Background
Malaria remains a major public health problem. Last global estimates of the malaria disease burden in 2006 indicate that at least 250 million clinical cases occurred each year, with around 1 million deaths of which 90% occurred in sub-Saharan Africa [
1,
2]. Recommendations of the World Health Organization (WHO-Roll Back Malaria programme) to combat malaria include artemisinin-based combination therapy (ACT) and long-lasting insecticidal nets (LLIN), supported by indoor residual spraying of insecticide (IRS) and intermittent preventive treatment in pregnancy (IPT) [
2]. Recent deployment of such strategies has showed important reduction in malaria-associated morbidity and mortality in settings with moderate to high transmission levels in sub-Saharan Africa [
3‐
5]. Eight LLINs are now recommended by WHOPES for malaria control [
6]. All of them contain pyrethroids because of their fast and high insecticidal properties on mosquitoes as well as their low mammalian toxicity [
7].
Unfortunately, pyrethroid resistance is now widespread in malaria vectors including Western [
8], Central [
9], Eastern [
10], and Southern Africa [
11,
12]. Resistance mechanisms are divided into two groups: metabolic (i.e. alterations in the levels or activities of detoxification proteins) and target site (i.e. non-silent point mutations within structural receptor genes) [
13]. Mutations (L1014F or L1014S) on the gene encoding for the sodium channel, known as knockdown resistance (
kdr), cause resistance to DDT and/or pyrethroid insecticides [
14,
15]. Over-expression of enzymes related to insecticide resistance involves the cytochrome P450-dependent monooxygenases (P450), the carboxylesterases (COE), and the glutathione-S transferases (GST) [
16]. Among these three families, P450s can play a primary role in pyrethroid detoxification and resistance in malaria vectors as recently shown in Benin [
17], Cameroon [
18], Ghana [
19] and South Africa [
20].
There are more and more evidences in the recent literature to support that pyrethroid resistance may seriously impact on malaria vector control [
21]. An experimental hut study carried out in southern Benin in 2004 (Ladji) showed a rather low insecticidal effect of permethrin-treated nets, at WHO recommended dosages against
Anopheles gambiae[
22]. A recent study carried out in the same locality with lambda-cyalothrin used for ITNs and IRS showed a major loss of efficacy associated with
kdr resistance [
23]. Reduced efficacy of permethrin-impregnated bed nets against
An. gambiae strain sharing oxidase-based pyrethroid tolerance was also reported in Cameroon [
24] and Kenya [
25,
26]. Moreover, an increasing number of countries (such as Benin, Ghana and Nigeria) reported the co-occurrence of the L1014F
kdr mutation and increased levels of P450s within the same Anopheline populations [
17,
19]. As demonstrated in
Culex quinquefasciatus, multiplicative interaction (epistasis) between these two types of resistance can lead to extremely high level of resistance to pyrethroids [
27,
28]. Thus, the challenge is not only to manage and control pyrethroid-resistant mosquitoes, but also to deal with the development of "multiple resistance" that may confer resistance to all insecticide classes used in public health (i.e. DDT, carbamates, etc.). Innovative tools are then urgently needed to ensure more effective control of resistant malaria vectors and to help developing countries to achieve the malaria-related Millennium Development Goals i.e. 75% reduction of malaria burden until 2015 [
2].
Among the new tools available in public health, PermaNet
® 3.0, has been designed to improving efficacy against pyrethroid-resistant mosquito populations [
29]. PermaNet
® 3.0 is a mosaic net combining deltamethrin-coated-polyester side panels and a deltamethrin plus piperonyl butoxide (PBO) incorporated-polyethylene roof. PBO has been incorporated to the net as it showed to enhance the effects of deltamethrin against insects by inhibiting metabolic defence systems, mainly P450s [
30].
