Detection of DENV-1, DENV-3 and DENV-4 serotypes in Ae. aegypti and Ae. albopictus, and epidemic risk in the departments of Oueme and Plateau, South-Eastern Benin

doi:10.21203/rs.3.rs-2990903/v1

Background

The present study conducted in the departments of Oueme and Plateau, aims to assess the presence of the dengue virus and its different serotypes in Ae. aegypti and Ae. albopictus, as well as the epidemic risk incurred by the populations.

Method

Collections of adult mosquitoes using human landing catches (HLC) were carried out in six communes, three of which (Porto-Novo, Adjarra and Avrankou) in the Oueme department, and the rest (Ifangni, Kétou and Pobè) in the Plateau department. Pools of ten Aedes mosquitoes were formed, and stored at − 80°C in RNA later. RT-PCR was used to detect dengue virus, and conventional PCR for the different setotypes. Inspection of water containers and collection of Aedes larvae were performed inside and around each house in order to calculate the stegomyan indices.

Results

In the six communes studied, the dengue virus was present both in Ae. Aegypti, and Ae. albopictus. Combined data of the two Aedes species at the communes level, revealed infection rates ranging from 80.00% (95% CI: 61.43–92.29) to 96.67% (95% CI: 82.78–99.92). In addition, the infection rates were similar for Aedes aegypti [88.19% (95% CI: 81.27–93.24)] and Aedes albopictus 86.79% [(95% CI: 74.66–94.52)]. The three virus serotypes detected in the pools of Aedes were: DENV-1, DENV-3 and DENV-4, with a high prevalence for the first two. Whatever the communes, the values of the stegomyan indices reached the threshold that indicates the existence of the risk of an arbovirosis epidemic.

Conclusion

The present study revealed that three serotypes (DENV-1, DENV-3 and DENV-4) of dengue virus circulate in Aedes aegypti and Aedes albopictus in the departments of Oueme and Plateau. Moreover, the risk of transmission of arboviruses was globally high and variable from commune to commune. These information are essential for informed decision-making in the preventive control of the disease.

Aedes aegypti

Aedes albopictus

serotype

DENV

Benin

Dengue fever is a haemorrhagic and potentially fatal disease transmitted by Aedes mosquitoes [1]. Its global incidence has increased over the past four decades [2]. In 2015, a 30-fold increase in the global incidence of dengue compared to 30 years earlier was observed [3]. Increasingly, many countries have started reporting their first outbreaks. The number of dengue cases reported to WHO has increased more than eightfold over the past two decades, from 505,430 cases in 2000 to over 2.4 million cases in 2010 and 4.2 million cases in 2019 [4]. The number of reported deaths shifted from 960 in 2000 to 4032 in 2015[4]. In 2019, an epidemiological rebound in dengue regardless of the continent was observed [5]. This disease, whose occurrence was limited to Latin America and Asia in the past, has now spread to West Africa with outbreaks in Burkina-Faso, Nigeria, Côte d'Ivoire, Niger and Senegal [6]. The disease is caused by an infection with dengue virus (DENV), which exists as four genetically distinct serotypes (DENV-1 to DENV-4) [7]. Although an infection with one of these serotypes confers long-term immunity, the latter is only acquired for this type, and not for the other three [8]. In 2019, an epidemiological rebound in dengue regardless of the continent was observed [5]. This disease, whose presence was limited to Latin America and Asia in the past, has now spread to West Africa with outbreaks in Burkina-Faso, Nigeria, Côte d'Ivoire, Niger and Senegal [6]. Several studies have shown that an infection with different serotypes can cause a severe and potentially life-threatening form of dengue fever, due to a reinforcement of antibodies [9–10]. The emergence of severe dengue fever in tropical environments can be partly attributed to the co-circulation of different serotypes in the same geographical area [8]. The main vectors that transmit the different serotypes of the virus are the mosquito’s commonly encountered in urban areas such as Aedes aegypti and Aedes albopictus [11]. In sub-Saharan Africa, existing information on dengue fever is unreliable due to unreported cases, low level of awareness among healthcare workers, and confusion of this ailment with other common febrile illnesses such as malaria [12].

