Infection
DOI 10.1007/s15010-015-0830-6
ORIGINAL PAPER
Biomarker candidates for the detection of an infectious etiology
of febrile neutropenia
Martin E. Richter1,2 · Sophie Neugebauer1,2 · Falco Engelmann1 · Stefan Hagel2,3,4 ·
Katrin Ludewig2 · Paul La Rosée5,2 · Herbert G. Sayer2,5,6 · Andreas Hochhaus2,5 ·
Marie von Lilienfeld‑Toal2,5 · Tom Bretschneider7 · Christine Pausch2,8 ·
Christoph Engel8 · Frank M. Brunkhorst9 · Michael Kiehntopf1,2 
Received: 26 May 2015 / Accepted: 31 July 2015
© Springer-Verlag Berlin Heidelberg 2015
Abstract  that differed in FNPI patients compared to patients with
Purpose  Infections and subsequent septicemia are major
complications in neutropenic patients with hematological
malignancies. Here, we identify biomarker candidates for
the early detection of an infectious origin, and monitoring
of febrile neutropenia (FN).
Methods  Proteome, metabolome, and conventional bio-
markers from 20 patients with febrile neutropenia without
proven infection (FNPI) were compared to 28 patients with
proven infection, including 17 patients with bacteremia.
Results  Three peptides (mass to charge ratio 1017.4–
1057.3; p-values 0.011–0.024), six proteins (mass to charge
ratio 6881–17,215; p-values 0.002–0.004), and six phos-
phatidylcholines (p-values 0.007–0.037) were identified
M. E. Richter and S. Neugebauer contributed equally to this
manuscript.
Electronic supplementary material  The online version of this
article (doi:10.1007/s15010-015-0830-6) contains supplementary
material, which is available to authorized users.
* Michael Kiehntopf
michael.kiehntopf@med.uni‑jena.de
1
Institut für Klinische Chemie und Laboratoriumsdiagnostik,
Universitätsklinikum Jena, Erlanger Allee 101, 07747 Jena,
Germany
2
Integriertes Forschungs‑ und Behandlungszentrum Sepsis
und Sepsisfolgen (CSCC), Universitätsklinikum Jena,
Erlanger Allee 101, 07747 Jena, Germany
3
Zentrum für Infektionsmedizin und Krankenhaushygiene,
Universitätsklinikum Jena, Erlanger Allee 101, 07747 Jena,
Germany
4
Klinik für Innere Medizin IV (Gastroenterologie,
Hepatologie, Infektiologie), Universitätsklinikum Jena,
Erlanger Allee 101, 07747 Jena, Germany
infection or bacteremia. Seven of these marker candidates
discriminated FNPI from infection at fever onset with
higher sensitivity and specificity (ROC-AUC 0.688–0.824)
than conventional biomarkers i.e., procalcitonin, C–reac-
tive protein, or interleukin–6 (ROC-AUC 0.535–0.672).
In a post hoc analysis, monitoring the time course of four
lysophosphatidylcholines, threonine, and tryptophan
allowed for discrimination of patients with or without
resolution of FN (ROC-AUC 0.648–0.919) with higher
accuracy compared to conventional markers (ROC-AUC
0.514–0.871).
Conclusions  Twenty-one promising biomarker candidates
for the early detection of an infectious origin or for moni-
toring the course of FN were found which might overcome
known shortcomings of conventional markers.
Keywords  Febrile neutropenia · Infection · Biomarkers ·
Proteome · Metabolome
5
Klinik für Innere Medizin II, Abt. Hämatologie und Intern.
Onkologie, Universitätsklinikum Jena, Erlanger Allee 101,
07747 Jena, Germany
6
Present Address: 4. Medizinische Klinik (Hämatologie
und internistische Onkologie, Hämostaseologie), HELIOS
Klinikum Erfurt, Nordhäuser Straße 74, 99089 Erfurt, Germany
7
Leibniz Institut für Naturstoff‑Forschung
und Infektionsbiologie, Hans-Knöll-Institut,
Adolf‑Reichwein‑Straße 23, 07747 Jena, Germany
8
Institut für Medizinische Informatik, Statistik und
Epidemiologie, Universität Leipzig, Härtelstraße 16‑18,
04107 Leipzig, Germany
9
Zentrum für Klinische Studien, Universitätsklinikum Jena,
Salvador‑Allende‑Platz 27, 07747 Jena, Germany
13

