Background
According to the latest World Malaria Report 2022 of the World Health Organization (WHO)
, there were 247 million malaria cases and 619,000 malaria deaths in 84 malaria endemic countries worldwide in 2021 [
1,
2]. This represents about 13.4 million more cases in 2021 compared to 2019 attributable to disruptions to essential malaria services during the COVID-18 pandemic [
1]. The WHO African Region, with an estimated 234 million cases in 2021, accounted for about 95% of global cases malaria [
1]. The recent decline of malaria burden from 2000 to 2019 was largely due to the massive distribution and use of long-lasting insecticidal nets (LLINs) (2.5 billion LLINs were delivered from 2004 to 2021) for
Anopheles mosquito vector control [
1]. The malaria burden reduction is now slowing down and is threatened by the spread of resistance to pyrethroids (78 of 88 endemic countries have detected resistance to at least one insecticide class reported to the WHO from 2010 to 2020) [
1]. The WHO Global Plan for Insecticide Resistance Management (GPIRM) has recommended the development of LLINs with insecticide mixtures of active ingredients with different modes of action to mitigate resistance [
3]. The WHO Global Technical Strategy (GTS) aims for a reduction of malaria case incidence and mortality rate of at least 40% by 2020, 75% by 2025 and 90% by 2030 from the 2015 baseline [
1,
4]. To meet these targets, GTS has called for the development of new tools, with combined or more effective insecticide molecules to control insecticide-resistant
Anopheles vectors [
4]. These new vector control tools must incorporate new insecticide molecules and/or insecticide mixtures containing at least two active ingredients with different modes of action for the management of malaria vector resistance to insecticides [
1,
3].
Malaria is endemic throughout Côte d’Ivoire and represents the leading cause of mortality and morbidity in the country. Several studies have shown a wide spread resistance in local
Anopheles to most of the insecticide classes currently used in malaria vector control (e.g. pyrethroids, DDT and carbamates) [
5‐
8]. The existence of multiple mechanisms of resistance in the main vector,
Anopheles gambiae sensu lato (
s.l.) threatens the efficacy of vector control tools currently used in Côte d’Ivoire, including LLINs [
9‐
11]. Meiwald et al. [
11] found that pyrethroid resistance is associated with significant overexpression of
CYP6P4,
CYP6P3, and
CYP6Z1 in
Anopheles coluzzii in Côte d’Ivoire. High allelic frequencies of knock-down resistance (
kdr)
L1014F mutation (range: 0.46–1), relatively low frequencies of the
ace-1R mutation in the acetylcholinesterase gene associated with target-site insensitivity to carbamates and organophosphates (< 0.5), and elevated activity of insecticide detoxifying enzymes (mainly mixed function oxidases (MFOs), esterase and glutathione S-transferase (GST)), have been reported in Côte d’Ivoire [
5,
7,
10]. Recent laboratory (WHO tube and CDC bottle) bioassays against local pyrethroid-resistant
An. gambiae from Côte d’Ivoire have shown that pyrethroids induce low mortality, whilst pre-exposure to the synergist piperonyl butoxide (PBO) increases the mortality but does not restore fully the susceptibility due to resistance [
9]. However, the pyrrole insecticide chlorfenapyr induces higher mortality in resistant
An. gambiae compared with pyrethroids alone or combined with PBO in Côte d’Ivoire [
9].
The present good laboratory practice (GLP) study evaluated the bio-efficacy of a new candidate LLIN, PermaNet
® Dual, against pyrethroid-resistant
An. gambiae s.l. in comparison with the WHO Prequalification Unit, Vector Control Product Assessment Team (PQT/VCP) listed standard LLINs, PermaNet
® 2.0 and PermaNet
® 3.0, through the conduct of an experimental hut trial and laboratory bioassays in Tiassalé, Côte d’Ivoire. PermaNet
® Dual has a mixture of the pyrrole chlorfenapyr and the pyrethroid deltamethrin coated onto a polyester fabric. PermaNet
® 3.0 is treated with deltamethrin and PBO, and PermaNet
® 2.0 is treated with deltamethrin only. Chlorfenapyr is a pro-insecticide activated by the oxygenase function of cytochrome P450s and oxidative removal of the N-ethoxymethyl group leads to a toxic form identified as CL 303,268. The toxic form uncouples oxidative phosphorylation in the mitochondria, resulting in disruption of the production of adenosine triphosphate and loss of energy, leading to cell dysfunction and ultimately death of the insect [
12]. The WHO has received some resistance monitoring data for chlorfenapyr, but these data are insufficient to assess the potential presence of resistance to this insecticide [
1]. Deltamethrin is a neurotoxic insecticide and has excito-repellent effects on mosquitoes. PBO, used on PermaNet
® 3.0, is a synergist which inhibits mixed function oxidases, blocking the detoxification of pyrethroids and at least partially restoring pyrethroid susceptibility [
1,
13]. Pyrethroid-resistant strains of
Anopheles have so far been found to be susceptible to chlorfenapyr [
9,
14‐
18]. Thus, the hypothesis of the current study was that chlorfenapyr would kill the pyrethroid-resistant
An. gambiae population in Tiassalé, and PermaNet
® Dual would induce higher mortality compared to both PermaNet
® 3.0 and PermaNet
® 2.0 in the experimental huts, and the supplementary laboratory cone and tunnel bioassays could predict these hut trial outcomes.