Indeed, the high malaria prevalence in West Africa makes it difficult to detect arboviroses and other non-malaria febrile illnesses, because they have similar symptoms. About 70% of febrile illnesses diagnosed in sub-Saharan Africa are mistakenly cared as malaria [6]. In Benin, the two main dengue vectors (Ae. aegypti and Ae. albopictus) live in sympatry [13. Aedes aegypti was found throughout the country, while Aedes albopictus was only collected in the Southern part [14]. A study by Padonou et al. [15] showed a strong infestation of the municipality of Abomey-calavi by Aedes aegypti. In control programs, isolation of dengue virus from patients is vital for monitoring the disease. Unfortunately, this approach detects the virus when it already strongly circulates in the community. Detecting viral activity in local mosquito populations is an essential step in dengue prevention and control [16]. This is why the present study was conducted to detect the different serotypes of the dengue virus (DENV) in the populations of Aedes aegypti and Aedes albopictus, and evaluate the epidemic risk in the departments of Oueme and Plateau. This information will enable to have a better understanding of the epidemiology of the disease, the risk incurred by local populations in order to implement effective control measures.

Study area

Mosquito samples were collected from six communes in the departments of Oueme and Plateau in southern Benin. These two departments border Nigeria, a dengue-endemic country. Indeed, collections were made in the communes of Porto-Novo, Adjarra, Avrankou, Ifangni, Pobè and Kétou as shown on the map in Fig. 1.

Mosquito collection

From July 2021 to October 2022, monthly rounds of human landing catches were organized to sample adult mosquitoes. In each of the 6 study communes, a central site and a peripheral site were selected for human landing catches between 7 a.m. and 6 p.m. During the collections, a first team of 8 people/communes worked from 7 a.m. to 1 p.m., and was replaced by another team of 8 people. In each house, a collector was installed inside and a second outside, which makes a total of 4 collectors/house/day. To reduce bias related to ability of collectors in capturing mosquitoes and their attractiveness, they were rotated between the different houses. At each capture point, the seated collectors who served as baits, collected the mosquitoes that landed on their bare legs or feet using hemolysis tubes before they bite. The collected mosquitoes were morphologically identified using a binocular magnifying glass and the identification key of Edwards [17] and Yiau-Min [18]. The main vectors of arboviroses (Aedes aegypti and Aedes albopictus) were kept aside and stored on RNA Later at − 80°C before the extraction of dengue virus RNA.

Extraction of the Viral RNA and synthesis of the complementary DNA

RNA extraction was performed on pools of 10 mosquitoes of Ae. aegypti or Ae. Albopictus, using Qiagen RNeasy Mini Kits® (Qiagen, UK). Thus, the grinding of the 10 mosquitoes of each pool was carried out in 150 µl of the RTL lysis buffer, using the Qiagen tissue lyser. 150 µl of 70% ethanol was added to the ground material and the supernatant obtained was transferred to RNeasy plates. The RNeasy plates were then sealed with the AirPore tape sheet and centrifuged at 6000 rpm (~ 5600 x g) for 2 minutes. Then, a series of 3 centrifugations at 6000 rpm (~ 5600 x g) for 2 min, 2 min and 4 min after respective addition of: 100 µl of buffer RW1, 100 µl of buffer RPE and an additional 100 µl of buffer RPE. After this step, 45 µl of RNase-free water was added to each well and incubated for 1 minute at room temperature then centrifuged at 6000 rpm (~ 5600 x g) for 2 minutes. The concentration of the extracted RNA was measured using the Nanodrop and stored at -80°C. Prior to cDNA synthesis, 2 mg of extracted RNA will be DNase treated using the Promega RQ1 RNase-Free DNase Kit (#M6101) as described below:

The DNase digestion solution was prepared with a reaction mix containing 2 mg of extracted RNA, 1 µl of Buffer RQ1, 2 µl of RQ1 DNase, and 10 µl of nuclease-free water. The mixture underwent a first incubation at 37oC for 4 minutes. After this step, 1 µl of the DNase stop solution was added then the mixture was incubated at 65°C for 10 minutes to inactivate the DNase. After this treatment, the cDNA was synthesized using large capacity cDNA reverse transcription kits (Applied Biosystems #4368814), according to the manufacturer's procedure. The cDNA concentration was measured using the Nanodrop, and stored at -80°C prior to the PCR that allowed to detect the dengue virus and its type.

