Evidence supporting deployment of next generation insecticide treated nets in Burkina Faso: bioassays with either chlorfenapyr or piperonyl butoxide increase mortality of pyrethroid-resistant Anopheles gambiae

Burkina Faso relies on mass distribution of insecticide-treated nets (ITNs) every three years as the primary method of vector control, while indoor residual spraying (IRS) has been implemented in two or three districts per year since 2018 [1]. Millions of pyrethroid ITNs have been distributed in Burkina Faso during the past 20 years which, along with agricultural use of pyrethroids, has increased the selection pressure on malaria vector populations. Pyrethroid resistance was observed in Anopheles gambiae sensu lato (s.l.) populations for the first time in Côte d’Ivoire in 1993 [2], and within a few years was detected throughout all countries in West Africa, including Burkina Faso, and is now found across all of sub-Saharan Africa [3, 4]. The voltage-gated sodium channel gene (Vgsc)-L1014F mutation (also known as kdr-west) was assumed to be the main mechanism conferring resistance to pyrethroids in West Africa [5, 6], while in East Africa, a different amino acid substitution at the same locus known as Vgsc-L1014S (also known as kdr-east) had been detected [7]. Voltage-gated sodium channel gene mutations were first identified within An. gambiae populations [5, 6] across West Africa before it was detected within Anopheles coluzzii [8], most likely as a result of introgression, while its occurrence in wild Anopheles arabiensis populations was identified as an independent mutation [9]. While Vgsc mutations are widespread and contribute to phenotypic pyrethroid resistance, metabolic resistance mechanisms are generally more important drivers of high intensity pyrethroid resistance [10].

Pyrethroid resistance poses a threat to the efficacy of ITNs, particularly when resistance is present at high intensity [11, 12]. In Burkina Faso, pyrethroid resistance has been observed nationwide for several years and there are fears that pyrethroid ITNs may no longer provide the desired levels of individual and community protection [6]. While there are several localized studies in Burkina Faso that have described the genetic mutations involved in pyrethroid resistance, including Vgsc-1014F and 1014S mutations in association with N1575Y mutations and metabolic resistance patterns [13, 14], there is limited bioassay information regarding the relative improvement in mortality provided by piperonyl butoxide (PBO) synergists within wild An. gambiae s.l. populations. Reduced mosquito mortality by pyrethroid ITNs was reported in experimental hut trials in Benin in 2007 [15] and more recently in Burkina Faso [16] with free-flying An. gambiae s.l. In order to preserve the efficacy of ITNs and other insecticide-based control methods such as IRS, the World Health Organization (WHO) has developed a global plan for insecticide resistance management (GPIRM) [17]. Among the key elements are: (i) rotation of insecticides; (ii) mixtures of at least two different insecticides; (iii) alternating use of at least two insecticides from different classes; and, (iv) mosaic use of insecticides.

A limiting factor preventing implementation of these strategies has been the lack of alternative classes of insecticide for ITNs. However, in recent years several PBO synergist nets have received prequalification (PQ) listing by the WHO [18]. PBO is a synergist that acts by inhibiting metabolic enzymes within the mosquito, particularly cytochrome P450s that detoxify or sequester pyrethroids [19]. Although synergists do not typically act as insecticides, they can increase or restore the potency of insecticides by overcoming existing resistance mechanisms [19, 20]. As a result, PBO nets should produce an increased killing effect of malaria vectors where the major pyrethroid resistance mechanisms are due to increased oxidase activity. Two randomized control trials in Tanzania and Uganda have demonstrated a clinical benefit of PBO nets in areas of moderate pyrethroid resistance, with a reduction in malaria prevalence detected for at least six months after net distribution compared to conventional pyrethroid nets [21, 22].

