Susceptibility of Anopheles gambiae from Côte d’Ivoire to insecticides used on insecticide-treated nets: evaluating the additional entomological impact of piperonyl butoxide and chlorfenapyr

The recent malaria control success recorded in several malaria endemic countries is largely attributed to the scale up of vector control tools, particularly the implementation and high coverage of insecticide-treated nets (ITNs) and indoor residual spraying (IRS) [1]. Pyrethroid insecticides have been used for several years both for ITNs and IRS because of their effectiveness in killing mosquitoes and also for their repellent properties, providing users additional personal protection and safety during sleeping hours [2, 3]. Due to their low cost, long residual activity and safety, pyrethroids remain the recommended insecticides for treating ITNs [4]. Unfortunately, the spread of target-site insensitivity and metabolic resistance mechanisms against pyrethroid insecticides occurring in malaria vectors are a threat to vector control and could lead to resurgence of malaria in endemic countries [5‐7].

Resistance to all the insecticides previously used in public health for adult malaria vector control has been reported across Côte d’Ivoire [8], limiting the country’s malaria vector control toolkit. Côte d’Ivoire was the first West African country to report the knockdown resistance (kdr) mutation in the 1990s, before it was detected all over the continent [9, 10]. Moreover, due to the intense exposure of Anopheles mosquitoes to insecticides used in both agriculture and public health, An. gambiae s.l. have developed resistance in several parts of the country [8, 11, 12].

A first attempt to manage insecticide resistance using ITNs was the development of ITNs that incorporate a pyrethroid and a synergist, piperonyl butoxide (PBO), which has been shown to increase the mortality of malaria vectors with metabolic resistance involving mono-oxygenases. Currently, five ITNs types with PBO have been developed, all of which received WHO pre-qualification and are available on the market. Three of these are treated with a combination of PBO and deltamethrin (PermaNet 3.0, Tsara Plus, Tsara Boost), one with PBO and permethrin (Olyset Plus), and one with PBO and alpha-cypermethrin (Veeralin) [13‐15]. The effectiveness of these ITNs in a given area depends on the extent of the involvement of metabolic resistance mediated by mono-oxygenase enzymes in vector populations. A countrywide distribution of such nets therefore requires baseline entomological studies to show the involvement of enzyme activities in pyrethroid resistance of An. gambiae s.l. and the effect of the synergist in restoring susceptibility of the targeted vector population to pyrethroids [16].

Recent research exploring other classes of insecticides identified chlorfenapyr-based tools as a promising option for vector control. Furthermore, WHO Pre-Qualification (WHO PQ) has approved chlorfenapyr-based nets and additional studies are ongoing under the UNITAID-funded New Nets Project to demonstrate the public health impact of those nets for wide use. The different mode of action of chlorfenapyr is expected to be effective in the presence of pyrethroid resistance developed by mosquitoes through several mutations [17, 18]. The oxidative removal of the N-ethoxymethyl group of the pro-insecticide chlorfenapyr leads to a toxic form of the molecule identified as CL 303268 which functions to uncouple oxidative phosphorylation and disrupt metabolic pathways of ATP production in mitochondria [19]. This molecule has low mammalian toxicity and is classified as a slightly hazardous insecticide by the WHO. Due to its novel mode of action, chlorfenapyr is unlikely to show any cross-resistance with standard neurotoxic insecticides as observed in Anopheles mosquitoes [20, 21]. Chlorfenapyr has been evaluated in laboratory bioassays and in experimental hut studies for ITNs in several countries including Côte d’Ivoire, Burkina Faso, Tanzania, and for both ITNs and IRS in Benin [22‐26]. Results from those studies suggest that chlorfenapyr is promising as a new molecule for ITNs and IRS to control populations of pyrethroid-resistant malaria vectors [27, 28].

With regards to widespread resistance to the insecticides previously used and the aim of introducing new generation vector control tools in Côte d’Ivoire, ITNs with chlorfenapyr and PBO represent options to overcome and manage widespread resistance. The current study was undertaken within a countrywide insecticide resistance monitoring effort to support the National Malaria Control Programme (NMCP) in making vector control decisions in Côte d’Ivoire and also to contribute to the knowledge base for chlorfenapyr more broadly.

