Study sites
Côte d’Ivoire is divided into four ecological zones including the forest zone in the South and West, the transitional zone in the Centre, the savanna zone and the Sudan savanna zone in the North. The climate in the southern forest zone is equatorial, with annual rainfall between 2,100 mm and 2,500 mm and subequatorial in the western forest with a range of average rainfall between 1,600 mm and 2,300 mm per year. In the central transitional zone of the country, the climate is tropical, with an average of 1,200 mm rainfall per year. The climate is also tropical in the northern savanna and Sudan Savanah zone, with an annual average rainfall of 900 mm. Insecticide resistance surveys were carried out in fifteen sites distributed across the country, including Aboisso, Adzopé, and San Pedro in the South, Bouaké, Béoumi, Dabakala, Sakassou and Yamoussoukro in the Centre, Odienné in the North-West, Bouna and Nassian in the North-East, Daloa and Gagnoa in the west and Abengourou and Bettié in the East (Fig. 1). Of Those sites, ten represented the NMCP sentinel sites were all malaria control activities and impact were monitored all year round. The additional sites (Bettie, Gagnoa, Sakassou, Dabakala and Béoumi) were surveyed based on their malaria endemicity and targeted for indoor residual spraying site selection. Furthermore, the country has a history of two mass campaigns of pyrethroid-only long lasting insecticide treated net (LLIN) distributions conducted in 2014 and 2017.
The study was conducted between June and September 2019. Anopheles gambiae s.l. larvae and pupae were collected from several urban and rural larval habitats in each site using the dipping method, pooled and reared to adulthood in a field laboratory at each site.
Insecticides and Synergist
Papers impregnated with deltamethrin (0.05%, 0.25% and 0.5%), permethrin (0.75%, 3.75% and 7.5%), alpha-cypermethrin (0.05%, 0.25% and 0.5%) and PBO (4%) were obtained from University Sains Malaysia (USM).
Pre-weighed preparations of technical-grade active ingredient of chlorfenapyr (BASF Corporation USA, Batch N 2130H070HV) provided by the Centers for Disease Control and Prevention (CDC), Atlanta, were used to prepare 50 ml of two doses (100 µg (a.i)/bottle and 200 µg (a.i) /bottle) by adding the prescribed volume of acetone solvent. The solution obtained was vortexed to ensure that the solution was uniformly mixed. All insecticide solutions and papers were kept at 4ºC before and after each test.
Prior to the field susceptibility testing, two impregnated papers received were randomly selected from each box and tested against a laboratory-maintained susceptible strain of An. gambiae Kisumu to verify the effectiveness of the paper. Both chlorfenapyr solutions (100 µg (a.i.)/bottle and 200 µg (a.i.)/bottle) were also tested using the CDC bottle assay against An. gambiae Kisumu to check the efficacy on a susceptible population of mosquitoes.
WHO susceptibility test, intensity and synergist assays
Insecticide susceptibility tests were conducted using WHO tube tests on 2-4 day old adult female An. gambiae s.l. [29, 30]. The diagnostic concentrations of deltamethrin (0.05%), permethrin (0.75%), alpha-cypermethrin (0.05%) were tested alone and in combination with PBO concurrently in all sites. For the synergist assays, mosquitoes were pre-exposed to PBO for one hour before being exposed to the pyrethroid insecticides for one hour. Resistance intensity at five and ten times the diagnostic concentration of deltamethrin, permethrin and alpha-cypermethrin was also tested in each site using WHO susceptibility kits. Two tubes lined with silicone oil treated papers were set in parallel during each test and served as controls. The start and end temperatures during all tests were recorded at 9 sites, and Hobo data loggers (HOBO UX100-003, Onset Computer Co., Bourne, MA, USA) were used to track temperatures every 10 minutes in the laboratory in two sites (Beoumi and Sakassou).
CDC bottle assay with chlorfenapyr
Due to non-availability of a WHO recommended protocol for testing chlorfenapyr, two doses (100 µg (a.i.)/bottle and 200 µg (a.i.)/bottle) were selected based on a literature review [21, 27, 28, 31] and used to coat the CDC bottles. The bottles were coated following the protocol described by Brogdon et al. [32], with 1 ml of chlorfenapyr diluted in acetone at the concentration of 100 µg (a.i.)/bottle and 200 µg (a.i.)/bottle. The coated bottles were wrapped in aluminum foil and dried overnight at room temperature. A modified protocol was designed following the procedures described by Brogdon et al. [32]. Fifteen to 20 adult mosquitoes (2-4 days old) that emerged from field collected larvae were aspirated into 250 ml chlorfenapyr-coated Wheaton bottles for 1 hour, after which the mosquitoes were released into a clean cage, aspirated back to paper cups and fed with 10% sugar solution. Mortality was recorded at 24, 48, and 72 hours after exposure. Two bottles coated with acetone only were prepared similarly to serve as controls.
Species identification of the An. gambiae s.l. population
A subsample of about fifty An. gambiae s.l. mosquitoes was randomly selected among the population tested per site and processed for molecular identification of the species of the An. gambiae complex following the SINE PCR protocol described by Santolamazza et al. [33]. Additionally, the presence of knocked-down resistance (kdr)-West and East resistance mechanisms was characterized within each An. gambiae s.l. population using the Taqman protocol described by Bass et al. [34] and the presence of acetylcholinesterase (ace-1) gene was determined following the protocol described by Weill et al. [35].
Statistical analysis
Insecticide resistance status was defined following WHO criteria [27] with mortality after 24 hours for pyrethroids and 72 hours for chlorfenapyr < 90% as confirmed resistance, between 90% and < 98% as possible resistance, and ≥ 98% as susceptible. Mortality was corrected using Abbott’s formula when the mortality of the control tubes was above 5% and less than 20%.
Corrected mortality of:
- 98-100% at 5x the diagnostic dose indicates low resistance intensity
- Less than 98% at 5x diagnostic dose implies testing the 10x diagnostic dose
- 98-100% at 10x the diagnostic dose confirms a moderate resistance intensity.
- Less than 98% at 10x the diagnostic doses indicates high resistance intensity.
For the synergist assays, an increase in the mortality after pre-exposure to PBO compared to the diagnostic dose of the insecticide alone indicates the involvement of enzymes such as P450s in the population tested. The mortality of mosquitoes exposed to PBO + pyrethroid was also compared with that of the insecticides alone and plotted using Graph Pad Prism 5 software. The total percentage mortality of all sites against each insecticide alone and those of the PBO + insecticides were analyzed and compared using a chi-squared test with Stata 14 (Stata Corporation, College Station, Texas, USA).