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Discovery and Characterization of Potential

Prognostic Biomarkers for Dengue Hemorrhagic
Fever

Article in The American journal of tropical medicine and hygiene October 2014
DOI: 10.4269/ajtmh.14-0193 Source: PubMed

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Am. J. Trop. Med. Hyg., 91(6), 2014, pp. 12181226
doi:10.4269/ajtmh.14-0193
Copyright 2014 by The American Society of Tropical Medicine and Hygiene

Discovery and Characterization of Potential Prognostic Biomarkers

for Dengue Hemorrhagic Fever
B. Katherine Poole-Smith,* Alexa Gilbert, Andrea L. Gonzalez, Manuela Beltran, Kay M. Tomashek, Brian J. Ward,
Elizabeth A. Hunsperger, and Momar Ndao
Division of Vector-Borne Diseases, Dengue Branch, Centers for Disease Control and Prevention, San Juan, Puerto Rico; National Reference Centre
for Parasitology, Research Institute of the McGill University Health Centre, Montreal General Hospital R3-137, Montreal, Quebec H3G 1A4,
Canada; 3FQRNT Centre for HostParasite Interactions, McGill University, Montreal, Quebec H3G 1A4, Canada

Abstract. Half a million patients are hospitalized with severe dengue every year, many of whom would die without
timely, appropriate clinical intervention. The majority of dengue cases are uncomplicated; however, 25% progress to
severe dengue. Severe dengue cases have been reported with increasing frequency over the last 30 years. To discover
biomarkers for severe dengue, we used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry
to analyze dengue virus positive serum samples from the acute phase of infection. Using this method, 16 proteins were
identified as candidate biomarkers for severe dengue. From these 16 biomarkers, three candidates were selected for
confirmation by enzyme-linked immunosorbent assay and Western blot: vitronectin (Vtn, 55.1 kDa), hemopexin (Hx,
52.4 kDa), and serotransferrin (Tf, 79.2 kDa). Vitronectin, Hx, and Tf best differentiated between dengue and
severe dengue.

INTRODUCTION tionation and proteomic analysis using the surface-enhanced

Dengue is caused by infection with any one of the four
mosquito-borne dengue viruses (family Flaviviridae, genus
Flavivirus; DENV14). Identifying patients with dengue early
in the course of illness can be challenging; initial symptoms
are often non-specific (fever, headache, retro-orbital pain,
and malaise) and may mimic other febrile illnesses such as
malaria, leptospirosis, and influenza. Dengue patients have a
25% risk of advancing to severe dengue, which manifests at
35 days post onset (DPO) of fever as a rapid drop in blood
platelets, and fluid accumulation or hemorrhaging.1 Currently,
there are no commercially available vaccines for prevention of
dengue. There are no anti-viral drugs for dengue but severe
dengue may be successfully treated with aggressive intravenous
rehydration. It is critical that patients with severe dengue
receive prompt treatment to manage shock, hemorrhage, and
organ impairment. Although methods for dengue diagnosis are
well established, there are no prognostic tests to help the clini-
cian evaluate the risk of progressing to severe dengue.2 This is
especially important because often the onset of severe disease
occurs as the patients fever is resolving.3
Previously identified markers of dengue severity include
cytokines (i.e., tumor necrosis factor alpha [TNFa] and Inter-
leukin 6 [IL-6]), vascular permeability proteins, clotting cas-
cade regulators, and gene expression profiles.417 These
severity markers are not, however, currently being used to
guide patient management.18,19 It is unclear why these sever-
ity markers are not being used, possibly because detection
methods are expensive, slow, or require sophisticated equip-
ment. This study focused on stable functional biomarkers,
specifically proteins, for dengue severity, as they could poten-
tially be incorporated into point-of-care (POC) tests, thus
ensuring their use. We used a classical proteomics approach
for biomarker discovery. We focused on high-throughput frac-
*Address correspondence to B. Katherine Poole-Smith, Immuno-
diagnostic, Development and Research Lab Centers for Disease
Control and Prevention, NCEZID, DVBD, Dengue Branch 1324
Calle Canada San Juan, PR 00920. E-mail: isd5@cdc.gov
These authors contributed equally to this work.
laser desorption/ionization time-of-flight mass spectrometry
(SELDI-TOF MS) platform to identify specific biomarkers
that can differentiate between dengue and severe dengue.
Biomarker discovery is an iterative process, which combines
several rounds of SELDI-TOF MS discovery with confirma-
tion of protein biomarkers by enzyme-linked immunosorbent
assay (ELISA) or Western blot. Early users of this technology
focused primarily on the identification of biomarkers for can-
cer; more recently, researchers have begun using SELDI-TOF
MS for biomarkers of infectious diseases such as human
immunodeficiency virus (HIV).20 Using this approach, we
have identified three unique biomarkers for severe dengue.
MATERIALS AND METHODS
Ethics statement. Protocol no. 6048.0 entitled The devel-
opment of a host biomarker diagnostic assay for dengue fever
and the differentiation of dengue hemorrhagic fever was
reviewed by the Centers for Disease Control and Prevention
(CDC) Human Research Protection Office and determined to
be exempt.
Study design. We used an iterative biomarker discovery
design of three groups of serum samples for three rounds of
experiments: discovery panel (Panel 1), serotype exclusion
panel (Panel 2), and confirmation panel (Panel 3). Figure 1
illustrates the methods for discovery, serotype exclusion, and
confirmation; Table 1 contains the patient characteristics for
each panel. Results from each round of experiments were
used to improve methods for the next round.
Samples. Serum samples were obtained from suspected
dengue cases submitted for testing during 20072010 to the
Passive Dengue Surveillance System (PDSS) of the CDC
Dengue Branch, at San Juan, Puerto Rico. Samples were
confirmed as DENV laboratory positive cases by reverse tran-
scriptase polymerase chain reaction or anti-DENV immuno-
globulin M (IgM) assay and clinically identified as dengue
hemorrhagic fever (DHF) or dengue fever (DF) based on a
dengue case investigation form (DCIF) containing clinical
data submitted with the specimen.2123 Clinical classification
of DF and DHF samples was based on the 1997 World Health