Discussion
The current study evaluated the efficacy of PermaNet® Dual, a new candidate net coated with a mixture of chlorfenapyr and deltamethrin, against An. gambiae in a small-scale GLP hut study in Tiassalé, Côte d’Ivoire, with supporting exploratory laboratory cone and tunnel bioassays. WHO-prequalified PermaNet® 3.0 (co-treated with deltamethrin and PBO) and PermaNet® 2.0 (treated with deltamethrin only) were used as the reference nets. In this GLP hut study, PermaNet® Dual performed better than PermaNet® 3.0 and PermaNet® 2.0, in terms of mortality and blood-feeding inhibition, for both unwashed and washed samples. Unlike the cone bioassays, the tunnel tests successfully confirmed the field efficacy of the nets. Briefly, this hut trial showed the benefit of mixing deltamethrin and chlorfenapyr together in a long-lasting net, PermaNet® Dual, for an effective control of pyrethroid-resistant Anopheles.
The current experimental hut study demonstrated that PermaNet
® Dual had increased efficacy against highly pyrethroid-resistant
An. gambiae. Indeed, hut mortality of
An. gambiae Tiassalé strain was significantly higher with PermaNet
® Dual (> 83%) in comparison with PermaNet
® 3.0 (< 38%) and PermaNet
® 2.0 (< 12%), for both unwashed and 20 times washed samples (Fig.
1B and Table
1). This PermaNet
® Dual efficacy was similar to chlorfenapyr and alpha-cypermethrin mixture-coated Intercept G2 in West and East Africa [
16,
17,
27‐
32]. The good performance of PermaNet
® Dual might be attributed to the susceptibility of the highly pyrethroid-resistant
An. gambiae s.l. to chlorfenapyr [
9,
11]. Due to the novel mode of action of chlorfenapyr, the present pyrethroid-resistance mechanisms did not provide any cross-resistance to this insecticide [
12]. Indeed, chlorfenapyr-treated tool has been shown to control a number of different multiple-insecticide-resistant
Anopheles populations [
14,
15,
27‐
32].
This hut study showed that the percentage blood-feeding in wild pyrethroid-resistant
An. gambiae population was lower for PermaNet
® Dual (unwashed and washed) compared with PermaNet
® 2.0 (unwashed and washed) and washed PermaNet
® 3.0, but slightly higher than unwashed PermaNet
® 3.0 (Fig.
1A and Table
1). Likewise, blood-feeding inhibition with PermaNet
® Dual was comparable or stronger than with PermaNet
® 2.0 and washed PermaNet
® 3.0. This PermaNet
® Dual blood-feeding inhibition outcome is consistent with that caused by Intercept G2 in earlier hut trials in West Africa [
27‐
31], and may be explained by the irritant effects of the deltamethrin [
32‐
35]. While unwashed PermaNet
® Dual and PermaNet
® 3.0 produced similar levels of blood-feeding inhibition, the 20 times washed PermaNet
® Dual induced a statistically greater blood-feeding inhibition in comparison with both PermaNet
® 3.0 and PermaNet
® 2.0 washed 20 times. This suggests that PermaNet
® Dual may have performed better than PermaNet
® 3.0 and PermaNet
® 2.0 over time. PermaNet
® Dual effectiveness for inhibiting blood-feeding could imply its good potential for reducing the human-biting and blood-feeding, adding value into its killing effects in malaria vectors.
In the present hut study, unwashed PermaNet® Dual deterrence effect against the wild pyrethroid-resistant
An. gambiae population was comparable to that recorded in PermaNet® 2.0 and PermaNet
® 3.0. The deltamethrin component of PermaNet
® Dual has a deterrence effect and may have induced exiting in mosquitoes. However, the 20 times washed PermaNet
® Dual did not have a deterrence effect (Table
1), probably due to a reduction of the chemical contents (Table
2). A dipped chlorfenapyr net did seem to have a deterrence effect, but did not have a significant effect on exiting rates in West Africa [
15,
16,
26‐
31]. The higher deterrence effect may be due to both the chlorfenapyr and deltamethrin components, but higher exiting rate would likely be due only to the deltamethrin component. Thus, PermaNet
® Dual may have both deterrent and excito-repellency effects, providing personal protection against pyrethroid-resistant
An. gambiae mosquitoes, and reducing human-vector contacts, which may, in turn, lead to an increased user acceptance [
36].
While standard laboratory cone bioassays failed to predict PermaNet
® Dual efficacy against pyrethroid-resistant
An. gambiae s.l., tunnel tests successfully predicted its efficacy against the same strain in the semi-field experimental huts. Both cone and tunnel bioassays met the WHO criteria [
44], with the susceptible
An. gambiae s.s. Kisumu strain. However,
An. gambiae s.s. Kisumu strain mortality against unwashed and used PermaNet® Dual (A) was lower (34%) with cone bioassay (Fig.
