Important Aedes Spp. Density Levels in Kinshasa, Democratic Republic of Congo

BACKGROUND: Dengue, yellow fever, chikungunya and Zika are among the most important emerging infectious vector-borne diseases worldwide. Besides sporadic dengue cases, yellow fever and chikungunya outbreaks have been increasingly reported in Democratic Republic of Congo (DRC) in the last decade. The main vectors of these arboviruses, Aedes aegypti and Aedes albopictus, were reported in DRC, but there is a lack of detailed information on their presence and spread hampering transmission risk assessments in the region. METHODS: In 2018, two cross-sectional surveys were realized in Kinshasa province (DRC), one in the rainy (January/February) and one in the dry season (July). Four hundred houses were visited in each of the four selected communes (N’Djili, Mont Ngafula, Lingwala and Kalamu). Breedings sites were recorded, larvae and pupae collected and reared to obtain adults for genus and species identication. A subset of specimens was DNA-barcoded for validation of the morphological species identication. RESULTS: The most rural commune (Mont Ngafula) had the highest density levels, with a Breteau Index of 82.2 and 19.5/100 houses in rainy and dry season, respectively. The Breteau Index in the other communes Kalamu, Lingwala and N’Djili elevated to 21.5 (4.7), 36.7 (9.8) and 41.7 (7.5) in the rainy (and dry) season. The House index was on average 27.5% and 7.6%; and the Container Index 15.0% and 10.0% in rainy and dry season, respectively. The vast majority of Aedes positive containers was found outside the houses (adjusted OR 27.4 (95%CI 14.9-50.1)). The main breeding sites were used tires, water storage containers and trash. Anopheles larvae were also found in Aedes breeding sites, especially during the rainy season. CONCLUSIONS: These results show that Kinshasa is highly infested with Aedes spp. which indicates a high potential for arbovirus transmission in the area. During the dry season, the most productive containers (for Aedes pupae production)

Brazzaville (Republic of the Congo), and in 2000 [16], 2012 [17] and 2019 [18] in Kinshasa, capital of the Democratic Republic of Congo (DRC). In Kenya it has been shown that a chikungunya outbreak infected 67% of the population [19]. In Kinshasa, several alpha-, avi-and bunyaviruses were found in mosquito samples (Aedes and Culex) in 2014 [20].There are reports that the dengue virus is circulating in DRC, but up to now there was no outbreak detected. The dengue infections found were in the majority of cases not clinically diagnosed, but detections upon analysis of stored samples. In 2012, the antigen dengue test was positive among three suspected chikungunya cases in Kinshasa [21]; in 2013-14 dried blood spots taken during a Demographic Health Survey were in 0.6% positive for dengue [22]; between 2002 and 2013, in 3.5% of stored samples of yellow fever suspect cases in the Bas Congo region, the dengue virus was identi ed [23]. More recently, in 2015-16 in an acute fever study in Mont Ngafula (suburban area of Kinshasa) dengue was the identi ed pathogen in 8.1% of the acute fever cases and chikungunya in 0.9% [24], however previous dengue infection was evidenced in 30.2% of the 342 study participants. In the neighboring country, Angola, a dengue outbreak with an estimated attack rate of 10% has been reported in 2013 [25]. Zika has been rarely detected in this African region [26], but several yellow fever outbreaks, the last one in 2016, were described [27].
Within the sub-Saharan African region, the information on the presence and distribution of the Aedes mosquito is even more di cult to nd than on the above pathogens. This lack of entomological data leads to the use of suitability maps, based on mathematical models, to estimate arbovirus transmission risk [28], however, real Aedes spp. density levels would give a more reliable estimate [29]. Both Aedes aegypti and Aedes albopictus are found in the region, where Ae. aegypti is native, whereas Ae. albopictus is native of South-East Asia [30]. The latter, nowadays considered omnipresent in Central Africa [8], was reported for the rst time in Central Africa in early 2000 [31] and in Kinshasa (DRC) in 2018 [32]. Both species can be found in human-domesticated environments [30], such as Kinshasa, a mega-city with a high population density and movement, where density levels remain unknown.
In this study, we evaluate the Aedes spp. density levels, together with the characteristics of the preferred breeding sites, in order to have an evidence basis for guidance of Aedes control efforts and for transmission estimation in Kinshasa, the capital of DRC.