1218
 BIOMARKERS FOR SEVERE DENGUE 1219

Figure 1. Iterative biomarker study design. (A) Panel 1 discovery methods and samples, (B) Panel 2 DENV serotype exclusion methods and
samples, and (C) Panel 3 confirmation methods and samples. All samples were obtained in the acute phase (05 days post onset of fever), and met
the 1997 World Health Organization (WHO) DF/DHF criteria. DF = dengue fever; DHF = dengue hemorrhagic fever; Fatal = confirmed fatal
dengue cases; HC = laboratory-negative healthy controls; OFI = other febrile illnesses.
1220 POOLE-SMITH AND OTHERS

Figure 1. Continued.

Organization (WHO) guidelines.1 Samples were randomly spectra. Protein spectra were analyzed by ProteinChip, Inte-
selected from the PDSS database then delinked and random- grated Biomarker Wizard (Bio-Rad Laboratories, Hercules,
ized for three separate panels for discovery, serotype exclu- CA), and Biomarker pattern software (BPS; Bio-Rad Labora-
sion, and confirmation. To aid in the discovery of biomarkers tories) to detect proteins, candidate biomarkers, which can
for dengue, all febrile samples were acute (days post onset of differentiate between DF and DHF. Candidate biomarkers
fever (DPO = 05). Overall, 401 samples were used, which were separated by gel electrophoresis and identified by tandem
included the following: DF, DHF, confirmed fatal dengue mass spectrometry (MS-MS).
cases (Fatal), laboratory-negative healthy controls (HC), and Panel 2: serotype exclusion. The purpose of this panel was
other febrile illnesses (OFI). to include only pan-dengue host biomarkers and exclude any
Panel 1: discovery. The purpose of this panel was to dis- DENV serotype specific biomarkers by SELDI-TOF MS.
cover host biomarkers that distinguish DHF from DF by A panel of 133 serum samples including OFI (N = 30), HC
SELDI-TOF MS. A panel of 115 serum samples, which (N = 30), and 73 DF (DENV-1 [N = 25], DENV-2 [N = 27],
included DHF (N = 68), DF (N = 11), dengue fatal cases DENV-3 [N = 13], DENV-4 [N = 8]), was used to exclude
(N = 6), and OFI (N = 30) was used for biomarker discovery. serotype-specific candidate biomarkers. After completing the
Classification of DF and DHF was based on single specimens. discovery study with Panel 1, an analysis revealed that 20%
Samples were analyzed by SELDI-TOF MS to generate protein of DF cases in PDSS were misclassified as DHF cases (CDC,
unpublished data). Therefore, to ensure that classification as
DF was correct, sample selection for Panel 2 used paired spec-
Table 1 imens. The use of paired specimens, acute phase specimens
Patient characteristics for each serum sample panel* (DPO = 05), and convalescent phase specimens (DPO > 5),
Panel 1 discovery Panel 2 serotype exclusion Panel 3 confirmation verified that the specimens were correctly clinically classified.
n % n % n % Panel 2 was analyzed by SELDI-TOF MS to generate protein
Age, years spectra, which were analyzed by ProteinChip, Integrated
< 15 22 19 35 26 36 24 Biomarker Wizard, and Biomarker pattern software. Panel 2
1529 24 20 29 22 35 23 results were used to exclude any serotype-specific candidate bio-
3044 10 9 21 16 19 12 markers discovered from Panel 1. Non-serotype-specific can-
4559 15 13 9 7 22 14
6074 9 8 8 6 13 9 didate biomarkers were further refined by literature mining.
75+ 5 4 1 1 3 2 Panel 3: confirmation. The purpose of this panel was to
Unknown 30 26 30 23 25 16 confirm the host biomarkers discovered by SELDI-TOF MS
Causative dengue virus and controls distinguish between DHF and DF in ELISA format. A panel
DENV-1 8 7 25 19 88 58
DENV-2 11 10 27 20 3 2
of 153 serum samples consisting of laboratory-confirmed DF
DENV-3 16 14 13 10 0 0 (N = 105), DHF (N = 23), and HC (N = 25) samples were
DENV-4 3 3 8 6 15 10 selected from the most recent samples submitted to PDSS.
HC 0 0 30 23 25 16 Sample selection for Panel 3 was improved by using the most
0FI 30 26 30 23 0 0 recent paired samples available, from the 2010 epidemic, to
DV UNKN 47 41 0 0 22 14
Dengue disease state ensure specimen integrity by minimizing any potential effects
DF 11 10 73 55 105 69 of storage.24 Samples were analyzed by quantitative anti-