In this paper, a multi centre study was carried out in western and central Africa to evaluate the performances of this new LLIN technology (PermaNet® 3.0) in comparison with the classical PermaNet® 2.0 recommended by WHO. Experimental hut trials were conducted in Malanville (Benin), Pitoa (Cameroon) and Vallée du Kou (Burkina Faso), where An. gambiae populations showed different levels and types of pyrethroid resistance (i.e. metabolic versus target site modification). Standard WHO procedures in phase II were followed to investigate the efficacy of unwashed and 20 times washed LLINs in terms of induced exophily, blood-feeding inhibition and mortality.
Discussion
A multi-centre experimental hut study was carried out to assess the efficacy of PermaNet
® 3.0 against wild pyrethroid-tolerant
An. gambiae s.s. (Malanville),
kdr-resistant
An. gambiae s.s. (Vallée du Kou) and pyrethroid-resistant
An. arabiensis s.l. showing metabolic resistance (Pitoa). The ABC network offers ideal conditions to address this objective based on the existence of eight experimental hut stations in different ecological and entomological settings (Figure
1). In the present study, three stations where
An. gambiae s.l. showed different level and type of resistance to pyrethroids were selected to assess whether PermaNet
® 3.0 may represent a more potent technology than PermaNet
® 2.0 against pyrethroid resistant mosquito populations.
Differences in behavioural responses between wild Anopheline populations
The results from the control huts showed that the behavioural preferences of Anopheline populations (in terms of endophily/exphily) significantly differ between the three sites as expected from literature on trophic behaviour of malaria vectors [
40‐
42]. It confirms that the behaviour of Anopheline populations depend on several factors including the species, the molecular forms, the resistance mechanisms and other environmental variables [
40‐
42]. Interestingly, there is a difference in mortality rate in the control huts between
An. arabiensis collected in Pitoa (12%) and the two others
An. gambiae populations from Malanville and Vallée du Kou (<5%). Unfortunately, this study did not allow to decipher on the causes of this difference of mortality (behavioural preference, environmental conditions, etc.) but other authors have already reported similar mortality rates of
An. arabiensis (10%) in experimental huts [
43]. Nevertheless these differences shed light on the need for further investigations on behavioural preferences of wild populations of
An. gambiae s.s. and
An. arabiensis.
Comparison between PermaNet® 2.0 and 3.0
The chemical analysis confirmed that in overall, unwashed nets were impregnated with the appropriate target dose of deltamethrin and PBO. Although efficacy of 20 times washed PermaNet
® 3.0 and PermaNet
® 2.0 was good, a rather high loss of insecticide was noted in the side panels (Table
3). Nevertheless, the deltamethrin and PBO retention in the roof was around 2.5 times higher than that of deltamethrin in the side panels, showing that the retention is better with incorporated polyethylene than with coated polyester [
39].
This study first demonstrated a better or equal impact of PermaNet® 3.0 washed 20 times on mortality and blood feeding inhibition of major malaria vectors compared with that of the conventionally treated polyester nets (25 mg/m2 AI) washed until just before exhaustion. This confirms that the PermaNet® 3.0 fulfils the WHOPES efficacy criteria of Phase II studies for LLIN.
Regarding the two LLINs, unwashed Permanet 3.0 induced significantly higher BFI and mortality than Permanet 2.0 in Vallée du Kou and Malanville. In the locality of Pitoa, the BFI was however higher with Permanet 2.0 than Permanet 3.0 but the mortality was still higher with Permanet 3.0. After 20 washes, the PermaNet® 3.0 also induced higher insecticidal effect than PermaNet® 2.0 in the pyrethroid resistance areas of Pitoa and Vallée du Kou, but performed equally in the area of Malanville.
One should note that in areas with high resistance levels (Vallée du Kou) 50% of resistant mosquitoes survived after exposure to PermaNet
® 3.0 relative to 75% survival after exposure to PermaNet
® 2.0. It remains to be seen if the gain of efficacy of PermaNet
® 3.0 over PermaNet
® 2.0 is enough to control highly pyrethroid-resistant malaria vector populations. Here, it is difficult to conclude on the benefit of using PBO on the roof because the deltamethrin content on PermaNet
® 3.0 was approximately twice higher than that of PermaNet
® 2.0. So the better efficacy on resistant mosquitoes could be impeded either to the higher deltamethrin concentration or to the PBO itself or both. Other field studies did not show an increase of efficacy on resistant
Culex and pyrethroid susceptible
An. gambiae s.s. [
44] as well as deltamethrin-resistant
Anopheles epiroticus[
45].
