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\begin{document}

\title{INTERLEUKIN 6 AS A MARKER OF SEVERE BACTERIAL INFECTION IN CHILDREN WITH
SICKLE CELL DISEASE AND FEVER}



\author[1]{Elena Rincon-Lopez}%
\author[1]{Maria Navarro Gómez}%
\author[1]{Teresa Hernández-Sampelayo Matos}%
\author[1]{David Aguilera-Alonso}%
\author[2]{Eva Dueñas-Moreno}%
\author[1]{Jesús Saavedra-Lozano}%
\author[1]{Begoña Santiago-Garcia}%
\author[2]{Maria Del Mar Santos}%
\author[1]{Marina García-Morin}%
\author[1]{Cristina Beléndez}%
\author[2]{Jorge Lorente-Romero}%
\author[1]{ELENA CELA}%
\affil[1]{Hospital General Universitario Gregorio Maranon}%
\affil[2]{Hospital General Universitario Gregorio Marañón}%


\vspace{-1em}



  
  \date{May 28, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Introduction Etiological diagnosis of fever in sickle cell disease (SCD)
children is often challenging. Objective: to analyze the pattern of
inflammatory biomarkers in SCD febrile children and controls, in order
to determine predictors of severe bacterial infection (SBI). Methods
Prospective, case-control study of febrile and steady-state SCD children
carried out during 3 years. Clinical characteristics and laboratory
parameters, including 10 serum proinflammatory cytokines (IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-2,
IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17a, IFN-\selectlanguage{greek}γ \selectlanguage{english}and TNF-\selectlanguage{greek}α\selectlanguage{english}) and
comparisons among study subgroups were analyzed. Results A total of 137
patients (78 cases and 59 controls) were included in the study; 78.5\%
males, median age 4.1 (1.7-7.5) years. Four cases were diagnosed with
SBI, 41 viral infection (VI) and 33 no proven infection (NPI). IL-6 was
significantly higher in patients with SBI than in patients with VI or
NPI (163 vs 0.7 vs 0.7 pg/ml, p \textless{} 0.001), and undetectable in
all controls. The rest of the cytokines analyzed did not show any
significant difference. The optimal cut-off value of IL-6 for the
diagnosis of SBI was 125 pg/mL, with high PPV and NPV (PPV of 100\% for
a prevalence of 5, 10 and 15\% and NPV of 98.7\%, 97.3\% and 95.8\% for
those prevalences, respectively). Conclusion We found that IL-6 (optimal
cut-off value of 125 pg/ml) was a very good marker for SBI in this
cohort of febrile SCD children, with high PPV and NPV. Therefore, IL-6
may be useful, alone or combined with other biomarkers, to guide the
management of these patients.%
\end{abstract}%



\sloppy


\textbf{INTERLEUKIN 6 AS A MARKER OF SEVERE BACTERIAL INFECTION IN
CHILDREN WITH SICKLE CELL DISEASE AND FEVER}

Elena Mar\selectlanguage{ngerman}ía Rincón-López¹ ², MD; María Luisa Navarro Gómez¹ ³, MD, PhD;
Teresa Hernández-Sampelayo Matos¹ ³, MD, PhD; David Aguilera-Alonso¹,
Eva Dueñas Moreno¹; MD; Jesús Saavedra-Lozano¹ ³ MD, PhD; Begoña
Santiago García¹, MD, PhD; María del Mar Santos Sebastián¹, MD; Marina
García Morín\selectlanguage{english}4, MD; Cristina Bel\selectlanguage{ngerman}éndez Bieler\selectlanguage{english}4, MD; Jorge Lorente Romero5,
\selectlanguage{english}MD; Elena Cela de Juli\selectlanguage{ngerman}án³ \selectlanguage{english}4, MD, PhD on behalf of F-DREP Study Group*.

*A list of other contributors of the F-DREP Study Group and their
affiliations is provided in the Collaborators section.