Detection of the dengue virus and its serotypes by PCR

These were performed following the protocol described by Pérez-Castro et al. [19]. This protocol provides for two series of amplifications: a first which detects the presence of the virus, and a second the serotype. The first PCR was performed using a thermocycler (Eppendorf Nexus) with a total reaction volume of 25 µl containing 10 pmol of each mD1 and D2 primers (Table 1) using a hotstartaque mastermix kit (Qiagen). PCR was performed by a denaturation step at 95°C for 15 minutes, followed by 35 cycles of 94°C for 15 seconds, 55°C for 15 seconds, 72°C for 30 seconds, and an extension of 72°C for 10 minutes. A fragment of 511 base pairs is expected for this first amplification.

The second PCR which allowed the detection of dengue serotypes was carried out with the same Hotstartaque mastermix kit, with 5 µl of PCR product from the previous reaction, 10 pmol of each primer (mD1, rTS1, mTS2, TS3 and rTS4) in a reaction volume of 25 µl. The amplification was composed of one cycle at 15 minutes, followed by 25 cycles at 95°C for 15 seconds, 55°C for 15 seconds and 72°C for 30 seconds and a final extension of 7 minutes at 72°C. The PCR product was analyzed by electrophoresis on a 2% agarose gel stained with Ethidium Bromide. The serotypes were determined according to the size of the amplicons. Indeed, fragments of 208bp, 119bp, 288bp and 260bp are expected respectively for DENV-1, DENV-2, DENV-3 and DENV-4 positive mosquitoes.

Evaluation of the risk of the epidemic of arboviruses

About 5% of the houses of the 2 villages [15] selected in each study commune were investigated. After the greeting the occupants of the houses, and the briefing of the objectives of the survey, the inspection of the water containers and the collection of the Aedes larvae were carried out inside and around each house. Each interviewer was provided with a tablet to record GIS data and general information. The data collected made it possible to describe the types of breeding sites encountered, and determine the following Stegomian indexes [20]:

Container Index (CI): number of containers containing water with the presence of larvae or pupae × 100 ∕ Number of visited containers;
Breteau index (BI): number of positive containers for 100 surveyed houses;
House index (HI): number of positive houses × 100 ∕ number of visited houses.

The results of the calculated indexes have been interpreted according to the table 2 below [21–22] (OMS, 1971; PAHO, 1994).

The field-collected larvae/pupae were transported to the CREC insectary for rearing until adult stage. After emergence, mosquitoes were morphologically identified per breeding site and per commune using taxonomic keys from Edwards [17] and Yiau-Min H [18], and stored on RNA Later at − 80°C before the extraction of the dengue virus RNA.

Statistical analysis

The binomial test was used to determine confidence intervals for the prevalence of infection to the dengue virus in Ae. aegypti and Ae. Albopictus. The Chi 2 test of comparison of multiple proportions was used to compare the proportions of the different breeding sites within the same village. The binomial test was used to generate the confidence intervals of the values obtained for the stegomyan indices. All statistical analyzes was performed the R software, version 4.13.

Ethical considerations

The protocol of this study was reviewed and approved by the Institutional Ethics Committee for Health Research of the Center for Research in Entomology of Cotonou (N°06–22/CREC/CIERS-CREC/SG). All the mosquito collectors were vaccinated against yellow fever and are regularly monitored. In case of confirmed fever, they are immediately taken care of by a medical doctor.

