Evaluation of the Dengue NS1 Ag Strip?for Detection
of Dengue Virus Antigen in Aedes aegypti (Diptera: Culicidae)
Cheong-Huat Tan, Pei-Sze Jeslyn Wong, Mei-Zhi Irene Li, Indra Vythilingam, and Lee-Ching Ng
Dengue fever is currently one of the most important mosquito-borne diseases that affect humans. With neither
vaccines nor treatment available, prevention of the disease relies heavily on surveillance and control of mosquito
vectors. In the present study, we have evaluated and showed the potential use of the Dengue NS1 Ag Strip?for
the detection of dengue virus (DENV) in Aedes aegypti. Initial results showed that the sensitivity of the test kit in
detecting DENV in wild-caught mosquitoes is comparable to that of real-time reverse transcriptase–polymerase
chain reaction. The detection of naturally infected Ae. aegypti with the NS1 rapid test kit in our dengue cluster
investigation further illustrates its potential use for surveillance of DENV in wild mosquito populations. The kit
can easily be used in a simple field station, and minimal training is required. The results can be obtained in less
than an hour. Employment of the kit in the field could help guide mosquito control operations in the prioriti-
zation of resources in controlling the transmission of DENV. In this study the potential of the kit for field
surveillance of infected dengue vectors, which are epidemiologically important, has been demonstrated.
Key Words: Aedes aegypti—Dengue NS1 Ag Strip?—Dengue virus—NS1 protein—Rapid test kit—Real-time
morbidity and mortality. Dengue fever has been reported in
over 100 countries and 2.5 billion people live in areas where
the disease is endemic (WHO 2009). It is caused by four
dengue virus (DENV 1–4), flaviviruses in the family flavivir-
idae. In Southeast Asia, Aedes aegypti is the main vector of
DENV and Aedes albopictus acts as a secondary vector (Gratz
2004). At present, there is neither effective vaccines nor spe-
cific treatment available for dengue; hence, prevention and
control of the disease rely heavily on the surveillance and
control of vectors to reduce transmission (Bangs et al. 2001).
Vector surveillance allows the timely implementation of
control measures, such as adulticiding, habitat destruction,
source reduction, and community participation, to limit an
impending outbreak. Active surveillance of infected mosqui-
toes may also prove valuable in defining the spatial and
temporal risk of acquiring dengue infection (Bangs et al.
Typically, the detection of DENV in Aedes spp. includes
isolation and propagation of viruses, reverse transcriptase–
engue fever is currently one of the most important
mosquito-borne diseases that affect humans in terms of
polymerase chain reaction (RT-PCR) assays, or enzyme-
linked immunosorbent assay or immunofluorescence assay
(Kuberski et al. 1977, Chan et al. 1994, Sithiprasasna et al.
1994, Rohani et al. 1997, Chow et al. 1998, Samuel and Tiyagi
2006). However, the need for expensive specialized labora-
tories and equipment and highly trained staff has made these
techniques impractical for regular surveillance of DENV-
Specialized rapid test kits have become available for de-
tecting arboviruses, such as West Nile and Saint Louis en-
cephalitis viruses in mosquito vectors (Nasci et al. 2002,
Burkhalter et al. 2006). These kits have the advantage in their
ease of use and require minimal laboratory facilities and
technical experience. However, to date, none has been vali-
dated for DENV detection in its mosquito vectors. Recently,
the Dengue NS1 Ag Strip?(BioRad Laboratories), a rapid
immunochromatographic assay, has been developed to de-
tect DENV NS1 antigen from acute patient serum samples
(Dussart et al. 2008, Chaiyaratana et al. 2009, Pok et al.
2010). NS1 is a structural protein that is produced in both
membrane-associated and secreted forms. It is involved
in viral replication and has been shown to correlate with
viremia (Libraty et al. 2002, Dussart et al. 2006, Thomas et al.
Environmental Health Institute, National Environmental Agency, Singapore, Singapore.
VECTOR-BORNE AND ZOONOTIC DISEASES
Volume 11, Number 6, 2011
ª Mary Ann Liebert, Inc.
2010). We report the usefulness of the NS1 rapid test in de-
tecting DENV in laboratory-infected Ae. aegypti and in wild-
caught mosquito population in Singapore.
