Monitoring the trend of insecticide resistance and understanding associated genetic mechanisms is important for designing efficient malaria vector control strategies. This study was conducted to provide temporal data on insecticide resistance status and mechanisms in the major malaria vector Anopheles gambiae s.l. from Ebolowa, Southern Cameroon. Methods: Larvae of An. gambiae s.l. were collected from typical breeding sites throughout the city and reared to adulthood. Emerging adults were morphologically identified and WHO tube assays were performed to determine their susceptibility to carbamate, organophosphate and pyrethroid insecticides at diagnostic doses. When resistance was observed, its intensity was determined by performing WHO tube tests using 5 and 10 times the concentration of the diagnostic dose. Metabolic resistance mechanisms were investigated using insecticide-synergist assays. Sibling species of the An. gambiae complex were identified using SINE-PCR protocol. TaqMan assay was used to genotype the L1014F and L1014S kdr mutations, and the N1575Y mutation, an amplifier of the resistance conferred by the L1014F mutation. Results: Anopheles coluzzii was by far the dominant (99%) member of the An. gambiae s.l. complex in Ebolowa. The species was fully susceptible to carbamates and organophosphates, but resistant to all pyrethroid insecticides tested. Resistance was of moderate intensity for deltamethrin (mortality: 37%, 70% and 99% for 1×, 5× and 10× insecticide concentration, respectively) but rather of high intensity for permethrin (5% for 1×; 62% for 5× and 75% for 10×) and for alphacypermethrin (4.4% for 1×; 57% for 5× and 80% for 10×). Pre-exposure to the synergist PBO resulted in a full recovery of the susceptibility to delthametrin, but this was not observed for the other two pyrethroids tested. L1014S (kdr-East) and the N1575Y mutations were absent, whereas the L1014F (kdr-West) mutation was present at a high frequency (75%), showing a significant association with resistance to permethrin (OR = 3.8; 95%; CI [1.9–7.4]; p < 0.0001) and alphacypermethrin (OR = 3; 95%; CI [1.6–5.4]; p = 0.0002). Conclusion: The increased resistance of An. gambiae s.l. to pyrethroid insecticides as observed in Ebolowa poses a threat to the efficacy of LLINs used to protect populations from the bites of Anopheles mosquitoes that transmit malaria parasites. The present study further highlights the urgent need to implement resistance management strategies in order to maintain the effectiveness of insecticide-based vector control interventions and prevent a rebound in malaria-related mortality.

BackgroundContinuous monitoring of insecticide resistance in malaria vector populations is crucial to help maintaining the efficacy of insecticide-based vector control strategies. The present study evaluated insecticide resistance in Anopheles gambaie s.l. and its impact on the efficacy of Long Lasting Insecticide-treated Nets (LLINs) in the city of Ebolowa, prior the next bednets mass distribution campaign in Cameroon.MethodsMosquitoes were collected as larvae and reared to adulthood. Females aged 3 to 4 days were exposed the insecticides according to the WHO tube test protocol, to determine their resistance profile. The bioefficacy of LLINs was determined using the WHO cone test protocol. Pyrethroids-synergist combined tests were used to search for metabolic resistance mechanisms. Molecular analysis helped investigating presence of mutations conferring resistance, as well as identifying mosquito species.ResultsAnopheles coluzzii, the dominant (99%) species of An. gambiae s.l complex found in Ebolowa, was full susceptible to Carbamates and Organophosphates, but resistant to all pyrethroids insecticides. Resistance was moderate for deltamethrin (mortality: 37%, 70% and 99% for 1x, 5x and 10x insecticide concentration, respectively) but rather high for permethrin (5% for 1x; 62% for 5x and 75% for 10x) and for alphacypermethrin (4.4% for 1x; 57% for 5x and 80% for 10x). All the LLINs tested were ineffective against the field strain with mortalities less than 50%. Synergist test indicated cytochrome P450 are fully involved in resistance against deltamethrin, but partially for the other two pyrethroids tested. The L1014F (kdr-West) mutation was present at a high frequency (75%) showing a significant association with resistance to permethrin (OR = 3.8; 95%; CI [1.9 - 7.4]; P < 0.0001) and alphacypermethrin (OR = 3; 95%; CI [1.6 - 5.4]; P = 0.0002). ConclusionThe high level of resistance to pyrethroids detected in An. colluzzii from Ebolowa is of concerns for malaria control as it threatens the efficacy of LLINs used in the city. For good management of resistance observed, the National Malaria Control Program should privilege distribution of new net formulations in this locality such as PBO-deltamethrin combined nets or new generation nets.