Resistance of An. gambiae s.l. mosquitoes to pyrethroids observed in this study is consistent with the previously described trends in Côte d’Ivoire involving various and high insecticide resistance mechanisms such as the kdr-West and East and the ace-1 mutations [8, 11, 12, 36, 37]. Testing the intensity of the resistance from 15 sites showed that resistance intensity to all three pyrethroid insecticides is high. This presents a challenge for malaria control as it limits the country’s options for insecticide-based vector control interventions. The level of resistance observed could be attributed to the different applications of insecticide in the country, either for agriculture or public health. Agriculture accounts for 24% of GDP in Côte d’Ivoire, one of the largest producers and exporters of coffee, cocoa, cashew, and palm oil worldwide. A recent survey by the ministry of agriculture revealed that more than 20,000 tons of chemicals including pesticides and insecticides were applied in the country in 2018 for agricultural purposes including 40% of this amount entering illegally [38]. This likely contributed to the increasing resistance of malaria vectors to insecticides commonly used for insect control in general and malaria vector control specifically [36, 37, 39]. In Côte d’Ivoire, the two major malaria vectors include An. gambiae and An. coluzzii [40]. Both species generally breed in fresh water pools resulting from human activities such as irrigated rice fields and vegetable gardens, which are often treated with insecticides [36, 37, 39, 41]. As described by Chouaibou et al. [42], agricultural pesticides contain several insecticide molecules and particularly pyrethroids and carbamates, which contribute to the increased and widespread resistance of the vectors [39]. Furthermore, studies conducted in Western and Eastern Africa have shown a significant increase in the frequency of genes associated with pyrethroid resistance immediately following the implementation of an ITN campaign [7]. Moreover, the National Strategic Plan (NSP) developed in 2018 by the Côte d’Ivoire National Malaria Control Programme (NMCP) prioritized universal ITN coverage in households across the country through mass distribution campaign of pyrethroid-only ITNs.

In this study, pre-exposure to PBO before the different pyrethroids tested showed statistically significant but still insufficient increases in the mortality of the An. gambiae s.l. populations. These results would support the use of PBO-treated vector control tools in select areas where significant reversal of resistance was observed after pre-exposure to PBO. In some locations, PBO has resulted in increased mortality of even highly resistant mosquito populations [43]. Out of several combinations of PBO ITNs, combinations with the type II pyrethroids (deltamethrin and alpha-cypermethrin) showed higher performance than those with the type I pyrethroid (permethrin), consistent with several other studies comparing the different types of PBO ITNs [44, 45]. These data could inform the selection and stratification of the PBO ITNs for resistance management in Côte d’Ivoire.

Higher mortality of mosquitoes was observed against chlorfenapyr particularly in areas of probable metabolic resistance where higher mortality was noted after pre-exposure of the mosquitoes to PBO before the different pyrethroids. It is known that significant increase in mortality after exposure to PBO indicates the involvement of P450 enzymes in the insecticide resistance of the vectors. The trends observed were consistent with previous observations, when assessing the lethality of chlorfenapyr in areas of high metabolic resistance in Aedes aegypti after mosquitoes are exposed or not to PBO [17].

Chlorfenapyr is a protoxin requiring activation, via cytochrome P450 monooxygenases, to exert its toxic effects, via uncoupling of oxidative phosphorylation [17, 19]. The expression in An. gambiae s.l. of some cytochrome P450s involved in oxidative metabolism are under circadian control and more strongly expressed at night when Anopheles flight and host-seeking activity are higher [46, 47]. Such a model could suggest that exposure at night could likely produce higher mortality results compared to what was observed using traditional testing methods. The supposition that laboratory-based exposures to technical grade active ingredient or any other formulation where behaviours are excluded, likely negates attributes of oxidative uncoupling by chlorfenapyr when compared to host-seeking behaviours which include free-flying physiologically heightened states known to enhance pro-insecticide chlorfenapyr intoxication to mosquitoes as demonstrated by Oxborough et al. [31].

Furthermore, previous work showed that testing completed during the night induces significantly greater mortality than daytime bioassays, indicating the possibility of higher effectiveness of chlorfenapyr-based ITNs such as Interceptor® G2 since the peak biting hours of the vectors are typically late in the night [31]. Moreover, several studies are still ongoing to understand and delineate the appropriate diagnostic doses and times for testing chlorfenapyr susceptibility [31, 48, 49]. This study will therefore contribute to the evidence base for supporting the determination of an appropriate diagnostic dose. With regards to the data generated, in ten out of 15 sites where the tests were conducted, 98% or greater mortality was recorded at the dose of 200 µg/bottle against An. gambiae s.l., indicating that chlorfenapyr could be considered as an option to manage the resistant population of malaria vectors in Côte d’Ivoire, where resistance has continuously increased over the past several decades.

Several trials and studies have been conducted on the Interceptor® G2 net. As described by Bayili et al., Camara et al. [22, 23] and Ngufor et al. [26] chlorfenapyr-treated nets evaluated in several areas with documented pyrethroid resistance have been proven effective for controlling pyrethroid-resistant malaria vectors and could contribute to malaria control decision-making and insecticide resistance management in Côte d’Ivoire.