DHF 68 59 0 0 23 15 biomarker ELISAs to determine biomarker concentrations
Fatal 6 5 0 0 0 0 in serum samples. In addition, Western blot analysis was used
HC 0 0 30 23 25 16
0FI 30 23 30 23 0 0
to further evaluate the Vtn biomarker.
Discovery: SELDI-TOF MS data acquisition and analysis.
*Panel 1 Discovery (N = 115), Panel 2 DENV serotype exclusion (N = 133), and Panel 3
confirmation of biomarkers by ELISA and Western blot (N = 153). All samples were Serum samples were fractionated using the ProteinChip Serum
obtained in the acute phase (05 days post onset of fever), and met the 1997 World Health
Organization (WHO) DF/DHF criteria. Fractionation kit (Bio-Rad Laboratories) by anion-exchange
DF = dengue fever; DHF = dengue hemorrhagic fever; Fatal = confirmed fatal dengue; chromatography and pH gradient as previously described by
OFI = other febrile illness; HC = healthy control; DV UNKN = dengue positive, serotype
unknown. Percentages are rounded up to a whole number. Ndao.25 Fractions 1, 5, and 6 were collected and applied
 BIOMARKERS FOR SEVERE DENGUE
to cationic (CM10), metal affinity (IMAC), and hydrophobic
(H50) ProteinChip arrays (Bio-Rad Laboratories). The frac-
tions were then washed to remove non-specific binding, and
the energy absorbing molecule was applied. Arrays were ana-
lyzed in a ProteinChip biology system reader series (PCS 4000)
equipped with an autoloader using ProteinChip software, ver-
sion 3.5 (Bio-Rad Laboratories). Ionization energy was opti-
mized on pooled human serum samples. Each spot of the
arrays was then read at low-energy intensity for low molecular
weight (LMW) and high-energy intensity for high molecular
weight (HMW).
Spectral analyses. Spectra were analyzed using ProteinChip
software and Ciphergen Express Data Manager (Bio-Rad Lab-
oratories). Spectra were externally calibrated using an equa-
tion generated from a spectrum of protein standards with
molecular weights ranging from 5733.6 Da (bovine insulin) to
147,300 Da (bovine IgG), which were collected at the same
SELDI-TOF MS settings. Spectra were baseline subtracted,
and normalized to total ion current within a mass/charge (m/z)
range corresponding to optimized LMW or HMW ranges, and
with an external normalization coefficient of 0.2 for both con-
ditions. As a quality control measure for the comparison of
spectra processed on different days, the average normalization
factor was first calculated for all spectra. Any spectra that did
not fall within the overall average normalization factor twice
were discarded from the analysis.
Two-step spectral analysis was performed with Integrated
Biomarker Wizard software (version 5, Bio-Rad Laborato-
ries). Initially automatic peak detection was used to deter-
mine qualified mass peaks (signal/noise [S/N] > 3; cluster
mass window at 0.3%) and (S/N > 3; cluster mass window at
2%) for LMW and HMW ranges, respectively. A peak clus-
ter was determined as a peak that was found in at least 10%
of the spectra for 1 condition (i.e., fraction 1 bound on CM10
chip read at LMW; F1CSL). The P values for these peaks
were calculated for differences between different groups
(DF, DHF, and OFI), and cluster peaks with a P value
0.05 (Mann-Whitney U test) were visually inspected and
manually relabeled. Second-pass peak detection was per-
formed on the user-defined peaks only, with the same set-
tings as the first-pass, but the cluster mass window was
increased to 2%. The P values for differences in average
peak intensity between groups (DF versus OFI, DHF versus
OFI, DF versus DHF) (Wilcoxon exact test) were deter-
mined; OFI comparison was used to exclude biomarkers rep-
resentative of other febrile illnesses. A candidate biomarker
was defined as a peak with receiver operator curve (ROC)
ranging from 0.30 or lower for downregulated proteins to
0.70 or greater for upregulated proteins with a P value
0.05 and an intensity ratio between the two compared groups
being at least two. Cluster data were analyzed with BPS
(Bio-Rad Laboratories) as previously described by Ndao
2010.25 The BPS used the classification and regression tree
