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.