Introduction
Neutropenia is a major risk factor for the development
of infections, particularly in cancer patients who receive
chemotherapy, which might progress to severe sepsis/sep-
tic shock—a substantial cause for therapy-related mortal-
ity. However, fever as a first sign of an infection, justifying
early causative therapy, might be also due to non-infectious
causes, i.e., drug fever, transfusion-dependent, or para-
neoplastic fever. Notably, it was demonstrated that only
49–67 % of febrile neutropenia (FN) episodes could be
traced back to clinically suspected or microbiologically
documented infections [1, 2]. Accordingly, the identifica-
tion of this group of patients is needed for a goal-directed
therapy of FN patients.
International guidelines recommend timely administra-
tion of broad spectrum antibiotics in FN patients regard-
less of whether an infection has been proven or not. This
concept is currently under debate considering a more risk-
adapted antibiotic treatment: Patients classified as low-risk
patients might benefit from outpatient treatment and from
oral or even no application of antibiotics [3]. Advantages
of this approach may be reduced development of bacterial
resistance, reduced hospitalization, and reduction of treat-
ment costs. Traditionally, risk stratification is performed
clinically using the “Multinational Association for Support-
ive Care in Cancer” (MASCC) score, which has a high pos-
itive predictive value for identifying patients at low risk for
serious complications [4]. Nonetheless, even patients strati-
fied as low risk are treated with empirical antibiotics. In
addition to clinical indices, biomarkers have been proposed
for risk stratification of FN patients. Current studies focus
on procalcitonin (PCT) as biomarker of an infectious cause
of fever as well as treatment response [5, 6]. Moreover,
interleukin-6 (IL–6) has been reported as an early and sen-
sitive predictor of bacterial infections in both neutropenic
and non-neutropenic febrile children [7]. However, the
positive predictive value of IL–6 was low with no impact
on treatment stratification. Furthermore, it was shown that
IL–6 and IL–8 concentrations are not predictive for bacte-
remia in FN patients [8] and that the MASCC index out-
competes PCT, C–reactive protein (CRP), IL–6, and IL–8
for the prediction of fever complications and death [9].
Likewise, novel biomarkers e.g., the angiopoietin 2/angi-
opoietin 1 ratio did not improve risk stratification of FN
patients [10]. However, the key step for risk stratification
is the early recognition of patients with an infectious cause
of FN. While blood cultures remain the gold standard,
their clinical utility is limited by several factors, primarily
their low sensitivity [11]. Taken together, there is still an
urgent need for an index or biomarker that allows for fast
and reliable discrimination of infectious from non-infec-
tious causes of FN at fever onset. “Omics-technologies”,
13
M. E. Richter et al.
in particular proteomic and metabolomic approaches have
the potential to unravel new biomarker candidates in infec-
tious diseases [12–16]. Accordingly, this study aims at
finding biomarker candidates through proteomic profiling
by SELDI- and MALDI-TOF and metabolomic profiling
by LC–MS/MS for the early identification of patients with
infectious origin of FN.
Methods
Study protocol and recruitment of patients
The study (German Clinical Trials Registry
DRKS00000647) was conducted in accordance with the
Declaration of Helsinki (2008) and has been approved by
the local ethics committee (no. 2970-11/10). Hospitalized
patients older than 18 years with malignancies, and chem-
otherapy-induced neutropenia were enrolled prospectively
at the Department of Hematology and Oncology, Jena
University Hospital, from December 2010 to August 2011
after written informed consent. Remaining plasma material
from routine laboratory diagnostics was used for analysis.
After blood drawing, samples were transported via pneu-
matic tube system to the routine clinical laboratory. Fol-
lowing automated measurement (e.g., differential blood
count in EDTA whole blood), samples were centrifuged for
10 min at 2500xg and 7 min at 4302xg for collection of
EDTA- and Lithium-heparin plasma, respectively. Plasma
was aliquoted and stored at −80 °C until analysis. Drugs
and blood products, which have been administered within
24 h prior to blood sampling were documented alongside
with demographic and clinical data. Only the patients first
neutropenic episode was analyzed.
Clinical classifications
Neutropenia was defined as an absolute neutrophil count
(ANC) in the peripheral blood of <500/mm3 or an ANC that
is expected to decrease to <500/mm3 during the next 48 h.
Fever was defined as a single ear temperature (tympanic
membrane) measurement of ≥38.3 °C, or a temperature of
≥38.0 °C sustained for a 1-h period [17]. Medical treatment
was at the discretion of the attending physician. All patients
with fever underwent a physical examination and blood
samples were drawn for routine laboratory analysis and bac-
terial and fungal cultures. Subsequently, patients received
broad spectrum i.v. antibiotics. In addition, cultures from
other sites (urine, sputum, feces, wound smears) and radio-
logical evaluation were performed when indicated. MASCC
risk index was calculated at the onset of FN [4].
Infections were defined as microbiologically docu-
mented if a pathogen had been isolated. A bacteremia
Biomarker candidates for the detection of an infectious etiology of febrile neutropenia
with common skin contaminants (e.g., Coagulase-negative
staphylococci) was diagnosed when at least two blood cul-
tures drawn on separate occasions were positive. A clini-
cally suspected infection was evidenced by one or more of
the following: systemic inflammatory response syndrome,
cough, new or increased sputum production, dysuria or
local signs or symptoms of skin infection like pain, tender-
ness or localized swelling. A febrile episode without micro-
biologically or clinically proven focus of infection was
considered febrile neutropenia without proven infection
(FNPI). To minimize reporting bias, the final clinical evalu-
ation was performed by a clinical infectious disease con-
sultant, who was not the attending physician. Severe sepsis
and septic shock were diagnosed according to the definition
of the German Sepsis Society [18].
Proteome analysis
Since it has been shown that sample handling, particu-
larly during blood coagulation, leads to variation in the
concentration of putative peptidome cancer biomarkers
[19], special care was taken to standardize the coagula-
tion process. Hence, coagulation was artificially induced
in EDTA plasma to reduce pre-analytical variation in the
heterogeneous clinical environment and to ensure repro-
ducible clotting of blood samples. Briefly, coagulation was
induced by the addition of Ca2+ to a final concentration of
6.67 mM. Coagulation was stopped after 2 h and the clot
was discarded after centrifugation. In one approach, pro-
teins originating from the supernatant were denatured and
analyzed by SELDI-TOF on an anion exchange surface.
Secondly, peptides were extracted from the supernatant
and desalted by solid phase extraction (SPE) on C18 mate-
rial before analysis with matrix-assisted laser desorption
ionization time-of-flight (MALDI-TOF) as well as normal
phase SELDI-TOF. After calibration and normalization of
the spectra, peak intensities were extracted and clustered to
afford intensity matrices.
Metabolome analysis
186 metabolites were quantified in Lithium-heparin plasma
using the AbsoluteIDQ™ kit p180 (Biocrates Life Science
AG, Innsbruck, Austria) as described previously [16].
Determination of conventional biomarkers
PCT, CRP, and IL–6 were measured using Lithium-hepa-
rin plasma. PCT was determined using a fully automated
sensitive KRYPTOR Random Access Analyser (Brahms,
Hennigsdorf, Germany) according to the manufactur-
er’s recommendations. The immunoassay CRP Vario®
from Abbott (Ludwigshafen, Germany) was used for the
measurement of CRP concentrations. IL–6 levels were
determined with the chemoluminescence immunoas-
say IMMULITE® from Siemens Healthcare Diagnostics
(Eschborn, Germany).
Statistical analyses
We focused on two comparisons, based on different subsets
of the study population: Patients with febrile neutropenia
without proven infection (FNPI group) were compared to
patients with fever due to infection (infection group) and to
patients with fever due to an infection with positive blood
culture (bacteremia group, a subgroup of infection group).
For each comparison, three time points were considered:
fever onset ±5 h, day 2 after fever onset and day 4 after
fever onset. We did not account for multiple testing, as the
study has an explorative character. p-values below 0.05 are
referred to as statistically significant in the following.
All statistical tests were performed using R [20]. Val-
ues of the MASCC score, CRP, PCT, and IL-6 were com-
pared by the Wilcoxon rank sum tests in the two groups of
patients and at the three time points each, as normal dis-
tribution cannot be assumed. For each metabolite concen-
tration, SELDI and MALDI protein/peptide-signal inten-
sity, we fitted a separate logistic regression model adjusted
for age, sex, and the presence of acute myeloid leukemia
(AML). Metabolites and peptides/proteins were considered
as specific for an early phase of infection, when significant
differences were found at fever onset; specificity for late
stage was defined when significant differences were found
2 and 4 days after fever onset.
Marker candidates were also checked by Wilcoxon
rank sum tests for different occurrences in groups when
patients were divided according to (a) their fever status
4 days after initial onset of FN (temperature >38.0 °C: yes
or no) and (b) the presence of an afebrile period for five
consecutive days beginning latest on day four after initial
onset of FN.
Changes in the concentrations of standard laboratory
parameters and metabolites from day 1 to day 2 (d1d2), day
1 to day 4 (d1d4), and day 2 to day 4 (d2d4) after initial
onset of FN were calculated. For each, a separate logistic
regression model adjusted for age, sex, and presence of
AML was fitted, using the occurrence of a stable afebrile
period as dependent variable.
Receiver operating characteristics (ROC) and accord-
ingly area under curves (AUC) were calculated using IBM®
SPSS® Statistics 19 (IBM, New Armonk, NY, USA). Box
plots were generated according to standard definitions:
bold lines depict median values, boxes interquartile ranges
(IQR) and whiskers extreme values within the 2.5-fold IQR
around the median; values outside this range are marked as
outliers [21].
13