Materials and Methods
Mosquito homogenization, NS1 antigen detection,
and sample preparation
Individual mosquito was homogenized with Retsch mixer
mill MM 200 (Germany) in 250mL of phosphate-buffered sa-
line. One hundred microliters of the mosquito homogenate
was used on Dengue NS1 AgStrip accordingto theprocedure
provided with the kit. To compare with RT-PCR, viral RNA
was extracted from 100mL of the remaining mosquito ho-
mogenate using the QIAmp viral RNA mini kit (Qiagen) ac-
cording to the manufacturer’s recommendation and stored at
?808C before its use.
Evaluation of NS1 for DENV detection
with laboratory-infected Ae. aegypti
To determine the feasibility of using Dengue NS1 Ag Strip
to detect all DENV serotypes antigen in infected mosquitoes,
5- to 7-day-old laboratory-colonized Ae. aegypti L. (F186) were
fed with an infectious blood meal, consisting of 1:1 washed
4 suspension, supplemented with 5mM ATP as a phagosti-
mulant. Feeding was performed using a Hemotek membrane
feeding system (Discovery Workshops) housed in anisolation
glove box within an Arthropod Containment Level 3 insec-
tary.Blood mealtiter for theinfectious mealused in thisstudy
was determined by titration to be 7.52, 6.95, 5.52, and 7.52
Log10tissue culture infectious dose50/mL for DENV sero-
types 1, 2, 3, and 4, respectively. Mosquitoes were incubated
at 308C and an RH of 80%.
To evaluate the specificity of the NS1 kit for all four sero-
types, four sets of eight mosquitoes, each infected with DENV
1, 2, 3, and 4, were sacrificed and homogenized at day-10
postinfectious (pi) blood meal. A dengue group-specific one-
step SYBR Green I-based one-step real-time RT-PCR assay
was performed as previously described (Lai et al. 2007).
To determine the sensitivity of NS1 Ag Strip, mosquitoes
infected with DENV 2 were sacrificed at 1, 2, 3, 4, 7, and 10
days pi blood meal and homogenized. A DENV 2–specific
one-step quantitative RT-PCR assay targeting a 177-bp re-
gion of the NS5 gene (Richardson et al. 2006) was per-
formed in a LightCycler 2.0 system using a LightCycler
RNA Master SYBR Green Kit I (Roche Diagnostics, GMbH).
Real-time quantitative RT-PCR assay conditions follow
that of Lai et al. 2007. Amplification of the target gene from
each individual mosquitoes was compared against a stan-
dard curve (Efficiency¼1.814, error¼0.0461) generated
from a 10-fold serial dilutions of NS5 DENV 2 RNA stan-
dard, and LightCycler software version 4.05 was used for
Comparison of Dengue NS1 Ag Strip and RT-PCR
in detecting DENV in wild-caught mosquitoes
A real-time RT-PCR assay is currently the standard
method at the Environmental Health Institute, a public
health laboratory,in screening field-collectedmosquitoesfor
the presence of arboviruses. The sensitivity of the Dengue
NS1 Ag Strip in detecting DENV antigen in field-caught
mosquitoes was thus compared with that of the molecular
Between July and August 2009, 83 in-house developed
sticky traps were deployed in 2 apartment blocks located
northeast of Singapore, where active DENV transmission was
on-going. Sticky traps were checked twice a week for 5 weeks
for the presence of mosquitoes. All Ae. aegypti caught in the
sticky trap were carefully removed and transferred individ-
ually into 1.7mL micro-centrifuge tubes for homogenization
for NS1 detection and RT-PCR according to Lai et al. 2007.
Results and Discussion
Specificity of Dengue NS1 Ag Strip to detect all four
The ability of the Dengue NS1 Ag Strip to detect all DENV
serotypes in infected Ae. aegypti and to detect DENV NS1
protein from a single infected mosquito at different time
points (days pi) was evaluated in the current study. The re-
sults obtained by the rapid test kit in detecting DENV 1, 2, 3,
and 4 in individual mosquitoes have been found to be con-
gruent to the results obtained by RT-PCR. All eight mosqui-
toes infected with each serotype were found to be positive for
both Dengue NS1 Ag Strip and the molecular method. The
rapid test kit did not show any positive results when tested
against uninfected mosquitoes. The results show the ability of
the rapid test kit in detection all DENV serotypes, and are in
agreement with studies conducted with human samples
(Dussart et al. 2008, Pok et al. 2010).