\textbf{Affiliations:} \selectlanguage{ngerman}¹Pediatric Infectious Diseases Unit, Hospital
General Universitario Gregorio Marañón, Instituto de Investigación
Sanitaria Gregorio Marañón, Madrid, Spain. ²PhD Program in Medicine,
Universidad Complutense de Madrid, Spain. ³Universidad Complutense de
Madrid, Spain. \selectlanguage{english}4Pediatric Hematology and Oncology Unit, Hospital General
Universitario Gregorio Mara\selectlanguage{ngerman}ñón, Madrid, Spain. \selectlanguage{english}5Pediatric Emergency
Unit, Hospital General Universitario Gregorio Mara\selectlanguage{ngerman}ñón, Madrid, Spain.

\textbf{Address correspondence to:} Elena M. Rincón-López. Servicio de
Pediatría, Sección Enfermedades Infecciosas, Hospital Materno-Infantil
Gregorio Marañón, c/ O'Donnell, 48-50, 28009 Madrid, Spain. Phone
number: 0034 650060285. E-mail: elenarinconlopez@hotmail.com

\textbf{Word count:} Abstract: 250 words. Main text: 2738 words.

\textbf{Figures and tables:} 2 figures and 4 tables. 1 supplemental
table.

\textbf{Short title:} IL-6 in sickle cell disease.

\textbf{Key words:} Sickle cell disease, infection, fever, cytokines,
interleukin-6.

\textbf{Data availability:} The data that support the findings of this
study are available on request from the corresponding author. The data
are not publicly available due to privacy or ethical restrictions.



\textbf{Abbreviations:}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
SCD & Sickle cell disease\tabularnewline
\midrule
\endhead
SBI & Severe bacterial infection\tabularnewline
VOC & Vaso-occlusive crisis\tabularnewline
HGUGM & Hospital General Universitario Gregorio Mara\selectlanguage{ngerman}ñón\tabularnewline
CRP & C-reactive protein\tabularnewline
Multiplex-PCR & Multiplex polymerase chain reaction\tabularnewline
RSV & Respiratory syncytial virus\tabularnewline
IL & Interleukin\tabularnewline
IFN & Interferon\tabularnewline
TNF & Tumor necrosis factor\tabularnewline
UTI & Urinary tract infection\tabularnewline
VI & Viral infection\tabularnewline
NPI & No proven infection\tabularnewline
CVC & Central venous catheter\tabularnewline
ACS & Acute chest syndrome\tabularnewline
PICU & Pediatric intensive care unit\tabularnewline
IQR & Interquartile range\tabularnewline
ROC & Receiver operating characteristic\tabularnewline
AUC & Area under the ROC curve\tabularnewline
PPV & Positive predictive value\tabularnewline
NPV & Negative predictive value\tabularnewline
PR & Prevalence rate\tabularnewline
CI & Confidence interval\tabularnewline
\bottomrule
\end{longtable}

\textbf{ABSTRACT}

\textbf{Introduction}

Etiological diagnosis of fever in sickle cell disease (SCD) children is
often challenging. Objective: to analyze the pattern of inflammatory
biomarkers in SCD febrile children and controls, in order to determine
predictors of severe bacterial infection (SBI).

\textbf{Methods}

Prospective, case-control study of febrile and steady-state SCD children
carried out during 3 years. Clinical characteristics and laboratory
parameters, including 10 serum proinflammatory cytokines (IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-2,
IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17a, IFN-\selectlanguage{greek}γ \selectlanguage{english}and TNF-\selectlanguage{greek}α\selectlanguage{english}) and
comparisons among study subgroups were analyzed.

\textbf{Results}

A total of 137 patients (78 cases and 59 controls) were included in the
study; 78.5\% males, median age 4.1 (1.7-7.5) years. Four cases were
diagnosed with SBI, 41 viral infection (VI) and 33 no proven infection
(NPI). IL-6 was significantly higher in patients with SBI than in
patients with VI or NPI (163 vs 0.7 vs 0.7 pg/ml, p \textless{} 0.001),
and undetectable in all controls. The rest of the cytokines analyzed did
not show any significant difference. The optimal cut-off value of IL-6
for the diagnosis of SBI was 125 pg/mL, with high PPV and NPV (PPV of
100\% for a prevalence of 5, 10 and 15\% and NPV of 98.7\%, 97.3\% and
95.8\% for those prevalences, respectively).