Settings
The study took place in Kinshasa, capital city of Democratic Republic of Congo (DRC), located in the Central-African region. Kinshasa lies at 279 m above sea level and is characterized by a tropical climate with a rainy season between October and May, and a dry season from June to September. The average temperature varies between 18°C and 32°C and the average monthly rainfall varies between 2 and 222 mm, in dry and rainy season respectively. Kinshasa is expanded over 9965 km² and has an estimated population of almost 12 million people. The city is administratively subdivided in 24 communes, which are grouped in four districts: Tshangu in the East, Lukunga in the North, Mont Amba in the South-East and Funa in the Center-West. In this study, four communes were selected in different geographical zones of the city having a different ecology, urbanization, water supply systems and history of arbovirus outbreaks ( gure 1).
(Insert here Figure 1) N'Djili is a peri-urban commune in the east of the city, pertaining to the Tshangu district, where many informal economic activities, speci cally vehicle repair shops, are located. Urban infrastructure, such as waste water infrastructure and garbage collection, is de cient. 97% of the houses have a water supply system in their compound, but an important proportion of them has problems with quality and amount of water availability. The population density of this area is estimated at 39 000 persons/km².
Kalamu II is a commune in the center of town, belonging to the Funa district, and is a residential place with as main economic activity technical service provision. It has an estimated population density of 47 000 persons/km². Mont Ngafula I is situated in the south of the city, bordering Mont Amba district, and is an example of a semi-urban area with an estimated population density of 730 persons/km². It is geographically characterized by the presence of hills (and accompanying erosions) and small valleys. The main economic activity is agriculture and the selling of agriculture products to Kinshasa city. The place is characterized by unplanned urbanization with a typical de cient water supply system -with in some areas supply frequency as low as two times/week -and a de cient waste water disposal.
Lingwala is a commune in the center of the town, pertaining to the Lukunga district, with a lot of informal markets. Population density is estimated at 33 000 persons/km².

Study design and data collection
Two cross-sectional surveys were done in the four selected communes, one in the rainy season (18 January -16 February, 2018) and one in the dry season (2 -27 July, 2018). In each of the four selected communes, one neighborhood has been randomly chosen (all neighborhoods per commune listed, followed by random number selection procedure) as study site. In each study site, 400 houses were randomly selected to be surveyed. The sample size was calculated to detect, with a power of 80%, 10% of the houses being positive for Aedes spp. mosquitoes with a precision of 3% and allowing for a 5% alfaerror. Each day, 80 houses were inspected, using a systematic sampling approach: on a landmark (roundabout or main road) random points were identi ed for each team as their starting point to enter the (smaller) avenues. With a sampling interval of three houses, starting on the right side of the avenue, each of the 4 teams inspected the selected houses up to reaching 20 houses/day. When the avenue came to an end and the sampling size of 20 was not yet reached, the team turned back entering the houses on the other side of the street up to reaching the daily sample size. In each selected house, the entire house was inspected inside and outside. If there was more than one house per compound, a random house was chosen to inspect, but the entire outside part of the compound was inspected. The next day, the next avenue (going left from the one of the previous day) was sampled. By this procedure, an extensive part of the neighborhood was covered with the survey. When one commune was nalized, the four entomological teams went to another commune and followed the same methodology. All communes were covered in four weeks' time. Each entomological survey team consisted of three persons, pre-trained by the entomology department of the 'Institut National de Recherche Biomédicale' (INRB), one entomologist of the INRB (supervisor) and one community health worker.
In each compound, all water holding containers were inspected and if immature stages (larvae or pupae) of mosquitoes were observed, they were collected in plastic bottles (one bottle per breeding site) and transported to the laboratory at INRB for genus identi cation (Anopheles, Aedes, Culex). The place, category and positivity/negativity of each container was reported. For larvae, only positivity and negativity was recorded; for pupae, the number of pupae was counted per positive breeding site. Both surveys were done in a similar way, but starting points of avenue and house selection differed and possibly a same house was visited in both surveys, but this was based on randomness and was not aimed at. Both surveys were largely realized by the same eld team members.

