Journal of Medical Entomology, XX(XX), 2023, 1–11

https://doi.org/10.1093/jme/tjad126
Research

Vector-Borne Diseases, Surveillance, Prevention

Dengue 1 outbreak in Rosso, northern Senegal, October

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2021: entomologic investigations
Babacar Diouf1,*, , Alioune Gaye1, Idrissa Dieng2, Cheikh Tidiane Diagne2,3,
El Hadj Ndiaye1, Moufid Mhamadi2, Assiyatou Gueye1, Oumar Ndiaye2,3,
Ndeye Marie Sene1, Faty Amadou Sy1, Oumar Faye2, Ibrahima Dia1, , Scott C. Weaver4,
Mawlouth Diallo1, Diawo Diallo1,
1
Pôle de Zoologie Médicale, Institut Pasteur de Dakar, Dakar 220, Senegal 2Pôle de Virologie, Institut Pasteur de Dakar, Dakar
220, Senegal 3DIATROPIX, Institut Pasteur de Dakar, Dakar 12900, Senegal 4World Reference Center for Emerging Viruses and
Arboviruses, Institute for Human Infections and Immunity, Department of Microbiology and Immunology, University of Texas
Medical Branch, Galveston, TX 77555, USA *Corresponding author: mail: babacar.DIOUF2@pasteur.sn

Subject Editor: William Reisen

Received on 11 May 2023; revised on 25 August 2023; accepted on 4 September 2023

Senegal has experienced periodic epidemics of dengue in urban areas with increased incidence in recent
years. However, few data are available on the local ecology of the epidemic vectors. In October 2021, a dengue
outbreak was reported in northern Senegal to the Institute Pasteur de Dakar. Entomologic investigations then
were undertaken to identify the areas at risk of transmission and to identify the vector(s). Adult mosquitoes
were collected indoors and outdoors at selected households, while containers with water were inspected
for mosquito larvae. All the Aedes aegypti (L.) collected were tested for dengue virus NS1 protein using a
rapid diagnostic test (RDT), and positive samples were confirmed by real-time RT–PCR. The qRT–PCR posi-
tive samples were subjected to whole genome sequencing using Nanopore technology. The majority of the
larvae-positive containers (83.1%) were used for water storage. The Breteau and Container indices exceeded
the WHO-recommended thresholds for the risk of dengue virus transmission except at 2 localities. Ae. aegypti,
the only reputed dengue vector, was collected resting indoors as well as outdoors and biting during the day
and night. The NS1 protein was detected in 22 mosquito pools, including one pool of females emerging from
field-collected larvae. All NS1-positive results were confirmed by RT-PCR. Virus serotyping showed that the
outbreak was caused by DENV-1. This study demonstrates the need for continuous control of adult and aquatic
stages of Ae. aegypti to prevent future dengue epidemics in Senegal. RDTs appear to be a promising tool for
dengue diagnostics and surveillance.

Key words: DENV-1, Rosso, outbreak, Aedes aegypti, Senegal

© The Author(s) 2023. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: 1
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2 Journal of Medical Entomology, 2023, Vol. XX, No. XX