The threat of insecticide resistance mechanisms
This multi-centre study provided also more evidence that pyrethroid resistance can seriously reduce the efficacy of pyrethroid -treated materials in malaria vectors [
21,
22]. Results obtained in Vallée du Kou showed a strong reduction of ITNs efficacy where the
kdr mutation frequency was high (e.g. personal protection of CTN washed to just before exhaustion ranged from 88% in Malanville to 24% in Vallée du Kou and the insecticidal effect ranged from 60% in Malanville to 25% in Vallée du Kou). The same trend was observed with PermaNet
® 2.0, confirming that the
Kdr mutation is an important predictor of pyrethroid resistance phenotype in malaria vectors as previously described [
23,
46]. Lower insecticidal activity and personal protection were already demonstrated in West Africa with pyrethroid resistant mosquito populations using either Olyset
® net or PermaNet
®[
47] and also insecticide treated plastic sheetings [
48]. Unfortunately, in most malaria endemic countries,
An. gambiae populations are sharing very high frequency of
Kdr mutation [
8,
49‐
51] alone or in combination with metabolic resistance [
16,
18]. In Pitoa, where
An. arabiensis show higher metabolism through elevated oxidase and esterase activity [
33], CTN efficacy was intermediate (PP and IE were 63.6% and 33.2%, respectively), suggesting that metabolic resistance could also reduce ITN efficacy [
24]. This finding supports the global warning about the spread of the pyrethroid resistance although there is no evidence yet for a malaria control failure using LLIN at an operational scale [
52].
Conclusion
To summarize, the present study showed that the new long-lasting bed nets PermaNet
® 3.0 caused better efficacy against both
Kdr and metabolic resistant malaria vectors than PermaNet
® 2.0. Nevertheless in areas of strong resistance like the Vallée du Kou, a large number of exposed mosquitoes survived after exposure to both LLINs. Then as a short term prospect, it seems essential to evaluate this tool in others areas of strong resistance like southern Benin, southern Nigeria and Côte d'Ivoire. It is also crucial to strengthen the collaboration between companies and Research Institutions to find alternative tools for malaria vector control (e.g. using mixtures of unrelated compounds for LLINs [
53‐
57] and/or the use of insecticide-treated plastic sheeting and LLINs [
58]), because the race towards an insecticide with a new mode of action will be long and expensive.
Acknowledgements
The experimental hut trial performed in Malanville (Benin) was carried out within the framework of the WHO Pesticide Evaluation Scheme (WHOPES) initiative for research and development of alternative insecticides for vector control. Trials carried out in Pitoa (Cameroon) and the Vallée du Kou (Burkina Faso) were supported by a grant from Vestergaard Frandsen SA, (Switzerland). We are grateful to the team of Malanville, Pitoa and the Vallée du Kou for their excellence during field work, and the people of these villages for their hospitality. We also thank Mr. Nicolas Moiroux for having set up of the map of the ABC network and Dr. Pennetier Cedric for revising the manuscript.
Competing interests
The authors received financial support from Vestergard Frandsen Company to carry out the experimental huts trials in Burkina Faso and Cameroon. However, the authors have strictly followed the WHOPES procedures for testing and evaluation of the efficacy of Permanet 3.0 against malaria vectors. The Research teams involve in this study (i.e. IRD, CREC, OCEAC, CM and CRA-W) have no competing and commercial interests with the manufacturer.
Authors' contributions
VC designs the study and drafted the manuscript. JC, RDD, JE, PN carried out bioassays and conducted the experimental hut trials. OP conducted the chemical analysis on nets. MA and JMH helped design the study and critically revised the manuscript. All authors read and approved the final manuscript.