The study had a cross-sectional design and took place in Soochow University Hospital in the city of Suzhou, Jiangsu province, China, from May 2014 to December 2017. Data were collected prospectively during that period from patients admitted consecutively to our department with recurrent wheezing. From the prospectively collected data, children aged < 36 months who had been admitted to the hospital consecutively with recurrent wheezing were considered eligible for the study if they had experienced wheezing for ≥ 4 weeks despite treatment with bronchodilators, inhaled corticosteroids or at least 15 days of treatment with systemic corticosteroids, and had experienced more than four episodes of wheezing in the previous 12 months. Patients were excluded from the study if any of the following were applicable: born prematurely or birthweight < 2500 g; family history of smoking; airway structural abnormalities (e.g. malacia, bronchial stenosis); endobronchial tuberculosis; gastroesophageal reflux; congenital or acquired immunodeficiencies; foreign-body aspiration; congenital heart disease, or; neuromuscular disorder.
Patients meeting the above eligibility criteria were enrolled on the study after informed and signed consent was received from their legal guardians. Enrolled patients underwent FFB and were tested for BALF CMV DNA. Flexible fiberoptic bronchoscopy was performed in accordance with the hospital guidelines. The study protocols were approved by the Ethics Committee of the Children’s Hospital of Soochow University.
Clinical definitions
Recurrent wheeze was defined as ≥4 episodes of wheeze within 1 year as documented in the patients’ medical records. A wheezing episode was defined as a respiratory episode with wheezing lasting for more than 1 day. The interval between two wheezing episodes was defined as a period of ≥7 days without respiratory symptoms.
A mAPI was considered positive when a patient had ≥4 wheezing episodes in a year while also exhibiting one of three defined major criteria (parental asthma, allergic sensitization to one or more aeroallergens, or physician-diagnosed dermatitis) or two of three defined minor criteria (wheezing unrelated to colds, peripheral blood eosinophils ≥4%, or allergic sensitization to milk, egg or peanuts) [4].
Baseline data collection
The following clinical characteristics were obtained from all recruited patients through interviews and reviews of their medical records: age; gender; exclusive breastfeeding during the first 4 months of life; other children in the household; previous wheezing episodes and duration of hospitalization. Laboratory data were collected from peripheral blood samples which were taken on admission to the hospital: peripheral blood eosinophil count was determined using an automated hematology analyzer (XE-2100, Sysmex, Japan); counts of subpopulations of lymphocytes were determined by a Coulter Epics XL MCL Flow Cytometer (Beckman Coulter, USA) after labelling with the following fluorochrome-antibody combinations: anti-CD45-fluorescein isothiocyanate/anti-CD4-phycoerythrin/anti-CD8-phycoerythrin-Texas red/anti-CD3-phycoerythrin-cyanin 5.1; anti-CD45- fluorescein isothiocyanate/anti-CD16CD56-phycoerythrin/anti-CD19-phycoerythrin-Texas red/anti-CD3- phycoerythrin-cyanin 5.1; anti-CD45-fluorescein isothiocyanate; anti-CD19-phycoerythrin-Texas red; anti-CD23-allophycocyanin; and anti-CD3-phycoerythrin. All fluorochrome-antibodies were purchased from Immunotech (France). Cells stained with antibodies were analyzed by flow cytometry using EXPO32™ ADC software. A CD45/SS gating strategy was used for the identification of T-, B- and NK-cell populations in peripheral blood; total IgG, IgA and IgM levels were determined using an Olympus AU400 analyzer (Olympus, Japan), and; total IgE was determined in a subset of 36 patients using the ImmunoCAP system (Pharmacia Diagnostics, Sweden) in accordance with the manufacturer’s instructions. Skin prick testing was performed in a total of 75 patients to 18 common allergens (food allergens and aeroallergens), using allergen extracts and testing devices manufactured by Becton Dickinson (USA).
Flexible fiberoptic bronchoscopy and bronchoalveolar lavage fluid testing
A flexible fiberoptic bronchoscope [Olympus CV260 (2.8 mm, 4.0 mm), Japan, or Fujinon EB-270P (3.6 mm), Japan] was chosen according to the age of the patient, and used to conduct FFB in accordance with previously described procedures [9]. During the procedure, BALF was collected for cell count analysis and CMV DNA detection.
Cell counts
Differential cell counts were obtained using a modified version of Wright-Giemsa staining (Wright-Giemsa Stain, Baso Diagnostics Inc., China). At least 500 cells were examined by the same observer for each specimen. Data were reported as percentages of the total cell counts.
Detection of CMV
The detection and quantification of the CMV DNA level was carried out by real-time PCR, using a diagnostic kit for the quantification of human CMV DNA (Sansure Biotech, China) and a LightCycler 480 system (Roche Applied Science, USA), according to the manufacturer’s instructions. The detection limit for the test was > 400 copies/mL.
Statistical analysis
Statistical analyses were performed using SPSS software, version 21.0 (IBM SPSS). The Kolmogorov-Smirnov test was used for testing data for normality. Continuous variables with non-normal distributions were expressed as medians and interquartile ranges (IQR) (25th to 75th percentile). Comparisons between groups were made using the Mann-Whitney U-test. Spearman’s rank correlation coefficient was used to evaluate correlations between the number of copies of CMV DNA and the various continuous variables. Comparisons of frequency distributions were performed using the Chi-squared test. A test result was deemed statistically significant if p < 0.05. A sample size estimation was calculated based on a likely sample proportion having the tested trait (P) of 50% [5], with 95% confidence (α = 0.05) and a 10% margin of error of the estimate. The minimum required sample size according to these parameters and the formula n = \( \frac{{\mathrm{Z}}_{\alpha /2}^2\mathrm{P}\left(1-\mathrm{P}\right)}{d^2} \), was n = 96.