Graphical Abstract

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Introduction in 2 neighborhoods of Dakar. With the continued non access of a
vaccine against DENV in Africa, mosquito vector surveillance and
In Africa, the existence of a sylvatic cycle of DENV-2 historically
control programs are essential to reduce human infections. Point-
was considered the principal mode of virus circulation, in contrast
of-care testing with antibody/antigen-detecting RDTs including the
to Asia or Latin America where mosquito-to-human transmis-
NS1 protein is increasingly available and has demonstrated consid-
sion predominates. Several epizootics of DENV-2 have occurred
erable potential for surveillance of DENV (Phommasone et al. 2015,
through amplification of the virus within a sylvatic cycle (involving
Choi et al. 2017). These RDTs also could be used to detect DENV
sylvatic mosquitoes and monkey populations), with human cases
in infected mosquitoes and would be a valuable addition to the sur-
representing “spillover” or tangential transmission (Cordellier et al.
veillance effort.
1983, Cordellier 1984, Diallo et al. 2003, 2022). The other DENV
Since 2011, in collaboration with the Senegal Ministry of Health
serotypes are believed to have only endemic human-to-human cir-
and Social Action (MOHSA), the Institut Pasteur de Dakar (IPD)
culation in Africa, with no evidence for the sylvatic cycle, which
has set up a national surveillance system for influenza and other
only has been identified in Asia (Diallo et al. 2022). Despite frequent
viruses associated with respiratory tract infections, namely the 4S
African DENV-2 epizootics and the existence in all bioclimatic areas
network (Thiam et al. 2014). This system was enhanced in 2015
of the epidemic vector, dengue epidemics rarely have been detected
to add surveillance of other pathogens such as dengue, Zika and
in Africa. However, during the last 2 decades, a major change in epi-
Rift Valley fever (Bob et al. 2022). During the following years,
demiology has been observed, with several urban epidemics reported
this human sentinel surveillance detected numerous sporadic cases
in Senegal (Faye et al. 2014, Dieng, Ndione, et al. 2021).
and outbreaks of dengue. In 2017, cocirculation of DENV-1 and
In Senegal, DENV-4 was first isolated in December 1981 from
-2 was reported in Louga, and DENV-2 in Rosso (Dieng, Cunha,
a patient who had just arrived from Haiti. In November 1983,
et al. 2021, Dieng, Ndione, et al. 2021). In 2018, different DENV
DENV-4 was isolated from both parents of a small European family
serotypes were reported in 8 regions of Senegal: Fatick (DENV-1
living in Dakar. Subsequently, a fourth case was diagnosed by se-
and -3), Diourbel (DENV-3 with only one death recorded in the
rology in Casamance (southwestern Senegal) (Saluzzo et al. 1986).
medical district of Touba), Saint Louis (DENV-2), Louga (DENV-1),
These cases were the first evidence of DENV-4 transmission in West
Thiès (DENV-1 and -3), Dakar (DENV-3), Matam (DENV-2), and
Africa. DEN-1 was first isolated in October 1979 from the sera of
Kaolack (DENV-3) (WHO 2017, Diagne et al. 2019, Sokhna and
febrile patients in the village of Bandia, 80 km southeast of Dakar
Gautret 2019, Dieng, Ndione, et al. 2021). The circulation of these
(Robin et al. 1980). Serological surveillance was conducted in this
different serotypes in several urban localities (Sokhna and Gautret
area from 1980 to 1982, when 21.5% of children showed anti-
2019, Dieng, Cunha, et al. 2021) comprised growing evidence of
DENV-1 antibodies. Until 2009, only DENV-1, -2, and -4 were
dengue hyperendemicity in Senegal.
detected or isolated in Senegal. Serotype 3 was first implicated in a
Despite the frequency of reported dengue cases over the past
2009 epidemic in Dakar with 196 people affected, including 5 cases
decade, data on the ecology of the mosquito vectors and their roles
of dengue hemorrhagic fever and one fatal case of dengue with shock
during urban epidemics have been limited. Usually, entomological
syndrome (Faye et al. 2014). Ae. aegypti, the only dengue vector col-
surveys have not been successful due to the delay between dengue
lected, were found at all sites, with high epidemic risk in all localities
detection from humans and the outbreak investigation response. To
infested. DENV-3 was detected from 3 pools of mosquitoes collected
Journal of Medical Entomology, 2023, Vol. XX, No. XX
fill this gap, a rapid entomological response was conducted after a
human case of DENV-1 was detected in the city of Rosso, northern
Senegal, in October 2021 (Keita et al. 2022). The objectives of this
study were to assess vector abundance and behavior and to estimate
the entomologic indicators for dengue transmission risk in the af-
fected areas and neighborhoods to generate recommendations for
the most appropriate control strategies.
Materials and Methods
Study Site Description
The investigation was conducted in the towns of Rosso (16°25ʹ13″N;
15°47ʹ54″W) and Richard-Toll (16°27ʹ44″N; 15°42ʹ02″ W) within
the medical district of Richard-Toll, located in northern Senegal
(about 388 km from Dakar), from October 15 to 25, 2021. Rosso is