\textbf{Conclusion}

We found that IL-6 (optimal cut-off value of 125 pg/ml) was a very good
marker for SBI in this cohort of febrile SCD children, with high PPV and
NPV. Therefore, IL-6 may be useful, alone or combined with other
biomarkers, to guide the management of these patients.



\textbf{INTRODUCTION}

Febrile episodes are common in children with sickle cell disease (SCD),
often with challenging etiological diagnosis. These patients are at risk
of severe bacterial infections (SBI), although they have become less
frequent in high-income countries in recent years\textsuperscript{1--5}.
Therefore, it would be important to find biomarkers that could help
distinguish the etiology of these febrile episodes early in order to
guide antimicrobial therapy. Several biomarkers {[}white blood count
(WBC), neutrophils, C-reactive protein (CRP) and procalcitonin, among
others{]} have been studied in SCD patients with bacteremia and other
bacterial infections with variable results\textsuperscript{1,6--9}.
Serum cytokines have also been studied in steady-state SCD patients and
those with vaso-occlusive crisis (VOC)\textsuperscript{10--15}, but they
have not been well studied in SCD patients with an infection.

The aim of this study was to analyze the pattern of inflammatory
biomarkers, including pro-inflammatory cytokines, in SCD children with
fever of different etiologies, in order to find useful parameters to
discriminate the patients at risk of SBI. Since SBI has become less
frequent in well-controled SCD children in recent years, this could help
to guide the management of these patients, reducing unnecessary
antibiotic treatments and hospital admissions.

\textbf{MATERIAL AND METHODS}

\textbf{Study design}

We conducted a prospective, case-control study with patients younger
than 18 years with SCD from June 2015 to June 2018. Children with SCD
and fever were eligible to participate in the study as cases and
asymptomatic steady-state SCD children were included as controls.
Exclusion criteria of cases and controls included age older than 18
years, hematopoietic stem cell transplantation, incomplete diagnostic
tests and patients whose parents or legal guardians did not sign the
informed consent form for the study. Controls were also excluded if they
had had a febrile episode in the previous month. The study received
approval from the Institutional Review Board.

\textbf{Study setting}

This study was conducted at the Hospital General Universitario Gregorio
Mara\selectlanguage{ngerman}ñón (HGUGM) in Madrid, Spain, a tertiary hospital with 120 pediatric
beds and around 7000 pediatric inpatient admissions per year. HGUGM is a
national reference center for children with SCD in Spain, with about 300
SCD pediatric patients upon follow-up.

\textbf{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\textbf{(}
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)-1\selectlanguage{greek}β, \selectlanguage{english}IL-2, IL-4,
IL-6, IL-8, IL-10, IL-12p70, IL-17a, interferon-\selectlanguage{greek}γ \selectlanguage{english}(IFN-\selectlanguage{greek}γ\selectlanguage{english}) and tumor
necrosis factor- \selectlanguage{greek}α \selectlanguage{english}(TNF-\selectlanguage{greek}α\selectlanguage{english}). The limit of detection for the different
cytokines was: IL-1\selectlanguage{greek}β \selectlanguage{english}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, TNF\selectlanguage{greek}α \selectlanguage{english}9.8 pg/mL and IFN-\selectlanguage{greek}γ \selectlanguage{english}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.

\textbf{Variables evaluated}

Data were collected in a database specifically designed for the study.
Data related to cases and controls included baseline characteristics
such as age, gender, SCD type, diagnostic method, parents' continent of
origin, immunization status (including vaccination
against\emph{Haemophilus influenzae} type b\emph{, Streptococcus
pneumoniae}and \emph{Neisseria meningitidis} serogroups A, B, C, W, Y),
penicillin prophylaxis, treatment with hydroxyurea, other treatments,
long-term central venous catheter (CVC), splenectomy and history of
previous hospital admissions. Clinical data during the episode (only in
cases) included duration of fever before admission, maximum axillary
temperature during the episode, respiratory symptoms, hemodynamic
instability (defined as hypotension or persistent tachycardia requiring
fluid resuscitation or inotropic drugs) and hypoxemia (\textless{}
92\%). Laboratory parameters included hemoglobin, WBC, neutrophils and
platelets on admission, initial and maximum CRP and procalcitonin during
the episode, blood cultures, as well as other cultures and viral tests.
Other recorded data were inpatient/outpatient management, antimicrobial
treatment, need for antibiotic change because of unfavorable clinical
course, diagnosis of VOC or acute chest syndrome (ACS), duration of
fever and of hospitalization, pediatric intensive care unit (PICU)
admission and final outcome (hospital discharge or exitus). Cytokine
levels in all cases and controls were also evaluated.