A new ‘dual active ingredient’ net with two different active ingredients (AIs) is Interceptor® G2 (combining chlorfenapyr and alpha-cypermethrin), which received WHO PQ listing in 2018 [18]. Chlorfenapyr is a pyrrole compound with a non-neurotoxic mode of action which shows no cross resistance to existing pyrethroid resistance mechanisms [23, 24]. Experimental hut studies of Interceptor G2 have shown high efficacy and wash durability against pyrethroid resistant malaria vectors in Benin [25], Burkina Faso [26] and Côte d’Ivoire [27].

To guide National Malaria Control Programme (NMCP) decision-making regarding choice of ITN type, it is crucial to regularly gather insecticide susceptibility data for those insecticides used on ITNs, namely pyrethroids with and without PBO synergist, and chlorfenapyr. This study evaluated the level of pyrethroid resistance intensity in An. gambiae s.l. against the main pyrethroids used on ITNs followed by synergist assays to determine whether the synergist PBO restores susceptibility. In addition, the study determined susceptibility of An. gambiae s.l. to chlorfenapyr.

This study have demonstrated for the first time, using standardized WHO protocols [28], that high intensity pyrethroid resistance is not geographically confined to areas of intense agriculture but is widespread throughout Burkina Faso. Given that the primary malaria vector control strategy in Burkina Faso is nationwide universal coverage with ITNs, of particular concern is the high intensity of resistance to deltamethrin and alpha-cypermethrin documented at every site tested. Pyrethroid resistance intensity is an important measure that may indicate the potential for reduced effectiveness of pyrethroid ITNs. WHO suggest that “confirmed levels of resistance, especially at ten times the discriminating concentration may indicate or predict operational control failure and highlight a particularly urgent need to develop an appropriate resistance management strategy” [28]. High intensity resistance was previously only documented in intensive agricultural areas, such as Vallée du Kou, an area of concentrated rice cultivation 25 km from Bobo-Dioulasso, where deltamethrin resistance up to 1,000 fold was reported in An. coluzzii in 2013 compared to the susceptible Kisumu strain of An. gambiae [16].

To supplement universal coverage with pyrethroid ITNs, which are deployed via mass campaigns every three years, US President’s Malaria Initiative (PMI)-funded IRS with non-pyrethroid insecticides (organophosphates and neonicotinoids) has also been conducted annually since 2018 in three high burden districts (Kampti, Solenzo, Kongoussi) in accordance with the WHO GPIRM recommendations. However, the relatively high cost of IRS makes the prospect of national use of this strategy low and PBO or dual active ingredient nets are likely to be the primary response for control of pyrethroid-resistant malaria vectors. A barrier limiting the purchase of PBO nets by NMCPs and donors is that PBO ITNs cost approximately 40% more than equivalent pyrethroid ITNs [33]. Therefore, it is important to target distribution to locations where PBO synergist bioassays indicate full or partial restoration of susceptibility to pyrethroids. Toé et al. [34] showed in Vallée du Kou and Tengrela (southwestern Burkina Faso) evidence of synergism but pre-exposure to PBO did not fully restore susceptibility to deltamethrin or permethrin in bioassays or in experimental hut studies. Bayili et al. [35] also showed in experimental hut studies in Vallée du Kou that deltamethrin plus PBO nets provided additional benefit over the equivalent pyrethroid-only LLINs, but mortality and blood-feeding inhibition rates were relatively low. While these studies provided important information, they were limited in geographical scope. Results from study showed that mosquito mortality rates nationwide significantly increased when An. gambiae s.l. were pre-exposed to PBO in association with permethrin, deltamethrin and alpha-cypermethrin even in areas of high resistance intensity in western part of country (Mangodara, Gaoua, Kampti). In all sites, pre-exposure to PBO also reduced the mean KDT compared to exposure to only pyrethroids. While randomized controlled trials have shown the benefit of PBO nets in Tanzania [21] and Uganda [22] in areas of moderate pyrethroid resistance, it is not clear what level of increased mortality in susceptibility bioassays is required to correlate with epidemiological impact. WHO provide limited guidance, stating that the added benefit of pyrethroid PBO nets compared to pyrethroid-only LLINs is expected to be the greatest where pyrethroid resistance is at “intermediate levels”, where mosquito mortality after exposure to a pyrethroid insecticide in WHO test kits ranges from 10 to 80%, but do not state what level of mortality increase is biologically significant [36]. The most recent PMI Malaria Operational Plan Guidance recommends that PBO nets only be considered in areas where PBO increases pyrethroid susceptibility by at least 10% (absolute terms) [37]. Based on results from study, PBO plus deltamethrin ITNs are the most promising option in Burkina Faso as greater than 70% mortality was reached from bioassays in 12 of 15 sites, with susceptibility restored in one site and a greater than 10% absolute increase in mortality in all sites.