a border town between Senegal and Mauritania, with an estimated
population of 10,717 living in 753 houses. The climate is Sahelian
with daytime temperatures ranging from 27 to 39 °C in October. The
Senegal River, the main source of surface water, as well as the Diama
anti-salt dam, have allowed the irrigation of agricultural areas along
the valley. The largest part of the commune of Rosso is located in
a basin with flat topography. Flooding occurs every rainy season
due to limited porosity of the clay soil. The localities visited were
Santhiaba 1, 2, 3, and 4, Diameguene, Rosso-Peulh, Médina, Cité
Niakh, and Mbagam (Fig. 1). Richard-Toll is located 21 km from
Rosso on both sides of a national road and is crossed by the Taouey
River which is the junction between the Senegal River and lake “Lac
de Guiers.” The population is estimated at 48,968. The predominant
soils of the town are clay and hydromorphic; they have low per-
meability, therefore, leading to flooding. It is warm all year round
with temperatures ranging from 23 to 37 °C in October. The rainiest
months are: July, August, and September. The only locality visited in
this town was Thiabakh (Fig. 1).
Fig. 1. Collection sites for aquatic and adult samples of Aedes aegypti, Richard-Toll district, October 2021.
3
Mosquito Sampling
During this entomological survey, which was carried out at the same
time as the epidemiological studies, 107 suspected cases were re-
corded, 102 of which were confirmed as DENV (Dieng et al. 2022).
Sampling of immature and adult mosquitoes was conducted in-
door and outdoor of houses of confirmed dengue cases, their imme-
diate neighborhoods (the 4 houses located in front of, behind, to the
left, and to the right of the confirmed cases’ houses), opportunisti-
cally selected houses, and shops for used tires, which are frequently
used by Ae. aegypti as larval habitats.
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Immature A. aegypti collections. All water storage or discarded
containers and all artificial containers likely to contain water found
indoors and outdoors (in the peridomestic environment) were
inspected at 830 housing units (number of bedrooms where people
slept) within 166 house compounds. Samples from each positive
container were collected using ladles and pipettes or, in the case
of large containers such as drums, the water was poured through
a sieve into a basin, and the larvae or pupae were collected from
the sieve using Pasteur pipettes. Only containers with water were
recorded for analysis. Indoor and outdoor water containers were
scored separately as either negative (with no Ae. aegypti immatures)
or positive (with at least one Ae. aegypti immature).
Sampling of resting and host-seeking adult mosquitoes. Adult
mosquitoes resting indoors and outdoors were collected for 10 min
in the morning from at least 15 houses in each locality surveyed
and at 10 used tires shops (6 in Rosso and 4 in Thiabakh) using a
Prokopack Aspirator (John W. Hock Co., Gainesville, Model 1419;
Maia et al. 2011). Active mosquitoes, considered host-seeking based
on their attraction to CO2 and human chemicals, were collected
in 3 confirmed case houses of each locality during one day (0600
to 1900h) and night (1900 to 0600h), using 2 BG-Sentinel traps
4 Journal of Medical Entomology, 2023, Vol. XX, No. XX
(Biogents AG, Regensburg, Germany) per house baited with dry
ice and BG-Lure, with one set indoors and one set outdoors in
bedrooms.
Sample Processing in the Field
The collected larvae were transferred to labeled bottles and transported
to the field station. Immature mosquitoes sampled from each type of
water container were reared to adults and identified morphologically
on a chill table using appropriate keys (Edwards 1941, Huang 2004).
Water containers where Ae. aegypti mosquitoes emerged were con-
firmed as positive. Ae. aegytpti mosquitoes were pooled by sex, phys-
iological status (fed or unfed), collection method, location, and day of
collection into 100 female pools and 51 male pools (10.9 mosquitoes
per pool), then stored in 2-ml microfuge tubes in liquid nitrogen or in
a freezer at −80 °C until processing at IPD.
Sample Processing in the Laboratory
Mosquito trituration. Mosquito pools were homogenized in 1.5-
ml cryogenic vials containing 500 μl of L-15 medium (GibcoBRL,
Grand Island, NY, USA) using sterile pestles in a biosafety level 2
laboratory. The homogenates were centrifuged at 8,000 rpm for 10
min at 4 °C to remove mosquito debris.
NS1 rapid detection test. The GADx prototype immunochrom­
atographic Dengue NS1 Ag kit (Reference: 12612102; Lot:
DNLFD-006-1) was used to detect DENV infection. The 151
A. aegypti pools were first combined into 24 “super pools” each
containing 6 to 7 pools sorted according to the physiological status
of the mosquitoes (blood fed or unfed). Each super pool contained
600 to 700 μl (i.e. 100 μl from each of the constituent 6 or 7 pools)
of supernatant for antigen testing. In each well, 40 µl of supernatant
was added followed 10 seconds later by 40 μl of buffer. Results were
read by eye after 10 min of fluid migration at room temperature
according to the manufacturer instructions. The pools within