(CART) method, to identify peaks that best discriminated
between groups.
Protein identification. Candidate biomarkers were initially
identified from spectra as proteins of a particular molecular
weight. To identify these proteins, samples from Panel 1 minus
OFI were pooled according to disease state: 11 DF, 68 DHF,
and 30 OFI. Samples were then fractionated using the ZOOM
Isoelectric Focusing Fractionator (Invitrogen, Carlsbad, CA)
according to the manufacturers instructions. Briefly, 5 ZOOM
1221
Disks were used to obtain pH 3.010.0 range (pH 3.0, pH 4.6,
pH 5.4, pH 7.0, pH 9.1, and pH 10.0) fractions that were stored
at 20C for further analysis by gel electrophoresis. Before
analysis, each fraction was precipitated using 3 volumes of
methanol, 1 volume of chloroform, and 4 volumes of distilled
water. Precipitated protein samples were dissolved in sodium
dodecyl sulfate (SDS) sample buffer, and separated at 200 V
for 45 min using a 412% Bis-Tris NuPAGE gel under dena-
turing conditions with Mark12 unstained protein standards
(Invitrogen). Gels were stained using Coomassie blue, and can-
didate biomarker bands were identified based on molecular
weight, excised, and stored in 2% acetic acid for MS-MS. The
proteins of interest were sequenced by MS-MS. The resulting
spectra were submitted to the database-mining tool MASCOT
(Matrix Sciences, Boston, MA) for identification.
Exclusion: SELDI-TOF MS. To exclude any serotype-specific
proteins identified in Panel 1, Panel 2 samples (representative
of the four dengue serotypes)were analyzed as described in the
SELDI-TOF MS Acquisition and Analysis methods and Spec-
tral Analyses methods. Candidate biomarkers classified as sero-
type specific were excluded from further analyses.
Literature mining. After refining the pool of candidate bio-
markers by exclusion of serotype-specific markers, we conducted
a literature review of the remaining candidate biomarkers. We
consulted the Protein Data Bank for basic protein characteris-
tics, PubMed to understand the role of candidate biomarkers in
healthy and diseased individuals, and Linscotts Directory to
determine availability of reagents for confirmation experi-
ments. From the literature data, we selected candidate bio-
markers that had not yet been evaluated for dengue severity,
and there were antibodies and/or ELISA reagents available for
confirmatory experiments.
Confirmation. Enzyme-linked immunosorbent assays. The
ELISAs were used to determine biomarker concentration in
serum samples from Panel 3 (Table 1). Serum samples were
tested for human vitronectin (Innovative Research, Novi, MI),
hemopexin, and transferrin, (MyBioSource, San Diego, CA)
according to the manufacturers instructions. Vitronectin
(Vtn) was further analyzed by Western blot because the Vtn
isoform (55 kDa) detected by SELDI-TOF MS was not
detected by the commercially available ELISA.
Gel electrophoresis and Western blot analysis. Each individ-
ual sample from Panel 3, 105 DF, 23 DHF, and 25 HC was
separated using Novex NuPAGE 412% Bis-Tris gels
(Invitrogen/Life Technologies Grand Island, NY) under
denaturing conditions with MagicMark XP Protein standards
(20220 kDa, Invitrogen) and 0.125 mg Vtn control (Sigma, St.
Louis, MO). Proteins were transferred to nitrocellulose mem-
branes (Invitrogen, 0.22 mm), and blocked at room temperature
overnight in 5% nonfat dry milk in phosphate-buffered saline
with 0.05% Tween20. Membranes were incubated with 1:1,000
anti-human Vtn antibody (Cedarlane, Hornby, Ontario), fol-
lowed by 1:10,000 anti-sheep peroxidase (KPL, Gaithersburg,
MD). SuperSignal West Pico solution (Pierce/Thermo Scien-
tific, Rockford, IL) was used to detect proteins.
Anti-Vtn western was used to compare Vtn isoforms of DF,
DHF, and HC samples from Panel 3 (Table 1). The Vtn con-
centration for all samples was determined using quantitative
ELISA as described in the ELISA analysis section (Figure 2C,
Table 2). Samples were divided by disease state (DF, DHF,
and HC), ranked according to total Vtn concentration, and gels
loaded based on the optimized detection of Vtn isoforms.
1222 POOLE-SMITH AND OTHERS