Results
Study population characteristics
Eighty-one patients (52 male, median age 54 years; range
21–73 years) with chemotherapy-induced or disease-asso-
ciated neutropenia were enrolled in the study. 33 patients
(40.7 %) had AML, 25 (30.9 %) multiple myeloma, 17
(21.0 %) lymphoma, 3 (3.7 %) acute lymphoblastic leu-
kemia (ALL), and 3 (3.7 %) other indications for chemo-
therapy (germ cell carcinoma, chronic myeloproliferative
neoplasm, and severe aplastic anemia). The median Charl-
son comorbidity index [22] was 4 (range 2–9). Two patients
died during hospitalization, one due to refractory septic
shock, and one due to the underlying hematological disease
(Table 1).
Forty-eight patients had at least one episode of febrile
neutropenia (FN), among them, 28 (58.3 %) were caused
by infection. Infections were documented clinically and
microbiologically in 8 (16.7 % of FN) and 20 (41.7 %
of FN) cases, respectively. Primary bacteremia was
observed predominantly in 14 cases (29.2 % of FN), fol-
lowed by pneumonia (8 cases, 16.7 % of FN), catheter
and wound infections (each 2 cases or 4.2 % of FN).
One patient (2.1 % of FN) developed a Clostridium dif-
ficile colitis and one patient (2.1 % of FN) presented a
BK virus that caused urinary tract infection. Detected
microorganisms in blood cultures were Staphylococ-
cus epidermidis (12 cases), Escherichia coli (2 cases),
Enterococcus faecalis (1 case), Staphylococcus haemo-
lyticus (1 case), Streptococcus oralis (1 case), and Pro-
teus mirabilis (1 case). In two patients, the infection
progressed to severe sepsis/septic shock: one clinically
documented pneumonia and one bacteremia caused by
Staphylococcus epidermidis. The remaining 20 febrile
episodes (41.7 % of FN) were febrile neutropenia with-
out proven infection (FNPI) including four cases (8.3 %
of FN) of non-microbial fever caused by preceding high-
dose cytarabine application (>500 mg/m2). Moreover,
ten patients with FNPI received cellular blood products
at fever onset, which are also known to cause elevated
temperatures in rare cases.
MASCC index and conventional biomarkers
MASCC scores at fever onset show a large overlap between
the patient groups investigated (Fig. 1a). However, sig-
nificant differences were observed between patients with
FNPI and those with infection (p  = 0.027), or patients
with bacteremia (p  = 0.013). For CRP, significant differ-
ences between patients with FNPI and patients with infec-
tion were found 2 days after fever onset (p  = 0.017) and
4 days after fever onset (p  = 0.016) (Fig. 1b); PCT was
13
M. E. Richter et al.
different 4 days after fever onset (p = 0.041) (Fig. 1c), and
IL–6 concentrations 2 days after fever onset (p  = 0.001)
and 4 days after fever onset (p = 0.029) (Fig. 1d). The only
significant difference between patients with FNPI and those
with bacteremia was observed for IL–6 2 days after fever
onset (p = 0.004). Remarkably, no significant difference of
either conventional biomarker could be found at fever onset
(Online Resource 1).
Marker candidates for fever origin
Proteome analysis detected a total of 717 unique peptide/
protein signals. In addition, 186 metabolites were quanti-
fied. According to the separate logistic regression analy-
ses for each signal corrected for age, sex, and the presence
of AML, 58 candidates (25 peptides/proteins (20 SELDI
signals, 5 MALDI signals) and 33 metabolites) were dis-
covered as potential biomarkers. 34 marker candidates (9
peptides/proteins (7 SELDI signals, 2 MALDI signals) and
25 metabolites) showed statistically significant differences
between patients with FNPI and bacteremia or infection at
an early stage (fever onset) and 26 marker candidates (16
peptides/proteins (13 SELDI signals, 3 MALDI signals)
and 10 metabolites) at a late stage (2 and 4 days after fever
onset). Two metabolites were identified as differentially
expressed in both early and late stage. Signals of biomarker
candidates did not correlate with the application of blood
products as well as therapeutic drugs.
Most of the 25 SELDI and MALDI signals showed
lower intensities in the infection and/or bacteremia group
compared to the FNPI group. The intensity of only two
SELDI signals (mass to charge ratio (m/z) 11,449 Da, m/z
11,603 Da) and one MALDI signal (m/z 6039.4 Da) was
higher in the infection and/or bacteremia group (Online
Resource 2). Metabolite changes were observed in five
substance classes (Online Resource 3); long-chain acylcar-
nitine C18, methionine sulfoxide, arginine, citrulline, gly-
cine, and proline increased in patients with FNPI compared
to patients with infection or bacteremia at a late stage and
glycine also at fever onset. Changes were also observed for
biogenic amines (acetylornithine, kynurenine) and for 17
glycerophospholipids, 4 sphingolipids that demonstrated
higher concentrations in patients with infection or bac-
teremia at fever onset. In contrast, concentrations of two
lysophosphatidylcholines and one acyl alkyl glycerophos-
pholipid decreased at a later stage.
For further validation of the discriminatory potential of
biomarkers for differentiation of FNPI from infection/bac-
teremia, 15 top candidates with the lowest p-value were
selected out of the 58 candidates initially found, compris-
ing three SELDI signals as well as six metabolites and
six SELDI signals at fever onset and a later stage of the
febrile episode, respectively (Fig. 2). ROC-AUC values
Biomarker candidates for the detection of an infectious etiology of febrile neutropenia

