Background
Malaria remains a serious public health issue although significant reduction in the disease burden has been observed over the last few years. The large-scale implementation of long-lasting insecticide-treated nets (LLIN and indoor residual spraying (IRS) have played a major role in this battle against malaria. Several studies have demonstrated the efficacy of both tools in curbing malaria incidence [
1‐
3]. During the last decade, the massive roll out of LNs has allowed a significant reduction in malaria-associated morbidity and mortality across sub-Saharan Africa [
4,
5]. Pyrethroids remain ideal insecticides for treating nets owing to their low cost, longer residual activity and safety [
4,
6,
7]. However, the emergence and subsequent spread of insecticide resistance in major mosquito vector species could jeopardize the success of malaria control programmes [
8,
9] relying on this mode of action to control them. Resistance to pyrethroids has now been reported in all major malaria vectors in 27 countries across sub-Saharan Africa [
5,
10].
In Burkina Faso, insecticide resistance in mosquito vector populations appeared as early as the 1960s when
Anopheles funestus and
Anopheles gambiae s.l. populations were demonstrated to have reduced mortality to dieldrin and DDT [
7,
11]. This resistance is quickly spreading across the country and has now been reported in the
An. gambiae species complex [
12‐
15]. Recently, it has been demonstrated that vector species
An. gambiae s.l. has developed high level resistance to pyrethroids and other classes of insecticides [
16]. Resistance through a combination of L1014F
kdr and CYP6P3P450 mechanisms and other metabolic enzymes were present in VK7 species, including two carboxylesterases (COEAE3G, COEAE4G) and a GST (GSTE5) [
18].
In Benin, a country close to Burkina Faso, the situation has become alarming in the southern part of the country where the
An. gambiae complex is reported resistant to pyrethroid LNs, and has demonstrated only limited personal protection that inadequately kill mosquitoes [
17]. Similarly, in Burkina Faso, a recent study has shown that resistance to pyrethroids has increased by more than 1000-fold over the last few years [
18]. These examples underscore the urgent need for alternative tools or new insecticide formulations to complement existing ones and preserve LNs effectiveness. In the short term, novel or repurposed insecticide classes with dissimilar modes of action could be used either alone or in combination with pyrethroids for IRS and bed nets.
Chlorfenapyr, a pyrrole insecticide (IRAC group 13) with a completely different mode of action, could be a viable alternative to pyrethroids [
19,
20]. Recent studies in experimental huts have shown that chlorfenapyr was more effective on resistant
Anopheles and
Culex populations than pyrethroids [
21,
22]. As chlorfenapyr acts as a metabolic toxin, it does not show the repellency and the knock down typical for neurotoxins like the pyrethroids. Repellency is crucial for reducing mosquito biting rates and providing personal protection to net users. Thus, the combination of chlorfenapyr with a pyrethroid should enhance LNs users’ protection and afford avoidance or reduction of resistance selection. This solo pyrethroid LNs have been proposed for these challenges. The combination of chlorfenapyr applied as IRS with LNs increased the protection against mosquito bites and enhanced the control of the disease transmission [
23]. More recently, studies carried out on mixtures of chlorfenapyr and pyrethroids (alpha-cypermethrin and permethrin) have shown effective control of resistant populations of
An. gambiae and
Culex quinquefasciatus [
24,
25]. In Burkina Faso, malaria vectors have posited selection and resistance to all available classes of insecticides in the vicinity to the rice growing area of Vallée du Kou, while also increasing their resistance intensity over 1000-fold in the last few years, thus threatening the future of pyrethroid LNs [
18]. The magnitude of pyrethroid resistance and the multi-mechanisms developed by mosquitoes, make this specific ecological setting an ideal place to test efficacy of new insecticides or new formulations. Indeed, it has been recently demonstrated that new LNs of different brands had almost no killing effect on field collected mosquitoes [
26].
The objective of this study was to assess the efficacy of Interceptor® G2, an LN with a mixture of chlorfenapyr and alpha-cypermethrin, in an area where pyrethroid nets have limited efficacy to known mosquito malaria vectors. This study will be among the first evaluations of a LN with two discrete insecticides with completely different modes of action that provide some indication of their suitability to control wild insecticide resistant mosquitoes.
Discussion
Country-wide surveys in Burkina Faso have documented increasing levels of insecticide resistance in malaria vectors with a dramatic rise in the frequency of the
kdr 1014F allele over the last decade, and the occurrence of the resistant
Ace-
1
R
119S allele in both
An. coluzzii and
An. gambiae [
13‐
16,
30,
31]. The aim of the study was to measure the efficacy of Interceptor
® G2 nets, treated with a mixture of alpha-cypermethrin and chlorfenapyr, in a such a complex environment where malaria vectors are highly resistant to pyrethroids and exhibit multiple mechanisms of resistance [
16,
18]. The aim of the current study was to determine if the Interceptor
®G2 nets could sustain the same level of efficacy after being washed 20 times as recommended by the WHOPES [
32]. According to WHO, LNs must be effective after 20 washing.
As such, we evaluated the potential of the nets to: (i) provide individual protection against mosquito biting; and (ii) restore the effective control of pyrethroid resistant An. gambiae s.l. populations at Vallée du Kou 7 (VK7), where resistance is now well-established to pyrethroids.