Figure 2. Enzyme-linked immunosorbent assay (ELISA) confirmation of biomarkers identified by surface-enhanced laser desorption ioniza-
tion time-of-flight mass spectrometry (SELDI-TOF MS). Mean micrograms per milliliter (mg/mL) were determined by quantitative ELISAs:
(A) hemopexin (Hx), (B) serotransferrin (Tf), (C) vitronectin (Vtn), and (D) Vtn by age. DF, DHF, and HC were compared for each biomarker
by the Kruskal-Wallis test with Dunns post hoc test (* = 0.01 to 0.05, ** = 0.001 to 0.01, *** < 0.001). DF = dengue fever; DHF = dengue hemorrhagic
fever; HC = healthy control.

Serum samples were diluted in Tris-buffered saline, and loaded
at the following total Vtn concentrations: 1) DF 500 ng (Vtn =
2,40010,000 mg) 125 ng (Vtn < 2,400 mg); 2) DHF 125 ng; 3)
HC 125 ng (Vtn = 5504,500 mg); and 4) 31.5 ng (Vtn < 550 mg).
Volume analysis of Vitronectin isoforms. Western blots
were imaged at 3-min exposures using a charge-coupled device
camera (Gel Doc Bio-Rad Laboratories), and analyzed using
version 4.6.9 Quantity One software (Bio-Rad Laboratories) as
previously described.26 Briefly, lanes were auto-framed and
bands auto-detected without background correction. All Lanes
Report function was used to determine the percentage of total
Vtn for each band per lane. Bands within molecular weights
125140 (oligomer), 80, 75, 65, 55, 52, 45, and 10 kDa were
selected for analysis, and the mean percentage of total Vtn
protein was determined (Table 2).
Statistical analysis. Integrated Biomarker Wizard software
(version 5, Bio-Rad Laboratories) used the Mann-Whitney U
test and Wilcoxian exact test for spectral analysis. The ELISA
concentration values for Hx, Tf, and Vtn from DF and DHF
samples were compared using Prism 5 software and P values
were calculated using a Kruskal-Wallis test with Dunns post
hoc test (GraphPad, La Jolla, CA).

Table 2
Candidate biomarkers identified by surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS)*
Predicted Actual mass Ratio of mean
Protein Accession no. mass (Da) (Da) Mean biomarker concentration (mg/mL) concentration P values

[95% CI]

NCBI UniProt DF DHF HC (DF/DHF)