each positive super pool then were tested individually for NS1 as
described earlier.
Molecular detection and serotyping of DENV. Super pools and their
constituent individual pools were retested by qRT-PCR. For each
sample, 100 μl of supernatant were used for RNA extraction with
the QiaAmp Viral RNA Extraction Kit (Qiagen, Heiden, Germany)
according to the manufacturer’s instructions. The DENV 3ʹUTR
region was amplified by qRT-PCR using a set of primers previously
described by Wagner et al. (2004) on a Biorad CFX96 instrument
using a QuantiTect kit (Qiagen, Hilden, Germany). The assay was
performed in a final reaction volume of 25 μl containing 5 μl of
extracted RNA, 10 μl of buffer (2× QuantiTect Probe), 6.8 μl of
RNase-free water, 1.25 μl of each primer, 0.5 μl of probe, and 0.2
μl of enzymes. To determine the DENV serotype by qRT–PCR,
real-time molecular serotyping was performed on the RNA extracts
using the commercial Tib-Molbiol Modular Dx Dengue Typing Kit
(Cat-No. 40-0700-24) on the Lightcycler 480 (Roche, Penzberg,
Germany) according to a previously published protocol (Dieng et
al. 2021).
Sequencing and phylogenetic analysis. Confirmed DENV-1-positive
samples were subjected to genome sequencing as previously described
by Dieng et al.(2022). Briefly, RNA samples were subjected to cDNA
synthesis using Lunascript RT supermix kit (New England Biolab,
County Road, Ipswich, MA, USA) according to the manufacture’s
recommendations. Using primer schemes specific to DENV-1, the full
coding sequence (CDS) was amplified in 2 separate reactions using
Q5 high fidelity 2× master mix (New England Biolabs County Road,
Ipswich, MA, USA). Amplicons of expected 1 kb size were visualized
on agarose gels and purified using Ampure beads (Beckman Coulter)
at a 1:0.8 ratio. For sequencing, the Rapid Barcoding Kit 96 (SQK-
RBK110.96) was used to tag purified amplicons following the
manufacturer’s instructions. A blank control sample was included.
The libraries were quantified, normalized, pooled, and loaded
onto Oxford Nanopore MinION platform R9.4.1 flow cells (FLO-
MIN106D). Guppy was used to “base-call” the sequencing reads,
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which were merged into a single Fastq file. Consensus sequences
were generated using genome detective. The newly assembled
DENV-1 genotype was defined using the genome detective dengue
typing tool. To create a dataset for analysis, nearly complete
genomes obtained during this work (n = 9) were combined with
a representative subset of DENV sequences available in Genbank
(n = 60) that were from Senegal, Africa, and global locations. The
final dataset (n = 69) was aligned using MAFFT (Katoh et al. 2002),
and a Maximum Likelihood (ML) tree was drawn using IQ-TREE
(Nguyen et al. 2018) with an automatic model selection argument
using a model finder (MF). The tree was visualized and annotated
using Figtree. Bootstrap values were determined for 1,000 replicates
to assess robustness of clades.
Data Analysis
The mosquito larval indices used were the Breteau Index (BI) or the
number of containers positive for immature stages of Ae. aegypti
per 100 housing units (Taufflieb 1972), and the Container Index
(CI) or the number of containers positive for immature Ae. aegypti
per 100 inspected water containers (WHO 2011). The epidemic
thresholds were set at 5% and 3% for the BI and CI, respectively,
according to WHO-documented thresholds. PooledInfRate (Version
4.0) software was used to calculate and compare viral infection
rates (IR) or, more accurately, viral RNA detection frequency from
mosquito pools tested (Biggerstaff 2016). The IR were reported per
1,000 mosquitoes tested and any pair of IRs (e.g. resting popula-
tion vs. host-seeking population IR) was considered not significantly
different if the confidence interval of the difference included zero.
Kruskal–Wallis tests were used to compare mosquito abundance at
different localities, and the Wilcoxon test was used for comparison
of resting or biting mosquito abundance indoors and outdoors.
Results
Typology of Infested Containers and Entomological
Risk Indices
A total of 166 houses (including 830 housing units) were visited, and
481 water containers were inspected (see Supplementary Table S1).
More than half of the 59 Ae. aegypti-positive containers (52.5%; N
= 31) were found indoors. Most positive containers (83.1%) were
water storage containers (plastic basins, plastic buckets, plastic/me-
tallic drums, plastic jerry cans, plastic bottles, and drinking water
pots), followed by used tires and puddles (5.1% each), discarded
containers (jerry cans, buckets, and basins), and flooded rooms (3.4%
each). Water storage containers were the only infested containers in
Santhiaba 4, Medina, and Thiabakh, whereas in the other localities,
although they were the majority, mosquito larvae also were collected
in other types of containers (see Supplementary Table S1).
No Ae. aegypti-positive containers were found in the localities
of Rosso-Peulh and Mbagam. The BI and CI in the other localities
exceeded the WHO-documented thresholds for dengue outbreak/
transmission risk (Fig. 2).
Journal of Medical Entomology, 2023, Vol. XX, No. XX 5