2B) probably due to the reduction of the chemical bioavailability on netting fibre surface of net samples tested, or the inappropriateness of cone bioassay method for evaluation of the chlorfenapyr-coated PermaNet
® Dual as the mortality was higher (100%) with tunnel test (Fig.
4B). With the wild pyrethroid-resistant
An. gambiae s.l. Tiassalé strain, only the tunnel tests achieved high efficacy that was consistent with that observed in the huts with PermaNet
® Dual. The difference in PermaNet
® Dual efficacy between both
Anopheles Kiumu and Tiassalé strains may be explained by the difference in the levels of their resistance to insecticides. However, the lower efficacy of PermaNet
® Dual against pyrethroid-resistant
An. gambiae s.l. with cone bioassays (daytime and 3-min exposure) compared with tunnel tests (night-time and 15-h exposure) may be attributable to the slow mode of action of chlorfenapyr and that mosquitoes need to be metabolically active for the activation of the chlorfenapyr pro-insecticide [
12,
13,
37]. Indeed, chlorfenapyr has a previously been observed to have a slower action and delayed toxic activity of 2–3 days post-exposure compared to other insecticides (pyrethroids and organophosphates) used in mosquito vector control [
14,
28,
29]. The cone bioassay method was developed to assess the bioefficacy of pyrethroid-only LLINs, and the use of this method to test LLINs containing non-neurotoxic insecticides, such as chlorfenapyr, may not necessarily be predictive of field impact, even with increased exposure time of mosquitoes inside the cones [
16,
27]. In contrast, the tunnel test results with pyrethroid-resistant
An. gambiae s.l. Tiassalé strain (field-collected F
0 generation) were more predictive of PermaNet
® Dual efficacy in huts, as observed for other chlorfenapyr-coated nets, such as Interceptor
® G2 [
16,
27,
28]. Tunnel tests provide an increased exposure time of mosquitoes to the LLIN samples being tested, and being an overnight exposure. As the female mosquitoes in the tunnel tests are also exhibiting host-seeking behaviour and are, therefore, metabolically active, the activation of chlorfenapyr following tarsal pickup by mosquitoes may be more effective. The tunnel test was a better predictor of PermaNet
® Dual field efficacy because exposure occurred at night when host-seeking mosquitoes are more vulnerable to chlorfenapyr. Tunnel test thus is a more reliable technique to assess the efficacy of a chlorfenapyr-treated net prior to field trials against free-flying mosquitoes [
16,
38,
39].
In PermaNet
® Dual, deltamethrin and chlorfenapyr contents varied slightly, but complied with the dose interval limits of target specification [
23]. This revealed a good homogeneity of the active ingredients’ distribution over and a good retention of these active ingredients in PermaNet
® Dual, thus resulting in high and similar mortality and blood-feeding inhibition between unwashed samples of PermaNet
® Dual (A) and PermaNet
® Dual (B). The reduction of chemical contents due to the loss of chlorfenapyr and deltamethrin with 20 washes had no apparent effect on PermaNet
® Dual efficacy as the washed samples were still producing high mortality against the pyrethroid-resistant mosquitoes. Overall, PermaNet
® Dual chemical bioavailability was sufficient and produced high mortality in pyrethroid-resistant
Anopheles mosquitoes after 20 standardized washes (corresponding to a 3-year use) and over the hut trial.
The high vector mortality and personal protection effect of the PermaNet
® Dual found in this study are the desired outcomes of any vector control tool. The high mortality effects and the additive blood-feeding inhibition impacts induced by PermaNet
® Dual in this hut trial are expected to substantially diminish the malaria vector density and biting rates in the field, and hence the transmission of malaria in the areas where vectors are resistant to insecticides [
40‐
47]. Additionally, PermaNet
® Dual performed better than the reference PermaNet
® 3.0 and PermaNet
® 2.0 nets, when washed 20 times, thus meeting the WHO hut trial criteria [
23].
However, these results need to be further validated in a large-scale field trial to assess the durability and acceptability of this new tool for malaria vector control [
48‐
50]. A community trial would be the best way to evaluate the community level effect of this promising candidate net (i.e. PermaNet
® Dual) against malaria transmission, by monitoring entomological infection rate,
Plasmodium prevalence and disease incidence [
48‐
50]. Such a community trial could be a randomized controlled trial as with dual-active-ingredient LLINs (e.g. Interceptor G2) in Tanzania [
49], soon in Benin [
48,
50], and the pilot deployments that are part of the New Nets Project led by PATH [
51]. Furthermore, PermaNet
® Dual should be tested against wild populations of
Anopheles funestus [
32], or other main or secondary vectors of malaria in Africa [
52‐
56]. Ultimately, in the present study, the series of hut and laboratory tests demonstrated that the chlorfenapyr component of PermaNet
® Dual could make a major contribution to controlling the pyrethroid-resistant
An. gambiae populations. Furthermore, there were no adverse effects reported among hut sleepers and mosquito collectors during the trial in the huts where PermaNet
® Dual were used, and this may possibly increase the rate of future user acceptability and improve the usage of this net for malaria vector control.