Laboratory analysis showed that the frequency of the Vgsc-L101F mutation is moderate or high in An. gambiae s.s., An. coluzzii and An. arabiensis. The frequency of the Vgsc-L1014S mutation is also increasing in frequency compared to prior years, particularly in An. gambiae s.s. [38]. Surprising results found also were the similar allele mutations frequencies in both species An. coluzzi and An. arabiensis. Indeed, this finding could be explained by the colonization of An. coluzzi breeding sites by An. arabiensis species particularly in urban sites exposed to insecticides pressure. Previous microarray analysis of An. coluzzii from Vallée du Kou has implicated several P450 genes including CYP6P3 and CYP6Z2 [39]. Similarly, Toé et al. [34] found CYP6Z2 and CYP6Z3 the most highly overexpressed genes in Vallée du Kou and Tengrela. This suggests that there are complex underlying resistance mechanisms involved, including target site resistance, metabolic resistance mechanisms and there may be other mechanisms not yet detected, such as increased cuticle thickness. It is important for further studies to be conducted over a wider geographical distribution to do molecular and genetic investigations to have a better understanding of the specific cytochrome P450 enzymes and mono-oxygenases.

While there is a good prospect that PBO plus deltamethrin ITNs will provide greater control than pyrethroid ITNs in most locations of Burkina Faso in the short term, there are already examples in parts of Côte d’Ivoire [40] and Mozambique [41] where PBO nets no longer provide additional benefit due to the high intensity of pyrethroid resistance and the resistance mechanisms present. Interceptor G2 may be a longer term alternative for improved control of pyrethroid-resistant mosquitoes as chlorfenapyr has a completely different mode of action and works through oxidative phosphorylation of the cell mitochondria [42]. Large-scale testing of susceptibility to chlorfenapyr at the interim diagnostic dose of 100 µg/bottle was conducted for the first time in Burkina Faso and showed susceptibility in nearly all sites. Experimental hut trials in Burkina Faso [26], Benin [25] and Côte d’Ivoire [27] have shown higher vector mortality with Interceptor G2 even when washed 20 times to simulate field use. The results of randomized controlled trials being conducted in Benin and Tanzania to determine the epidemiological impact of Interceptor G2 nets compared to pyrethroid nets are eagerly awaited before widespread distribution can be recommended by WHO. In the interim, as part of the ‘New Nets Project’, pilot distribution of PBO and Interceptor G2 nets was conducted in parts of Burkina Faso in 2019 [43]. Districts were selected to receive PBO and Interceptor G2 nets based on the results of insecticide resistance monitoring, along with other operational criteria. Given the increased costs of PBO and Interceptor G2 nets it is important to continue monitoring their lifetime durability including physical integrity, residual insecticidal efficacy and chemical retention over three years.

High pyrethroid resistance intensity in malaria vector species is widespread across Burkina Faso and may be a predictor of reduced pyrethroid ITN effectiveness. Pre-exposure to the synergist PBO significantly increased vector mortality, resulting in partial or full restoration of susceptibility in most sites. PBO plus deltamethrin ITNs should be prioritized for distribution in Burkina Faso as this combination provided the highest levels of mortality increase. However, susceptibility was not restored in most sites and dual active ingredient nets, such as Interceptor G,2 may be a better long-term solution.