Prevalence of dengue virus infection in Ae. aegypti and Ae. albopictus

A total of 180 pools including 127for Ae. aegypti and 53 for Ae. albopictus were analyzed. Our results showed the presence of the dengue virus in the two Aedes species in the six study communes. In Aedes aegypti, the infection rate varied from 76.19% (95% CI: 52.83–91.78) in Adjarra, to 95.24% (95% CI: 76.18–99.88) in Ifangni and Pobè, with an average of 88.19% (95% CI: 81.27–93.24) for all 6 communes. In Aedes albopictus, the lowest infection rate was 66.67% (95% CI: 29.93–92.51) in Kétou and Pobè and the highest of 100% (95% CI: 66.37–100) in Ifangni, Porto -Novo and Adjarra, with an average of 86.79% (95% CI: 74.66–94.52) for the study area. No difference was observed between the infection rate of Aedes aegypti (88.19%; 95% CI: 81.27–93.24) and Aedes albopictus (86.79%; 95% CI: 74.66–94.52). The combination of data from the two species (Ae. aegypti and Ae. albopictus) showed an infection rate varying between 80% (95% CI: 61.43–92.29) in Kétou and 96.67% (95% CI: 82.78–99.92) in Ifangni, with an average of 87.78% (95% CI: 82.08–92.18) for the study area.

Distribution of dengue virus serotypes in the study communes

Three dengue virus serotypes have been detected. These are the DENV_1, DENV_3, and DENV_4 serotypes (Table 2). DENV_1 and DENV_3 serotypes are present throughout the study area, while the DENV_4 serotype was found only in Adjarra and Ifangni. Overall, high frequencies of the DENV_1 serotype were observed, with prevalences varying from 66.67% (47.19–82.71) in Kétou, to 83.33% (65.28–94.36) in Porto-Novo and Avrankou, and an average of 77.22% (70.39–83.13) for the entire study area (Table 4). DENV-3 serotype was present in all the study communes with relatively low prevalences ranging from 10% (2.11; 26.53) in Adjarra and Ifangni, to 30% (14.73; 49.40) in Porto-Novo, and an average of 17.22% (12.01–23.55) for all the study communes.

Furthermore, the DENV_4 serotype was only present in Adjarra and Ifangni with a prevalence of 3.33% (0.08; 17.22), and an average of 1.11% (0.13–3.96) for all the study area (Table 4).

Distribution of dengue fever serotypes within species and by study commune

The Dengue serotypes were diversely distributed among the main Aedes species present in the different study communes table 5. In Aedes aegypti, the prevalence of the DENV_1 serotype varied from 71.43% (95% CI: 47.82–88.72) in Adjarra and Ifangni, to 86.36% (95% CI: 65.09–97.09) in Avrankou, with an average of 77.95% (69.74–84.82) for all 6 the study communes. The DENV_1 serotype was the most frequent in the infected pools compared to the DENV_3 serotype (16.54%; 95% CI: 10.54–24.16) and the DENV_4 serotype (0.79%; 95% CI: 0.02–4.31).

Similarly, in Aedes albopictus, the DENV_1 serotype presented the higher prevalence (75.47%; 95% CI: 61.72–86.24), followed by the DENV_3 serotype (18.87%; 95% CI: 9.44–31.97), and the DENV_4 serotype (1.89%; 95% CI: 0.05–10.07) in the whole study area. Overall, no significant difference was observed between the distribution of the different serotypes within the main dengue vectors (Ae. aegypti and Ae. albopictus).

Type of breeding sites

Figures 2 and 3 show the variety of Aedes breeding sites and the proportion of positive sites (containing Aedes larvae) inside and outside the houses in Ifangni and Porto-Novo districts.

Figure 2 shows that in the villages of Ifangni, the proportions of house hives differed from one village to another (p < 0.05). The highest proportions of household pits are found in Doke and Igolo. These household containers are the positive sites encountered. A small proportion (5.56%) of positive natural sites were found in Gbaodjo. No positive household sites were found in Lokossa. Domestic containers, tires, and other types of lodges were the most common outdoor lodges encountered with a high proportion of domestic containers (p < 0.05). No positive outdoor sites were found in Doke; domestic containers were the only positive sites found outside the houses in Lokossa and Gbaodjo.