Table 1  Patients’ Group Total (n = 81) FNPI (n = 20) Infection (n = 28) Bacteremiaa (n = 17)
characteristics
Age, median [IQR], year 54 (47–60) 50.5 (39–56) 54.5 (39–61) 54 (47–63)
Male sex, n (%) 52 (64.2 %) 12 (60.0 %) 17 (60.7 %) 12 (70.6 %)
Diagnosis, n (%)
 Acute myeloid leukemia 33 (40.7 %) 12 (60.0 %) 18 (64.3 %) 8 (47.1 %)
 Acute lymphoblastic leukemia 3 (3.7 %) 0 (0.0 %) 1 (3.6 %) 1 (5.9 %)
 B-cell malignancies 42 (51.9 %) 6 (30.0 %) 9 (32.1 %) 8 (47.1 %)
  Hodgkin lymphoma 3 (3.7 %) 0 (0.0 %) 1 (3.6 %) 1 (5.9 %)
  Follicular lymphoma 3 (3.7 %) 1 (5.0 %) 1 (3.6 %) 1 (5.9 %)
  Multiple myeloma 25 (30.9 %) 2 (10.0 %) 4 (14.3 %) 4 (23.5 %)
 Otherb 3 (3.7 %) 2 (10.0 %) 0 (0.0 %) 0 (0.0 %)
Comorbidities, n (%)
 Arterial hypertension 30 (37.0 %) 8 (40.0 %) 12 (42.9 %) 8 (47.1 %)
 Diabetes mellitus (DM) 9 (11.1 %) 1 (5.0 %) 5 (17.9 %) 3 (17.6 %)
  DM (Insulin dependent) 3 (3.7 %) 0 (0.0 %) 3 (10.7 %) 2 (11.8 %)
 Chronic renal disease 6 (7.4 %) 0 (0.0 %) 1 (3.6 %) 1 (5.9 %)
  Renal replacement therapy 1 (1.2 %) 0 (0.0 %) 0 (0.0 %) 0 (0.0 %)
 Chronic pulmonary disease 5 (6.2 %) 0 (0.0 %) 1 (3.6 %) 1 (5.9 %)
ECOG performance status, n (%)
 Grade 0 10 (12.3 %) 4 (20.0 %) 4 (14.3 %) 2 (11.8 %)
 Grade 1 65 (80.2 %) 14 (70.0 %) 20 (71.4 %) 14 (82.4 %)
 Grade 2 4 (4.9 %) 2 (10.0 %) 2 (7.1 %) 0 (0.0 %)
 Grade 3 1 (1.2 %) 0 (0.0 %) 1 (3.6 %) 1 (5.9 %)
 Grade 4 1 (1.2 %) 0 (0.0 %) 1 (3.6 %) 0 (0.0 %)
Therapeutic characteristics, n (%)
 Conventional chemotherapy 40 (49.4 %) 15 (75.0 %) 14 (50.0 %) 6 (35.3 %)
 Stem cell transplantation 41 (50.6 %) 4 (20.0 %) 14 (50.0 %) 11 (64.7 %)
  Autologous 29 (35.8 %) 2 (10.0 %) 8 (28.6 %) 7 (41.2 %)
  Allogeneic 12 (14.8 %) 2 (10.0 %) 6 (21.4 %) 4 (23.5 %)
   Incl. irradiation 6 (7.4 %) 0 (0.0 %) 3 (10.7 %) 2 (11.8 %)
Duration of neutropenia, n (%)
 <7 days 12 (14.8 %) 3 (15.0 %) 0 (0.0 %) 0 (0.0 %)
 ≥7 days 69 (85.2 %) 17 (85.0 %) 28 (100.0 %) 17 (100.0 %)
a
  Patients with bacteremia are a subgroup of patients with infection
b
  Other diagnoses are: germ cell carcinoma, chronic myeloproliferative neoplasm, and severe aplastic ane-
mia