\textbf{Statistical analysis}

Qualitative variables were expressed as frequencies or rates.
Quantitative variables were reported as means (standard deviation) or
medians {[}interquartile range (IQR){]}, according to their
distribution\emph{.} The \selectlanguage{greek}χ \selectlanguage{english}test was used to compare categorical
variables; Yates correction was applied to any contingency table and
Fisher's exact test was used when at least one cell had a value
\textless{}5. T-test and ANOVA were used to compare the means of
normally distributed variables whereas Mann-Whitney U test and
Kruskal-Wallis test were performed to compare the medians of variables
not normally distributed. Receiver operating characteristics (ROC)
curves and area under the ROC curve (AUC) were calculated for
significant parameters. The optimal cut-off point of each biomarker was
selected using the Youden Index, based on the maximum sum of sensitivity
and specificity. Sensitivity, specificity, positive predictive values
(VPP) and negative predictive values (NPV) were calculated according to
various possible prevalence rates (PR) of SBI. A p value \textless{}0.05
was considered significant. Statistical analysis was performed using IBM
SPSS Statistics software version 22.0 (IBM Corp., Armonk, N.Y., USA).

\textbf{RESULTS}

A total of 137 patients were included in the study: 78 cases and 59
controls (Figure 1). Median age of the patients was 4.1 (1.7-7.5) years
for cases and 5.2 (2.1-9.3) for controls (p = 0.193), with a higher
percentage of males in the group of cases (78.5\% vs 62.1\%, p = 0.035).
The majority of the children were born in Spain and had been diagnosed
by newborn screening, while their parents were predominantly from Africa
and South/Central America. Most patients were appropriately immunized
and receiving penicillin prophylaxis, while approximately half of them
were on hydroxyurea treatment. Baseline characteristics of cases and
controls are summarized in Table 1.

Four out of 78 cases (5.1\%) were diagnosed with SBI: two of them were
catheter-related bacteremia (caused by \emph{Staphylococcus aureus}
and\emph{Enterobacter cloacae} ), one \emph{Streptococcus
pneumoniae}(serotype 9N) bacteremic pneumonia and one \emph{Escherichia
coli} UTI. Another patient was also diagnosed with \emph{Escherichia
coli} UTI, but she had a viral detection in a respiratory sample at the
same time (influenza B) and, therefore, she was considered a
bacterial-viral coinfection and excluded from the subanalyses. A virus
was detected by multiplex-PCR in 41/78 (52.6\%) of the respiratory
samples. The most frequently detected virus was influenza (A or B) in
23.3\% of the cases, followed by rhinovirus in 20.9\%, adenovirus in
14\% and enterovirus in 11.6\%. When comparing the baseline
characteristics among cases with SBI, VI and the rest of patients with
NPI (Supplemental Table 1), no statistically significant differences
were found, except for the presence of CVC and hypertransfusional
regimen, which were more frequent in the SBI group (50\% vs 9.8\% vs
36.4\%, p = 0.011 and 50\% vs 4.9\% vs 15.2\%, p = 0.018, respectively).

Data on clinical presentation, laboratory parameters and outcome during
the febrile episodes in all cases, and comparisons among groups, are
summarized in Table 2. Most patients were hospitalized (81\%) and
received at least one dose of empiric antibiotic treatment (96.2\%).
Three patients (3.8\%) required PICU admission and no patient died.
Patients with SBI had more often hemodynamic instability compared to
patients with VI or NPI (25\% vs 2.4\% vs 0, respectively; p = 0.012)
and significantly higher inflammatory parameters (neutrophils, CRP and
procalcitonin). They required more frequently a change in the antibiotic
therapy (75\% vs 7.5\% vs 15.6\%; p = 0.001) and longer hospital
admissions (7.5 vs 3 vs 5 days; p = 0.013). Upper respiratory symptoms
were more frequent in patients with VI compared to patients with SBI or
NPI (80.5\% vs 50\% vs 42.4\%, respectively; p = 0.003).