Mosquitoes Collected as Adults
A total of 11,936 adult mosquitoes belonging to 4 genera and 14
species was collected by all sampling methods. A. aegypti, the only
reputed dengue vector collected in the area, accounted for 13.4% of
the mosquitoes collected (Table 1).
Abundance and Behavior of A. aegypti
In the district Richard-Toll, 92.6% (673 females and 560 males, N
= 1,331) of adult Ae. aegypti were collected in 53.3% of the 166
houses investigated. Statistically more Ae. aegypti were collected
resting indoors (0.9 ± 1.6 females per house) than outdoors (0.5
± 2.1 females per house; Fig. 3A; W = 7303, P < 0.0001). Females
were collected host-seeking in statistically similar numbers in-
doors (38.1 ± 109.5 females per trap) and outdoors (5.1 ± 7.3
females per trap; W = 49, P = 0.5). A. aegypti appeared to seek
blood meals during both the day and night, mainly within indoor
rooms (Fig. 3B). The epidemic vector was also collected resting
indoors (4.7 ± 4.1 females per shop) and outdoors (4.2 ± 5.0
females per shop) in comparable numbers at tire repair shops
(W = 49, P = 0.7).

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Fig. 2. Epidemic risk indices, Richard-Toll district, October 2021. The lines indicate the WHO thresholds for epidemic risk. A. CI; Container index; B. BI: Breteau
index; where PC: Positive containers; TC: Total containers inspected; HU: Habitation units.

Table 1. Adult mosquitoes collected by aspiration, BG traps, and emerging from larvae, Richard-Toll District, October 2021

Sampling methods

Species Aspiration EFL BG traps Total

Males Females Males Females Males Females

Aedes aegypti (Linnaeus, 1762) 260 259 129 141 343 469 1601
Anopheles coustani Laveran, 1900 — — — — 0 5 5
Anopheles gambiae Giles, 1902 51 142 — — 23 19 235
Anopheles pharoensis Theobald, 1901 1 3 — — 0 74 78
Anopheles rufipes Gough, 1910 0 1 — — 0 1 2
Anopheles sp - - — — 0 2 2
Anopheles ziemanni Grünberg, 1902 0 2 — — 0 2 4
Culex antennatus (Becker, 1903) 0 6 — — 0 151 157
Culex neavei Theobald, 1906 0 3 — — 0 1 4
Culex poicilipes (Theobald, 1903) 0 1 — — 0 22 23
Culex quinquefasciatus Say, 1823 2184 2168 44 91 2701 1534 8722
Culex tritaeniorhynchus Giles, 1901 64 152 0 2 27 826 1071
Mansonia africana (Theobald, 1901) — — — — 0 17 17
Mansonia uniformis (Theobald, 1901) 0 2 — — 0 13 15
Total 2560 2739 173 234 3094 3136 11936

EFL: emerging from larvae.

6 Journal of Medical Entomology, 2023, Vol. XX, No. XX
Fig. 3. Resting behavior (A) and blood meal seeking (B) of Aedes aegypti collected in the Richard-Toll District, October 2021.
DENV Detection by RDTs and RT-PCR
The results of the antigenic tests showed that 14 of the 24 super
pools (comprising 57 female and 29 male pools) were positive for
dengue NS1 protein. Individual pool tests showed that all of the
male pools were negative, whereas 22 of the 57 female pools were
positive for DENV NS1, including 15 unfed + gravid female pools,
6 blood-fed pools and 1 pool adult females that emerged from field-
collected larvae. All of these NS1-positive samples were confirmed
by real-time DENV RT-PCR.
Among 22 DENV-positive samples, 15 were successfully
serotyped as DENV-1; 7 failed to be serotyped. Serotypes 2, 3, or 4
of DENV were not detected.
Phylogenetic Analysis
The Maximum Likelihood (ML) tree (Fig. 4) revealed that all the
sequences obtained during this entomological investigation belonged
to DENV-1/Genotype V and to the same clade as determined for
samples from human cases detected in Rosso during this outbreak
(Dieng et al. 2022). Interestingly strains from this cluster were dif-
ferent from strains sequenced during previous DENV-1/Genotype
V outbreaks in Senegal which occurred in the Louga area. These
results confirmed that DENV-1 was responsible of the Rosso epi-
demic. Furthermore, our results showed that circulating strains in
Rosso are closely related to strains circulating in Burkina Faso (ac-
cession number: MW243050.1) and Cote d’Ivoire (MW243052.1)
in West Africa.
Infection Rates of Mosquitoes
Table 2 shows the IRs of Ae. aegypti mosquitoes tested by RT-PCR
and collected as adults in houses and tire repair shops. Overall,
the IR was 2.5 infected mosquitoes/1,000 mosquitoes tested in the
communes of Rosso Senegal.
Household IR ranged from 39 infected mosquitoes/1,000
mosquitoes tested in Santhiaba 4 to 1 infected mosquito/1,000
mosquitoes tested in Santhiaba 1. These IRs were statistically
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comparable with the exception of Santhiaba 3 and Santhiaba 4,
which had a higher IRs compared to Santhiaba 1 (Table 2). All the
resting DENV-infected females were collected indoors. Statistically,
more DENV-infected females Ae. aegypti were collected host-seeking
outdoors (13 infected mosquitoes/1,000 mosquitoes tested) than in-
doors (1 infected mosquitoes/1,000 mosquitoes tested).
In tire workshops, the 3-positive pools came from 2 of the 6
neighborhoods investigated: Diamagène (2 pools collected indoors)
and Santiaba 1 (1 pool collected outdoors) with IRs of 54 and 170
infected mosquitoes/1,000 mosquitoes tested, respectively. IRs in tire
workshops (IR = 75.4 infected mosquitoes/1,000 mosquitoes tested)
were statistically higher compared to houses (IR = 2.1 infected
mosquitoes/1,000 mosquitoes tested).
The IR of blood-fed females (IR = 2.2 infected mosquitoes/1,000
mosquitoes tested) was significantly higher than that of unfed +
gravid females (IR = 0.5 infected mosquitoes/1,000 mosquitoes
tested).
Analysis of 22 pools comprised of 270 adult Ae. aegypti emerging
from larvae collected in Cité Niakh, Diameguene, Medina, Santhiaba
1, 2, 3, and 4 detected NS1 protein in 1 pool of 8 female mosquitoes
collected in a water storage container in Santhiaba 4. These results
were confirmed by RT–PCR detection of DENV-1 in the same pool.
The IR was 0.2 infected mosquitoes/1,000 mosquitoes tested.
Discussion
A. aegypti, currently the only known vector of dengue in urban areas
of Senegal, was the only Aedes species collected in this study. This
species immature stages frequently were collected in water storage
containers that are known to be suitable larval habitats in domestic
environments (Koenraadt and Harrington 2008, Guindo‐Coulibaly
et al. 2022). Indeed, the long-term storage of drinking water or water
for domestic use from these containers that are constantly filled and
poorly sealed accounted for their high infestation rate compared to
other domestic aquatic sites (Konan et al. 2013). Although water
storage containers were the main sites infested, the presence of
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Fig. 4. Maximum likelihood (ML) tree based on nearly complete genome sequencing (coding region without UTRs) showing phylogenetic relationships among
DENV-1 strains from field collected mosquitoes during the Rosso outbreak in 2021 and DENV sequences from GenBank. Tips are labelled using Sequence
identifier, Region/ Country of sampling and the Year of collection. DENV serotypes labels are placed on each serotype parental nodes.