were calculated for these markers and compared to stand-
ard laboratory parameters. Conventional biomarkers per-
formed inferior at fever onset, with the highest ROC-AUC
value of 0.672 for IL–6, whereas all three peptides and
four of the six metabolites among the new marker candi-
dates showed higher ROC-AUC values, with a maximum
of 0.824 for phosphatidylcholine PC aa C34:1 (Fig. 3a,
Online Resource 4). However, this difference did not yet
reach significance (p  = 0.107) according to Hanley et al.
[23]—likely due to small case numbers. At later time
points the differences in ROC-AUC were less substantial,
e.g., 2 days after fever onset, only two of the six protein
marker candidates at m/z 13,812 (ROC-AUC = 0.802) and
m/z 13,937 (ROC-AUC = 0.798) showed equal discrimi-
natory potential, compared to IL–6 with the highest value
(ROC-AUC  = 0.791) among the conventional biomarkers
(Fig. 3b, Online Resource 4).
Marker candidates for defervescence
In addition to the primary goal of the study, we asked the
clinically relevant question whether candidate biomarkers
could be used for the prediction of defervescence. Defer-
vescence was defined as temperature <38 °C 4 days after
fever onset. Remarkably, six of the nine peptide-/protein-
based marker candidates were significantly different

13
 M. E. Richter et al.

Fig.  1  a “Multinational Asso-

a MASCC score b CRP (µg/mL)
ciation for Supportive Care in
* fever onset +2d +4d
Cancer” (MASCC) score at 25 * 600
fever onset and concentrations * *
of b C–reactive protein (CRP),
c procalcitonine (PCT), and d 20
400
interleukin (IL)–6 in patients
with febrile neutropenia without 15
proven infection (“FNPI”), 200
fever caused by infection
(“infection”) and fever caused 10
by bacteremia (“bacteremia”) 0
at the time points of fever

infection

infection

infection

infection
bacteremia

bacteremia

bacteremia

bacteremia
FNPI

FNPI

FNPI

FNPI
onset, 2 days after fever onset
(“+2d”) and 4 days after fever
onset (“+4d”). Significance
levels are denoted with asterisks
(*p < 0.05; **p < 0.01). Note
the logarithmic scale of PCT c PCT (ng/mL) d IL−6 (pg/mL)
(c) and IL–6 concentrations (d). fever onset +2d +4d fever onset +2d +4d
For detailed values see Online 10 1000 **
Resource 1 * ** *