Hemopexin NP_000604 52,385 51,600 3,593 2,963 1,571 1.31 < 0.0001
[3,346, 3,841] [2,375, 3,550] [1,327, 1,814]
Serotransferrin NP_001054.1 79,280 76,500 11,163 9,674 11,536 1.15 0.0309
[10,501, 11,825] [8,252, 11,095] [10,405, 12,668]
Vitronectin NP_000629 55,165 75,000, 65,000 + 3,011 2,089 704 1.44 < 0.0001
10,000 [2,720, 3,302] [1,561, 2,616] [370, 1,038]
*Accession nos., predicted mass by SELDI-TOF MS, actual mass in Daltons (Da), and biomarkers identified as hemopexin (Hx), serotransferrin (Tf), and vitronectin (Vtn). Mean micrograms
per milliliter (mg/mL) and 95% confidence interval were determined for each biomarker by disease state (DF = dengue fever, DHF = dengue hemorrhagic fever, HC = healthy control).
Overall P values calculated using Kruskal-Wallis test.
Deglycosylated vitronectin ~55,000 Da.
 BIOMARKERS FOR SEVERE DENGUE
RESULTS
Discovery of 16 candidate biomarkers discriminating DF
from DHF. The SELDI-TOF MS analysis of 115 samples
from Panel 1 yielded 2,070 spectra. Using the ProteinChip
software analysis, we identified 251 unique biomarkers that
discriminated between DF and DHF. These unique bio-
markers were selected based on differences in average mass
spectrometry peak intensity between DF and DHF samples
(P values < 0.05 and ROC analysis 0.9). Further analysis was
performed using BPS, which allowed selection of the most
significant biomarkers by generating decision trees thus iden-
tifying 16 candidate biomarkers.
Serotype exclusion: biomarkers were consistent across all
four DENV serotypes. The SELDI-TOF MS analysis of 133
samples from Panel 2 yielded 2,394 spectra, from which
ProteinChip software analysis determined no candidate bio-
markers that differed by DENV serotypes 14. From these
16 DENV biomarkers that were non-serotype-specific can-
didate biomarkers, Hx, Tf, and Vtn were selected for confir-
mation analysis because their role in dengue severity had
never been evaluated. For Vtn, the predicted size based on
the NCBI accession number differed from the actual size
observed in SELDI-TOF MS (Table 2).
Confirmation. The ELISA analysis of 153 serum samples
(Table 1, Panel 3) was used to determine the average concen-
tration of the three candidate biomarkers based on disease
state (DF versus DHF). On the basis of a Kruskal-Wallis test
followed by Dunns post hoc test, Vtn (DF = 3,011 mg/mL
versus DHF = 2,089 mg/mL, Ratio DF/DHF 1.44), Hx (DF =
3,593 mg/mL versus DHF = 2,963 mg/mL, Ratio DF/DHF 1.31),
Tf (DF = 11,163 mg/mL, DHF = 9,674 mg/mL, Ratio DF/DHF
1.15) were significantly different (P 0.05) between DF and
DHF patients. The Vtn, Hx, and Tf showed a decrease in con-
centration between DF and DHF patients (Table 2, Figure 2).
The Hx and Vtn were elevated in dengue-infected individuals
(DF and DHF) relative to HCs. There were no significant dif-
ferences in Vtn levels between DF and DHF by age.
Although the dominant Vtn isoforms in humans were
75 kDa and 65 + 10 kDa, we identified one Vtn isoform
(55 kDa) by SELDI-TOF MS; Vtn isoforms have different
functions in coagulation. Because the ELISA used for Vtn
confirmation only detected the 75 and 65 kDa isoforms, we
also analyzed samples by Western blot. Western blot analysis
confirmed the 75, 65, and 55 kDa isoforms, and identified five
Figure 3. Western blot analysis of vitronectin (Vtn) isoforms. Serum samples from dengue fever (DF), dengue hemorrhagic fever (DHF), and
healthy controls (HCs) were separated under denaturing, reduced conditions (Panel 3). Representative blots are presented. Lane 1: MagicMark
XP protein standard, Lane 2: control of 0.125 mg Vtn isolated from human plasma (75 and 65 kDa), Lanes 312 DF, DHF, and HC, loaded based
on 0.125 mg Vtn as determined by quantitative ELISA. The 8 isoforms identified per lane were oligomer (125 or 140 kDa), 80, 75, 65, 55, 52, 45, and
10 kDa.
1223
other isoforms not observed by SELDI-TOF MS (oligomer
[125140] 80, 52, 45, and 10 kDa) (Figure 3). For all disease
states (DF and DHF), the most prevalent isoforms identified
by Western blot analysis were the 75, 65, and 55 kDa iso-
forms. An ~2-fold decrease (DF versus DHF) of the 80, 52,
and 10 kDa isoforms was observed by Western blot. Unfortu-
nately, the 52- and 80-kDa Vtn isoforms are less commonly
found in the general population, limiting their utility as diag-
nostics. None of the Vtn isoforms identified by Western blot,
alone or in combination, provided improved differential diag-
nosis between disease state and non-disease state compared
with total Vtn concentration measured by ELISA (Table 3).
DISCUSSION
Analysis of SELDI-TOF MS results identified 16 unique
proteins that discriminated between DF and DHF across all
DENV serotypes. We selected three of these, Vtn, Hx, and
Tf, which operate to maintain the hemostatic balance between
coagulation and fibrinogenesis, for further analysis. By ELISA,
three of these (Vtn, Hx, Tf) were elevated in DF relative to
DHF cases; Hx and Vtn were elevated in dengue cases relative
to healthy controls. Currently the molecular mechanisms of
dengue coagulopathology, which occur during DHF are attrib-
uted to increased viral replication, apoptosis, complement
activation, and proinflammatory cytokines. Although many
molecular mechanisms of coagulation malfunction have been
proposed, exact roles of effector proteins, such as Vtn, Hx, and
Tf in pathology are not yet known.27
Based on ELISA confirmation of SELDI-TOF MS results,
Vtn is our single most statistically promising biomarker for
distinguishing between DHF and DF. The Hx and Tf require
further analysis to determine why our data differed from other
researchers.17,28,29 After further characterization of Vtn, Hx,
and Tf, we may find that a combination of biomarkers has the
greatest utility as a prognostic test for severe dengue as noted
by other researchers.7
Vitronectin is a multifunctional glycoprotein that circulates
in the blood, usually as an inactive monomer, until it is
recruited to regulate coagulation and platelet aggregation.30,31
In our study, the Vtn levels of DF patients and DHF patients
were much higher compared with HCs. There was an overall
increase in Vtn after dengue compared with HCs, with overall
Vtn concentrations decreasing in DHF cases compared with
DF cases. The recruitment of Vtn monomers to oligomers
1224 POOLE-SMITH AND OTHERS

Table 3
Analysis of vitronectin (Vtn) isoforms by dengue disease state*
Dengue fever (N = 105) Dengue hemorrhagic fever (N = 23) Healthy control (N = 25)