In Igolo, in addition to domestic containers, tires were found outside the houses but in unequal proportions (P < 0.05). According to Fig. 3, the dominant household sites are domestic containers, regardless of the neighborhood considered in Porto-Novo (p < 0.05). No positive household sites were found in Gbodjè. Domestic containers were the only positive ones in Anavié and Tokpota. In Dowa a small proportion of tires (5.88%) and other sites (5.88%) were positive. In Porto-Novo the proportions of the different outdoor sites also varied within the same neighborhood (p < 0.05). Tires were also found as outdoor sites in all neighborhoods except in Anavié where none of the outdoor sites found were positive. In Dowa only the domestic containers were positive. In Gbodjè and Tokpota, tires and domestic containers are the two types of positive outdoor sites but with domestic containers more dominant (p < 0.05).

Assessment of the risk of an arboviross epidemic

Table 6 present the results of the Stegomyan indices that served to estimate the risk of arbovirosis in the context of entomological surveillance. According to the World Health Organization, there is a risk of epidemic when the Breteau, container, and house indices exceed 5%, 3%, and 4% respectively. Considering the container index, our results showed a high risk of epidemic in Avrankou (71.43% [51.33–86.77]), Kétou (83.33% [35.87-99, 57]), and Ifangni (13.24% [18.85-28.094]). With the same index, the risk of an epidemic is low in Porto-Novo (11.07% [7.74–15.19]). With the Breteau index, there was a high rish for an outbreak in Avrankou (153.85% [93.97–237.60]), Ifangni (179.55% [142.14-223.76]) and Porto-Novo (55% [37.85–77.24]). On the other hand, an average risk was noted in Kétou (41.67% [13.52–97.23]). Moreover, the house index showed a high risk in Avrankou (84.62% [54.55–98.07]) and Ifangni (59.09% [54.55–98.07]), while a medium risk was seen in Porto-Novo (33.33% [21.68–46.68]) and Kétou (33.33% [9.92–65.11]). Overall, taking into account the three indices, there was a high risk of arbovirose epidemics.

Longitudinal entomological surveillance of dengue fever is an important means for detecting and preventing epidemic outbreaks, understanding the spatio-temporal dynamics of diseases, and evaluating control interventions [23]. In Benin, the clinical diagnosis of dengue fever is made difficult by the existence of malaria, which presents the same clinical picture as the latter. Dengue fever is therefore mistakenly confused with malaria. Furthermore, there is no effective early warning system for possible dengue epidemics. This study is one of the first in Benin to investigate the presence of dengue virus in mosquitoes. Our results showed a strong mosquito infection (commune range: 80.00%-96.67%) in all the communes of the study. These results could be explained by the closeness of these districts to Nigeria, a country endemic to dengue fever. No difference is observed between the infection rate of Aedes aegypti (88.19%; 81.27–93.24) and Aedes albopictus (86.79%; 74.66–94.52), these two vector mosquitoes being reported as highly competent in the transmission of arboviral pathogens [24]. Similarly several studies showed the involvement of Aedes albopictus and 20 in dengue epidemics in Africa [25]. Anthropogenic practices of drinking water conservation, alcohol production, and palm wine requiring the conservation of water in many domestic containers could justify the proliferation of Aedes mosquitoes.

Our results revealed a high prevalence of DENV-1 followed by DENV-3, while DENV-4 was weakly represented. A similar trend was previously observed by Ayolabi et al. [26] (2019) in Nigeria. The presence of the three dengue serotypes in the departments of Oueme-Plateau in Benin is worrying. Indeed, it has been shown that infections with different serotypes can cause a severe and potentially fatal form of dengue, due to a reinforcement of antibodies [9–10]. The emergence of severe dengue in tropical environments can therefore be partly attributed to the co-circulation of different serotypes [8]. Unlike the present study, which detected no DENV-2 infection, this serotype was found in several other West African countries, either alone (Mali and Ghana), or in co-circulation with the other serotypes (Nigeria, Senegal, Burkina, Ivory Coast) [27].

