MIP-1α were measured by flow cytometry. The patients were followed every 3 months for a duration of 2 years by telephone or at outpatient appointments.
Patients
Infants diagnosed with bronchiolitis and hospitalized at the Department of Respiratory Disease at Children’s Hospital Soochow University, China, from November 2016 through March 2017, were enrolled in this study. Recurrent wheezing was defined as two or more episodes following initial bronchiolitis for two years. We also enrolled 24 healthy infants with no history of wheezing from the department of children's health prevention (age 3.5±1.2 months; 15 (62.5%) males) as a control group. The individuals had no immune deficiency and no signs of respiratory tract infection in the four weeks preceding the study. There were no differences in gender and age between the patients and the control group. The study was approved by the ethics committees of the Children’s Hospital Soochow University (Approval No.:2016050). Informed consent was obtained from the parents of all children enrolled in this study.
Inclusion criteria: 1) Age: 1–24 months; 2) Patients were hospitalized with bronchiolitis; 3) Bronchiolitis was defined as the first wheezing episode characterized by cough, tachypnea and chest retractions.
Exclusion criteria: Neuromuscular disease, congenital airway deformity, congenital heart disease, gastroesophageal reflux disease, bronchial foreign body inhalation, primary or secondary immune deficiency or other immune-associated diseases were excluded.
Disease severity criteria: according to Wang expiratory flow limitation (EFL) scoring[18], the severity of disease was graded as follows (Table 1): 0–4.9 was mild; 5–8.9 was moderate; 9–12 was severe.
Sample collection
Nasopharyngeal aspirates were obtained using a suction catheter passed through the nose into the lower part of the pharynx. Peripheral venous blood (2 ml) was also collected.
Detection of seven common viruses by direct immunofluorescent assay (DFA)
DFA was used to detect respiratory syncytial virus (RSV), influenza virus A (IVA), influenza virus B (IVB), parainfluenza virus (PIV) I, PIV II, PIV III, and adenovirus (ADV). All assay kits were purchased from Chemicon (USA) and all staining procedures were performed according to the manufacturer’s instructions. Immunostained preparations were viewed with a fluorescence microscope (Leica 020-518.500, Germany).
Detection of the metapneumovirus (hMPV), rhinovirus (hRV), bocavirus (hBoV) gene by real-time polymerase chain reaction (RT-PCR)
For hMPV detection, primers were designed to specifically amplify the N gene (213 base pairs [bps]). The forward and reverse primers were hMPV-F:5’- AACCGTGTACTAAGTGA
TGCACTC-3’ and hMPV-R:5’- CATTGTTTGACCGGCCCCATAA-3’, respectively. The cyclic temperature settings were 94 °C, 30 s; 55 °C, 30 s; 68 °C, 30 s; amplified by 45 cycles with the last at 68 °C for 7 min.
For hRV detection, the primers and probe sequences were HRV-F: 5’-TGGACAGGGTGTGAAGAGC-3′;HRV-R:5’-CAAAGTAGTCGGTCCCATCC-3′;HRV-probe:FAM-TCCTCCGGCCCCTGA ATG-TAMRA. The cyclic temperature settings were 94℃, 30 s; 56℃, 30 s; 72℃, 30 s; amplified, 40 cycles.
For hBoV detection, the primers and probe sequences were HBoV-F:5’-TGACATTCAACTACCAACAACCTG-3’;HBoV-R:5’CAGATCCTTTTCCTCCTCC
AATAC-3′; HBoV-probe: FAMAGCACCACAAAACACCTCAGGGG-TAMRA. The cyclic temperature settings were 94℃,30 s; 56℃, 30 s; 72℃, 30 s; amplified for 40 cycles.
Blood tests
Peripheral venous blood (2 ml) was collected and anticoagulated with EDTA. The blood was detected by an automatic five classification hematology analyzer.
Testing of humoral immunity
Detection indexes: IgG, IgM and IgA. IgG and IgM were determined by transmission immunoassay, and IgA was determined by immunoturbidimetry.
Testing of cellular immunity
Peripheral venous blood (2 ml) was collected and anticoagulated with heparin. Flow cytometry of Beckman Coulter company was used for analysis. The kit was purchased from Immunotech (France). Detection indexes: CD3+, CD3+ CD4+, CD4+ / CD8+, CD3+CD8+, CD19+CD23+, CD3-CD16+CD56+ and CD3-CD19+.
Detection of serum levels of cytokine
The peripheral blood was centrifuged at 2500r/min for 5 minutes. Supernatants were frozen at -80℃. Serum levels of cytokines (TNF-a, IL-2, IL-13, IL-4, IL-5, IL-25, IL-33, TSLP, TIMP-1, MMP-9 and MIP-1α) were measured by flow cytometry. Flow cytometry (Beckman Coulter, Brea, CA, USA) was performed according to the manufacturer's instructions (Immunotech, Marseille, France). All assay kits were purchased from BEIJING TONGSHENG SHIDAI BIOTECH CO., LTD. Data were automatically processed and analyzed using FCAP Array 3.0 with the standard curve produced from the cytokine standard.
Data collection
Each patient’s data, including age, gender, gestational age at delivery, birth weight, feeding patterns, history of eczema, family history of asthma, exposure to smoking, and pet contact were recorded.
Follow-up of patients
After discharge from hospital, the patients were followed up every 3 months for a 2‑year period by outpatient visits or telephone consultations.
Statistical analysis
SPSS version 18.0 software was used for data analysis. Distribution normality of continuous data was tested by the P-P plots methods before comparison. Data with normal distribution were represented as mean ± standard deviation (SD) and analyzed by t tests. Continuous data with non-normal distribution were represented as median (minimum-maximum) and analyzed with the Mann-Whitney U test. Categorical data were represented as frequency and analyzed with Chi square examination. Predictors of recurrent wheezing were analyzed using a stepwise logistic regression model.