numerous used tires and dry abandoned containers in the domestic Our study showed that, more than 15 days after the last rain
environment suggests that they may have played an important role and during high ambient temperatures, BI and CI were above
in Ae. aegypti ecology during the rainy season that ended two weeks the epidemic risk threshold, with the exception of Rosso-peulh
before our survey. and Mbagam, indicating that most neighborhoods were at risk
8 Journal of Medical Entomology, 2023, Vol. XX, No. XX

Table 2. Infection rates of resting and host-seeking Aedes aegypti populations infected with dengue virus, Richard-Toll District, October
2021

Position

Indoor Outdoor

Sites Localities Method N.M N.P P.P IR CI 95% N.M N.P P.P IR CI 95% Global IR [CI]

Houses Cité Niakh Prokopack 32 3 1 10.5 [0.7;57] 7 2 0 0 — 9.3 [0.6;49]

BG-trap 0 0 0 NA NA 0 0 0 NA NA
Medina Prokopack 22 6 3 27.9 [7.7;81] 7 3 0 0 — 19.9 [6.5;49,3]

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BG-trap 18 4 1 14 [0.9;72] 5 2 0 0 —
Santhiaba 1 Prokopack 118 3 1 2.9 [0.2;16] 87 3 0 0 — 1 [0.5;1,9]
BG-trap 683 14 6 0.8 [0.3;2] 52 7 2 6 [1.1;20]
Santhiaba 3 Prokopack 0 0 0 NA NA 4 2 0 0 — 29.5 [7.9-80,2]
BG-trap 11 4 1 22.8 [1.4;114] 10 6 2 36.4 [6.7;121]
Santhiaba 4 Prokopack 7 2 0 0 — 1 1 0 0 — 39 [2.4;178]
BG-trap 2 1 0 0 — 4 2 1 127.7 [8.2;575]
Workshops Diamagene Prokopack 15 3 2 56 [11;228] 1 1 0 0 — 54 [10.5;211]
Santiaba1 Prokopack 0 0 0 NA NA 3 2 1 170 [11;681] 170 [11;681]
Total Houses Prokopack 274 28 5 10 [4;21] 147 21 0 0 — 2.1 [1.4;3.1]
BG-trap 718 25 8 1 [0.5;2] 94 24 5 13 [5.1;28]
Total Workshops Prokopack 40 16 2 56 [11;228] 58 15 1 149 [9;583] 75.4 [22;215]

NM: number of mosquitoes tested; NP: number of pools tested; PP: positive pools, IR: infection rate; IC 95%: confident interval at 95%.
Totals represent all 151 mosquito pools tested.