Mean Vtn/lane Isoform absent Mean Vtn/lane Isoform absent Mean Vtn/lane Isoform absent
Isoform (kDa) % Range (%) % Range (%) % Range (%)

Oligo 4 311 61 (57) 3 71 12 (52) 5 131 2 (8)

80 9 401 72 (67) 5 63 17 (74) 3 61 13 (52)
75 16 601 2 (2) 20 442 0 (0) 15 527 1 (4)
65 44 703 0 (0) 50 7214 0 (0) 50 628 0 (0)
55 30 731 0 (0) 25 711 1 (4) 25 525 0 (0)
52 7 312 86 (81) 2 32 20 (87) 7 191 21 (84)
45 4 161 26 (25) 4 110 11 (48) 3 81 10 (40)
10 3 200 25 (24) 2 51 2 (9) 3 70 0 (0)
*Western blots were analyzed using Bio-Rad Laboratories Quantity One software. Isoform bands were auto-detected and reported as the percentage of total vitronectin (Vtn) protein/lane for
each isoform. The mean and range of the percentage of total Vtn protein/lane from each molecular weight isoform (oligomer 125 or 140, 80, 75, 65, 55, 52, 45, 10 kDa) and the percentage of absent
bands was calculated by disease state. Percentages are rounded up to a whole number.

occurs after the initiation of the clotting cascade.32 In addition
to its role in the clotting cascade, Vtn also assists in the regen-
eration of the vascular extracellular matrix. The decline of Vtn
levels in DHF cases might be caused by Vtn recruitment to
damaged vascular tissues.33,34 A decline in Vtn levels has been
observed in other hemorrhagic viral infections, such as hemor-
rhagic fever renal syndrome (HFRS), which is caused by
Hantaan virus, showing that Vtn is essential for vascular integ-
rity. The Vtn levels were lower in HFRS patients during all
phases of the disease, except the convalescent phase, and
changes in serum Vtn correlated with disease severity.35
The Vtn monomeric pool is composed of at least seven
isoforms, which might determine Vtn function during the clin-
ical course of dengue (Table 3). In our study, DHF patients
exhibited an overall decrease in Vtn oligomer (125140), 80-,
52-, and 10-kDa isoforms compared with DF. The two most
functionally relevant Vtn isoforms are 10 kDa and the oligo-
mer. The 10-kDa Vtn band was detected in 82% of DF and
DHF patients, indicating that this isoform could be used as a
biomarker for severe dengue.36 The Vtn oligomer has two
distinct functions that could have implications for DENV
infection and pathogenesis. First, the oligomer binds to hepa-
rin, a known receptor for DENV.37 Second, it regulates coag-
ulation by forming a bridge between integrin and fibrin to
induce platelet aggregation to inhibit plasma leakage.38,39
Western blot analysis did not identify any single isoform as
having a greater association with dengue severity compared
with the overall Vtn concentration measured by ELISA. To
determine the role of Vtn, fine discriminatory analysis of Vtn
isoforms after infection may require sequential sampling
throughout the clinical course of severe dengue.
Hemopexin is a glycoprotein that binds free heme, pre-
venting oxidative tissue damage.4042 Elevated Hx levels were
observed in DF and DHF patients relative to healthy con-
trols. Our findings differ from some earlier studies. Our
healthy controls had 2-fold higher Hx (1,571 mg/mL) com-
pared with a previous report (770 mg/mL).40 Ray and col-
leagues28 observed higher Hx levels in healthy controls
compared with DF. This discrepancy could be caused by a
difference in methods (Western blot versus ELISA), or to
sample size (HC = 6, DF = 6). Kumar and colleagues17
observed elevated levels of Hx in DHF cases relative to DF
cases over time, in contrast to our observation of lower Hx
levels in DHF relative to DF (DPO = 05). This discrepancy
may be caused by age differences between the samples ana-
lyzed or quantitation methodology (SELDI-TOF MS and
ELISA versus isobaric tagging). The potential of Hx as a
biomarker will depend on resolution of these differences
through further studies.
Serotransferrin, an iron-binding and transport protein that
maintains hemostasis by transferring iron from sites of heme
degradation, thus preventing tissue damage; it also stimulates
cell proliferation.43,44 We observed approximately equivalent
Tf levels in DF (11,163 mg/mL) and HC (11,536 mg/mL); how-
ever, Tf levels were lower in DHF cases (9,674 mg/mL). Inad-
equate levels of Tf in DHF cases may lead to vascular tissue
damage and plasma leakage. The Tf levels were higher in
classical swine fever compared with uninfected swine, con-
trary to our observation with DENV in humans. Clinical man-
ifestations of classical swine fever, which is also a flavivirus,
pathologically resemble DHF, including thrombocytopenia
and the hemorrhage of skin, mucosa, and organs. This differ-
ence in Tf levels after a flavivirus infection may be explained
by virus or host-specific protein differences. A protein basic
local alignment search tool (BLAST) search of human and
swine Tf predicted 98% homology; therefore functional dif-
ferences between human and swine Tf may exist.
The fact that our samples had been collected as part of
PDSS somewhat limits our knowledge of the course of disease
in the donors. Biomarker expression may vary during the
clinical course of dengue; hence, serially prospectively col-
lected patient serum samples would be preferred to the single
samples obtained from PDSS. It is not possible retrospec-
tively to re-examine patients or charts to determine whether
any patient in the study developed symptoms of severe den-
gue later in the course of their illness. An estimated 20% of
DF cases in PDSS are misclassified; these cases are actually
DHF cases (CDC, unpublished data). This misclassification
may have reduced our ability to identify biomarkers during
the initial discovery study. Another limitation of our retro-
spective analysis is that the samples had been classified as
DF and DHF using the 1997 WHO case classifications rather
than those published in 2009, which categorize all infections
as either dengue, dengue with warning signs, or severe den-
gue. Finally, although age-related differences for one of our
biomarkers (Vtn) have been reported in healthy individuals;
we did not have age-matched DF and DHF specimens in the
serum panels for analysis by age.4548
Further validation is required before any of these biomarkers
could be formulated as prognostic tests. Future studies should
verify the prognostic utility of the biomarkers by analyzing
biomarker levels over time and using the 2009 WHO severity
classifications. Diagnostic cut-off values should then be devel-
oped for each biomarker. After diagnostic cut-off values have
 BIOMARKERS FOR SEVERE DENGUE
been determined, the use of combinations of biomarkers could
be evaluated. Finally, the development of these biomarkers or
their combinations into prognostic tests can be initiated.
Ideally, a prognostic POC test for severe dengue would be
used in combination with a dengue diagnostic test. Patients
that test positive for dengue can then be tested for severity.
Those patients with high risk for severe disease can be hospi-
talized and closely monitored by a physician. Whereas
patients without clinical warning signs and at low risk for
severe disease can be more confidently sent home to recover.
Received March 31, 2014. Accepted for publication August 25, 2014.
Published online October 27, 2014.
Acknowledgments: We thank Elizabeth Cartozian and Raziel Rojas-
Rodriguez at Centers for Disease Control and Prevention, and
Christine Straccini at McGill University, for their technical expertise
and support.
Financial support: This work was supported by the Centers for Dis-
ease Control and Prevention, the Sandler Foundation, the Founda-
tion of the Montreal General Hospital and the Research Institute of
the McGill University Health Centre.
Disclosure: B. Katherine Poole-Smith, Kay M. Tomashek, Elizabeth
A. Hunsperger, and Momar Ndao are inventors on a patent Detec-
tion of subject biomarker diagnostic assay for dengue fever and the
differentiation of dengue hemorrhagic fever. United States Patent
WO/2013/130029 Sept 6, 2013. Momar Ndao and Brian J. Ward are
inventors on a patent entitled Biomarkers for Dengue. United States
Patent US 2012/0021936 A1 Jan 26, 2012. B. Katherine Poole-Smith,
Manuela Beltran, Kay M. Tomashek, and Elizabeth A. Hunsperger
are employees of the U.S. Government. This work was prepared as
part of their official duties.
Disclaimer: The views expressed in this article are those of the author
and do not necessarily reflect the official policy or position of the
Centers for Disease Control and Prevention or the U.S. Government.
Authors addresses: B. Katherine Poole-Smith, Manuela Beltran,
Kay M. Tomashek, and Elizabeth A. Hunsperger, Dengue Branch,
Centers for Disease Control and Prevention, San Juan, PR, E-mails:
isd5@cdc.gov, mvb6@cdc.gov, kct9@cdc.gov, and enh4@cdc.gov.
Alexa Gilbert, AssureRx Canada, Toronto, Ontario, Canada, E-mail:
alexa.gilbert@mail.mcgill.ca. Andrea L. Gonzalez, Environmental
Health, University of Puerto Rico Graduate School of Public Health,
San Juan, PR, E-mail: andrea.l.gonzalez@upr.edu. Brian J. Ward,
National Reference Centre for Parasitology, Research Institute of
the McGill University Health Centre, Montreal, Quebec, Canada,
E-mail: brian.ward@mcgill.ca. Momar Ndao, National Reference
Centre for Parasitology, Research Institute of the McGill University
Health Centre, Montreal, Quebec, Canada, and FQRNT Centre for
HostParasite Interactions, McGill University, Montreal, Quebec,
Canada, E-mail: momar.ndao@mcgill.ca.
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