Our results report a high infestation of a variety of domestic and peridomestic containers (notably tires) in all the study sites. Similar results were obtained in Benin in 2020 where Aedes larvae were found in different categories of breeding sites in both domestic and peri-domestic environments [15]. These results suggest that it is important to involve populations in the control of Aedes spp. vectors.

Indeed, some habits and practices of populations create breeding sites for these vectors that multiply very rapidly. These habits that favor the creation of breeding sites for Aedes spp vectors in the domestic or peri-domestic environment could be avoided if this population is aware of the danger that these vectors constitute and has knowledge of good habits to avoid the reproduction of these vectors. Overgaard et al [28] associated infestation of domestic containers with low frequency of cleaning. Good housekeeping would contribute to the decrease of household infestation rate by arbovirosis vectors. These facts further emphasize the importance of population involvement in successful vector control.

Regardless of the locality and the stegomyen risk index considered, our results showed values above the reference thresholds established for dengue and yellow fever [21–22]. These results show that there is a serious risk of transmission of arboviroses in this region of the country. Moreover, the risk is higher for dengue because the vectors are already carrying the dengue virus in this region of the country. This area of the country deserves special attention and regular surveillance to prevent it from being a focus for dengue emergence in Benin. Similar results were obtained by Padonou et al [15] in Abomey-Calavi and Hêvié where stegomyen indices exceeded the reference thresholds indicating a threat of arbovirosis in these two districts.The present study could have reported the variations in the seasonal circulation of the different dengue virus serotypes, which was not done and constitutes a limitation. However, the information collected will be useful in the implementation of control programs that will prevent a possible outbreak of dengue fever.

Different dengue virus serotypes (DENV-1, DENV-3 and DENV-4) are actively circulating among the main vectors of arboviroses in the departments of Oueme and Plateau. In addition, a strong infection was observed in the pools of tested vectors. In addition, the calculated stegomyan indices showed that there is a high risk of transmission of arboviroses in the area. These results indicate the need to pay a particular attention to Aedes vectors by implementing an effective surveillance and alert system in order to prevent outbreaks of dengue epidemics.

Acknowledgements

We thank the Beninese government through its Ministry of Health for funding this study. We also thank the researchers of the Centre de Recherche Entomologique de Cotonou (CREC) who supported the study through our critical discussions. Finally, we would like to thank the laboratory technicians of the CREC.

Author contributions

The study was designed and its protocol written by GGP, AKK, ASS and MCA. Data collection was performed by AKK, DMZ, ASS, AS, OO and JA. Molecular analyses were performed by AKK, AS, JA, LT, and DMZ. AKK, ASS, AS, GGP, and MCA wrote the manuscript. KAK, DMZ, and ASS performed the statistical analysis of the data. GGP, AS, RO, LB, and MCA provided intellectual input for the scientific quality of the content. The final version was approved by all authors.

Funding

Not applicable.

Availability of data and materials

The datasets that were analyzed in this study are available from the corresponding author and the second author.

Ethics for approval and consent to participate

The protocol of this study was reviewed and approved by the Institutional Ethics Committee for Health Research of the Center for Research in Entomology of Cotonou (N°06-22/CREC/CIERS-CREC/SG). All the mosquito collectors were vaccinated against yellow fever and are regularly monitored. In case of confirmed fever, they are immediately taken care of by a medical doctor.