100
1
10
0.1
1
infection

infection

infection

infection

infection

infection
bacteremia

bacteremia

bacteremia

bacteremia

bacteremia

bacteremia
FNPI

FNPI

FNPI

FNPI

FNPI

in patients with/w.o. defervescence (p < 0.05) (Online
Resource 5). However, from a more clinical point of view,
it might be of interest whether candidate biomarkers could
be used for the prediction of a stable afebrile period for
more than 5 days, starting no later than 4 days after fever
onset. In this setting it might be more informative to ana-
lyze the changes of biomarker concentrations over time
rather than absolute values. Thus, we calculated the differ-
ences of candidate markers from day 1 to day 2 (d1d2),
day 1 to day 4 (d1d4), and day 2 to day 4 (d2d4) after
fever onset. Each of them was used as independent varia-
ble to predict a stable afebrile period (dependent variable),
using logistic regression models adjusted for age, sex, and
presence of AML. Of interest, none of the changes of the
standard laboratory parameters PCT, CRP, and IL–6 were
associated with a stable afebrile period (data not shown).
In contrast, the differences—both d1d4 and d2d4—of
four lysophosphatidylcholines (lysoPC a C16:1, lysoPC a
C17:0, lysoPC a C18:1, lysoPC a C20:3) and the amino
acids threonine and tryptophan showed statistically signifi-
cant changes in the regression models (Online Resource 6).
Patients with a subsequent afebrile period showed a higher
increase of lysophosphatidylcholines (d1d4 and d2d4)
FNPI
compared to patients without a stable afebrile period,
while threonine and tryptophan increased in patients with
an afebrile period, and decreased in patients with no reso-
lution of FN (Fig. 4). ROC analyses showed highest AUC
values from day 1 to day 4 after fever onset (lysoPC a
C17:0 AUC = 0.895 and tryptophan AUC = 0.919, Online
Resource 6, Fig. 3c, d).
Discussion
Infections and subsequent septicemia remain a major risk
for neutropenic patients. At fever onset, discrimination of
infectious from non-infectious causes of inflammation is a
prerequisite for early goal-directed therapy, stratification of
patients at risk, and prognosis for the development of seri-
ous complications.
In our patient cohort, fever was observed in about 60 %
of neutropenic episodes. 42 % of these episodes were
classified as FNPI, whereas 58 % were caused by infec-
tion. This is in accordance with similar studies describing
FNPI rates of 33–40 % [1, 2, 24]. In 35 % infections were
proven by blood culture, identifying coagulase-negative

13
Biomarker candidates for the detection of an infectious etiology of febrile neutropenia

Fig. 2  Selected top candi- a m/z 1017.4 m/z 1042.3 m/z 1057.3

date markers in patients with
infectious fever normalized to
patients with febrile neutrope- 1.0 1.0 1.0
nia without proven infection
(FNPI), a at fever onset and
b 2 days after fever onset.
Intensity of SELDI signals
0.1
[defined by mass to charge ratio 0.1 0.1
(m/z)] (a top row, and b) and
FNPI infection FNPI infection FNPI infection
concentrations of metabolites (a
central and bottom row) were PC aa C34:1 PC aa C34:2 PC aa C36:3
normalized to FNPI group and ● ●
log-scale plotted. aa diacyl; ae
acyl-alkyl, Cx:y where x is the ● 1.0
number of carbons in the fatty 1.0 ● 1.0
acid side chain; y is the number
of double bonds in the fatty acid
side chain, PC phosphatidyl-
choline 0.1 0.1 0.1
FNPI infection FNPI infection FNPI infection
PC aa C36:4 PC aa C42:2 PC ae C34:1
●
1.0 1.0 1.0

0.1 0.1 0.1

FNPI infection FNPI infection FNPI infection

b m/z 6881.2 m/z 11603 m/z 13812

1.0
1.0
1.0
●

0.1 0.1 0.1

FNPI infection FNPI infection FNPI infection
m/z 13937 m/z 14397 m/z 17215

1.0 1.0 1.0

0.1 0.1 0.1

FNPI infection FNPI infection FNPI infection

staphylococci and Escherichia coli in most of the cases (76
and 12 %, respectively). This is comparable to former stud-
ies also identifying coagulase-negative staphylococci as the
most frequent causative microorganism [1, 25, 26].
By comparing patients with FNPI and infection or bacte-
remia univariate statistical analyses showed only moderate
differences for conventional markers. In previous studies,
CRP has been shown to be predictive for the development
of fever in neutropenic patients and of severe sepsis in FN
patients [27, 28]. However, in accordance with our results
CRP reached no significance when comparing bacteremic
and non-bacteremic febrile episodes on day 2 after fever

13
 M. E. Richter et al.

Fig. 3  Receiver operating a 1.0 b 1.0

characteristic (ROC) curves
for patient classification using
selected markers and standard 0.8 0.8
laboratory parameters to dis-
criminate patients with febrile