\textbf{Analysis of proinflammatory cytokine levels and other
biomarkers}

IL-6 was significantly higher in patients with SBI than in those with VI
or NPI {[}median (IQR) pg/ml: 163 (70.4-459.5) vs 0.7 (0.7-0.7) vs 0.7
(0.7-0.7), respectively; p \textless{} 0.001{]}, being undetectable in
all controls. To note, the patient with the highest level of IL-6 was
the case of confirmed bacterial pneumonia (733 pg/ml). However, in the
patient with the bacterial-viral coinfection, IL-6 was undetectable.
Figure 2 represents the values of IL-6 and other significant biomarkers
(neutrophils, CRP and PCT) of the study subgroups, including the optimal
cut-off point for each parameter calculated by the Youden index.
IL-12p70 was not detected in any of the cases or controls. The rest of
the cytokines analyzed (IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-2, IL-4, IL-8, IL-10, IL-17a, TNF\selectlanguage{greek}α,
\selectlanguage{english}IFN-\selectlanguage{greek}γ\selectlanguage{english}) did not show any significant difference between cases and
controls or among different groups of cases. No differences were found
in any of the cytokines analyzed between the cases diagnosed with VOC
and the rest of the patients. None of the cytokine levels significantly
correlated with possible confounders as the age or the number of
previous admissions.

The AUC of IL-6 to discriminate confirmed SBI was 0.87 (95\% CI
0.77-0.93), similarly to the other significant parameters {[}neutrophils
0.87 (0.78-0.94); CRP 0.89 (0.81-0.96); procalcitonin 0.84
(0.73-0.91){]}. The optimal cut-off value of IL-6 for the diagnosis of
SBI was 125 pg/mL. Data of sensibility, specificity, PPV and NPV (the
last two according to various possible PR of SBI) of IL-6 and other
significant biomarkers are represented in Table 4. IL-6 had very high
values of PPV and NPV, NPV values being similar to other biomarkers,
with significantly higher PPV values (PPV of 100\% for a PR of 5, 10 and
15\%; NPV of 98.7\%, 97.3\% and 95.8\% for a PR of 5, 10 and 15\%,
respectively).

\textbf{DISCUSSION}

In this prospective, case-control study we compared the clinical
characteristics and laboratory parameters, including 10 serum
proinflammatory cytokines, in SCD children with fever of different
etiologies and asymptomatic steady-state controls. We found that IL-6
was a very good biomarker for the diagnosis of SBI in these patients,
with similar NPV to other biomarkers and better PPV. To our knowledge,
this is the first study describing IL-6 as a marker of SBI in febrile
SCD children.

Most of the children included in this study had been diagnosed with SCD
by newborn screening, had a complete vaccination status and were
receiving penicillin prophylaxis. In the group of SCD patients with
fever, we found a low proportion of confirmed SBI (5.1\%), similarly to
other studies performed in high-income
countries\textsuperscript{1,3--5}. However, in more than a half of the
cases (52.6\%) a virus was detected by multiplex-PCR from respiratory
samples, as it has been observed in recent pediatric
studies\textsuperscript{16,17}. In the remaining 42.8\% of cases an
infectious etiology was not confirmed. The only statistically
significant differences among the baseline characteristics of study
subgroups were a higher percentage of patients with CVC and on
hypertransfusional regimen in the group of SBI, suggesting a poorer
clinical baseline condition of these patients and also highlighting the
presence of long-term CVC as a predisposing factor for
bacteremia\textsuperscript{6}.