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests

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Table 1: Sequences of the primers for detecting the dengue virus and its serotypes

Primers	Sequences of primers
mD1	5’-TCAATATGCTGAAACGCGAGAAACCG-3’
D2	5’-TTGCACCAACAGTCAATGTCTTCAGGTTC-3’
rTS1	5’-CCCGTAACACTTTGATCGCT-3’
mTS2	5’-CGCACAAGGGCATGAACAGTTT-3’
TS3	5’-TAACATCATCATGAGACAGAGC-3’
rTS4	5’-TTCTCCCGTTCAGGATGTC-3’

Table 2: Intervals of interpretation of stegomyan indexes in relation to larval density and the risk of arbovirus circulation in the human population

Risk level	CI	HI	BI
low	< 3	< 4	< 5
Medium	3-20	4-35	5-50
High	> 20	> 35	> 50

CI : Container Index HI : House Index BI : Breteau Index

Table 3. Dengue virus Infection rate in Ae. aegypti and Ae. albopictus

		*Aedes aegypti*			*Aedes albopictus*			Total
Departements	Districts	N tested pools	N positifs pools	Infection % (95%-CI)	N tested pools	N positifs pools	Infection % (95%-CI)	N tested pools	N positifs pools	Infection % (95%-CI)
Oueme	Adjarra	21	16	76.19 (52.83-91.78)	9	9	100 (66.37-100)	30	25	83.33 (65.28-94.36)
	Avrankou	22	20	90.91 (70.84-98.88)	8	7	87.5 (47.35-99.68)	30	27	90 (73.47-97.89)
	Porto-Novo	21	18	85.71 (63.66-96.95)	9	9	100 (66.37-100)	30	27	90 (73.47-97.89)

Plateau	Ifangni	21	20	95.24 (76.18-99.88)	9	9	100 (66.37-100)	30	29	96.67 (82.78-99.92)
	Kétou	21	18	85.71 (63.66-96.95)	9	6	66.67 (29.93-92.51)	30	24	80(61.43-92.29)
	Pobè	21	20	95.24 (76.18-99.88)	9	6	66.67 (29.93-92.51)	30	26	86.67 (69.28-96.24)

	All area	127	112	88.19 (81.27-93.24)	53	46	86.79 (74.66-94.52)	180	158	87.78 (82.08-92.18)

N : Number ; CI : Confidence interval

Table 4: Distribution of dengue serotypes in the surveyed districts

		N _	N_DENV 1 (%, 95% CI)	N_DENV 3 (%, 95% CI)	N_DENV 4 (%, 95% CI)
Departements	Communes	Total	N_DENV 1 (%, 95% CI)	N_DENV 3 (%, 95% CI)	N_DENV 4 (%, 95% CI)
Oueme	Adjarra	30	24(80 ; 61.43-92.29)	3(10 ; 2.11-26.53)	1(3.33 ; 0.08-17.22)
	Avrankou	30	25(83.33 ; 65.28-94.36)	6(20 ; 7.71-38.57)	0(0 ; 0-11.57)
	Porto-Novo	30	25(83.33 ; 65.28-94.36)	9(30 ; 14.73-49.4)	0(0 ; 0-11.57)
Plateau	Ifangni	30	24(80 ; 61.43-92.29)	3(10 ; 2.11-26.53)	1(3.33;0.08-17.22)
	Kétou	30	20(66.67 ; 47.19-82.71)	4(13.33 ; 3.76-30.72)	0(0 ; 0-11.57)
	Pobè	30	21(70 ; 50.6-85.27)	6(20 ; 7.71-38.57)	0(0 ; 0-11.57)
	All area	180	139(77.22 ; 70.39-83.13)	31(17.22 ; 12.01-23.55)	2(1.11 ; 0.13-3.96)

N_Total : number of pools; N_DENV 1: number of positive pools for DENV1; N_DENV 2: number of positive pools for DENV2; N_DENV 3: number of positive pools for DENV3.