Sensitivity

Sensitivity
neutropenia without proven 0.6 0.6
infection from those with infec-
tion at fever onset a and 2 days 0.4 0.4
after fever onset b or using
changes of metabolites and Variable Area Variable Area
standard laboratory parameters 0.2 PC aa C34:1 0.824 0.2 m/z 13812 0.784
CRP 0.535 CRP 0.713
from day 1 to 4 c and day 2 to 4 PCT 0.635 PCT 0.620
IL-6 0.672 IL-6 0.791
after fever onset d to recognize Reference Reference
patients with a subsequent
0 0
stable afebrile period. See also 0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0
Online Resources 4 and 6. aa 1-specificity 1-specificity
diacyl, Cx:y where x is the
number of carbons in the fatty c 1.0 d 1.0
acid side chain; y is the number
of double bonds in the fatty 0.8 0.8
acid side chain, CRP C-reactive
protein, IL interleukin, PC
Sensitivity

Sensitivity
phosphatidylcholine, PCT 0.6 0.6
procalcitonin
0.4 0.4

Variable Area Variable Area

0.2 Tryptophan 0.919 0.2 Threonine 0.725
CRP 0.871 CRP 0.757
PCT 0.732 PCT 0.532
IL-6 0.612 IL-6 0.514
0 Reference 0 Reference

0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0
1-specificity 1-specificity

onset [1]. Aimoto et al. detected elevated levels of PCT in
FN patients with septic shock compared to those with FNPI
[29]. However, this study observed no differences between
patients with FNPI and bacteremia or local infection.
Moreover, findings are comparable to our results, since dif-
ferences were observed only within 3 days after the onset
of FN, limiting usability of PCT for timely risk stratifica-
tion in FN patients. In our study, IL–6 showed moderate
potential (AUC = 0.791) for the discrimination of FNPI
from infection 2 days after fever onset, which is in contrast
to the results of Buyukberger and colleagues who found
that IL–6 was not suitable for the prediction of bacteremia.
However, 14 of the 22 patients of that study suffered from
solid tumors as underlying disease making direct compari-
son with our results questionable [8].
To identify reliable biomarkers in FN for discrimina-
tion of FNPI from infection/bacteremia, logistic regression
models were calculated resulting in 58 possible biomarker
candidates. As this pilot study aimed at generating hypoth-
eses and marker candidates that of course have to be vali-
dated in further studies, we ranked and selected the poten-
tial marker candidates according to their p-values at fever
onset and 2 or 4 days after fever onset. Interestingly, the
top candidates for discrimination at fever onset comprise
exclusively lipid metabolites and small peptides, whereas
markers for discrimination at day 2 or 4 solely include
protein signals in the m/z-range of 6.8–17.2 kDa. Eight of
the nine peptide/protein marker presented higher intensi-
ties in FNPI patients and three of them in the m/z-range
of 1.017–1.057 kDa performed better (AUC = 0.81–0.76)
than conventional laboratory markers (AUC = 0.53–0.67)
in identifying patients with infection at fever onset (Online
Resource 4). More strikingly, the peptide at 1.057 kDa
was also different at fever onset comparing patients with
and without defervescence at day 4 (Online Resource 5),
underlining the prognostic potential of this peptide. This
might reflect an increased proteolytic activity in patients
with infection that we have previously demonstrated for
e.g., Alpha-1 antitrypsin (AAT) in patients with severe
sepsis/septic shock [15]. Proteases released by a variety of
relevant pathogens such as staphylococci [30–32], gram-
negative bacteria [33–35], and Candida albicans [36] have
been shown to interact with the host’s immune or hemo-
static systems. Pathogen-secreted proteases might therefore
contribute to the formation of infection-specific peptide
signals.
In addition to altered protein expression, meta-
bolic changes have been observed that might reflect the

13
Biomarker candidates for the detection of an infectious etiology of febrile neutropenia

Fig. 4  Changes in concentra- d1d4 d2d4 d1d4 d2d4

tions (given as µmol/L) of four 75

L ys oP C a C 17:0
L ys oP C a C 16:1
* * ** *
lysophosphatidyl-cholines, ● 1.0 ●
threonine, and tryptophan in 50
patients with or without a subse- 0.5
25
quent afebrile period (“ap”, “no ●
ap”, resp.) between day 1 and 0 ● 0.0
day 4 (“d1d4”) and day 2 and ●
day 4 (“d2d4”) after initial onset −25
of FN. Significance levels are ap no ap ap no ap ap no ap ap no ap
denoted with asterisks (p-values
according to logistic regression d1d4 d2d4 d1d4 d2d4

L ys oP C a C 18:1

L ys oP C a C 20:3
analyses *p < 0.05; **p < 0.01). * * * *
20 3
Lysophosphatidylcholines ● ● ● ●
showed a higher increase in ● 2 ●
patients with a stable afebrile 10 ● ●
●
1
period than in those without ●
(upper four panels). Threonine 0 0 ●
● ● ●
and tryptophan (lower two pan- −1
els) increased in patients with ap no ap ap no ap ap no ap ap no ap
subsequent fever resolution and
decreased or remained static in
patients with persisting elevated
d1d4 d2d4 d1d4 d2d4
* ** 40 ** *
temperatures. See also Online