Species identi cation: morphology and DNA-based
Each day, a random sample of 50 Aedes genus larvae/pupae were reared to adults in the insectarium to allow species identi cation using morphological keys [33,34]. F0 adults were stored at -20°C for DNA barcoding to validate the morphological identi cation of Aedes aegypti and Aedes albopictus and con rm the presence of the identi ed species in Kinshasa. Therefor ve specimens of each species were randomly selected per study site. DNA barcoding is a technique based on the ampli cation of a standard barcode -the partial mitochondrial cytochrome c oxidase subunit I gene for animals. Sanger sequencing of the 658 bp COI standard barcode was performed using the LCO1490 and HCO2198 universal primers [35,36]. Ampli cations were carried out in a 20 µl reaction mixture containing 2 µl of DNA template, 2 µl of 10X buffer, 1.5 mM MgCl2, 0.2 mM dNTP, 0.4 µM of each primer, and 0.03 units/µl of PlatinumTM Taq DNA Polymerase (InvitrogenTM). PCR products and negative controls were checked on a 1.5% agarose gel, using a UV transilluminator and the MidoriGreenTM Direct (NIPPON Genetics Europe) method.
Positive amplicons were puri ed using the ExoSAP-ITTM protocol and sequenced in both directions on an ABI 3230xl capillary DNA sequencer using BigDye Terminator v3.1 chemistry (ThermoFisher Scienti c). Subsequently, the generated sequences were compared to a library of reference sequences. A specimen was identi ed by analyzing its percentage sequence similarity with these reference sequences under the assumption that genetic diversity is lower within than between species. A rooted Neighbour-Joining tree was constructed including a sub-selection of the Ae. albopictus and Ae. aegypti barcodes available from online repositories, together with the newly generated haplotypes (full details of the protocol can be found in Additional File 2).

Data analysis
Data were entered in an Access database and 5% of the data were manually checked to evaluate inconsistencies. Data were cleaned and types of recipients regrouped into categories, adapted from guidelines used in dengue-endemic regions [37]: big deposits used for water storage (> 15 L); small water deposits used for daily kitchen and cleaning activities (< 15 L); arti cial containers, mainly trash that has no speci c use or goal; natural sites, such a tree holes and bamboo; arti cials that are used in the households and cannot be destroyed (for example animal drinking pots); tires; water evacuation systems/ponds. Data were analyzed using IBM SPSS Statistics, version 25. We calculated per round and per commune House Index (number of houses positive for at least one container with immature stages of Aedes spp. per 100 inspected houses), Breteau Index (number of containers positive for immature stages of Aedes spp. per 100 inspected houses), Container Index (number of containers positive for immature stages of Aedes spp. per 100 inspected containers), and Pupal Index (number of Aedes spp. pupae per 100 inspected houses). The relative contribution to pupal productivity was calculated and de ned as the total number of pupae of Aedes spp. per category of breeding site divided by the total number of pupae of Aedes spp. collected per commune and per survey round. A descriptive analysis was done. In order to evaluate the factors determining Aedes spp. immature stage positivity, a logistic regression model was made and associated variables were identi ed based on a backwards conditional model, taking into account the clustering at household-level by inserting the household identi cation variable as a random factor in the model.
The number of breeding sites with at least one immature stage of Anopheles spp. was enumerated and its proportional importance calculated for each season and respective commune.