for DENV transmission. With the increasing burden of dengue
worldwide, identification of specific areas with the highest risk of
epidemics is of paramount importance in prioritizing resource-
intensive interventions (Bowman et al. 2014). This requires a good
knowledge of the resting and host-seeking behavior of the epidemic
vector in the affected area. In our study, Ae. aegypti was found
resting both indoors and outdoors, as previously described in south-
eastern Senegal (Diallo and Diallo 2020); however, all the resting
DENV-infected females were collected indoors. The host-seeking
females were collected both indoors and outdoors, with higher IRs
outdoors. These resting and host-seeking behaviors of the epidemic
vector, which may vary among geographical areas (Diarrassouba
and Dossou-Yovo 1997, Chadee 2013, Dzul-Manzanilla et al. 2016,
Labbo et al. 2019, Captain-Esoah et al. 2020), may guide efficient
and effective vector control interventions for dengue risk reduction
and outbreak mitigation.
The higher IR in blood-fed compared to unfed females indicated
that a portion of the positive females likely had DENV in their blood
meal and were not truly infected. This finding suggests the possibility
of using blood-fed female mosquitoes (all species) for early detection
of DENV and other pathogens circulating in human populations
(Cameron and Ramesh 2021).
Resting Ae. aegypti adults were collected inside and outside of
workshops. These locations possibly were critical sources of prolif-
eration, maintenance, and dispersal of the vector. Indeed, the tires
exposed outside shops are often productive oviposition and larval
habitats for Ae. aegypti (Lounibos et al. 2002, Bennett et al. 2019,
González et al. 2020) and indoor tire storage also provides a good
resting place for adult mosquitoes (Diallo and Diallo 2020). The
detection of DENV in mosquitoes collected in these abundant tire
workshops with higher IRs compared to those observed in houses,
indicated that they may play an important role in the spread of
DENV in Senegal. In addition, these tires act as mobile reservoirs of
mosquito eggs and larvae, and their trade could be responsible for
the introduction of DENV into disease-free areas considering the
vertical transmission that was detected in our study (Pliego Pliego et
al. 2018). Therefore, used tires shops should be included in surveil-
lance and control programs for Ae. aegypti-borne viruses.
Molecular serotyping confirmed DENV-1 as the etiologic agent
of this outbreak, suggesting that DENV-1 replaced DENV-2, that
was responsible for the 2018 outbreak in Rosso (Dieng et al. 2022).
The introduction of this new serotype combined with other factors
such as the increasing urbanization of Rosso and the strong pres-
ence of Ae. aegypti could explain, in part, the high prevalence of
dengue (Keita et al. 2022) during this outbreak. Results from our
phylogenetic analysis confirmed previous findings, highlighting
that the 2021 Rosso epidemic was caused by DENV-1 (Dieng et al.
2022). Clustering of human strains and those from field-collected
mosquitoes highlights the occurrence of ongoing active transmission
at the time of our investigation, as well as the role of Ae. aegypti in
DENV transmission in Rosso. These findings corroborate previous
results indicating the rising regional endemic circulation of DENV,
as our sequences grouped with limited DENV-1 sequences available
from other parts of West Africa.
Our results also showed that the GADx prototype DENGUE-NS1
rapid test had very good sensitivity (i.e. 100%) towards DENV NS1
protein in mosquitoes collected directly in the field when using
RT–PCR as a reference method. These results were comparable to
previous data obtained by Tan et al. (2011), who showed that the
Dengue NS1 Ag Strip kit to was able to detect all 4 DENV serotypes
in laboratory-infected and wild-collected Ae. aegypti and was as
sensitive as real-time RT–PCR. In another study, Voge et al. (2013)
showed that detection of DENV infection using the Platelia Dengue
NS1 Ag kit was more sensitive than RT–PCR and laboratory virus
isolation. Abraham et al. (2021) also showed that a commercially
available Dengue NS1 antigen kit (J. Mitra & Co. Pvt. Ltd) was
highly sensitive and specific towards for the detection of recombi-
nant dengue virus-2 (rDENV-2) NS1 protein in serum of dengue-
infected patients or spiked Ae. aegypti pools. Our results strongly
indicate that DENV RDTs are cheaper and easier to use and are
promising tools for the surveillance of DENV in areas where special-
ized research laboratories are not available. Thus, these RDTs could
Journal of Medical Entomology, 2023, Vol. XX, No. XX
allow vector control programs to better target interventions to areas
of highest risk or of ongoing epidemics, and to respond quickly and
more effectively to the emergence of DENV in new areas with sus-
ceptible human populations.
The detection of DENV-1 in adult mosquitoes emerging from
larvae in domestic settings indicates that virus is transmitted vertic-
ally to the offspring of infected female mosquitoes, which act not