Despite the low proportion of confirmed SBI in this cohort, almost all
patients received at least one dose of antibiotic (96.2\%), in agreement
to current guidelines\textsuperscript{18--20}. This high use of
antibiotics emphasizes the importance of defining laboratory parameters
that could early discriminate the etiology of fever in these patients,
in order to optimize the antimicrobial prescription. Children with SBI
presented more frequently with hemodynamic instability and had higher
inflammatory parameters (neutrophils, CRP and procalcitonin) than the
rest of the patients, similarly to what was described in other
studies\textsuperscript{1,5--9}. We also found that children with SBI
had significantly higher IL-6 levels when compared with those with VI,
NPI and controls, with the highest value observed in the only patient
with confirmed pneumococcal pneumonia. The optimal cut-off value of IL-6
for the diagnosis of SBI was 125 pg/ml, with a very high PPV
(significantly better than other biomarkers) and NPV applying to
different PR. In a setting with a PR of SBI of 5\% in children with SCD
(average PR in high-income countries\textsuperscript{1,4,5}), the PPV
would be 100\% and NPV 98.7\%. Therefore, we consider that IL-6 could be
a very useful marker for the diagnosis of SCD patients with confirmed
SBI.

IL-6 is an important factor in systemic and local immune response. It
can be secreted by almost all stromal cells and cells of the immune
system at an early stage of inflammation (with rapid elevation in 1-3
hours), and further promote the synthesis and release of CRP by the
liver \textsuperscript{21,22}. IL-6 has been previously described as a
marker of different types of SBI (alone or in combination with other
biomarkers)\textsuperscript{23,24}, such as early-onset neonatal
sepsis\textsuperscript{25,26}, sepsis and septic
shock\textsuperscript{21,23,27}, bacteremia in patients with febrile
neutropenia\textsuperscript{28,29} or pneumococcal
pneumonia\textsuperscript{30--33}. The early elevation of IL-6 that
occurs in the presence of SBI is an important advantage of this cytokine
over other biomarkers.

In SCD patients, it has previously been described that IL-6 and other
proinflammatory cytokines are elevated in asymptomatic steady-state
patients when compared to healthy individuals\textsuperscript{10--13}.
However, in our study all controls (steady-state SCD patients) had
undetectable levels of IL-6. A possible explanation for this could be
that almost all children included in this study were receiving vitamin D
supplements. According to the study performed by Adegoke et al.,
children with SCD had a maintained vitamin D deficiency with a high
level of proinflammatory cytokines (IL-6, IL-8 and IL-18), which
normalizes after vitamin D supplementation\textsuperscript{34}. Other
previous studies also found higher levels of IL-6 in patients with
VOC\textsuperscript{14,15}, a finding that we did not observe in our
study.

The strengths of this study were its prospective design, the homogeneity
of the patients and that it was carried out in a reference center for
patients with SCD in Spain; therefore, this cohort may be quite
representative of the pediatric population with SCD in high-income
countries. However, it has also several limitations. Most importantly,
the sample size was relatively small, with few cases of confirmed
bacterial infection, and the categorization in several study groups
might have decreased the statistical power. Blood samples were collected
regardless the previous duration of fever and serial samples were not
collected, so it was not possible to evaluate the trend over time of the
different biomarkers during the febrile episode. Finally, only confirmed
SBI were included in the study to avoid bias in the analysis, but maybe
some not-confirmed bacterial infections (e. g. pneumonia) were included
in the NPI group.

Conclusion

In this prospective study, we found that IL-6 (with an optimal cut-off
of 125 pg/ml) was a good marker of SBI with a high PPV and NPV.
Therefore, given its rapid elevation, IL-6 may be useful (alone or in
combination with other biomarkers) to early discriminate SCD children at
risk of SBI, in order to guide their management. In settings with a low
PR of SBI, its use could help to reduce unnecessary antibiotic
treatments and hospital admissions. These findings should be confirmed
in larger cohorts and multicenter studies.

\textbf{ACKNOWLEDGEMENTS}

We thank HGUGM nurses for their help in collecting samples and the
Spanish HIV HGM Biobank for storing and processing blood samples for
cytokine analysis. We also thank Ana Mar\selectlanguage{ngerman}ía Rincón-López, official
translator, for her careful review of the manuscript.