Results
The survey in the rainy and dry season allowed to sample a total of 1 678 and 1 598 houses, respectively.
In the rainy season, 5 079 water-holding containers, which were potential breeding sites, were inspected against 1 657 in the dry season. The average number of containers per household was different across communes (p < 0.001): for example in the rainy season, an average of 1.4 (Standard deviation SD 1.3) in Kalamu, 2.0 (SD 1.7) in Lingwala, 2.9 (SD 2.3) in Mont Ngafula and 5.3 (SD 2.6) in N'Djili. In rainy and dry season, 65.9% and 78.3% of the containers, respectively, were observed outside the sampled houses, i.e. in the open space around the house within the compound, (p < 0.001). In the Additional File 1 (table 1), the distribution of type of containers per location, commune and season is detailed.
Aedes density levels were higher in the rainy than in the dry season (p < 0.001, see  (Table 1). Mont Ngafula, a rural sub-urban area in the Southern edge of Kinshasa had the highest density levels amongst all visited communes with a BI of 82.21 and 19.50/100 houses in the rainy and dry season respectively, which was about four times higher than Kalamu, a commune that lies within the heart of the center of town. The number of Aedes pupae present per 100 houses reached in the rainy season 246 pupae/100 houses in Mont Ngafula, 126 in N'Djili, 90 in Lingwala, and 50 in Kalamu (Table 1). In the rainy season 99.3% of the positive breeding sites were outdoors against 96.4% in the dry season. A wide variety of containers is used by the Aedes mosquito to breed: big water deposits, small water deposits, arti cials type trash, natural sites, non-destroyable arti cial containers, tires, water evacuation/ponds ( Figure 2). Tires were taken as a separate group, as they are frequently present and it is di cult to know if they are just put aside for reuse/temporary storage or to be destroyed.
When analyzing the pupal productivity of breeding sites, we observed a statistically signi cant difference between rainy and dry season (aOR 3.73, 95% CI (2.21-6.31); p < 0.001). In the dry season, we observed that big water deposits were producing 20.3% of the pupae against 5.5% in the rainy season, indicating seasonal variability in breeding preference of the vector (Figure 3). In the rainy season 64.3% of all inspected containers were small water deposits, but they were only responsible for 46.4% of the pupae production, whereas tires, representing only 11.1% of the inspected containers, were responsible for 35.0% of the pupae production. The breeding containers used for water storage (big and small ones together) contributed relatively more to the pupal productivity in the dry season compared to the rainy season. Furthermore, we observed that productivity of arti cial containers (mainly trash) was different across communities (p < 0.001) and season (p < 0.001) (Figure 4).
Based on the morphological identi cation of F0 adults, Ae. aegypti and Ae. albopictus were found in both seasons. The morphological identi cations were validated by comparing the generated sequences of a subset of specimens against the Identi cation System of BOLD, with Species Level Barcode Records. The obtained similarity percentages ranged from 99.69 to 100%. The ve and 14 haplotypes of Ae. albopictus and Ae. aegypti, respectively, are clustering only with conspeci c sequences from specimens collected worldwide, supported with maximum bootstrap support (Figure 1 of Additional File 2). The generated sequences were deposited in GenBank with following accession numbers: MT345349-MT345426. 9.46% and 9.06% of the total number of containers positive for Aedes spp. immature stages, contained also immature stages of other genera, such as Culex and Anopheles, in the rainy and dry season respectively. This was in 99.3% of the cases observed in outdoor recipients and speci cally in big water deposits in the rainy season and in trash in the dry season. Of them, in the rainy season, a total of 32 Aedes breeding sites were positive for Anopheles against only two in the dry season. Anopheles were found in big and small water deposits, trash and tires ( Figure 6). In the rainy season Anopheles were observed in all communes whereas in the dry season Anopheles larvae were only found in small water deposits in Mont Ngafula, the most rural commune of the four study sites (Figure 7).