Sensitivity of Dengue NS1 Ag Strip in detecting DENV
Simple dilution of homogenate of infected mosquitoes may
not appropriately reflect the biological ratio of viral NS1:viral
RNA copy. Sensitivity of the rapid kit was thus determined
time points, which resulted in mosquitoes having varied viral
loads (Table 1). All infected mosquitoes were tested positive
by the molecular assay; however, two infected mosquitoes
sampled at day 2pi (Table 1) were found to be negative by the
rapid test kit. The viral load in these two mosquitoes corre-
sponds to <2?105DENV RNA copy per mosquito, whereas
the NS1-positive mosquitoes were all above 4?105DENV
RNA copy per mosquito. As illustrated in Table 1, Dengue
NS1 Ag Strip assay reported in this study has a detection limit
of 106DENV copy per mosquito. Mosquitoes that have in-
gested low DENV dose and in its early stage of infection
(eclipse phase) may escape detection, usually on day 2pi.
Nevertheless, among the 48 infected mosquitoes collected at
different time points (1, 2, 3, 4, 7, and 10pi), the rapid test kit
had a sensitivity of 95.8%.
Out of 44 field-caught Ae. aegypti, two (4.54%) were found
to be infected with DENV by both the real-time RT-PCR
assay and the Dengue NS1 Ag Strip tests (Table 2). All field-
caught mosquitoes that tested negative by the NS1 rapid test
kit were also negative by the real-time molecular detection
assays. As a result of this study, NS1 rapid is currently being
used to detect DENV in mosquitoes caught from dengue
cluster areas in Singapore, as part of vector control assessment.
Among 10 clusters, 634 mosquitoes were caught and pooled.
790 TAN ET AL.
Of the 126 pools, 6 pools were positive for NS1, and confirmed
by RT-PCR (Vythilingam et al. unpublished document).
Surveillance for dengue-infected mosquito populations
may assist in risk assessment and evaluation of vector control
measures. The NS1 rapid test kit can easily be performed with
minimal training in a simple field station. The results can be
obtained in less than an hour. We have shown the potential of
the kit for field surveillance of infected Aedes spp. mosquitoes,
which are epidemiologically important.
However, it is now well understood that the extrinsic in-
cubation period and the replication rate of DENV in mos-
quitoes are influenced by the genotypes of the virus, the
infectious dose, and the strain of Ae. aegypti (Anderson and
kit will be influenced by the virus–mosquito partnership.
Only through more field studies from different countries can
we determine the usefulness of the kit in detecting transmis-
sion in different epidemiological setting.
Table 1. Comparison Between Dengue NS1 Ag Strip (BioRad Laboratories)
and Real-Time Quantitative Reverse Transcriptase–Polymerase Chain Reaction in Detecting Dengue Virus-2
in Laboratory-Infected Mosquitoes Sampled at Different Time Points
blood mealMosquito no.
copy number/mL template)
Viral copy in
DENGUE NS1 KIT FOR DETECTION OF DENV IN Aedes aegypti
We thank colleagues at EHI, especially Ms. Lai Yee Ling
and Ms. Pang Sook Cheng, for their assistance during the
laboratory aspect of the study, and Mr. Caleb Lee, Ms. Du-
langi Subadrage, Mr. Md.Alif Abdul Razak, and Mr. Leong
Chee Kong for their assistance in the collection of field mos-
quitoes. We also thank the Ministry of Finance for the Re-
investment Fund made available for the study.
virus infection, and molecular and immunological aspect of
the laboratory study, and prepared the article. I.V. led the col-
No competing financial interests exist.
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Table 2. Number of Aedes aegypti Caught Per
Week and Number of Mosquitoes Found to be Infected
with Dengue Virus by Dengue NS1 Ag Strip
(BioRad Laboratories) and Real-Time Reverse
Transcriptase–Polymerase Chain Reaction Assay
No. of positive mosquitoes
No. of mosquitoes
RT-PCR, reverse transcriptase–polymerase chain reaction.
792 TAN ET AL.