only as vectors but also as reservoirs of the virus (Fontenille et al.
1997, Ibáñez‐Bernal et al. 1997). This mechanism of maintaining
DENV in nature via Aedes eggs is a well-known phenomenon in
dengue-endemic areas, particularly in South America and Asia
(Arunachalam et al. 2008, Le Goff et al. 2011, Ferreira-de-Lima and
Lima-Camara 2018). Vertical transmission of DENV has already
been demonstrated in laboratory through studies involving the 4
serotypes of DENV (Rosen et al. 1983, Joshi et al. 1996, Buckner
et al. 2013, da Costa et al. 2017, Sánchez-Vargas et al. 2018, Danis-
Lozano et al. 2019). However, more field-based studies are needed
to better define the contribution of vertical DENV transmission to
dengue epidemiology.
Our results showed that the current dengue outbreak was wide-
spread, and highlighted a high risk of DENV transmission in Rosso.
This indicated the need for continuous control of adult and aquatic
stages of Ae. aegypti to prevent future dengue epidemics in the area.
Based on the entomological results obtained and the evidence
of DENV circulating in Rosso, short term recommendations were
formulated: immediate insecticide spraying both inside and outside
houses, as well as at tire workshops, to reduce the abundance of
infected adult mosquitoes; raise public awareness about the biology
of Ae. aegypti (water storage containers are mosquito breeding
habitat) and the risks of prolonged water storage; recommend that
people change the water in storage containers every 3 days; removal
of abandoned containers and tires from the domestic environment
through community sanitation measures; sanitation of abandoned
buildings and rooms.
In the long term, the aim will be to carry out a study of the vector’s
sensitivity to the insecticides available to the health authorities; carry
out additional entomological surveys in other localities to better de-
fine the affected area and avoid reintroduction of the virus through
population movements; monitor vector control actions using a par-
ticipatory approach, to ensure long-term collaboration between
local populations and authorities; and above all a transfer of skills in
terms of surveillance and response.
Acknowledgments
The authors would like to thank the Medical Officer of the Richard-
Toll district, the Regional Hygiene Brigade Officer of St.Louis, and
the community health workers for their technical assistance and
collaboration.
Author Contributions
Babacar Diouf (Data curation [Equal], Formal analysis [Equal],
Investigation [Equal], Methodology [Equal], Software [Equal],
Supervision [Equal], Validation [Equal], Visualization [Equal],
Writing – original draft [Equal], Writing – review & editing [Equal]),
Alioune Gaye (Data curation [Equal], Formal analysis [Equal],
Investigation [Equal], Methodology [Equal], Supervision [Equal],
Validation [Equal], Visualization [Equal], Writing – original draft
[Equal], Writing – review & editing [Equal]), Idrissa DIENG (Formal
analysis [Equal], Resources [Equal], Software [Equal], Writing – re-
view & editing [Equal]), Cheikh DIAGNE (Resources [Equal],
9
Supervision [Equal], Writing – review & editing [Equal]), El hadj
Ndiaye (Investigation [Equal], Methodology [Equal], Validation
[Equal], Writing – review & editing [Equal]), Moufid Mhamadi
(Methodology [Equal], Resources [Equal], Writing – review & editing
[Equal]), Assiyatou Gueye (Methodology [Equal], Writing – review
& editing [Equal]), Oumar Ndiaye (Resources [Equal], Validation
[Equal], Writing – review & editing [Equal]), Ndeye Sene (Writing –
original draft [Equal], Writing – review & editing [Equal]), Faty SY
(Writing – original draft [Equal], Writing – review & editing [Equal]),
Oumar Faye (Resources [Equal], Validation [Equal], Writing – review
Downloaded from https://academic.oup.com/jme/advance-article/doi/10.1093/jme/tjad126/7273086 by guest on 14 September 2023
& editing [Equal]), Ibrahima Dia (Software [Equal], Supervision
[Equal], Writing – original draft [Equal], Writing – review & ed-
iting [Equal]), Scott Weaver (Funding acquisition [Lead], Project
administration [Equal], Visualization [Equal], Writing – review &
editing [Equal]), Mawlouth Diallo (Conceptualization [Equal], Data
curation [Equal], Funding acquisition [Lead], Methodology [Equal],
Project administration [Equal], Resources [Equal], Supervision
[Equal], Validation [Equal], Visualization [Equal], Writing – original
draft [Equal], Writing – review & editing [Equal]), and Diawo Diallo
(Conceptualization [Equal], Data curation [Equal], Formal anal-
ysis [Equal], Investigation [Equal], Software [Equal], Supervision
[Equal], Validation [Equal], Visualization [Equal], Writing – original
draft [Equal], Writing – review & editing [Equal])
Funding
This work was supported by the West African Center for
Emerging Infectious Diseases (NIH/NIAID) under Grant number
U01AI151801 and NIH under Grant number R24 AI120942.
Conflict of Interest
The authors declare no competing of interests.
Supplementary Material
Supplementary material is available at Journal of Medical
Entomology online.
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