\textbf{COLLABORATORS}

\textbf{Collaborators of F-DREP Study Group:}

\begin{itemize}
\tightlist
\item
  Carmen Garrido Colino. Pediatric Hematology and Oncology Unit,
  Hospital General Universitario Gregorio Marañón, Madrid, Spain.
\item
  Jorge Huerta Aragonés. Pediatric Hematology and Oncology Unit,
  Hospital General Universitario Gregorio Marañón, Madrid, Spain.
\item
  Cristina Mata Fernández. Pediatric Hematology and Oncology Unit,
  Hospital General Universitario Gregorio Marañón, Madrid, Spain.
\item
  Concepción Míguez Navarro. Pediatric Emergency Unit, Hospital General
  Universitario Gregorio Marañón, Madrid, Spain.
\item
  Andrea Mora Capín. Pediatric Emergency Unit, Hospital General
  Universitario Gregorio Marañón, Madrid, Spain.
\item
  Rafael Marañón Pardillo. Pediatric Emergency Unit, Hospital General
  Universitario Gregorio Marañón, Madrid, Spain.
\item
  Arístides Rivas García. Pediatric Emergency Unit, Hospital General
  Universitario Gregorio Marañón, Madrid, Spain.
\item
  Paula Vázquez López. Pediatric Emergency Unit, Hospital General
  Universitario Gregorio Marañón, Madrid, Spain.
\item
  Jose Luis Jiménez Fuentes. Immunology Section, Laboratorio
  InmunoBiología Molecular, Instituto Investigación Sanitaria Gregorio
  Marañón, Hospital General Universitario Gregorio Marañón, Spanish HIV
  HGM BioBank, Madrid, Spain.
\item
  Maria Ángeles Muñoz Fernández. Immunology Section, Laboratorio
  InmunoBiología Molecular, Instituto Investigación Sanitaria Gregorio
  Marañón, Hospital General Universitario Gregorio Marañón, Spanish HIV
  HGM BioBank, Madrid, Spain.
\item
  Rosario Zamarro Arranz. Health educator of parents and children with
  sickle cell disease. Nurse in Hospital General Universitario Gregorio
  Marañón, Madrid, Spain.
\end{itemize}

\textbf{FUNDING:} This work was supported by an unrestricted ESPID
(European Society for Pediatric Infectious Diseases) Grant Award. The
sponsor did not participate in the study design or interpretation of the
results.

\textbf{CONFLICT OF INTEREST:} The authors have no conflicts of interest
or financial relationships relevant to this article to disclose.

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\textbf{LEGENDS}

\begin{itemize}
\tightlist
\item
  Figure 1. Flow diagram.
\item
  Figure 2. Biomarkers' values in the study subgroups and optimal
  cut-off points.
\item
  Table 1. Baseline characteristics of patients.
\item
  Table 2. Characteristics of cases during the febrile episode and
  differences among study subgroups.
\item
  Table 3. Cytokines values and comparisons among study subgroups.
\item
  Table 4. Diagnostic tests of IL-6 and other significant biomarkers for
  the prediction of SBI.
\item
  Supplemental table 1. Baseline characteristics of cases and
  comparisons among study subgroups.
\end{itemize}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1-Flow-diagram/Figure-1-Flow-diagram}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

\end{center}
\end{figure}

\textbf{Hosted file}

\verb`TABLE 1. Baseline characteristics of patients.docx`  available at \url{https://authorea.com/users/469331/articles/562775-interleukin-6-as-a-marker-of-severe-bacterial-infection-in-children-with-sickle-cell-disease-and-fever}

\textbf{Hosted file}

\verb`TABLE 2. Characteristics of cases during the febrile episode and differences among study subgroups.docx`  available at \url{https://authorea.com/users/469331/articles/562775-interleukin-6-as-a-marker-of-severe-bacterial-infection-in-children-with-sickle-cell-disease-and-fever}

\textbf{Hosted file}

\verb`TABLE 3. Cytokines values and comparisons among study  subgroups.docx`  available at \url{https://authorea.com/users/469331/articles/562775-interleukin-6-as-a-marker-of-severe-bacterial-infection-in-children-with-sickle-cell-disease-and-fever}

\textbf{Hosted file}

\verb`TABLE 4. Diagnostic tests of IL-6 and other significant biomarkers for the prediction of SBI.docx`  available at \url{https://authorea.com/users/469331/articles/562775-interleukin-6-as-a-marker-of-severe-bacterial-infection-in-children-with-sickle-cell-disease-and-fever}

\selectlanguage{english}
\FloatBarrier
\end{document}