Discussion
In both surveys and in all communes, the larval indices (HI, CI, and BI) were higher than the arbovirus transmission threshold values (BI of 5 set out by the World Health Organization) [38,39]. The Breteau Index was on average 45 per 100 houses in the rainy season, and in comparison to a House Index of on average 27%, it is clear that one house can have different Aedes spp. positive breeding sites. In case an arbovirus is introduced in Kinshasa, the high larval and pupal Aedes densities suggest that transmission can rapidly occur and cause a major outbreak, such as the one caused by chikungunya in 2019 [18].
Aedes aegypti originated from Africa and is the main vector of arboviruses outside Africa. Yet, the vector competence, unknown for Kinshasa, of this species in Africa seems to be highly variable depending on the vector population, the virus isolate and the ecological context [28]. The presence of Aedes albopictus, which is an exotic species for Africa, might change the epidemiology for a number of arboviruses in Africa. Considered as a secondary vector for dengue virus, it can drive transmission of the chikungunya virus, especially the one of the ECSA lineage with the A226A, as shown in Kinshasa in the recent outbreak [40].
Having done surveys, following standardized procedures, in four different communes of Kinshasa during the rainy and dry season is the major strength of this study. The entomological team was trained beforehand and was largely the same for both surveys. A weakness is that the study took place over only one year time and only once per season. As inspection of breeding sites depends on the rigor and professionality of the team doing the eldwork, quality control was established, namely a xed supervisor was available in the eld site during the survey and there was regular extra control from the international integrant of the study-team. Due to operational issues we were not in a position to identify all larvae to species level, which was another weakness, hence we could not calculate the relative importance of Ae. aegypti and Ae. albopictus, neither which species has predilection for which container type.
In a place like Kinshasa, where dengue is rarely reported [21][22][23][24] and chikungunya and yellow fever cause sporadic outbreaks [16,18,41], we didn't expect to nd such high Aedes densities. The observed densities are comparable to the ones of other African settings: south-eastern Tanzania has a HI of 4.9 -6.6, CI of 14.6-18.9 [42]; Burkina Faso a HI of 70, CI of 35 and BI of 10 [43]; north-west Ethiopia a HI of 25.5, CI of 32.9 and BI of 48.4 [44]; Mozambique a CI of 22 [45]; and Angola a HI of 4.3 -27.9, CI of 2.1-9.3 and BI of 5.8-42.2 [46]. However, the densities were much lower than the one observed in Kenya during a dengue outbreak in 2013-14, where BI reached a value of 270/100 houses [47].
In contrast to ndings in Latin-America [48], in Kinshasa Aedes breeding sites were mainly found outdoors, a characteristic also seen in other African countries [49]. The prevalence of Aedes breeding sites outdoors, together with the behavior in this context (to stay during daytime in the backyard or in the open place in front of the house), suggests a close human-mosquito contact, favoring the development of the Aedes spp. cycle, by blood feeding during daytime [50]. The low presence of Aedes immature stages inside the homes can also be due to rapid use and cleaning of the few containers found there.
These results indicate that for controlling Aedes in Kinshasa, management strategies need to target outdoor spaces for breeding sites destruction or reduction.
Used car tires, water storage containers and arti cial breeding sites (type trash) were the main containers chosen by Aedes mosquitoes for the oviposition coinciding with other studies conducted in the African continent [43][44][45][46][47]49,51]. The water storage containers were also found to be the most productive for Aedes pupae, which is a stage in the mosquito cycle which does not need nutrients and which is just before the adult stage of the mosquito [52]. These containers used to store water are always lled (partially or fully) with water due to a de cient water supply system, not depending on rain, which makes them a preferred breeding site, especially in the dry season, even despite being constantly subject to anthropogenic action. In the rainy season, in all study sites, breeding sites are favored by rain and containers typically lled with rain water are the most productive ones for Aedes pupae. A nice example are the tires, while they only represent 11% of the potential breeding sites, in the rainy season, about 35% of all pupae are found in them. Temperature, humidity and reduced light inside tires create a suitable environment for Aedes mosquito breeding and when tires are stored or discarded for long duration, it makes them a proli c breeding site [53][54][55]. In these conditions, eggs can be attached to the tires for a long time, playing their role in the preservation of the Aedes mosquito population throughout the dry season [56]. Small containers lled with water for kitchen/cleaning purposes were found in large numbers in all study sites and were also highly productive, especially in the rainy season.
In this study, Aedes species, which transmits among others chikungunya, dengue, Zika and yellow fever, were dominant in the inspected potential breeding sites, in and around the houses. Also other mosquito genera were found, such as Culex and Anopheles. Culex is common in urban settings using similar breeding sites as the urban Aedes species. It is of note that Anopheles species was found together with Aedes in the same breeding sites [57]. Anopheles usually prefers other types of breeding sites, such as ponds with static fresh water and are not particularly attracted to small containers [58]. The presence of Anopheles in urban settings is primarily associated with urban agriculture, as in Mont Ngafula [59], though we found Anopheles in all four communes in the rainy season, also in the center of Kinshasa. The observation of Anopheles larvae in man-made containers suggest that also Anopheles species adapt to those kind of containers, which is important in the light of transmission risk of urban malaria in Kinshasa.

Conclusion
Aedes spp. seem to be well established in all four study communes of Kinshasa and are especially abundant in the sub-urban area of Mont Ngafula. This study -the rst in its kind in Kinshasaevidences that an Aedes control strategy needs to target outdoor containers, speci cally containers for water storage in the dry season and tires in the rainy season. Additional insights in the ecology of the adult Aedes mosquito and its insecticide susceptibility will support the design of a comprehensive Aedes control strategy to be implemented to prevent a next outbreak of arboviral infections in Kinshasa. The study protocol was approved by the 'Comité d'éthique de l'Université de Kinshasa' (authorization number: ESP/CE/032/2018). Before starting the survey in each commune, the study was presented to the 'Médecin Chef de Zone' and the local mayor, in order to have their approval for realizing the study in their area of responsibility. An informed consent was asked to the head of the households of the sampled houses and an oral approval was obtained. Different quality control measures were put in place: in each commune an entomological expert supervised the work of the eld teams, the project-leader veri ed at the end of each day a subset of the data collection forms on completeness and an external entomological expert (Cuban expert) did ad hoc supervisions of the eld work and of the laboratory activities.

List Of Abbreviations
Consent for publication NA Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests