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Corresponding author. Tel.: +1 510 642 4845; fax: +1 510 642 6350.
E-mail address: eharris@berkeley.edu (E. Harris).
wide, with tens of millions of dengue cases annually.
1
DENV
infection can be asymptomatic, can manifest as classic dengue
fever (DF), or can evolve into the most serious form, dengue
hemorrhagic fever/dengue shock syndrome (DHF/DSS). Tradition-
ally, diagnosis and epidemiologic surveillance relies on detection
of DENV-specic IgM antibodies in serum of suspected dengue
cases,
2,3
while the incidence of DENV infection is determined by
detecting DENV-specic IgG or total antibody in serum via ELISA
or the time-consuming and cumbersome hemagglutination inhi-
bition or plaque reduction neutralization test (PRNT) assays.
48
A
few studies have evaluated the use of anti-DENV IgA as a dengue
diagnostic marker
911
; others have assessed alternatives to serum
1386-6532/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jcv.2008.07.016
288 A. Balmaseda et al. / Journal of Clinical Virology 43 (2008) 287291
as diagnostic specimens.
9,1113
However, no comprehensive studies
of multiple immunoglobulin classes and different types of biologi-
cal samples have been reported, especially in relation to measuring
incidence of DENV infection. This study evaluates the utility of dif-
ferent immunological markers for diagnosis of acute illness and for
measuring the incidence of DENV infection. For dengue diagnosis,
IgM and IgA in acute- and convalescent-phase serum, lter-paper
blood spots, and saliva were studied; for determining DENV infec-
tion over a 6-month period of time, changes in levels of IgM, IgA
and IgG in serum and lter-paper blood spots, as well as IgM and
IgG in saliva, were evaluated.
2. Methods
2.1. Study population
Two distinct populations participated in this study. For assess-
ing immunological markers for diagnosis of symptomatic dengue
cases, childrenandinfants ages 014years oldwhopresentedtothe
Hospital Infantil Manuel de Jess Rivera (HIMJR) from September
2003 to February 2004 with suspected acute dengue within 4 days
of symptomonset were invitedto participate inthe study. Inclusion
criteria included meeting the WHO criteria for a suspected case of
dengue,
14
less thanor equal to4days since symptomonset, consent
froma parent or legal guardian, and assent for children over 5 years
of age. Exclusion criteria included the presence of a hematologic or
other illness that increased risk to study participants. Acute-phase
samples (days 14 post-symptom onset) and convalescent-phase
samples (days 1014 post-symptom onset) were collected from
each participant.
The second population was comprised of healthy children from
non-contiguous homes living in District II of Managua, whose fam-
ilies were participating in an entomological study fromJuly 2003 to
January 2004. Consent to participate in the serological survey was
required from a parent or guardian, and assent was required from
all subjects 5 years of age and older. Samples evaluated inthis study
were collected in July 2003 and January 2004 (6 months apart).
These studies were approved by the Institutional ReviewBoards
at the University of California Berkeley andthe NicaraguanMinistry
of Health.
2.2. Sample collection
To minimize inconvenience to participants, blood obtained via
standard venipuncture was used both for the serumsample and for
the lter-paper blood spot. Blood was collected into a glass tube
(without anti-coagulants), and prior to its coagulation, 0.20.4cc
of blood was transferred via microcapillary to 20-weight What-
man 3 paper. The paper, cut into the shape of microscope slides,
was placed upright in a slide case at ambient temperature until
dry (up to 2h). The lter-paper samples were then placed in
ziplock bags and kept at 4
C until processing.
Samples fromthe same participant were processed simultaneously
and within 12 months of their collection. For saliva, participants
dripped saliva from their mouths into a 1-in. diameter speci-
men cup. For very young children, nurses used plastic disposable
pipettes to gently suction saliva from under the tongue. After
the specimen containers were sealed, samples were immediately
placed at approximately 4
C
until processing. Again, samples from the same participant were
processed simultaneously and within 12 months of their collec-
tion.
2.3. Laboratory methods
For all assays, serumand saliva frompreviously laboratory con-
rmed dengue and non-dengue cases were used as positive and
negative controls, respectively.
2.3.1. Capture ELISAIgM and IgG in serum, lter-paper blood
spots and saliva; IgA in serum
To perform capture ELISA of anti-DENV IgM, IgA or IgG,
9
polystyrene wells were xed with 100l of anti-human IgM, IgA
or IgG antibodies diluted in carbonatebicarbonate buffer and
were incubated overnight at 4
C
for 30min with 50l of serum diluted 1/20 in PBS-T, 50l of PBS
eluted overnight from lter-paper blood spots (approximately a
1:20 dilution; one 6-mm diameter circle in 200l PBS-T), or 50l
of undiluted saliva. Wells were washed four times with PBS-T after
each incubation. After a 1-h incubation with 50l of a mixture
of antigens from all four DENV serotypes obtained from infected
C6/36 cells,
9
wells were incubated for 30min at 37
C with 50l of
pooled polyclonal human anti-DENV immunoglobulin (with high
inhibition ELISA titer directed against all serotypes) conjugated to
horseradishperoxidase and diluted inPBS-T plus 5%normal human
serum. This was followed by incubation with 50l of tetra-methyl-
bencidine (TMB) substrate for 10min, andthe reactionwas stopped
with50l of 12.5%sulfuric acid. Optical density(OD) was measured
at 450nm in a Humanreader ELISA reader (Human Gesellschaft,
Taunsstein, Germany). For diagnosis of suspected cases, samples
withODgreater than2times (IgMELISA) or 1.8times (IgAELISA) the
mean of the OD of negative controls were considered positive. For
measuring incidence, a >twofold increase in IgM, IgA (serumonly),
and IgG antibody units between pre- and post-season samples was
considered positive; antibody units were calculated as follows:
units =
OD sample OD negative control
OD positive control OD negative control
100.
2.3.2. Sandwich ELISAIgA in saliva
To measure anti-DENV IgA in saliva, wells were xed with
100l of human anti-DENV immunoglobulin at 10g/ml in
carbonatebicarbonate buffer, pH 9.5, and incubated overnight at
4
C for
30min with PBS-T containing 1% bovine serumalbumin, then incu-
bated at 37