Tryptophan
●
Threonine

Resource 6. a acyl, Cx:y where 50 20 ●

x is the number of carbons in ●
●
the fatty acid side chain, y is the
0 0
●
number of double bonds in the ●
fatty acid side chain, lysoPC:
● −20
lysophosphatidylcholine −50
ap no ap ap no ap ap no ap ap no ap

pathophysiology of inflammation [37–39] e.g., we have
already shown that alterations in lipid metabolism can be
used for stratification of patients with non-infectious sys-
temic inflammation from patients with sepsis [16]. In the
present study, glycerophospholipids and sphingolipids
increased in infection or bacteremia at fever onset com-
pared to patients with FNPI confirming our former data in
the patient cohort mentioned above. Cytokines, particularly
IL–1β, stimulate hepatic ceramide and sphingomyelin syn-
thesis [40]. Increased sphingolipids and glycerophospho-
lipids as major components of lipoproteins might also be
explained by highly elevated LDL and HDL [41] and their
enrichment in sphingolipids during inflammation [42].
Moreover, they play a crucial role in host defense during
endotoxemia by binding and neutralizing lipopolysaccha-
ride (LPS) [43, 44]. Remarkably, all six metabolites that
were selected as top candidate markers belong to the group
of glycerophospholipids.
Moreover, lysophosphatidylcholines decreased in
patients with infection compared to patients with FNPI
at day 2 and 4 after fever onset. Notably, patients with a
subsequent stable resolution of fever exhibited a higher
increase in lysophosphatidylcholine concentrations than in
patients with ongoing fever (Fig. 4). This is in accordance
with previous studies, showing that low lysophosphatidyl-
choline levels are associated with a poor outcome in sepsis
patients [45]. The enhanced conversion to lysophosphatidic
acid by plasmatic lysophospholipase D or the exertion of
immune-suppressive function by binding to immune-regu-
latory receptor G2A might contribute to low lysophosphati-
dylcholine levels [45, 46].
Furthermore, we observed that concentrations of argi-
nine, citrulline, glycine, and proline decreased in patients
with infection 2 and 4 days after fever onset. This is in
accordance with former studies in patients with sepsis as
well as corresponding animal models [47, 48]. Arginine,
a key substrate in NO-metabolism, plays a crucial role in
microcirculatory control, immune defense, wound heal-
ing, and energy balance as well as cell growth and differ-
entiation [49]. During inflammation, endogenous de novo
production from citrulline and food intake are reduced.
Additionally, arginine catabolism is markedly increased
by enhanced use of arginine in the urea and citrulline-NO
cycle [50].
Further focusing on prognostic markers for resolution of
fever we have shown that the essential amino acids thre-
onine and tryptophan increased from day 1 to day 4 after
fever onset in patients with stable defervescence compared
to patients who remained febrile (ROC-AUC = 0.919 and
0.789 for tryptophan and threonine, respectively; Fig. 4).
Pro-inflammatory signals such as interferons, LPS, and
tumor necrosis factor–α result in an increased expression

13

of the indoleamine 2,3-dioxygenase 1, thus leading to
decreased tryptophan concentrations at the site of inflam-
mation. Decrease of tryptophan and concomitant increase
of tryptophan degradation products are known to modu-
late innate and adaptive immunity in multiple ways [51].
It seems plausible that a sustainably resolved inflammatory
reaction, reflected by defervescence, can be recognized by
increased tryptophan concentrations, which is in line with
our results.
However, this explorative study has some limitations.
First, due to the relatively small number of patients it was
not possible to validate our findings in a separate independ-
ent test cohort. Nonetheless, this novel approach compris-
ing a combination of proteomics and metabolomics might
give rise to new diagnostic tools for the highly demanded
timely detection of fever of infectious etiology. This would
require confirmation of our results in a preferentially pro-
spective patient cohort. Second, although we included
several covariates of patient characteristics (age, sex, and
the presence of AML) in our logistic regression models,
excluded correlation of medications and candidate mark-
ers and minimized reporting bias, we cannot exclude other
confounders typical for observational studies, such as nutri-
tion and comorbidities, which may result in bias difficult
to approximate. Third, proteome analysis was based on
peak intensities only, without the identification of underly-
ing proteins of highly significant peptide signals. Finally,
since FNPI is still a diagnosis of exclusion, patients might
be misclassified.
Conclusions
This proof-of-principle study identified promising metab-
olomic as well as proteomic biomarker candidates with
higher diagnostic capacity compared to conventional bio-
markers, for detection of infections and/or prognosis of
defervescence in patients with FN, using untargeted as
well as a targeted mass spectrometry. This may help to gain
deeper insights into the complex biological processes of
inflammation and more importantly may facilitate effective
stratification of patients with FN. This will ultimately allow
for timely initiation of early goal-directed therapy and thus
improvement of clinical outcome.
Acknowledgments  We wish to thank Viola Bahr, Cora Richert,
Julia Köhler, Kay Stötzer, Sandra Wacker, Andrea Naumann, Heike
Simon, and Sandra Pöschmann for technical assistance. This study
was supported by the Federal Ministry of Education and Research
(BMBF), Germany, FKZ: 01EO1002. The project was further sup-
ported by the Paul-Martini-Sepsis Research Group, funded by the
Thuringian Ministry of Education, Science and Culture (ProExcel-
lence; grant PE 108-2); the public funded Thuringian Foundation for
Technology, Innovation and Research (STIFT), and the German Sep-
sis Society (GSS).
13
M. E. Richter et al.
Compliance with ethical standards 
Conflict of interest  The authors declare that they have no conflict
of interest.
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