Page 121 - Journal of Special Operations Medicine - Fall 2014

of personal protective measures, drive effective preven- the determination of the presence of dengue antibodies.
tive countermeasure (vaccine and rapid assay) develop- There are no other approved assays available to deter-
ment, and prompt further investigation. mine specific dengue neutralizing antibody levels, and
the only location in the Department of Defense (DoD)
for this assay is the WRAIR.
Methods
The first phase was a retrospective seroprevalence study The assay is a high-throughput dengue enzyme-linked
using archived Armed Forces Serum Repository (AFSR) immunosorbent assay (ELISA) microneutralization test
samples collected from USASOC personnel deployed for that identifies and measures dengue (DENV-1–4) neu-
at least 30 days to Asia or South or Central America tralizing antibodies from primary and secondary dengue
from 2006 to 2008. The second phase is a multicenter, virus infections with good specificity and sensitivity. The
prospective serosurveillance project that includes up assay takes advantage of a 96-well plate format that uses
to 1000 US Army Special Forces personnel deployed the WHO’s Vero recommended cells as an infection cell
to dengue-endemic areas. The population consists of culture substrate. Briefly, in a polypropylene U-bottom
healthy US Army personnel (male and female, aged 18 96-well plate, a calibrated concentration (50–100PFU/
to 62 years), who represent a high-risk dengue exposure well) of the dengue virus is added to seven 3-fold se-
group among military populations. They are active duty rum sample dilutions (or one initial dilution [e.g., 1:10]
US Army personnel who are engaged in special opera- for screening purposes), which after 2 hours at 35ºC
tions missions in dengue endemic areas globally. are transferred to healthy monolayers of Vero WHO
cells and then incubated at 35ºC, 5% CO , 95% rela-
2
For the first phase, a random, unit-stratified sample of tive humidity for 4 days. The cells are then fixed with
500 anonymous serum specimens from personnel as- ethanol:methanol for 1 hour at –20ºC. After fixation, an
signed to units in USASOC most frequently deployed to ELISA is performed on the fixed plates using 4G2 mono-
dengue endemic areas were provided from the Armed clonal as the detecting primary antibody followed by a
Forces Serum Repository (AFSR) for dengue screening. horseradish perioxidase (HRP)-conjugated anti-mouse
Samples were shipped to the VDB at the WRAIR to test secondary antibody to detect and quantify dengue cell–
the humoral immune response (i.e., test for antibodies based associated viral antigens in the virus dose and the
via microneutralization assay) to each dengue virus se- serum:virus dose dilutions inoculated monolayers. The
rotype (DENV-1–4). An aliquot (maximum amount: resulting optical densities (tetramethylbenzidine [TMB])
0.5mL) was prepared from the original archived sample is used as an HRP substrate followed by a stop solution
and labeled with a unique barcode label (S plus nine-digit of 1:25 phosphoric acid) are automatically transferred
number, generated by AFSR). The samples were frozen and processed to a linear curve-fitting model to obtain
to –70ºC and shipped on dry ice to the WRAIR VDB the 50% reduction of viral infection titers, referred to
Compliance Management Unit, where it was in-pro- as the MN titer. The MN titer is the reciprocal of
50
50
cessed (documented, recorded, and stored until testing). the serum dilution that neutralizes 50% or more of the
dengue virus dose, leading to a reduction of 50% of the
In the second phase of the project, as part of their pre- optical density measured by ELISA on fixed cells after 4
deployment Soldier Readiness Processing (SRP), volun- days of incubation. Seropositivity is defined as a MN
50
tary subjects completed a short questionnaire querying titer ≥ 10.
them on demographics, preventive health measures,
vaccinations received and provided a clotting tube of Statistics for both studies were descriptive; the per-
blood (10mL) for dengue neutralizing antibody testing. centage of seronegative and seropositive subjects was
Whenever possible, they repeated these procedures post- determined for each serotype. The seroprevalence of
deployment but for the purpose of this publication re- anti bodies to DENV-1–4 was calculated and reported.
sults will represent only overall exposure not exposures
attributed to a specific location or deployment.
Results
The serum and blood specimens were analyzed for the
presence of neutralizing antibody against all four sero- Phase 1
types of dengue virus (DENV-1–4) by using the dengue Of the 500 repository specimens tested screening re-
microneutralization antibody assay performed at the sulted in 56 (11%) of 500 samples with neutralizing
WRAIR Pilot Bioproduction Facility. The virus neu- titers (MN ≥ 10) against at least one DENV serotype.
50
tralization titer is the amount of virus-specific antibody Subsequent sample titration resulted in 85% (48 of
that must be administered to an organism to block the 56) of the samples with neutralizing titers (MN ≥ 10)
50
establishment of that virus 99% of the time. Neutral- against at least one DENV serotype for an overall den-
izing antibody assays are the gold standard assay for gue exposure rate of 9.6% (48 of 500).

Dengue Seroprevalence in USASOC 113