Study protocol and definitions
Patients included in the study as cases were those with an axillary temperature of 38ºC or more, documented at home or within the first 24 hours of hospitalization. Controls were asymptomatic steady-state SCD patients recruited during their follow-up attendance in the Pediatric Hematology clinic. Patients recruited as cases could also be included in the study as controls if at least one month had passed since their last fever episode. The following samples were collected in all cases: blood tests (including complete blood count, biochemistry, CRP and procalcitonin), blood cultures and nasopharyngeal samples for viral detection by a multiplex polymerase chain reaction( multiplex-PCR) assay [including influenza A, B and C, respiratory syncytial virus (RSV), human metapneumovirus, adenovirus, human bocavirus, rhinovirus, human parainfluenza virus and coronavirus]. Other diagnostic tests and the patients’ management were performed under the criteria of the treating physicians. In all cases and controls a blood sample was collected and stored at the HGUGM Biobank for subsequent cytokine analysis. This analysis was performed by the DIAplex Human Th1 / Th2 / Inflammation kit (bioNova científica S. L., Spain), a multiplexed fluorescent bead-based immunoassay for the quantification of multiple human cytokines in serum and culture supernatants by flow cytometry, including the following: interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17a, interferon-γ (IFN-γ) and tumor necrosis factor- α (TNF-α). The limit of detection for the different cytokines was: IL-1β 3.5 pg/mL, IL-2 12.4 pg/mL, IL-4 4.3 pg/mL, IL-6 1.4 pg/mL, IL-8 1.3 pg/mL, IL-10 1.7 pg/mL, IL-12p70 3.4 pg/mL, IL-17a 8.7 pg/mL, TNFα 9.8 pg/mL and IFN-γ 0.8 pg/mL. For statistical analysis, when a single value was non-detectable, it was considered to be half the limit of detection of that cytokine.
SBI was defined as bacteremia, meningitis, pneumonia, osteomyelitis, urinary tract infection (UTI) or any other severe infection with the identification of a plausible microorganism in a normally sterile site (only confirmed SBI were included in this group). For the diagnosis of UTI, pyuria in urine analysis was required in addition to the positive urine culture. Viral infection (VI) was defined as a positive result in the multiplex-PCR from respiratory samples. Patients with a bacterial infection and a viral detection in the respiratory sample at the same time were classified in the group of bacterial-viral coinfections and excluded from the subanalyses. Patients with no proven infection (NPI) were those not included in the previous groups.