Table 5: Distribution of dengue serotypes within species and by study site

		*Aedes aegypti*				*Aedes albopictus*
		N _	N_DENV 1 (%, 95% CI)	N_DENV 3 (%, 95% CI)	N_DENV 4 (%, 95% CI)	N _	N_DENV 1 (%, 95% CI)	N_DENV 3 (%, 95% CI)	N_DENV 4 (%, 95% CI)
Departements	Communes	Total	N_DENV 1 (%, 95% CI)	N_DENV 3 (%, 95% CI)	N_DENV 4 (%, 95% CI)	Total	N_DENV 1 (%, 95% CI)	N_DENV 3 (%, 95% CI)	N_DENV 4 (%, 95% CI)
Oueme	Adjarra	21	15(71.43;47.82-88.72)	3(14.29;3.05-36.34)	0(0;0-16.11)	9	9 (100;66.37-100)	0 (0;0-33.63)	1(11.11;0.28-48.25)
	Avrankou	22	19(86.36;65.09-97.09)	5(22.73;7.82-45.37)	0(0;0-15.44)	8	6 (75;34.91-96.81)	1(12.5;0.32-52.65)	0(0;0-36.94)
	Porto-Novo	21	17(80.95;58.09-94.55)	5(23.81;8.22-47.17)	0(0;0-16.11)	9	8 (88.89;51.75-99.72)	4(44.44;13.7-78.8)	0(0;0-33.63)
Plateau	Ifangni	21	15(71.43;47.82-88.72)	3(14.29;3.05-36.34)	1(4.76;0.12-23.82)	9	9 (100;66.37-100)	0 (0;0-33.63)	0(0;0-33.63)
	Kétou	21	16(76.19;52.83-91.78)	2(9.52;1.17-30.38)	0(0;0-16.11)	9	4 (44.44;13.7-78.8)	2(22.22;2.81-60.01)	0(0;0-33.63)
	Pobè	21	17(80.95;58.09-94.55)	3(14.29;3.05-36.34)	0(0;0-16.11)	9	4 (44.44;13.7-78.8)	3(33.33;7.49-70.07)	0(0;0-33.63)
	All area	127	99(77.95;69.74-84.82)	21(16.54;10.54-24.16)	1(0.79;0.02-4.31)	53	40(75.47;61.72-86.24)	10(18.87;9.44-31.97)	1(1.89;0.05-10.07)

N_Total : number of positive pools; N_DENV 1: number of positive pools for DENV1; N_DENV 2: number of positive pools for DENV2; N_DENV 3: number of positive pools for DENV3.

Table 6. Assessment of epidemic risk with the Container Index (CI), Breteau Index (BI) and House Index (HI) in the departments of Oueme and Plateau

Departements	Districts	Container Index % (95%-CI)	Level of risk with C Ind	Breteau Index % (95%-CI)	Level of risk with BI	House Index % (95%-CI)	Level of risk with HI
Oueme	Avrankou	71.43 [51.33-86.77]	High	153.85 [93.97-237.60]	High	84.62 [55.55-98.07]	High
Oueme	Porto-Novo	11.07 [07.74-15.19]	Medium	55.00 [37.85-77.24]	High	33.33 [21.68-46.68]	Medium
	Ifangni	23.24 [18.84-28.09]	High	179.55 [142.14-223.76]	High	59.09 [43.24-73.66]	High
Plateau	Kétou	83.33 [35.87-99.57]	High	41.67 [13.52-97.23]	Medium	33.33 [09.92-65.11]	Medium

CI : Confidence interval ; Container Index : C Ind ; Breteau Index : BI ; House Index : HI.

No competing interests reported.

GraphicalabstractPadonouetal..tif

Detection of DENV-1, DENV-3 and DENV-4 serotypes in Ae. aegypti and Ae. albopictus, and epidemic risk in the departments of Oueme and Plateau, South-Eastern Benin

Status:

Version 1

Abstract

Figures

Introduction

Method

Mosquito collection

Extraction of the Viral RNA and synthesis of the complementary DNA

Detection of the dengue virus and its serotypes by PCR

Evaluation of the risk of the epidemic of arboviruses

Statistical analysis

Ethical considerations

Results

Distribution of dengue virus serotypes in the study communes

Distribution of dengue fever serotypes within species and by study commune

Type of breeding sites

Assessment of the risk of an arboviross epidemic

Discussion

Conclusion

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1