Several studies have recently questioned the efficacy of pyrethroid treated nets where rapidly developing insecticide resistance is being observed. In such a context, there is an urgent need for managing insecticide resistance with new tools that can complement existing ones [
25,
33]. Mixtures of carbosulfan and a pyrethroid were evaluated on mosquito nets, but results of these previous studies were not advanced due to mammalian toxicity issues associated with the carbamate [
34].
In this study, superior performances of Interceptor
® G2 nets were achieved compared to the standard WHOPES recommended Interceptor
® in experimental hut trials. The significance of this study can be emphasized enough, where Interceptor
® G2 not only sustained higher mortality rates of wild mosquitoes compared to the WHOPES recommended standard Interceptor
® net, but Interceptor
® G2 effectively controlled well-documented, highly pyrethroid resistant mosquito populations. Although concentrations of alpha-cypermethrin (200 mg/m
2) in the standard Interceptor
® net is higher than that of Interceptor
® G2 (100 mg/m
2), the latter nets have an improved protective effect in terms of biting reduction. The combinational effects of two discrete and completely different modes of action to a single vector target have only begun to be investigated. Although the mode of action for chlorfenapyr is known to be slower, owing to the mitigation of protons across the inner mitochondrial membranes [
36], it remains relatively unclear how intoxication and/or conversation via metabolic detoxification (by mosquitoes) in the presence of another insecticide like alpha-cypermethrin is influencing the observed behaviour. Another finding in this study demonstrated that there was a significant increase of exiting rate with Interceptor
® G2 washed 20 times compared to standard Interceptor
® washed 20 times. These results demonstrate that combining alpha-cypermethrin with chlorfenapyr on the same net afford benefits from the unique properties of each insecticide: the protective (excito-repellent) effect of the alpha-cypermethrin and the enhanced mortality to resistant mosquitoes through a completely novel mode of action in chlorfenapyr. The long-lasting formulation which combines these dual modes of action on a single net that is wash resistant and adheres to the WHOPES criteria for durability is profound, and underscores one of the more daunting reasons other modes of actions have not been routinely applied to LNs—namely the incompatibility of formulation(s), limits to physical-chemistry and solubility needed to sustain both mortality and wash resistance on or in nets. The protective effect of the pyrethroid and the killing effect of the chlorfenapyr against pyrethroid resistant
Anopheline and
Culicine mosquitoes confirm the potential of the mixture of pyrethroid and a pyrrole on the same net as an alternative ITN treatment [
33]. In experimental huts the Interceptor
® G2 provided high mortality against wild pyrethroid resistant
An. gambiae s.l. Interestingly, the mortality with Interceptor
® G2 unwashed was not significantly different from that of the 20 times washed of the same nets. Results from the tunnel test also confirm the superior killing effect and blood-feeding inhibition of the unwashed and washed Interceptor
® G2 nets compared to the standard Interceptor
® nets.
Among the more significant findings recently reported in literature, it is clear that testing modality for non-neurotoxic compounds like chlorfenapyr can be highly influential [
35]. In the present study, it was observed that standard WHOPES cone tests, which principally measure the biological impact of a chemical on mosquitoes through forced direct exposures, posited high mortality to adult mosquitoes and provides evidence that the combination of chlorfenapyr with alpha-cypermethrin on a net has a real potential to control pyrethroid resistant mosquitoes. This should be considered carefully in all future studies, as cone bioassays can be problematic for mosquito exposures to a physiological toxin like chlorfenapyr, as identified by Oxborough et al. [
35].
The active ingredient content recovered by analytical determination for chlorfenapyr exhibited only moderate loss of active ingredient over 20 washes, and clearly had no observable effect on the level of control chlorfenapyr exacted on mosquitoes in this study. The loss of Interceptor® G2 alpha-cypermethrin active ingredient content was proportionately reduced compared to active alpha-cypermethrin ingredient loss (from washing) reported from WHOPES recommended Interceptor® nets. The chlorfenapyr control (chlorfenapyr-dipped net) was effective in killing pyrethroid resistant mosquitoes. Interceptor® G2 nets afford protection that cannot be realized with Interceptor® nets. Because higher levels of mortality were observed from exposures to chlorfenapyr control and Interceptor® G2 nets with chlorfenapyr, the two positive controls clarify the relative contribution of chlorfenapyr compared to that which alpha-cypermethrin alone can contribute. Alpha-cypermethrin has lost its killing effect at this location, but the combination of both in Interceptor® G2 were effective against mosquitoes even given the pervasive nature of resistant alleles at the VK7 site. The nets evaluated in this study clearly demonstrated improved performance of Interceptor® G2 with a good personal protective rate, and an improved ability to kill pyrethroid resistant mosquito populations.
Under the present experimental conditions, Interceptor® G2 LN outperformed the WHOPES recommended Interceptor® LN washed 20 times and hence meets the WHO criteria for LNs. Our work suggests that long-lasting mixture of chlorfenapyr and alpha-cypermethrin on nets has a real potential in controlling pyrethroid resistant mosquitoes in Africa and should be urgently developed and used as a pyrethroid IRM tool in areas relevant to its need. It also marks the first LN with two discrete modes of action (two adulticides) which are complementary to each other and afford improved user protection while maintaining safety and utility.
Authors’ contributions
KB, AD, DM conceived and designed the experiments, SN, KB, MN, AD wrote the paper, RS, SN, AO, KB participated in the sample collections and performed the experiments under supervision of AGO, RKD, AD. BK, MN analysed the data under supervision of AD. All authors read and approved the final manuscript.