Study population
Thirty-one infants, aged 0–11 months, diagnosed with RSV bronchiolitis were enrolled in this study. None of them were born prematurely (at or before 35 weeks of gestation) or had any history of diseases, including bronchopulmonary dysplasia, hemodynamically significant congenital heart diseases, neuromuscular disorders, immunocompromise, or Down syndrome. They had never received palivizumab injection before admission. The patients’ demographic characteristics are presented in Table 1. They were admitted to the Akita Kousei Medical Center between August 2018 and January 2020, and their diagnosis of RSV infection was made performing antigen detection tests on nasopharyngeal discharges (ImunoAce RSV Neo, Shizuoka, Japan). The clinical diagnosis of RSV bronchiolitis was made based on criteria that comprised cough, increase in respiratory rate, chest retraction, prolongation of expiratory time, sibilant rhonchi, and hyperinflated lungs on chest X-ray10. Blood was collected on admission and serum was stored frozen at <−20°C.
Eight healthy infants and the nine other RSV-negative hospitalized febrile infants, including three diagnoses of acute bronchitis, two of adenovirus infections, one of hand-foot-and-mouth disease, one of febrile convulsion, one of mycoplasma infection, and one of otitis media, all of whom presented with fever and respiratory symptoms, were also included as controls in this study. RSV-negative infections were confirmed using an antigen detection test.
Patients with RSV bronchiolitis were scored from 0 to 3 points for each of four factors (oxygen saturation of peripheral artery, respiratory rate, wheezing, and depressed breathing) for a maximum of 12 points, every day during hospitalization. We modified a previously reported scoring system (Table 2)11,12. Medical records were accessed for age, sex, clinical symptoms, complications, need for oxygen administration, and laboratory data including white blood cell count, platelet count, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, hemoglobin, and C-reactive protein (CRP) levels.
Determination of serum S-ASM activity, interferon-γ (IFN-γ), and interleukin-18 (IL-18)
Serum S-ASM activity was measured as previously described13. Briefly, serum S-ASM activities were assayed using a substrate of 14C-labeled sphingomyelin (PerkinElmer, MA, USA) and a buffer containing Zn2+ as Zn2+-dependent S-ASM. A standard 200-μL assay mixture was made up of 100μL serum, 50μL assay buffer (1.0M sodium acetate, pH 5.0) with 4% Triton X-100 as a final concentration of 1%, and 50μL of substrate (20 nmol of 14C-labeled sphingomyelin; 0.08 μCi/20nmol) in 0.2% taurodeoxycholic acid and then incubated at 37℃ for 6h. The reaction was stopped with 200 μL ice-cold 30% trichloroacetate and 400 μL 2.5% bovine serum albumin. The mixtures were briefly vortexed and allowed to settle for 5 min before centrifugation (3,000 rpm for 5 min). The supernatant (500μ) was carefully transferred to glass scintillation pre-filled with 4.5 mL of Clearsol II (Nakalai Tesque, Kyoto, Japan). Radioactivity was measured using a liquid scintillation counter LSC 950 (Aloka, Tokyo, Japan).
Levels of serum INF-γ and IL-18 were determined using the Human IFN gamma ELISA Kit (Thermo Fisher Scientific, Waltham, MA, USA) and Human IL-18 ELISA Kit (MBL Inc., Tokyo, Japan), respectively.
Statistical Analysis
Data were analyzed using the IBM SPSS statistics 26.0 software package, and the results are presented as the mean ± standard deviation. The Kruskal–Wallis test was used to compare the mean differences between the two groups. Spearman’s rank correlation coefficient was used to examine the correlation between serum S-ASM levels with the clinical study measures. Statistical significance was set at p < 0.05.